G2Cdb::Gene report

Gene id
G00001419
Gene symbol
MAPK1 (HGNC)
Species
Homo sapiens
Description
mitogen-activated protein kinase 1
Orthologue
G00000170 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000030508 (Vega human gene)
Gene
ENSG00000100030 (Ensembl human gene)
5594 (Entrez Gene)
57 (G2Cdb plasticity & disease)
MAPK1 (GeneCards)
Literature
176948 (OMIM)
Marker Symbol
HGNC:6871 (HGNC)
Protein Expression
4229 (human protein atlas)
Protein Sequence
P28482 (UniProt)

Synonyms (4)

  • ERK
  • ERK2
  • MAPK2
  • p41mapk

Literature (906)

Pubmed - other

  • RNAi-mediated MEK1 knock-down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo.

    Gailhouste L, Ezan F, Bessard A, Frémin C, Rageul J, Langouët S and Baffet G

    EA 4427-SeRAIC, IFR 140, Université de Rennes 1, F-35043 Rennes, France.

    The mitogen-activated protein kinases MEK/ERK pathway regulates fundamental processes in malignant cells and represents an attractive target in the development of new cancer treatments especially for human hepatocarcinoma highly resistant to chemotherapy. Although gene extinction experiments have suggested distinct roles for these proteins, the MEK/ERK cascade remains widely considered as exhibiting an overlap of functions. To investigate the functionality of each kinase in tumorigenesis, we have generated stably knock-down clones for MEK1/2 and ERK1/2 isoforms in the human hepatocellular carcinoma line HuH7. Our results have shown that RNAi strategy allows a specific disruption of the targeted kinases and argued for the critical function of MEK1 in liver tumor growth. Transient and stable extinction experiments demonstrated that MEK1 isoform acts as a major element in the signal transduction by phosphorylating ERK1 and ERK2 after growth factors stimulation, whereas oncogenic level of ERK1/2 phosphorylation appears to be MEK1 and MEK2 dependent in basal condition. In addition, silencing of MEK1 or ERK2 abolished cell proliferation and DNA replication in vitro as well as tumor growth in vivo after injection in rodent. In contrast, targeting MEK2 or ERK1 had no effect on hepatocarcinoma progression. These results strongly corroborate the relevance of targeting the MEK cascade as attested by pharmacologic drugs and support the potential application of RNAi in future development of more effective cancer therapies. Our study emphasizes the importance of the MEK/ERK pathway in human hepatocarcinoma cell growth and argues for a crucial role of MEK1 and ERK2 in this regulation.

    International journal of cancer 2010;126;6;1367-77

  • Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo.

    Zittermann SI, Capurro MI, Shi W and Filmus J

    Division of Molecular and Cellular Biology, Sunnybrook Research Institute and Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.

    The heterogeneity of the molecular pathology of HCC poses a formidable obstacle to the development of non-cytotoxic therapies. Several pro-tumorigenic signaling pathways can be aberrantly activated in HCC, including those triggered by Wnts. Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan that is overexpressed in most HCCs, promotes the growth of these tumors by stimulating Wnt signaling. Because GPC3 binds with high affinity to Wnts, and its growth-promoting activity requires attachment to the cell membrane, we have hypothesized that a mutated GPC3 lacking the GPI anchoring domain (sGPC3) will block Wnt signaling and inhibit the growth of Wnt-dependent tumors. In addition, because sGPC3 displays heparan sulfate chains, this secreted glypican could also inhibit HCC growth by blocking the activity of other heparin-binding growth factors. To test this hypothesis, HCC cell lines were infected with an sGPC3-expressing lentivirus or virus control, and the effect of sGPC3 on the in vitro and in vivo growth was investigated. In addition, the signaling pathways targeted by sGPC3 were identified. We observed that sGPC3-expressing cells had lower proliferation rate. In addition, sGPC3 significantly inhibited the in vivo growth of the Huh6, HepG2 and Huh7 HCC cell lines. sGPC3 blocked Wnt signaling in Huh6- and Huh7-derived tumors and Erk1/2 and Akt phosphorylation in tumors generated by Huh7 and HepG2 cells, respectively. An anti-angiogenic effect in Huh7 and HepG2-derived tumors was also observed. We conclude that sGPC3 can inhibit HCC tumorigenicity by blocking the activity of several pro-tumorigenic growth factors.

    International journal of cancer 2010;126;6;1291-301

  • Redox proteomic analysis of carbonylated brain proteins in mild cognitive impairment and early Alzheimer's disease.

    Sultana R, Perluigi M, Newman SF, Pierce WM, Cini C, Coccia R and Butterfield DA

    Department of Chemistry, University of Kentucky , Lexington, KY, USA.

    Previous studies indicated increased levels of protein oxidation in brain from subjects with Alzheimer's disease (AD), raising the question of whether oxidative damage is a late effect of neurodegeneration or precedes and contributes to the pathogenesis of AD. Hence, in the present study we used a parallel proteomic approach to identify oxidatively modified proteins in inferior parietal lobule (IPL) from subjects with mild cognitive impairment (MCI) and early stage-AD (EAD). By comparing to age-matched controls, we reasoned that such analysis could help in understanding potential mechanisms involved in upstream processes in AD pathogenesis. We have identified four proteins that showed elevated levels of protein carbonyls: carbonic anhydrase II (CA II), heat shock protein 70 (Hsp70), mitogen-activated protein kinase I (MAPKI), and syntaxin binding protein I (SBP1) in MCI IPL. In EAD IPL we identified three proteins: phosphoglycerate mutase 1 (PM1), glial fibrillary acidic protein, and fructose bisphospate aldolase C (FBA-C). Our results imply that some of the common targets of protein carbonylation correlated with AD neuropathology and suggest a possible involvement of protein modifications in the AD progression.

    Funded by: NIA NIH HHS: AG-05119, AG-10836

    Antioxidants & redox signaling 2010;12;3;327-36

  • The dopamine-somatostatin chimeric compound BIM-23A760 exerts antiproliferative and cytotoxic effects in human non-functioning pituitary tumors by activating ERK1/2 and p38 pathways.

    Peverelli E, Olgiati L, Locatelli M, Magni P, Fustini MF, Frank G, Mantovani G, Beck-Peccoz P, Spada A and Lania A

    Dept. of Medical Sciences, University of Milan, Fondazione IRCCS Ospedale Maggiore Policlinico, Milano, Italy.

    The study investigated the effects of the dopamine-somatostatin chimeric compound BIM-23A760 on cell proliferation and apoptosis in cultured cells from human non-functioning pituitary tumors (NFPTs). Both BIM-23A760 and the dopaminergic agonist BIM-53097 induced a significant inhibition of cell proliferation associated with increased p27 expression, together with a significant increase in caspase-3 activity. Conversely, null or marginal effects were elicited by somatostatin analogs. Moreover, BIM-23A760 and BIM-53097 induced ERK1/2 and p38 phosphorylation and the blockade of these pathways prevented both the antiproliferative and the pro-apoptotic effects of these drugs. In conclusions the chimeric compound BIM-23A760 is able to exert cytostatic and cytotoxic effects in NFPTs, these phenomena being mainly mediated by DR2D and involving ERK1/2 and p38 pathways activation.

    Cancer letters 2010;288;2;170-6

  • Bile acid reflux contributes to development of esophageal adenocarcinoma via activation of phosphatidylinositol-specific phospholipase Cgamma2 and NADPH oxidase NOX5-S.

    Hong J, Behar J, Wands J, Resnick M, Wang LJ, Delellis RA, Lambeth D and Cao W

    Department of Medicine, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.

    Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA.

    Funded by: NIDDK NIH HHS: R01 DK080703, R01 DK080703-01A1

    Cancer research 2010;70;3;1247-55

  • Biological effects induced by insulin-like growth factor binding protein 3 (IGFBP-3) in malignant melanoma.

    Oy GF, Slipicevic A, Davidson B, Solberg Faye R, Maelandsmo GM and Flørenes VA

    Department of Tumor Biology, Institute for Cancer Research, Oslo, Norway.

    The insulin like growth factor (IGF) signaling pathway has been shown to contribute to melanoma progression, but little is known about the role of the IGF binding protein 3 (IGFBP-3) in melanoma biology. The aim of the present study was to characterize expression, function and regulation of IGFBP-3 in malignant melanomas and study its potential as a biomarker. The expression of IGFBP-3 varied between different human melanoma cell lines and reintroduction of the protein in non-expressing cells led to induction of apoptosis. Interestingly, in cell lines expressing endogenous IGFBP-3, siRNA silencing of the protein led to a cell line-dependent decrease in proliferation, but had no effect on apoptosis and invasion. Examination of patient material showed that IGFBP-3 is unexpressed in benign nevi while a slight increase in protein expression was seen in primary and metastatic melanoma. However, expression of the protein was low and no correlation was found with circulating levels of IGFBP-3 in serum, suggesting that IGFBP-3 has limited potential as a predictive marker in malignant melanoma. We showed that promoter methylation of IGFBP-3 occurred in both melanoma cell lines and patient material, implicating epigenetic silencing as a regulation mechanism. Furthermore, expression of the protein was shown to be regulated by the PI3-kinase/AKT and MAPK/ERK1/2 pathways. In summary, our findings suggest that IGFBP-3 can exert dual functional effects influencing both apoptosis and proliferation. Development of resistance to the antiproliferative effects of IGFBP-3 may be an important step in progression of malignant melanomas.

    International journal of cancer 2010;126;2;350-61

  • c-Kit mutants require hypoxia-inducible factor 1alpha to transform melanocytes.

    Monsel G, Ortonne N, Bagot M, Bensussan A and Dumaz N

    INSERM U976, Hôpital Saint Louis, Paris, France.

    Many studies have highlighted the critical role of c-Kit in normal melanocyte development but its role in melanoma development remains unclear. Although c-Kit expression is often lost during melanoma progression, a subset of melanoma has been found to overexpress c-Kit and mutations activating c-Kit have recently been identified in some acral and mucosal melanoma. To address the role of these c-Kit mutants in the transformation of melanocytes, we characterized the physiological responses of melanocytes expressing the most frequent c-Kit mutants found in melanoma (K642E and L576P) and a novel mutant we identified in an acral melanoma. We analysed signaling pathways activated downstream of c-Kit and showed that all three mutants led to a strong activation of the phosphatidyl-inositol-3 kinase (PI3K) pathway but only weak activation of the Ras/Raf/Mek/Erk pathway, which was not sufficient to promote uncontrolled melanocyte proliferation and transformation. However, in hypoxic conditions or coexpressed with a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha), c-Kit mutants activate the Ras/Raf/Mek/Erk pathway, stimulate proliferation and transform melanocytes. Proliferation of melanocytes transformed by these mutants was specifically inhibited by imatinib. These results show for the first time that melanocytes require a specific epigenetic environment to be transformed by c-Kit mutants and highlight a distinct molecular mechanism of melanocyte transformation.

    Oncogene 2010;29;2;227-36

  • Novel nuclear nesprin-2 variants tether active extracellular signal-regulated MAPK1 and MAPK2 at promyelocytic leukemia protein nuclear bodies and act to regulate smooth muscle cell proliferation.

    Warren DT, Tajsic T, Mellad JA, Searles R, Zhang Q and Shanahan CM

    British Heart Foundation Centre, Division of Cardiovascular Medicine, King's College London, London SE5 9NU, United Kingdom. derek.warren@kcl.ac.uk

    Nuclear and cytoplasmic scaffold proteins have been shown to be essential for temporal and spatial organization, as well as the fidelity, of MAPK signaling pathways. In this study we show that nesprin-2 is a novel extracellular signal-regulated MAPK1 and 2 (ERK1/2) scaffold protein that serves to regulate nuclear signaling by tethering these kinases at promyelocytic leukemia protein nuclear bodies (PML NBs). Using immunofluorescence microscopy, GST pull-down and immunoprecipitation, we show that nesprin-2, ERK1/2, and PML colocalize and bind to form a nuclear complex. Interference of nesprin-2 function, by either siRNA-mediated knockdown or overexpression of a dominant negative nesprin-2 fragment, augmented ERK1/2 nuclear signaling shown by increased SP1 activity and ELK1 phosphorylation. The functional outcome of nesprin-2 disruption and the resultant sustained ERK1/2 signal was increased proliferation. Importantly, these activities were not induced by previously identified nuclear envelope (NE)-targeted nesprin-2 isoforms but rather were mediated by novel nuclear isoforms that lacked the KASH domain. Taken together, this study suggests that nesprin-2 is a novel intranuclear scaffold, essential for nuclear ERK1/2 signaling fidelity and cell cycle progression.

    Funded by: British Heart Foundation: RG/05/001, RG/09/008/27923

    The Journal of biological chemistry 2010;285;2;1311-20

  • S100B interaction with the receptor for advanced glycation end products (RAGE): a novel receptor-mediated mechanism for myocyte apoptosis postinfarction.

    Tsoporis JN, Izhar S, Leong-Poi H, Desjardins JF, Huttunen HJ and Parker TG

    Division of Cardiology, Department of Medicine, Keenan Research Centre, Li Ka Shing Knowledge Institute, St Michael's Hospital, University of Toronto, Canada.

    Rationale: Post-myocardial infarction ventricular remodeling is associated with the expression of a variety of factors including S100B that can potentially modulate myocyte apoptosis.

    Objective: This study was undertaken to investigate the expression and function of S100B and its receptor, the receptor for advanced glycation end products (RAGE) in both postinfarction myocardium and in a rat neonatal myocyte culture model.

    In a rat model of myocardial infarction following coronary artery ligation, we demonstrate in periinfarct myocytes, upregulation of RAGE, induction of S100B, and release into plasma with consequent myocyte apoptosis. Using a coimmunoprecipitation strategy, we demonstrate a direct interaction between S100B and RAGE. In rat neonatal cardiac myocyte cultures, S100B at concentrations > or = 50 nmol/L induced myocyte apoptosis, as evidenced by increased terminal DNA fragmentation, TUNEL, cytochrome c release from mitochondria to cytoplasm, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p53, increased expression and activity of proapoptotic caspase-3, and decreased expression of antiapoptotic Bcl-2. Transfection of a full-length cDNA of RAGE or a dominant-negative mutant of RAGE resulted in increased or attenuated S100B-induced myocyte apoptosis, respectively. Inhibition of ERK1/2 by U0126/PD-98059 or overexpression of a dominant negative p53 comparably inhibited S100B-induced myocyte apoptosis.

    Conclusions: These results suggest that interaction of RAGE and its ligand S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53 signaling. This receptor-mediated mechanism is uniquely amenable to therapeutic intervention.

    Funded by: Canadian Institutes of Health Research

    Circulation research 2010;106;1;93-101

  • Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling.

    Yalcin A, Clem B, Makoni S, Clem A, Nelson K, Thornburg J, Siow D, Lane AN, Brock SE, Goswami U, Eaton JW, Telang S and Chesney J

    Department of Biochemistry and Molecular Biology, James Graham Brown Cancer Center (Molecular Targets Program), University of Louisville, Louisville, KY 40202, USA.

    Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras(V12) in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.

    Funded by: NCI NIH HHS: 1 R01 CA11642801

    Oncogene 2010;29;1;139-49

  • Betuletol 3-methyl ether induces G(2)-M phase arrest and activates the sphingomyelin and MAPK pathways in human leukemia cells.

    Rubio S, Quintana J, Eiroa JL, Triana J and Estévez F

    Department of Biochemistry and Molecular Biology, University of Las Palmas de Gran Canaria, Spain.

    Betuletol 3-methyl ether (BME) is a natural phenylbenzo-gamma-pyrone that inhibits cell proliferation in human tumor cell lines and induces apoptotic cell death in HL-60 cells. Here we show that BME displays strong cytotoxic properties in several human leukemia cell lines (U937, K-562, THP-1, Jurkat, and Molt-3) and in cells that over-express two anti-apoptotic proteins, namely Bcl-2 and Bcl-x(L). BME arrested HL-60 cells at G(2)-M phase of the cell cycle, which was associated with the accumulation of cyclin B1 and p21(Cip1). Fluorescence microscopy experiments suggest that BME blocked the cell cycle in mitosis. The in vivo tubulin polymerization assay shows that BME inhibits tubulin polymerization and causes similar changes of cellular microtubule network as colchicine. Our results demonstrate that BME-induced cell death is (i) triggered in human myeloid leukemia cell that over-express Bcl-2 and Bcl-x(L), and (ii) associated with loss of inner mitochondrial membrane potential (DeltaPsim) and an increase in reactive oxygen species (ROS). Although ROS increased in response to BME, this did not seem to play a pivotal role in the apoptotic process since the anti-oxidant trolox was unable to provide cell protection. The treatment of HL-60 cells with BME induces the activation of mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinases, p38 mitogen-activated protein kinases and extracellular signal-regulated kinases (ERK)1/2 and stimulates the acid sphingomyelinase with concomitant ceramide generation. The findings of this study suggest that BME could be useful in the development of novel anticancer agents.

    Molecular carcinogenesis 2010;49;1;32-43

  • Co-engagement of alpha(4)beta(1) integrin (VLA-4) and CD4 or CD8 is necessary to induce maximal Erk1/2 phosphorylation and cytokine production in human T cells.

    Kim TK, Billard MJ, Wieder ED, McIntyre BW and Komanduri KV

    Adult Stem Cell Transplant Program, University of Miami, Sylvester Comprehensive Cancer Center, Miami, Florida 33136, USA.

    The alpha(4)beta(1) integrin VLA-4 (very-late activation antigen-4) and the lineage-specific CD4 and CD8 receptors have been proposed as putative co-stimulatory receptors on T cells. To assess the relative contribution of signaling through the TCR, CD28 and these accessory molecules, we activated human T cells using soluble antibodies recognizing all four of these T-cell receptor classes (CD3, CD28, CD4/CD8, and VLA-4), and we assessed the degree of activation using higher-order flow cytometry detecting intracellular Erk1/2 phosphorylation and production of IL-2 and IFN-gamma. We found that: (1) co-stimulation via CD4/CD8, in addition to CD28, is required for optimal T-cell activation; (2) VLA-4 binding consistently potentiates CD4(+) and CD8(+) T-cell activation; (3) augmentation of T-cell activation through VLA-4 binding is most pronounced following engagement of CD4/CD8. These results confirm that multiple signals, including VLA-4 engagement, are necessary for maximal T-cell activation beyond that induced via the TCR and CD28.

    Funded by: NCI NIH HHS: R01 CA109326, R01 CA109326-04; NHLBI NIH HHS: R01 HL091749, R01 HL091749-01

    Human immunology 2010;71;1;23-8

  • Helicobacter pylori lipopolysaccharides upregulate toll-like receptor 4 expression and proliferation of gastric epithelial cells via the MEK1/2-ERK1/2 mitogen-activated protein kinase pathway.

    Yokota S, Okabayashi T, Rehli M, Fujii N and Amano K

    Department of Microbiology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan. syokota@sapmed.ac.jp

    Helicobacter pylori is recognized as an etiological agent of gastroduodenal diseases. H. pylori produces various toxic substances, including lipopolysaccharide (LPS). However, H. pylori LPS exhibits extremely weakly endotoxic activity compared to the typical LPS, such as that produced by Escherichia coli, which acts through Toll-like receptor 4 (TLR4) to induce inflammatory molecules. The gastric epithelial cell lines MKN28 and MKN45 express TLR4 at very low levels, so they show very weak interleukin-8 (IL-8) production in response to E. coli LPS, but pretreatment with H. pylori LPS markedly enhanced IL-8 production induced by E. coli LPS by upregulating TLR4 via TLR2 and the MEK1/2-ERK1/2 pathway. The transcription factor NF-Y was activated by this signal and promoted transcription of the tlr4 gene. These MEK1/2-ERK1/2 signal-mediated activities were more potently activated by LPS carrying a weakly antigenic epitope, which is frequently found in gastric cancers, than by LPS carrying a highly antigenic epitope, which is associated with chronic gastritis. H. pylori LPS also augmented the proliferation rate of gastric epithelial cells via the MEK1/2-ERK1/2 pathway. H. pylori LPS may be a pathogenic factor causing gastric tumors by enhancing cell proliferation and inflammation via the MEK1/2-ERK1/2 mitogen-activated protein kinase cascade in gastric epithelial cells.

    Infection and immunity 2010;78;1;468-76

  • Hypoxia switches glucose depletion-induced necrosis to phosphoinositide 3-kinase/Akt-dependent apoptosis in A549 lung adenocarcinoma cells.

    Kim CH, Ko AR, Lee SY, Jeon HM, Kim SM, Park HG, Han SI and Kang HS

    Department of Molecular Biology, College of Natural Sciences, and Research Institute of Genetic Engineering, Pusan National University, Pusan 609-735, Korea.

    In solid tumours, necrosis is commonly found in the core region in response to metabolic stress that results from oxygen and glucose depletion (OGD) due to insufficient vascularization and has been implicated in tumour progression. We have previously shown that metabolic stress due to glucose depletion (GD) induces necrosis and HMGB1 release through mitochondrial ROS production in A549 lung adenocarcinoma cells. In this study, we examined the effects of hypoxia on GD-induced necrosis and show that hypoxia prevented GD-induced mitochondrial ROS production, HMGB1 release, and necrosis and switched the cell death mode to apoptosis that is dependent on caspase-3 and -9. We further found that inhibition of ERK1/2 by U0126 abolished the effects of hypoxia to switch the cell death mode and to suppress mitochondrial ROS production, indicating an important role(s) of the ERK pathway in cell death mode determination. We also found that during OGD-induced apoptosis the prosurvival protein kinase Akt is activated and inhibition of Akt by the phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and wortmannin prevent OGD-induced apoptosis, caspase-3 and -9 activation, and nuclear translocation of AIF and EndoG. Similar inhibitory effects of PI3K inhibitors were observed in A549 cells that underwent apoptosis when treated with GD in the presence of NAC (a general antioxidant) or catalase (a H(2)O(2) scavenger), or in the presence of active PKC by treatment with phorbol-12-myristate-13-acetate, indicating a crucial role(s) of the PI3K-Akt pathway in OGD-indcued apoptosis. In conclusion, our results demonstrate that hypoxia switches GD-induced necrosis to apoptosis and ERK1/2 and PI3K-Akt exert anti-necrotic and pro-apoptotic activities in the cell death, respectively.

    International journal of oncology 2010;36;1;117-24

  • Induction of mitogen-activated protein kinases is proportional to the amount of pressure overload.

    Esposito G, Perrino C, Schiattarella GG, Belardo L, di Pietro E, Franzone A, Capretti G, Gargiulo G, Pironti G, Cannavo A, Sannino A, Izzo R and Chiariello M

    Division of Cardiology, Federico II University, Via Pansini 5, 80131 Naples, Italy. espogiov@unina.it

    Pressure overload has been shown to induce mitogen activated protein kinases (MAPKs) and reactivate the atrial natriuretic factor in the heart. To test the sensitivity of these signals to pressure overload, we assayed the activity of MAPKs extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 in protein lysates from the left ventricle (LV) or white blood cells (WBC) isolated from aortic banded mice with varying levels of pressure overload. In separated mice we measured atrial natriuretic factor mRNA levels by Northern blotting. As expected, a significant induction of atrial natriuretic factor mRNA levels was observed after aortic banding, and it significantly correlated with the trans-stenotic systolic pressure gradient but not with the LV weight:body weight ratio. In contrast, a significant correlation with systolic pressure gradient or LV weight:body weight ratio was observed for all of the MAPK activity detected in LV samples or WBCs. Importantly, LV activation of MAPKs significantly correlated with their activation in WBCs from the same animal. To test whether MAPK activation in WBCs might reflect uncontrolled blood pressure levels in humans, we assayed extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 activation in WBCs isolated from normotensive volunteers, hypertensive patients with controlled blood pressure values, or hypertensive patients with uncontrolled blood pressure values. Interestingly, in hypertensive patients with controlled blood pressure values, LV mass and extracellular signal-regulated kinase phosphorylation were significantly reduced compared with those in hypertensive patients with uncontrolled blood pressure values. These results suggest that MAPKs are sensors of pressure overload and that extracellular signal-regulated kinase activation in WBCs might be used as a novel surrogate biomarker of uncontrolled human hypertension.

    Hypertension (Dallas, Tex. : 1979) 2010;55;1;137-43

  • Phosphorylation of myocardin by extracellular signal-regulated kinase.

    Taurin S, Sandbo N, Yau DM, Sethakorn N, Kach J and Dulin NO

    Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.

    The contractile phenotype of smooth muscle (SM) cells is controlled by serum response factor (SRF), which drives the expression of SM-specific genes including SM alpha-actin, SM22, and others. Myocardin is a cardiac and SM-restricted coactivator of SRF that is necessary for SM gene transcription. Growth factors inducing proliferation of SM cells inhibit SM gene transcription, in a manner dependent on the activation of extracellular signal-regulated kinases ERK1/2. In this study, we found that ERK1/2 phosphorylates mouse myocardin (isoform B) at four sites (Ser(812), Ser(859), Ser(866), and Thr(893)), all of which are located within the transactivation domain of myocardin. The single mutation of each site either to alanine or to aspartate has no effect on the ability of myocardin to activate SRF. However, the phosphomimetic mutation of all four sites to aspartate (4xD) significantly impairs activation of SRF by myocardin, whereas the phosphodeficient mutation of all four sites to alanine (4xA) has no effect. This translates to a reduced ability of the 4xD (but not of 4xA) mutant of myocardin to stimulate expression of SM alpha-actin and SM22, as assessed by corresponding promoter, mRNA, or protein assays. Furthermore, we found that phosphorylation of myocardin at these sites impairs its interaction with acetyltransferase, cAMP response element-binding protein-binding protein, which is known to promote the transcriptional activity of myocardin. In conclusion, we describe a novel mode of modulation of SM gene transcription by ERK1/2 through a direct phosphorylation of myocardin.

    Funded by: NHLBI NIH HHS: K08 HL093367, R01 HL071755, T32 HL007237, T32 HL007605, T32 HL07237, T32 HL07605; NICHD NIH HHS: T32 HD007009, T32 HD07009; NIGMS NIH HHS: GM085058, R01 GM085058

    The Journal of biological chemistry 2009;284;49;33789-94

  • Early response of mucosal epithelial cells during Toxoplasma gondii infection.

    Ju CH, Chockalingam A and Leifer CA

    Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

    The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from i.p. infection models, more recent studies have revealed the importance of studying immunity following infection through the natural peroral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-kappaB nuclear translocation, and secretion of IL-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses.

    Funded by: NCI NIH HHS: K22 CA113705; NIAID NIH HHS: R01 AI076588, R01 AI076588-01A2S1

    Journal of immunology (Baltimore, Md. : 1950) 2009;183;11;7420-7

  • The rho guanosine 5'-triphosphatase, cell division cycle 42, is required for insulin-induced actin remodeling and glucagon-like peptide-1 secretion in the intestinal endocrine L cell.

    Lim GE, Xu M, Sun J, Jin T and Brubaker PL

    Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

    Rho GTPases, such as cell division cycle 42 (Cdc42) and ras-related C3 botulinum toxin substrate 1 (Rac1), have been identified as regulators of F-actin dynamics and hormone release from endocrine cells; however, their role in secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1), from the enteroendocrine L cell is unknown. Insulin induced a 1.4-fold increase in L cell GLP-1 release; however, secretion was potentiated to 2.1-fold in the presence of the F-actin depolymerizing agent, latrunculin B, suggesting that F-actin functions as a permissive barrier. In murine GLUTag L cells, insulin stimulated F-actin depolymerization and Cdc42 activation simultaneously, and these events occurred prior to detectable increases in insulin-induced GLP-1 release. After insulin treatment, Cdc42-dependent p21-activated kinase-1 (PAK1) activation was also detected, and transfection of small-interfering RNA against Cdc42 or of dominant-negative Cdc42(T17N) impaired insulin-stimulated PAK1 activation, actin remodeling, and GLP-1 secretion. Overexpression of kinase-dead PAK1(K299R) or PAK1 small interfering RNA similarly attenuated insulin-induced GLP-1 secretion. Knockdown or inhibition of Cdc42 and PAK1 activities also prevented activation of MAPK/ERK (MEK)-1/2-ERK1/2 by insulin, which was previously identified as a critical pathway for insulin-regulated GLP-1 release. Taken together, these data identify a novel signaling pathway in the endocrine L cell, whereby Cdc42 regulates actin remodeling, activation of the cannonical 1/2-ERK1/2 pathway and PAK1, and GLP-1 secretion in response to insulin.

    Funded by: Canadian Institutes of Health Research

    Endocrinology 2009;150;12;5249-61

  • Bovine colostrum promotes growth and migration of the human keratinocyte HaCaT cell line.

    Kovacs D, Cardinali G, Aspite N and Picardo M

    San Gallicano Dermatological Institute, IRCCS, Rome, Italy.

    Bovine colostrum represents a rich source of growth factors, which are known to play a central role in wound healing. The aim of our study was to investigate the possible mitogenic and motogenic effects induced by colostrum on human keratinocytes. Cell proliferation evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test and 5-Bromo-2'-deoxyuridine incorporation revealed that colostrum exerts a growth promoting activity. Scratch assay and immunofluorescence of actin cytoskeleton showed its effectiveness also in inducing cell migration. Furthermore, colostrum treatment increases the levels of tyrosine phosphorylated proteins and the activated forms of the extracellular signal-regulated kinases 1 and 2 and such effects appear to be repressed by the tyrosine kinase inhibitor genistein. Our results indicate that the biological activities of colostrum are specifically mediated by the growth factor-induced activation of tyrosine kinase receptors and underline the relevance of the synergistic action exerted by the growth factors in stimulating keratinocyte proliferation and migration essential for tissue repair.

    Growth factors (Chur, Switzerland) 2009;27;6;448-55

  • Down-regulation of Cdx2 in colorectal carcinoma cells by the Raf-MEK-ERK 1/2 pathway.

    Krueger F, Madeja Z, Hemberger M, McMahon M, Cook SJ and Gaunt SJ

    Laboratory of Developmental Genetics & Imprinting, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

    Cdx2 is a homeodomain transcription factor that regulates normal intestinal cell differentiation. Cdx2 is frequently lost during progression of colorectal cancer (CRC) and is widely viewed as a colorectal tumour suppressor. A previous study suggested that activation of protein kinase C (PKC) may be responsible for Cdx2 down-regulation in CRC cells. Here we show that activation of PKC does indeed promote down-regulation of Cdx2 at both the mRNA and protein levels. However, PKC-dependent loss of Cdx2 is dependent upon activation of the Raf-MEK-ERK1/2 pathway. Indeed, specific activation of the ERK1/2 pathway using the conditional kinase DeltaRaf-1:ER is sufficient to inhibit Cdx2 transcription. The Raf-MEK-ERK1/2 pathway is hyper-activated in a large fraction of colorectal cancers due to mutations in K-Ras and we show that treatment of CRC cell lines with MEK inhibitors causes an increase in Cdx2 expression. Furthermore, activation of the ERK1/2 pathway promotes the phosphorylation and proteasome-dependent degradation of the Cdx2 protein. The inhibitory effect of ERK1/2 upon Cdx2 in CRC cells is in sharp contrast to its stimulatory effect upon Cdx2 expression in trophectoderm and trophoblast stem cells. These results provide important new insights into the regulation of the Cdx2 tumour suppressor by linking it to ERK1/2, a pathway which is frequently activated in CRC.

    Funded by: Biotechnology and Biological Sciences Research Council: BBS/E/B/0000C162, BBS/E/B/0000C224, BBS/E/B/0000C225, BBS/E/B/0000C230; Medical Research Council: G0800784, G120/824; NCI NIH HHS: R01 CA131261

    Cellular signalling 2009;21;12;1846-56

  • hnRNP-K is a nuclear target of TCR-activated ERK and required for T-cell late activation.

    Chang JW, Koike T and Iwashima M

    Department of Medicine, Immunotherapy Center, Medical College of Georgia, Augusta, GA 30912-2600, USA.

    Sustained extracellular signal-regulated kinase (ERK)-signaling plays a critical role in T-cell-mediated IL-2 production. Although many downstream targets are known for ERK, details remain unknown about which molecules play functional roles in IL-2 production. Here, we addressed this question using proteomic analysis of nuclear proteins from TCR-activated T cells and identified hnRNP-K as one of the ERK targets essential for IL-2 production. hnRNP-K was previously shown by others to be a direct substrate of ERK and form complexes with multiple signaling proteins as well as DNA and RNA. Our data showed a clear ERK-dependent increase in one form of hnRNP-K after TCR stimulation. Small interfering RNA-mediated gene knockdown of hnRNP-K expression abrogated IL-2 production by T cells. Moreover, reduction of hnRNP-K expression caused a notable increase in proteolysis of Vav1, a binding target of hnRNP-K. Since Vav1 is an essential molecule for T-cell activation, the data suggest that ERK signaling is required for T-cell activation partly by inhibiting activation-induced proteolysis of Vav1.

    Funded by: NIAID NIH HHS: AI47266, AI49398

    International immunology 2009;21;12;1351-61

  • Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2.

    García Lopez MA, Aguado Martínez A, Lamaze C, Martínez-A C and Fischer T

    Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Darwin 3, 28049 Madrid, Spain.

    The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca2+ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor beta-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrin-dependent and -independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.

    Cellular signalling 2009;21;12;1748-57

  • Stimulation of the bradykinin B(1) receptor induces the proliferation of estrogen-sensitive breast cancer cells and activates the ERK1/2 signaling pathway.

    Molina L, Matus CE, Astroza A, Pavicic F, Tapia E, Toledo C, Perez JA, Nualart F, Gonzalez CB, Burgos RA, Figueroa CD, Ehrenfeld P and Poblete MT

    Laboratorio de Patologia Celular, Instituto de Anatomia, Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile.

    Kinin peptides exert multiple biological effects by binding to two types of G protein-coupled receptors known as B(1) (B(1)R) and B(2) receptors. Expression of the B(1)R in human breast cancer was recently reported, but up to now the consequences of its stimulation are unknown. Our aims were (1) to investigate the capacity of B(1)R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B(1)R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B(1)R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B(1)R that once stimulated triggered cell proliferation at nanomolar concentrations of the ligand. Inhibitor studies suggested that the proliferative effects depend on the activity of epidermal growth factor receptor and subsequent ERK1/2 mitogen-activated protein kinases phosphorylation. B(1)R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B(1)R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B(1)R may be a valuable alternative for the treatment of breast cancer.

    Breast cancer research and treatment 2009;118;3;499-510

  • Noncatalytic function of ERK1/2 can promote Raf/MEK/ERK-mediated growth arrest signaling.

    Hong SK, Yoon S, Moelling C, Arthan D and Park JI

    Department of Biochemistry, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

    Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21(CIP1) up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90(RSK) and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling.

    The Journal of biological chemistry 2009;284;48;33006-18

  • Protein kinase Cdelta supports survival of MDA-MB-231 breast cancer cells by suppressing the ERK1/2 pathway.

    Lønne GK, Masoumi KC, Lennartsson J and Larsson C

    Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, and Malmö University Hospital, Sweden.

    Mechanisms that mediate apoptosis resistance are attractive therapeutic targets for cancer. Protein kinase Cdelta (PKCdelta) is considered a pro-apoptotic factor in many cell types. In breast cancer, however, it has shown both pro-survival and pro-apoptotic effects. Here, we report for the first time that down-regulation of PKCdelta per se leads to apoptosis of MDA-MB-231 cells. Inhibition of MEK1/2 by either PD98059 or U0126 suppressed the induction of apoptosis of PKCdelta-depleted MDA-MB-231 cells but did not support survival of MCF-7 or MDA-MB-468 cells. Basal ERK1/2 phosphorylation was substantially higher in MDA-MB-231 cells than in the other cell lines. PKCdelta depletion led to even higher ERK1/2 phosphorylation levels and also to lower expression levels of the ERK1/2 phosphatase MKP3. Depletion of MKP3 led to apoptosis and higher levels of ERK1/2 phosphorylation, suggesting that this may be a mechanism mediating the effect of PKCdelta down-regulation. However, PKCdelta silencing also induced increased MEK1/2 phosphorylation, indicating that PKCdelta regulates ERK1/2 phosphorylation both upstream and downstream. Moreover, PKCdelta silencing led to increased levels of the E3 ubiquitin ligase Nedd4, which is a potential regulator of MKP3, because down-regulation led to increased MKP3 levels. Our results highlight PKCdelta as a potential target for therapy of breast cancers with high activity of the ERK1/2 pathway.

    The Journal of biological chemistry 2009;284;48;33456-65

  • Regulation of protein kinase A activity by p90 ribosomal S6 kinase 1.

    Gao X and Patel TB

    Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.

    Previously, we reported that the catalytic subunit of cAMP-dependent protein kinase (PKAc) binds to the active p90 ribosomal S6 kinase 1 (RSK1) (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell. Biol. 26, 4586-4600). Herein, by overexpressing hemagglutinin-tagged RSK1 fragments in HeLa cells we have identified the region of RSK1 that is responsible for the interaction with PKAc. PKAc bound to the last 13 amino acids of RSK1, which overlaps the Erk1/2 docking site. This interaction between PKAc and RSK1 required the phosphorylation of Ser-732 in the C terminus of RSK1. Depending upon its phosphorylation status, RSK1 switched interactions between Erk1/2 and PKAc. In addition, a peptide corresponding to the last 13 amino acids of RSK1 with substitution of Ser-732 with Glu (peptide E), but not Ala (peptide A), decreased interactions between endogenous active RSK1 and PKAc. RSK1 attenuated the ability of cAMP to activate PKA in vitro and this modulation was abrogated by peptide E, but not by peptide A. Similarly, in intact cells, cAMP-mediated phosphorylation of Bcl-xL/Bcl-2-associated death promoter on Ser-115, the PKA site, was reduced when RSK1 was activated by epidermal growth factor, and this effect was blocked by peptide E, but not by peptide A. These findings demonstrate that interactions between endogenous RSK1 and PKAc in intact cells regulate the ability of cAMP to activate PKA and identify a novel mechanism by which PKA activity is regulated by the Erk1/2 pathway.

    Funded by: NIGMS NIH HHS: GM079226, R01 GM079226

    The Journal of biological chemistry 2009;284;48;33070-8

  • EGF-induced ERK activation promotes CK2-mediated disassociation of alpha-Catenin from beta-Catenin and transactivation of beta-Catenin.

    Ji H, Wang J, Nika H, Hawke D, Keezer S, Ge Q, Fang B, Fang X, Fang D, Litchfield DW, Aldape K and Lu Z

    Brain Tumor Center and Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, 77030, USA.

    Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation remains unclear. We demonstrate here that EGFR activation results in disruption of the complex of beta-catenin and alpha-catenin, thereby abrogating the inhibitory effect of alpha-catenin on beta-catenin transactivation via CK2alpha-dependent phosphorylation of alpha-catenin at S641. ERK2, which is activated by EGFR signaling, directly binds to CK2alpha via the ERK2 docking groove and phosphorylates CK2alpha primarily at T360/S362, subsequently enhancing CK2alpha activity toward alpha-catenin phosphorylation. In addition, levels of alpha-catenin S641 phosphorylation correlate with levels of ERK1/2 activity in human glioblastoma specimens and with grades of glioma malignancy. This EGFR-ERK-CK2-mediated phosphorylation of alpha-catenin promotes beta-catenin transactivation and tumor cell invasion. These findings highlight the importance of the crosstalk between EGFR and Wnt pathways in tumor development.

    Funded by: NCI NIH HHS: 5R01CA109035, R01 CA109035, R01 CA109035-05

    Molecular cell 2009;36;4;547-59

  • C-Raf inhibits MAPK activation and transformation by B-Raf(V600E).

    Karreth FA, DeNicola GM, Winter SP and Tuveson DA

    Li Ka Shing Centre, Cambridge Research Institute, Cancer Research UK, Robinson Way, Cambridge CB2 0RE, UK.

    Activating B-Raf mutations that deregulate the MAPK pathway commonly occur in cancer. Whether additional proteins modulate the enzymatic activity of oncogenic B-Raf is unknown. Here we show that the proto-oncogene C-Raf paradoxically inhibits B-Raf(V600E) kinase activity through the formation of B-Raf(V600E)-C-Raf complexes. Although all Raf family members associate with oncogenic B-Raf, this inhibitory effect is specific to C-Raf. Indeed, a B-Raf(V600E) isoform with impaired ability to interact with C-Raf exhibits elevated oncogenic potential. Human melanoma cells expressing B-Raf(V600E) display a reduced C-Raf:B-Raf ratio, and further suppression of C-Raf increases MAPK activation and proliferation. Conversely, ectopic C-Raf expression lowers ERK phosphorylation and proliferation. Moreover, both oncogenic Ras and Sorafenib stabilize B-Raf(V600E)-C-Raf complexes, thereby impairing MAPK activation. This inhibitory function of C-Raf on B-Raf(V600E)-mediated MAPK activation may explain the lack of co-occurrence of B-Raf(V600E) and oncogenic Ras mutations, and influence the successful clinical development of small molecule inhibitors for B-Raf(V600E)-driven cancers.

    Funded by: Cancer Research UK

    Molecular cell 2009;36;3;477-86

  • Prolonged cigarette smoke exposure decreases heme oxygenase-1 and alters Nrf2 and Bach1 expression in human macrophages: roles of the MAP kinases ERK(1/2) and JNK.

    Goven D, Boutten A, Leçon-Malas V, Boczkowski J and Bonay M

    Inserm U700, Université Paris 7, Faculté de Médecine Denis Diderot-site Bichat, Paris, France.

    Tobacco may be involved in the decreased macrophage heme oxygenase-1 (HO-1) expression described in smoking-induced severe emphysema, via the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-BTB and CNC homology 1, basic leucine zipper transcription factor 1 (Bach1) pathway. We assessed in vitro effects of cigarette smoke condensate (CS) in the human monocyte/macrophage cell line (THP-1). CS exposure led to increased HO-1 and nuclear Nrf2 expression (6 h) followed by decreased HO-1 expression concomitantly with nuclear Nrf2/Bach1 ratio decrease (72h). CS-induced mitogen-activated protein kinase (MAPK) phosphorylation. Extracellular-signal-regulated kinase(1/2) (ERK(1/2)) and c-Jun NH2-terminal kinase (JNK) inhibition completely abrogated CS effects on HO-1 expression and nuclear Nrf2/Bach1 translocation. These results suggest that ERK(1/2) and JNK are involved in CS-induced biphasic HO-1 expression by a specific regulation of Nrf2/Keap1-Bach1.

    FEBS letters 2009;583;21;3508-18

  • Mycobacteria exploit p38 signaling to affect CD1 expression and lipid antigen presentation by human dendritic cells.

    Gagliardi MC, Teloni R, Giannoni F, Mariotti S, Remoli ME, Sargentini V, Videtta M, Pardini M, De Libero G, Coccia EM and Nisini R

    Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Roma, Italy.

    Group I CD1 proteins are specialized antigen-presenting molecules that present both microbial and self lipid antigens to CD1-restricted alpha/beta T lymphocytes. The production of high levels of gamma interferon and lysis of infected macrophages by lipid-specific T lymphocytes are believed to play pivotal roles mainly in the defense against mycobacterial infections. We previously demonstrated that Mycobacterium tuberculosis and bacillus Calmette-Guérin (Mycobacterium bovis BCG) induce human monocytes to differentiate into CD1- dendritic cells (DC), which cannot present lipid antigens to specific T cells. Here, we show that in human monocytes mycobacteria trigger phosphorylation of p38 mitogen-activated protein kinase to inhibit CD1 expression in DC derived from infected monocytes. Pretreatment with a specific p38 inhibitor renders monocytes insensitive to mycobacterial subversion and allows them to differentiate into CD1+ DC, which are fully capable of presenting lipid antigens to specific T cells. We also report that one of the pathogen recognition receptors triggered by BCG to activate p38 is complement receptor 3 (CR3), as shown by reduced p38 phosphorylation and partial reestablishment of CD1 membrane expression obtained by CR3 blockade before infection. In conclusion, we propose that p38 signaling is a novel pathway exploited by mycobacteria to affect the expression of CD1 antigen-presenting cells and avoid immune recognition.

    Funded by: PHS HHS: 5303

    Infection and immunity 2009;77;11;4947-52

  • Signaling through cholesterol esterification: a new pathway for the cholecystokinin 2 receptor involved in cell growth and invasion.

    Paillasse MR, de Medina P, Amouroux G, Mhamdi L, Poirot M and Silvente-Poirot S

    INSERM 563, Equipe Métabolisme, Oncogenèse et Différenciation cellulaire, Centre de Physiopathologie de Toulouse Purpan, Institut Claudius Regaud, Toulouse France.

    Several studies indicate that cholesterol esterification is deregulated in cancers. The present study aimed to characterize the role of cholesterol esterification in proliferation and invasion of two tumor cells expressing an activated cholecystokinin 2 receptor (CCK2R). A significant increase in cholesterol esterification and activity of Acyl-CoA:cholesterol acyltransferase (ACAT) was measured in tumor cells expressing a constitutively activated oncogenic mutant of the CCK2R (CCK2R-E151A cells) compared with nontumor cells expressing the wild-type CCK2R (CCK2R-WT cells). Inhibition of cholesteryl ester formation and ACAT activity by Sah58-035, an inhibitor of ACAT, decreased by 34% and 73% CCK2R-E151A cell growth and invasion. Sustained activation of CCK2R-WT cells by gastrin increased cholesteryl ester production while addition of cholesteryl oleate to the culture medium of CCK2R-WT cells increased cell proliferation and invasion to a level close to that of CCK2R-E151A cells. In U87 glioma cells, a model of autocrine growth stimulation of the CCK2R, inhibition of cholesterol esterification and ACAT activity by Sah58-035 and two selective antagonists of the CCK2R significantly reduced cell proliferation and invasion. In both models, cholesteryl ester formation was found dependent on protein kinase zeta/ extracellular signal-related kinase 1/2 (PKCzeta/ERK1/2) activation. These results show that signaling through ACAT/cholesterol esterification is a novel pathway for the CCK2R that contributes to tumor cell proliferation and invasion.

    Journal of lipid research 2009;50;11;2203-11

  • Profiling the human protein-DNA interactome reveals ERK2 as a transcriptional repressor of interferon signaling.

    Hu S, Xie Z, Onishi A, Yu X, Jiang L, Lin J, Rho HS, Woodard C, Wang H, Jeong JS, Long S, He X, Wade H, Blackshaw S, Qian J and Zhu H

    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We identified a large number of unanticipated PDIs for known TFs, as well as for previously uncharacterized TFs. We also found that over three hundred unconventional DNA-binding proteins (uDBPs)--which include RNA-binding proteins, mitochondrial proteins, and protein kinases--showed sequence-specific PDIs. One such uDBP, ERK2, acts as a transcriptional repressor for interferon gamma-induced genes, suggesting important biological roles for such proteins.

    Funded by: NCRR NIH HHS: RR020839, U54 RR020839; NIGMS NIH HHS: GM076102, R01 GM076102, R01 GM076102-01A1, R01 GM076102-02, R01 GM076102-03, R01 GM076102-04

    Cell 2009;139;3;610-22

  • Genetical genomic determinants of alcohol consumption in rats and humans.

    Tabakoff B, Saba L, Printz M, Flodman P, Hodgkinson C, Goldman D, Koob G, Richardson HN, Kechris K, Bell RL, Hübner N, Heinig M, Pravenec M, Mangion J, Legault L, Dongier M, Conigrave KM, Whitfield JB, Saunders J, Grant B, Hoffman PL and WHO/ISBRA Study on State and Trait Markers of Alcoholism

    Department of Pharmacology, University of Colorado, Denver, Aurora, CO, USA. boris.tabakoff@ucdenver.edu

    Background: We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.

    Results: In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption.

    Conclusion: Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: HL35018, P01 HL035018; NIAAA NIH HHS: AA006420, AA013162, AA013517-INIA, AA013522-INIA, AA016649-INIA, AA016663-INIA, AA16922, K01 AA016922, P50 AA006420, P60 AA006420, R01 AA013162, R24 AA013162, R24 AA015512, U01 AA013517, U01 AA013522, U01 AA016649, U01 AA016663, U24 AA013517, U24 AA013522, U24 AA015512; NIDDK NIH HHS: R01 DK100340; Wellcome Trust

    BMC biology 2009;7;70

  • Radial spoke protein 3 is a mammalian protein kinase A-anchoring protein that binds ERK1/2.

    Jivan A, Earnest S, Juang YC and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIalpha and RIIbeta, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes.

    Funded by: NIDDK NIH HHS: R37 DK034128, R37 DK34128

    The Journal of biological chemistry 2009;284;43;29437-45

  • Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism.

    Pricola KL, Kuhn NZ, Haleem-Smith H, Song Y and Tuan RS

    Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.

    Adult human mesenchymal stem cells (MSCs) hold promise for an increasing list of therapeutic uses due to their ease of isolation, expansion, and multi-lineage differentiation potential. To maximize the clinical potential of MSCs, the underlying mechanisms by which MSC functionality is controlled must be understood. We have taken a deconstructive approach to understand the individual components in vitro, namely the role of candidate "stemness" genes. Our recent microarray gene expression profiling data suggest that interleukin-6 (IL-6) may contribute to the maintenance of MSCs in their undifferentiated state. In this study, we showed that IL-6 gene expression is significantly higher in undifferentiated MSCs as compared to their chondrogenic, osteogenic, and adipogenic derivatives. Moreover, we found that MSCs secrete copious amounts of IL-6 protein, which decreases dramatically during osteogenic differentiation. We further evaluated the role of IL-6 for maintenance of MSC "stemness," using a series of functional assays. The data showed that IL-6 is both necessary and sufficient for enhanced MSC proliferation, protects MSCs from apoptosis, inhibits adipogenic and chondrogenic differentiation of MSCs, and increases the rate of in vitro wound healing of MSCs. We further identified ERK1/2 activation as the key pathway through which IL-6 regulates both MSC proliferation and inhibition of differentiation. Taken together, these findings show for the first time that IL-6 maintains the proliferative and undifferentiated state of bone marrow-derived MSCs, an important parameter for the optimization of both in vitro and in vivo manipulation of MSCs.

    Funded by: Intramural NIH HHS: Z01 AR041131-06, Z99 CA999999; NIAMS NIH HHS: Z01 AR041131, Z01 AR041137

    Journal of cellular biochemistry 2009;108;3;577-88

  • Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2.

    Kumari G and Mahalingam S

    Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076, India.

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates nucleo-cytoplasmic transport and cell growth arrest activity of RASSF2. Taken together, the present study suggests that active transport between nucleus and cytoplasm may constitute an important regulatory mechanism for RASSF2 function.

    Experimental cell research 2009;315;16;2775-90

  • Detection of two novel mutations and relatively high incidence of H-RAS mutations in Vietnamese oral cancer.

    Murugan AK, Hong NT, Cuc TT, Hung NC, Munirajan AK, Ikeda MA and Tsuchida N

    Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. amuruga1@jhmi.edu

    Oral squamous cell carcinoma is the sixth most common cancer in the world and the seventh most common cancer in Vietnam. The RAS and PI3K-AKT signaling pathways play an important role in oral carcinogenesis. Our previous study on PI3K signaling pathway showed the absence of PIK3CA and PTEN gene mutations in Vietnamese oral cancer. We thus hypothesized that the RAS could be more likely activated as an upstream effector. However, the status of RAS mutations in Vietnamese oral cancer had not been studied. In the present study, Fifty six primary tumor DNA samples were screened for mutations of hot spots in exons 1 and 2 of H-RAS and a part of the samples for exon 7 of ERK2 gene in which we previously reported a mutation in an OSCC cell line. The H-RAS mutations were detected in 10 of 56 tumors (18%). Two novel mutations were found, one was an insertion of three nucleotides (GGC) between codons 10 and 11 resulting in in-frame insertion of glycine (10(Gly)11) and the other was a missense mutation in codon 62 (GAG>GGG). We also found T81C single nucleotide polymorphism in 12 of 56 tumors (22%) and there was no mutation in exon 7 of ERK2 gene. The H-RAS mutation incidence showed significant association with advanced stages of the tumor and also with well-differentiated tumor grade. Our study is the first to report H-RAS mutation from Vietnamese ethnicity, with two novel mutations and relatively high incidence of H-RAS mutations. The results suggest that RAS is an important member in the PI3K-AKT signaling and could play an important role in the tumorigenesis of oral carcinoma.

    Oral oncology 2009;45;10;e161-6

  • Involvement of mitogen-activated protein kinases and nuclear factor kappa B pathways in signaling COX-2 expression in chronic rhinosinusitis.

    Wang Z, Zhang Q, Li Y, Li P, Zhang G and Li Y

    Department of Otolaryngology-Head and Neck Surgery, Xuan Wu Hospital, Capital Medical University, 100053, Beijing, People's Republic of China. wzl1812@yahoo.com.cn

    Objective: To investigate the signal pathways involved in cyclooxygenase-2 (COX-2) expression in chronic rhinosinusitis (CRS).

    Methods: The expressions of COX-2, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK), and nuclear factor kappa B (NF-kappaB) in nasal mucosa were detected by immunohistological stain and polymerase chain reaction (PCR). Their expressions and prostaglandin E2 (PGE(2)) release were determined by PCR, Western blot and enzyme immunoassay (EIA) in human nasal epithelia (HNE) cells after lipopolysaccharide (LPS) induction, and/or small interfering RNA (siRNA) transfection.

    Results: Positive protein expressions of COX-2, p38MAPK, ERK, NF-kappaB subunits were detected in epithelial and inflammatory cells. Their mRNA levels were significantly higher in CRS than controls (P < 0.05). The expressions varied in time and concentration-dependent manner in LPS-induced HNE cells. COX-2 expression was suppressed by siRNAs of P38MAPK, ERK, and NF-kappaB; however, COX-2-specific siRNA had no blocking effect on them. SiRNAs of P38MAPK or ERK could block NF-kappaB, but NF-kappaB-specific siRNA had no blocking effect on the former. SiRNA of p38MAPK, or ERK did not inhibit each other.

    Conclusion: Upregulation of COX-2 expression suggested its role as a mediator in CRS. ERK and p38MAPK pathways were involved in signaling COX-2 through NF-kappaB pathway.

    Inflammation research : official journal of the European Histamine Research Society ... [et al.] 2009;58;10;649-58

  • Osteopontin increases migration and MMP-9 up-regulation via alphavbeta3 integrin, FAK, ERK, and NF-kappaB-dependent pathway in human chondrosarcoma cells.

    Chen YJ, Wei YY, Chen HT, Fong YC, Hsu CJ, Tsai CH, Hsu HC, Liu SH and Tang CH

    School of Pharmacy, China Medical University, Taichung, Taiwan.

    Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, alphavbeta3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)-9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, alphavbeta3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN-induced increase of the migration and MMP-9 up-regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, treatment of JJ012 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited OPN-induced cell migration and MMP-9 up-regulation. Stimulation of JJ012 cells with OPN also induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OPN-mediated increases in MMP-9 and kappaB-luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP-9 expression through the alphavbeta3 integrin, FAK, MEK, ERK and NF-kappaB signal transduction pathway.

    Journal of cellular physiology 2009;221;1;98-108

  • Stromal cell-derived factor-1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF-kappaB-dependent pathways.

    Huang CY, Lee CY, Chen MY, Yang WH, Chen YH, Chang CH, Hsu HC, Fong YC and Tang CH

    Department of Orthopaedic Surgery, China Medical University Beigang Hospital, Yun-Lin County, Taiwan.

    Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF-1 and CXCR4 (SDF-1 receptor). Treatment of osteosarcoma cells with SDF-1alpha increased the migration and cell surface expression of alphavbeta3 integrin. CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1alpha-induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF-1alpha-mediated migration and integrin expression. Stimulation of cells with SDF-1alpha increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited SDF-1alpha-mediated cell migration and integrin up-regulation. Stimulation of cells with SDF-1alpha induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the SDF-1alpha-mediated increasing kappaB-luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKalpha and IKKbeta mutants. Taken together, these results suggest that the SDF-1alpha acts through CXCR4 to activate MEK and ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of alphavbeta3 integrins and contributing the migration of human osteosarcoma cells.

    Journal of cellular physiology 2009;221;1;204-12

  • Syndecan-1 and syndecan-4 are involved in RANTES/CCL5-induced migration and invasion of human hepatoma cells.

    Charni F, Friand V, Haddad O, Hlawaty H, Martin L, Vassy R, Oudar O, Gattegno L, Charnaux N and Sutton A

    INSERM U698, CNRS, UMR 7033, Université Paris 13, Bobigny, France.

    Background: We previously demonstrated that the CC-chemokine Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)/CCL5 exerts pro-tumoral effects on human hepatoma Huh7 cells through its G protein-coupled receptor, CCR1. Glycosaminoglycans play major roles in these biological events.

    Methods: In the present study, we explored 1/ the signalling pathways underlying RANTES/CCL5-mediated hepatoma cell migration or invasion by the use of specific pharmacological inhibitors, 2/ the role of RANTES/CCL5 oligomerization in these effects by using a dimeric RANTES/CCL5, 3/ the possible involvement of two membrane heparan sulfate proteoglycans, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) in RANTES/CCL5-induced cell chemotaxis and spreading by pre-incubating cells with specific antibodies or by reducing SDC-1 or -4 expression by RNA interference.

    The present data suggest that focal adhesion kinase phosphorylation, phosphoinositide 3-kinase-, mitogen-activated protein kinase- and Rho kinase activations are involved in RANTES/CCL5 pro-tumoral effects on Huh7 cells. Interference with oligomerization of the chemokine reduced RANTES/CCL5-mediated cell chemotaxis. This study also indicates that SDC-1 and -4 may be required for HepG2, Hep3B and Huh7 human hepatoma cell migration, invasion or spreading induced by the chemokine. These results also further demonstrate the involvement of glycosaminoglycans as the glycosaminoglycan-binding deficient RANTES/CCL5 variant, in which arginine 47 was replaced by lysine, was devoid of effect.

    The modulation of RANTES/CCL5-mediated cellular effects by targeting the chemokine-syndecan interaction could represent a new therapeutic approach for hepatocellular carcinoma.

    Biochimica et biophysica acta 2009;1790;10;1314-26

  • S100A4 and bone morphogenetic protein-2 codependently induce vascular smooth muscle cell migration via phospho-extracellular signal-regulated kinase and chloride intracellular channel 4.

    Spiekerkoetter E, Guignabert C, de Jesus Perez V, Alastalo TP, Powers JM, Wang L, Lawrie A, Ambartsumian N, Schmidt AM, Berryman M, Ashley RH and Rabinovitch M

    Department of Pediatrics, Stanford University School of Medicine, Stanford, Calif 94305-5162, USA.

    Rationale: S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMCs), through an interaction with the RAGE (receptor for advanced glycation end products).

    Objective: We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension.

    Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho-extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells.

    Conclusions: Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

    Funded by: British Heart Foundation: PG/06/125/1633; NHLBI NIH HHS: 1-R01-HL074186-01, R01 HL074186-01A1, R01 HL074186-02, R01 HL074186-03, R01 HL074186-04, R01 HL074186-05, R01 HL087118-04, T32-HL007708-14

    Circulation research 2009;105;7;639-47, 13 p following 647

  • Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells.

    Dai J, Peng L, Fan K, Wang H, Wei R, Ji G, Cai J, Lu B, Li B, Zhang D, Kang Y, Tan M, Qian W and Guo Y

    International Joint Cancer Institute and General Hospital Cancer Center, The Second Military Medical University, Shanghai, PR China.

    Angiogenesis is a key step in tumor growth and metastasis. The mechanism by which osteopontin (OPN) induces the angiogenesis of endothelial cells remains unclear. Here, we show that OPN confers cytoprotection through the activation of the PI3K/Akt pathway with subsequent upregulation of Bcl-xL and activation of nuclear factor-kappaB. OPN enhances the expression of vascular endothelial growth factor (VEGF) through the phosphorylation of AKT and extracellular signal-regulated kinase (ERK). In turn, OPN-induced VEGF activates PI3K/AKT and the ERK1/2 pathway as a positive feedback signal. Blocking the feedback signal by anti-VEGF antibody, PI3-kinase inhibitor or ERK inhibitor can partially inhibit the OPN-induced human umbilical vein endothelial cell (HUVEC) motility, proliferation and tube formation, while blocking the signal by anti-OPN or anti-alphavbeta3 antibody completely abrogates the biological effects of OPN on HUVECs. In addition, blood vessel formation is also investigated in vivo. The antiangiogenesis efficacy of anti-OPN antibody in vivo is more effective than that of anti-VEGF antibody, which only blocks the feedback signals. These data show that OPN enhances angiogenesis directly through PI3K/AKT- and ERK-mediated pathways with VEGF acting as a positive feedback signal. The results suggest that OPN might be a valuable target for developing novel antiangiogenesis therapy for treatment of cancer.

    Oncogene 2009;28;38;3412-22

  • Coxsackie adenovirus receptor (CAR) regulates integrin function through activation of p44/42 MAPK.

    Farmer C, Morton PE, Snippe M, Santis G and Parsons M

    Division of Asthma, Allergy and Lung Biology, King's College London School of Medicine, 5th Floor Tower Wing, Guy's Hospital Campus, London, UK.

    The coxsackie B virus and adenovirus receptor (CAR) is an attachment receptor for Adenovirus serotype 5 (Ad5) and in many cell types forms homodimers with neighbouring cells as part of a cell adhesion complex. CAR co-operates with cell surface integrin receptors to enable efficient viral entry, but little is known about the mechanism of crosstalk between these two receptor types. Here we show that overexpression of CAR in human epithelial cells leads to increased basal activation of p44/42 MAPK and this is required for efficient Ad5 infection. We demonstrate that CAR forms homodimers in cis and that this dimerisation is enhanced in the presence of Ad5 in a phospho-p44/42-dependent manner. CAR-induced p44/42 activation also leads to increased activation of beta1 and beta3 integrins. Analysis of CAR mutants demonstrates that the cyto domain of CAR is required for CAR-induced p44/42 activation, integrin activation and localisation to cell junctions. This data for the first time demonstrates that signalling downstream of CAR can have a dual effect on integrins and CAR itself in order to promote efficient viral binding to cell membranes.

    Funded by: Biotechnology and Biological Sciences Research Council; Medical Research Council

    Experimental cell research 2009;315;15;2637-47

  • Emodin enhances gefitinib-induced cytotoxicity via Rad51 downregulation and ERK1/2 inactivation.

    Chen RS, Jhan JY, Su YJ, Lee WT, Cheng CM, Ciou SC, Lin ST, Chuang SM, Ko JC and Lin YW

    Molecular Genetics of Microorganisms Laboratory, Department of Biochemical Science and Technology, National Chiayi University, Chiayi, Taiwan.

    Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It reportedly exhibits an anticancer effect on lung cancer. Gefitinib (Iressa) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). However, the molecular mechanism of how emodin combined with gefitinib decreases NSCLC cell viability is unclear. The recombinase protein Rad51 is essential for homologous recombination repair, and Rad51 overexpression is resistant to DNA double-strand break-inducing cancer therapies. In this study, we found that emodin enhanced the cytotoxicity induced by gefitinib in two NSCLC cells lines, A549 and H1650. Emodin at low doses of 2-10 microM did not affect ERK1/2 activation, mRNA, and Rad51 protein levels; however, it enhanced a gefitinib-induced decrease in phospho-ERK1/2 and Rad51 protein levels by enhancing Rad51 protein instability. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescued the reduced phospho-ERK1/2 and Rad51 protein levels as well as cell viability on gefitinib and emodin cotreatment. Blocking of ERK1/2 activation by U0126 (an MKK1/2 inhibitor) lowered Rad51 protein levels and cell viability in emodin-treated H1650 and A549 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA enhanced emodin cytotoxicity. In contrast, Rad51 overexpression protected the cells from the cytotoxic effects induced by emodin and gefitinib. Consequently, emodin-gefitinib cotreatment may serve as the basis for a novel and better therapeutic modality in the management of advanced lung cancer.

    Experimental cell research 2009;315;15;2658-72

  • Phosphatidylinositol 3-kinase/Akt pathway targets acetylation of Smad3 through Smad3/CREB-binding protein interaction: contribution to transforming growth factor beta1-induced Epstein-Barr virus reactivation.

    Oussaief L, Hippocrate A, Ramirez V, Rampanou A, Zhang W, Meyers D, Cole P, Khelifa R and Joab I

    UMR542 INSERM-Université Paris 11, Hôpital Paul Brousse, 94807 Villejuif Cedex, France.

    Epstein-Barr virus, a ubiquitous human herpesvirus, is associated with the development of carcinomas and lymphomas. We previously showed that transforming growth factor beta1 (TGF-beta1) mediated the virus to enter the lytic cycle, which is triggered by expression of Z Epstein-Barr virus replication activator (ZEBRA), through the ERK 1/2 MAPK signaling pathway. We report here that Akt, activated downstream from ERK 1/2, was required for TGF-beta1-induced ZEBRA expression and enabled Smad3, a mediator of TGF-beta1 signaling, to be acetylated by direct interaction with the co-activator CREB-binding protein and then to regulate TGF-beta1-induced ZEBRA expression.

    The Journal of biological chemistry 2009;284;36;23912-24

  • CRP regulates the expression and activity of tissue factor as well as tissue factor pathway inhibitor via NF-kappaB and ERK 1/2 MAPK pathway.

    Chen Y, Wang J, Yao Y, Yuan W, Kong M, Lin Y, Geng D and Nie R

    Department of Cardiology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, China.

    It was found that C-reactive protein (CRP) could significantly increase the expression and activity of tissue factor (TF), but decrease that of tissue factor pathway inhibitor (TFPI) in human umbilical vein endothelial cells (HUVECs) in dose- and time-dependent manners, which could be antagonized by PDTC and U0126. CRP could also increase protein expression of phosphorylated nuclear factor-kappaB (NF-kappaB), IkappaB-alpha and ERK1/2 in dose- and time-dependent manner. In addition, neutralizing antibody to CD32 (FcgammaR II) could significantly attenuate the expression and activity of TF and TFPI induced by CRP. These results suggest that CRP may promote coagulation by enhancing the expression and activity of TF and reducing that of TFPI by activating NF-kappaB and extracellular signal-regulated kinase via FcgammaR II.

    FEBS letters 2009;583;17;2811-8

  • The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors.

    Landa I, Ruiz-Llorente S, Montero-Conde C, Inglada-Pérez L, Schiavi F, Leskelä S, Pita G, Milne R, Maravall J, Ramos I, Andía V, Rodríguez-Poyo P, Jara-Albarrán A, Meoro A, del Peso C, Arribas L, Iglesias P, Caballero J, Serrano J, Picó A, Pomares F, Giménez G, López-Mondéjar P, Castello R, Merante-Boschin I, Pelizzo MR, Mauricio D, Opocher G, Rodríguez-Antona C, González-Neira A, Matías-Guiu X, Santisteban P and Robledo M

    Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

    In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

    PLoS genetics 2009;5;9;e1000637

  • Adiponectin-stimulated CXCL8 release in primary human hepatocytes is regulated by ERK1/ERK2, p38 MAPK, NF-kappaB, and STAT3 signaling pathways.

    Wanninger J, Neumeier M, Weigert J, Bauer S, Weiss TS, Schäffler A, Krempl C, Bleyl C, Aslanidis C, Schölmerich J and Buechler C

    Department of Internal Medicine I, University Hospital of Regensburg, Regensburg, Germany.

    Adiponectin is believed to exert hepatoprotective effects and induces CXCL8, a chemokine that functions as a survival factor, in vascular cells. In the current study, it is demonstrated that adiponectin also induces CXCL8 expression in primary human hepatocytes but not in hepatocellular carcinoma cell lines. Knock down of the adiponectin receptor (AdipoR) 1 or AdipoR2 by small-interfering RNA indicates that AdipoR1 is involved in adiponectin-stimulated CXCL8 release. Adiponectin activates nuclear factor (NF)-kappaB in primary hepatocytes and pharmacological inhibition of NF-kappaB, the p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase (ERK) 1/ERK2 reduces adiponectin-mediated CXCL8 secretion. Furthermore, adiponectin also activates STAT3 involved in interleukin (IL)-6 and leptin-mediated CXCL8 induction in primary hepatocytes. Inhibition of JAK2 by AG-490 does not abolish adiponectin-stimulated CXCL8, indicating that this kinase is not involved. Pretreatment of primary cells with "STAT3 Inhibitor VI," however, elevates hepatocytic CXCL8 secretion, demonstrating that STAT3 is a negative regulator of CXCL8 in these cells. In accordance with this assumption, IL-6, a well-characterized activator of STAT3, reduces hepatocytic CXCL8. Therefore, adiponectin-stimulated induction of CXCL8 seems to be tightly controlled in primary human hepatocytes, whereas neither NF-kappaB, STAT3, nor CXCL8 are influenced in hepatocytic cell lines. CXCL8 is a survival factor, and its upregulation by adiponectin may contribute to the hepatoprotective effects of this adipokine.

    American journal of physiology. Gastrointestinal and liver physiology 2009;297;3;G611-8

  • Compartmentalizing VEGF-induced ERK2/1 signaling in placental artery endothelial cell caveolae: a paradoxical role of caveolin-1 in placental angiogenesis in vitro.

    Liao WX, Feng L, Zhang H, Zheng J, Moore TR and Chen DB

    Department of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093, USA.

    On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase Calpha, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

    Funded by: NHLBI NIH HHS: HL64703, HL70562, HL74947, R01 HL064703, R01 HL070562, R01 HL074947

    Molecular endocrinology (Baltimore, Md.) 2009;23;9;1428-44

  • Role of extracellular signal-regulated kinase for endothelial progenitor cell dysfunction in coronary artery disease.

    Friedrich EB, Werner C, Walenta K, Böhm M and Scheller B

    Klinik für Innere Medizin III (Kardiologie, Angiologie, Internistische Intensivmedizin), Universitätsklinikum des Saarlandes, Homburg, Saar, Germany. erik.friedrich@uks.eu

    In patients with coronary artery disease (CAD), number and function of endothelial progenitor cells (EPCs) are down-regulated. The relevant intracellular signalling mechanisms responsible for dysfunction of EPCs in CAD remain poorly characterized. Our goal was to examine the regulation of ERK-1/2 by SDF-1 and the role of ERK-1/2 for adhesion in EPCs. Western analysis revealed that the chemokine SDF-1 (SDF-1, 100 nM) mediates phosphorylation of ERK-2 after 90 s with a maximum after 180-300 s in EPCs isolated from healthy control subjects, while EPCs from patients with CAD are characterized by a temporally delayed and quantitatively markedly attenuated SDF-1-triggered ERK-2-phosphorylation. Functionally, EPCs isolated from patients with CAD display reduced SDF-1-induced adhesion under flow conditions, while augmenting ERK-2 signalling using an activating MEK-2 cDNA construct restores adhesion to control levels and rescues the adhesion defect of CAD-EPCs. These data indicate that defects in SDF-1-triggered EPC-adhesion contribute to the functional impairment of EPCs in CAD, and that ERK-2 represents a new therapeutic target for functional improvement of EPC adhesion in CAD.

    Basic research in cardiology 2009;104;5;613-20

  • Sorafenib induces growth inhibition and apoptosis in human synovial sarcoma cells via inhibiting the RAF/MEK/ERK signaling pathway.

    Peng CL, Guo W, Ji T, Ren T, Yang Y, Li DS, Qu HY, Li X, Tang S, Yan TQ and Tang XD

    Musculoskeletal Tumor Center, Peking University Peoples' Hospital, Beijing, China.

    Synovial sarcoma is a soft tissue sarcoma with poor prognosis and lack of response to conventional cytotoxic chemotherapy. The regulatory mechanisms for the rapid proliferation of synovial sarcoma cells and the particular aggressiveness of this sarcoma remain poorly understood. Mitogen-activated protein kinase (MAPK) cascades have been shown to play important roles in synovial sarcoma survival. Sorafenib (Nexavar, BAY 43-9006), a potent recombinant activated factor (RAF) inhibitor, inhibits the MAPK signaling pathway both in vitro and in vivo. In this study, we examined the inhibitory proliferation effects of sorafenib in synovial sarcoma growth and evaluated whether sorafenib modulates MAPK and tumor apoptosis cascades in human synovial sarcoma cell lines SW982 and HS-SY-II. Our results indicated that sorafenib effectively inhibited cellular proliferation and induces apoptosis of these two cells. Sorafenib inhibited the phosphorylation of MEK and ERK, downregulated cyclin D1 and Rb levels, caused G(1) arrest and S phase decrease, and induced apoptosis as confirmed by flow cytometry and the TUNEL assay. Furthermore, Bcl-xl and Mcl-1 levels significantly decreased, whereas expression levels of the proteins bcl-2 and bax were unchanged in response to sorafenib treatment in SW982 and HS-SY-II cells. In conclusion, our findings demonstrate that sorafenib is effective for growth inhibition of synovial sarcoma cell lines in vitro and suggest that sorafenib may be a new therapeutic option for patients with synovial sarcoma.

    Cancer biology & therapy 2009;8;18;1729-36

  • Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling.

    Cottrell GS, Padilla BE, Amadesi S, Poole DP, Murphy JE, Hardt M, Roosterman D, Steinhoff M and Bunnett NW

    Department of Surgery, University of California, San Francisco, CA 94143-0660, USA.

    Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

    Funded by: British Heart Foundation: FS/08/017/25027; NIDDK NIH HHS: DK39957, DK43207, R01 DK039957, R01 DK043207, R37 DK039957, R56 DK043207

    The Journal of biological chemistry 2009;284;33;22411-25

  • Tumor suppressor density-enhanced phosphatase-1 (DEP-1) inhibits the RAS pathway by direct dephosphorylation of ERK1/2 kinases.

    Sacco F, Tinti M, Palma A, Ferrari E, Nardozza AP, Hooft van Huijsduijnen R, Takahashi T, Castagnoli L and Cesareni G

    Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy.

    Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.

    The Journal of biological chemistry 2009;284;33;22048-58

  • LFA-1 and CD2 synergize for the Erk1/2 activation in the Natural Killer (NK) cell immunological synapse.

    Zheng X, Wang Y, Wei H, Sun R and Tian Z

    Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.

    Natural killer (NK) cell recognition and formation of a conjugate with target cells, followed by intracellular signal pathway activation and degradation of cytolytic granules, are essential for NK cell cytotoxicity. In this study, NK92 cells were used to investigate synapse formation and subsequent signaling after binding to the target cell. The binding rate of the NK92-target cell was associated with NK92 cell cytotoxicity. Confocal results showed that adhesion molecules, LFA-1 (CD11a) and CD2, accumulated at the interface of the NK92-K562 contact. Ligation with K562 cells activated the Erk1/2 signal pathway of NK92 cells. The blocking of the NK-target conjugate by EDTA or anti-CD11a or/and anti-CD2 antibody decreased the phosphorylation of Erk1/2 and NK cell cytotoxicity. Inhibition of Erk1/2 phosphorylation by the chemical inhibitor U0126 suppressed the cytolytic activity of NK92 cells, but had no effect on NK-target conjugate formation. Thus, conjugate formation of the NK92-target cell was prerequisite to NK cell activation, and subsequent signal transduction was also required for NK cell cytotoxicity.

    The Journal of biological chemistry 2009;284;32;21280-7

  • Gonadotropin-releasing hormone (GnRH)-I and GnRH-II induce cell growth inhibition in human endometrial cancer cells: involvement of integrin beta3 and focal adhesion kinase.

    Park DW, Choi KC, MacCalman CD and Leung PC

    Department of Obstetrics and Gynecology, Child and Family Research Institute, The University of British Columbia, Vancouver, British Columbia, V6H 3V5, Canada. gypsyroad@empal.com

    Endometrial carcinoma is the most common neoplasm of the female genital tract, accounting for nearly one half of all gynecologic cancers in the Western world. Although intensive research on pathological phenomena of endometrial cancer is currently going on, but exact cause and biological aspects of this disease are not well described yet. In addition to well-documented roles of gonadotropin-releasing hormone (GnRH) in hypopituitary ovarian (HPO) axis, the agonistic or antagonistic analogs (or both) of GnRH have been shown to inhibit the proliferation of a variety of human gynecologic cancers. Thus, in the present study, we further examined the possibility that GnRH induces integrin beta3 and activation of focal adhesion kinase (FAK) through mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, to inhibit the growth of HEC1A endometrial cancer cell line. As a result, both GnRH-I and GnRH-II resulted in a significant increase in integrin beta3 expression and evoked the activation of FAK in a time-dependent manner in these cells. In addition, these analogs induced an activation of ERK1/2 and p38 MAPK in a time-dependent manner as downstream pathways of FAK. It appears that GnRH-II has much greater effect on the activation of FAK, ERK1/2 and p38 compared to GnRH-I in these cells. Further, we demonstrated that the growth inhibition of HEC1A cells by GnRH-I or GnRH-II is involved in the activation of integrin-FAK and ERK1/2 and p38 MAPK pathways. Taken together, these results suggest that GnRH may be involved in the inhibition of endometrial cancer cell growth via activation of integrin beta3 and FAK as a direct effect. This knowledge could contribute to a better understanding of the mechanisms implicated in the therapeutic action of GnRH and its biomedical application for the treatment against endometrial cancer.

    Reproductive biology and endocrinology : RB&E 2009;7;81

  • Metformin decreases angiogenesis via NF-kappaB and Erk1/2/Erk5 pathways by increasing the antiangiogenic thrombospondin-1.

    Tan BK, Adya R, Chen J, Farhatullah S, Heutling D, Mitchell D, Lehnert H and Randeva HS

    Endocrinology and Metabolism Group, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

    Aims: Polycystic ovary syndrome (PCOS) is associated with insulin resistance (IR), obesity, and cardiovascular complications. Thrombospondin-1 (TSP-1) is a novel antiangiogenic adipokine highly expressed in obese insulin-resistant subjects. We sought to assess TSP-1 levels in adipose tissue (AT) from PCOS women and matched controls. The effects of metformin treatment on circulating TSP-1 levels in PCOS subjects, the effects of serum from normal and PCOS women on in vitro migration and angiogenesis before and after metformin treatment, and ex vivo regulation of AT TSP-1 by D-glucose were also studied.

    Serum TSP-1 (ELISA), subcutaneous and omental AT TSP-1 mRNA (reverse transcriptase-polymerase chain reaction), and protein (western blotting) were significantly lower in PCOS women (P < 0.05). Corresponding plasminogen activator inhibitor-1 (PAI-1) and PAI-1 activity were significantly higher (P < 0.01). After 6 months of metformin treatment, there was a significant increase in serum TSP-1 (P < 0.05) and a corresponding decrease in PAI-1 and PAI-1 activity (P < 0.01). In vitro migration and angiogenesis were significantly increased in serum from PCOS women (P < 0.01); these effects were significantly attenuated by metformin treatment (P < 0.01) through the regulation of TSP-1 levels via nuclear factor-kappaB (NF-kappaB), extracellular regulated-signal kinase 1/2 (Erk1/2) and Erk5 pathways. Importantly, changes in the intima media thickness were predictive of changes in serum TSP-1 (P = 0.049). In AT explants, glucose significantly decreased TSP-1 protein production and secretion into conditioned media (ELISA) (P < 0.05, P < 0.001).

    Conclusion: TSP-1 levels are lower in PCOS women. Metformin treatment increases serum TSP-1 in these women. Our findings provide novel insights into the relationship between obesity, IR, and angiogenesis.

    Cardiovascular research 2009;83;3;566-74

  • Specific phosphorylation and activation of ERK1c by MEK1b: a unique route in the ERK cascade.

    Shaul YD, Gibor G, Plotnikov A and Seger R

    Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.

    Extracellular signal-regulated kinases (ERKs) are key signaling molecules that regulate a large number of cellular processes, including mitosis. We showed previously that ERK1c, an alternatively spliced form of ERK1, facilitates mitotic Golgi fragmentation without the involvement of ERK1 and ERK2. Here we demonstrate that activation of ERK1c is mainly mediated by mitogen-activated protein kinase (MAPK)/ERK kinase 1b (MEK1b), which is an alternatively spliced form of MEK1 that was previously considered an inactive kinase. MEK1b phosphorylation and activity are preferentially stimulated by nocodazole, to induce its specific activity toward ERK1c. MEK1/2, on the other hand, preferentially target ERK1/2 in response to growth factors, such as EGF. As previously demonstrated for ERK1c, also MEK1b expression and activity are elevated during mitosis, and thereby enhance Golgi fragmentation and mitotic rate. MEK1 activity is also increased during mitosis, but this isoform facilitates mitotic progression without affecting the Golgi architecture. These results illustrate that the ERK cascade is divided into two routes: the classic MEK1/2-ERK1/2 and the splice-variant MEK1b-ERK1c, each of which regulates distinct cellular processes and thus extends the cascade specificity.

    Genes & development 2009;23;15;1779-90

  • Activation of PKCdelta and ERK1/2 in the sensitivity to UV-induced apoptosis of human cells harboring 4977 bp deletion of mitochondrial DNA.

    Liu CY, Lee CF and Wei YH

    Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, 155 Li-Nong St., Sec. 2, Peitou, Taipei 112, Taiwan.

    The 4977 bp deletion of mitochondrial DNA (mtDNA), often found in patients with chronic progressive external ophthalmoplegia (CPEO), has been demonstrated to increase the susceptibility to apoptosis of human cells. We investigated the mechanism underlying the apoptotic susceptibility of the Delta4977 cybrid harboring about 80% 4977 bp-deleted mtDNA. The production of hydrogen peroxide (H(2)O(2)) and phosphorylation of PKCdelta and ERK1/2 were increased in the Delta4977 cybrid, which was more susceptible to UV-induced apoptosis. Moreover, treatment with N-acetyl-l-cysteine (NAC) or blocking of activation of PKCdelta by rottlerin or PKCdelta-siRNA, and inhibition of ERK1/2 by PD98059 or ERK1/2-siRNA significantly attenuated the susceptibility of the Delta4977 cybrid to apoptosis. Furthermore, the increase of PKCdelta expression in the Delta4977 cybrid also amplified the apoptotic signal through caspase 3-mediated proteolytic activation of PKCdelta. In addition, PKCdelta and ERK1/2 were hyperphosphorylated in skin fibroblasts of CPEO patients harboring 4977 bp-deleted mtDNA. We suggest that the activation of PKCdelta and ERK1/2 elicited by 4977 bp-deleted mtDNA-induced oxidative stress plays a role in the susceptibility of the mutant cells to apoptosis. This may explain, at least in part, the degenerative manifestation of brain and muscle in patients with mitochondrial encephalomyopathies such as CPEO syndrome.

    Biochimica et biophysica acta 2009;1792;8;783-90

  • Defects in cell spreading and ERK1/2 activation in fibroblasts with lamin A/C mutations.

    Emerson LJ, Holt MR, Wheeler MA, Wehnert M, Parsons M and Ellis JA

    The Randall Division of Cell and Molecular Biophysics, King's College, New Hunts House, Guy's Campus, London, SE1 1UL, UK.

    In-frame mutations in nuclear lamin A/C lead to a multitude of tissue-specific degenerative diseases known as the 'laminopathies'. Previous studies have demonstrated that lamin A/C-null mouse fibroblasts have defects in cell polarisation, suggesting a role for lamin A/C in nucleo-cytoskeletal-cell surface cross-talk. However, this has not been examined in patient fibroblasts expressing modified forms of lamin A/C. Here, we analysed skin fibroblasts from 3 patients with Emery-Dreifuss muscular dystrophy and from 1 with dilated cardiomyopathy. The emerin-lamin A/C interaction was impaired in each mutant cell line. Mutant cells exhibited enhanced cell proliferation, collagen-dependent adhesion, larger numbers of filopodia and smaller cell spread size, compared with control cells. Furthermore, cell migration, speed and polarization were elevated. Mutant cells also showed an enhanced ability to contract collagen gels at early time points, compared with control cells. Phosphotyrosine measurements during cell spreading indicated an initial temporal lag in ERK1/2 activation in our mutant cells, followed by hyper-activation of ERK1/2 at 2 h post cell attachment. Deregulated ERK1/2 activation is linked with cardiomyopathy, cell spreading and proliferation defects. We conclude that a functional emerin-lamin A/C complex is required for cell spreading and proliferation, possibly acting through ERK1/2 signalling.

    Biochimica et biophysica acta 2009;1792;8;810-21

  • Genistein inhibits cell growth by modulating various mitogen-activated protein kinases and AKT in cervical cancer cells.

    Kim SH, Kim SH, Kim YB, Jeon YT, Lee SC and Song YS

    Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.

    Genistein, a soy-derived isoflavone, inhibits growth of tumor cells from various malignancies. Here we investigated the effect of genistein on the growth of cervical cancer cells (HeLa and CaSki) and its possible mechanism. Genistein significantly suppressed cell growth of HeLa and CaSki cells at concentrations of 20 and 60 micromol/L, respectively, for 24 h. Western blotting analysis showed that genistein reduced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)-1/2 and induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Moreover, inhibition of ERK1/2 activity enhanced cell growth inhibition by genistein, whereas inhibition of p38 MAPK activity rescued from genistein-mediated growth inhibition. Interestingly, inhibition of AKT activity recovered genistein-induced growth inhibition in CaSki cells but did not in HeLa cells. However, inhibition of JNK activity seemed to have little effect on cell growth inhibition by genistein. Taken together, these results suggest that genistein could inhibit cell growth by inhibiting ERK1/2 activity and activating p38 MAPK.

    Annals of the New York Academy of Sciences 2009;1171;495-500

  • Selective inhibition of prostaglandin E2 receptors EP2 and EP4 induces apoptosis of human endometriotic cells through suppression of ERK1/2, AKT, NFkappaB, and beta-catenin pathways and activation of intrinsic apoptotic mechanisms.

    Banu SK, Lee J, Speights VO, Starzinski-Powitz A and Arosh JA

    Reproductive Endocrinology and Cell Signaling Laboratory, Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843, USA.

    Endometriosis is a benign chronic gynecological disease of reproductive-age women characterized by the presence of functional endometrial tissues outside the uterine cavity. It is an estrogen-dependent disease. Current treatment modalities to inhibit biosynthesis and actions of estrogen compromise menstruation, pregnancy, and the reproductive health of women and fail to prevent reoccurrence of disease. There is a critical need to identify new specific signaling modules for non-estrogen-targeted therapies for endometriosis. In our previous study, we reported that selective inhibition of cyclooxygenase-2 prevented survival, migration, and invasion of human endometriotic epithelial and stromal cells, which was due to decreased prostaglandin E(2) (PGE(2)) production. In this study, we determined mechanisms through which PGE(2) promoted survival of human endometriotic cells. Results of the present study indicate that 1) PGE(2) promotes survival of human endometriotic cells through EP2 and EP4 receptors by activating ERK1/2, AKT, nuclear factor-kappaB, and beta-catenin signaling pathways; 2) selective inhibition of EP2 and EP4 suppresses these cell survival pathways and augments interactions between proapoptotic proteins (Bax and Bad) and antiapoptotic proteins (Bcl-2/Bcl-XL), facilitates the release of cytochrome c, and thus activates caspase-3/poly (ADP-ribose) polymerase-mediated intrinsic apoptotic pathways; and 3) these PGE(2) signaling components are more abundantly expressed in ectopic endometriosis tissues compared with eutopic endometrial tissues during the menstrual cycle in women. These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential therapy, including nonestrogen target, to expand the spectrum of currently available treatment options for endometriosis in women.

    Molecular endocrinology (Baltimore, Md.) 2009;23;8;1291-305

  • Suppression of urokinase plasminogen activator receptor inhibits proliferation and migration of pancreatic adenocarcinoma cells via regulation of ERK/p38 signaling.

    Xue A, Xue M, Jackson C and Smith RC

    Department of Surgery, The University of Sydney, Royal North Shore Hospital, St Leonards, NSW, Australia.

    Pancreatic ductal adenocarcinoma (PDAC) expresses high levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor (PAI)-2, which may play an important role in PDAC progression. The overexpression of uPAR predicted short survival in PDAC patients. In this study, two different PDAC cell lines were used to examine the effect of small interfering (si) RNAs to uPAR, uPA and PAI-2 on proliferation, apoptosis, migration and MAP kinase activation. In both PDAC cell lines, siRNA to uPAR significantly inhibited cell proliferation and migration and stimulated apoptosis, to a greater extent than uPA siRNA. When either PDAC cell line was treated with uPAR siRNA, the level of phosphorylated ERK (p-ERK) decreased substantially, whereas phosphorylated p38 (p-p38) increased when compared to non-silencing control, uPA siRNA or PAI-2 siRNA treatment. This resulted in enhancement of the p-p38/p-ERK ratio which favors cancer cell arrest. Interestingly, uPAR protein expression was suppressed by p-ERK inhibition and stimulated with p-p38 inhibition, suggesting the presence of a positive feedback loop between uPAR and ERK. In summary, our data indicate that, of the uPA system, uPAR exerts the strongest effects on PDAC cells, by acting through the ERK signaling pathway via a positive feedback loop. Disruption of this loop with uPAR siRNA or inhibitor of p-ERK, inhibits PDAC proliferation and migration and promotes apoptosis. These findings suggest that uPAR strongly contributes to PDAC progression and may be considered as a potential anti-pancreatic cancer target.

    The international journal of biochemistry & cell biology 2009;41;8-9;1731-8

  • Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells.

    Chen QW, Edvinsson L and Xu CB

    Division of Experimental Vascular Research, Institute of Clinical Science in Lund, Lund University, Lund, Sweden. cherqw@hotmail.com

    Unlabelled: muscle cells (VSMCs) through activation of endothelin type A (ETA) and type B (ETB) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ETA and ETB receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.

    Results: Subconfluent human VSMCs were stimulated by ET-1 at different concentrations (1 nM-1 microM). The activation of ERK1/2 was examined by immunofluorescence, Western blot and phosphoELISA using specific antibody against phosphorylated ERK1/2 protein. ET-1 induced a concentration- and time- dependent activation of ERK1/2 with a maximal effect at 10 min. It declined to baseline level at 30 min. The ET-1-induced activation of ERK1/2 was completely abolished by MEK1/2 inhibitors U0126 and SL327, and partially inhibited by the MEK1 inhibitor PD98059. A dual endothelin receptor antagonist bosentan or the ETA antagonist BQ123 blocked the ET-1 effect, while the ETB antagonist BQ788 had no significant effect. However, a selective ETB receptor agonist, Sarafotoxin 6c (S6c) caused a time-dependent ERK1/2 activation with a maximal effect by less than 20% of the ET-1-induced activation of ERK1/2. Increase in bosentan concentration up to 10 microM further inhibited ET-1-induced activation of ERK1/2 and had a stronger inhibitory effect than BQ123 or the combined use of BQ123 and BQ788. To further explore ET-1 intracellular signaling, PKC inhibitors (staurosporin and GF109203X), PKC-delta inhibitor (rottlerin), PKA inhibitor (H-89), and phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) were applied. The inhibitors showed significant inhibitory effects on ET-1-induced activation of ERK1/2. However, blockage of L-type Ca2+ channels or calcium/calmodulin-dependent protein kinase II, chelating extracellular Ca2+ or emptying internal Ca2+ stores, did not affect ET-1-induced activation of ERK1/2.

    Conclusion: The ETA receptors predominate in the ET-1-induced activation of ERK1/2 in human VSMCs, which associates with increments in intracellular PKC, PKA and PI3K activities, but not Ca2+ signalling.

    BMC cell biology 2009;10;52

  • Protein kinase SGK1 enhances MEK/ERK complex formation through the phosphorylation of ERK2: implication for the positive regulatory role of SGK1 on the ERK function during liver regeneration.

    Won M, Park KA, Byun HS, Kim YR, Choi BL, Hong JH, Park J, Seok JH, Lee YH, Cho CH, Song IS, Kim YK, Shen HM and Hur GM

    Department of Pharmacology, Research Institute for Medical Science, Infection Signaling Network Research Center, Daejeon Regional Cancer Center, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon 301-131, Republic of Korea.

    Based on the observation of biphasic induction of SGK1 expression in the regenerating liver, we investigated the role of SGK1 in the regulation of MEK/ERK signaling pathway which plays a crucial role in regulating growth and survival signaling.

    Methods: To determine the role of SGK1 in the activation of MEK/ERK signaling cascade, we infected primary hepatocytes with recombinant adenoviral vector encoding SGK1, and assessed its effect on the MEK/ERK signaling pathway.

    Results: Partial hepatectomy resulted in the biphasic transcriptional induction of SGK1 in regenerating liver tissues. Infection of primary hepatocytes with an adenoviral vector encoding SGK1 enhanced the ERK phosphorylation under serum-starved conditions and this was blocked by the expression of kinase-dead SGK1. SGK1 was found to physically interact with ERK1/2 as well as MEK1/2. Furthermore, SGK1 mediated the phosphorylation of ERK2 on Ser(29) in a serum-dependent manner. Replacement of Ser(29) to aspartic acid, which mimics the phosphorylation of Ser(29), enhanced the ERK2 activity as well as the MEK/ERK complexes formation.

    Conclusions: SGK1 expression during liver regeneration is a part of a signaling pathway that is necessary for enhancing ERK signaling activation through modulating the MEK/ERK complex formation.

    Journal of hepatology 2009;51;1;67-76

  • [The expression of MKP-1 and p-ERK(1/2) in primary ovarian epithelial tumor tissues].

    Zhou JW, Gan NY and Zhang WJ

    Department of Gynecology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China. jianwei-zhou@163.com

    To investigate the expression of mitogen activated protein kinase phosphatase-1 (MKP-1) and phosphorylation extracellular signal-regulated kinases (p-ERK(1/2)) in primary ovarian epithelial tumor tissues, and provide experiment's foundation on the new treatment in ovarian cancer. Expression of MKP-1 and p-ERK(1/2) in tissues from 64 patients with primary ovarian epithelial tumor, 35 patients with ovarian epithelial bordline tumor, 32 patients with ovarian epithelial benign tumor and 26 normal ovarian tissues was detected by immunohistochemistry. Western-blot was also used for detecting the expression of MKP-1 and p-ERK(1/2) protein in these tissues. Immunohistochemistry and Western-blot assay showed that the expression of MKP-1 was gradually decreased in normal ovarian tissues, benign tumor, bordline tumor and carcinoma respectively, and there were significant differences among them (P < 0.01). The MKP-1 expression level in the carcinoma tissues of stage III/IV patients was significantly lower than that of stage I/II patients. However, the expression of p-ERK(1/2) was gradually increased in normal ovarian tissues, benign tumor, bordline tumor and carcinoma respectively, and there were also significant differences among them (P < 0.01), the p-ERK(1/2) expression level in the carcinoma tissues of stage III/IV patients was significantly higher than that of stage I/II patients. Expression of MKP-1 and p-ERK(1/2) in same ovarian carcinoma tissues detected by immunohistochemistry and Western-blot assay showed significant negative correlation (r = -0.90, P < 0.01 and r = -0.78, P < 0.01 respectively). The expression changes of MKP-1 and ERKs may play a role in the development of ovarian carcinoma. The abnormal expression of MKP-1 and p-ERK(1/2) probably assists in promoting the development and progression of ovarian carcinoma.

    Fen zi xi bao sheng wu xue bao = Journal of molecular cell biology 2009;42;3-4;224-30

  • Cilostazol protects endothelial cells against lipopolysaccharide-induced apoptosis through ERK1/2- and P38 MAPK-dependent pathways.

    Lim JH, Woo JS and Shin YW

    Department of Internal Medicine, Pusan National University College of Medicine, Seo-gu, Busan, Korea.

    We examined the effects of cilostazol on mitogen-activated protein kinase (MAPK) activity and its relationship with cilostazol-mediated protection against apoptosis in lipopolysaccharide (LPS)-treated endothelial cells.

    Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS and cilostazol with and without specific inhibitors of MAPKs; changes in MAPK activity in association with cell viability and apoptotic signaling were investigated.

    Results: Cilostazol protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c, and subsequent activation of caspases, stimulating extracellullar signal-regulated kinase (ERK1/2) and p38 MAPK signaling, and increasing phosphorylated cAMP-responsive element-binding protein (CREB) and Bcl-2 expression, while suppressing Bax expression. These cilostazol-mediated cellular events were effectively blocked by MAPK/ERK kinase (MEK1/2) and p38 MAPK inhibitors.

    Conclusions: Cilostazol protects HUVECs against LPS-induced apoptosis by suppressing mitochondria-dependent apoptotic signaling. Activation of ERK1/2 and p38 MAPKs, and subsequent stimulation of CREB phosphorylation and Bcl-2 expression, may be responsible for the cellular signaling mechanism of cilostazol-mediated protection.

    The Korean journal of internal medicine 2009;24;2;113-22

  • Cross talk initiated by endothelial cells enhances migration and inhibits anoikis of squamous cell carcinoma cells through STAT3/Akt/ERK signaling.

    Neiva KG, Zhang Z, Miyazawa M, Warner KA, Karl E and Nör JE

    Angiogenesis Research Laboratory, Department of Restorative Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA.

    It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL- 6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

    Funded by: NCI NIH HHS: P50 CA097248, P50-CA97248; NIDCR NIH HHS: R01 DE014601, R01 DE015948, R01 DE016586, R01-DE14601, R01-DE15948, R01-DE16586, R21 DE019279, R21-DE19279

    Neoplasia (New York, N.Y.) 2009;11;6;583-93

  • Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation.

    Huang Q, Cheung AP, Zhang Y, Huang HF, Auersperg N and Leung PC

    Dept. of Obstetrics and Gynecology, Univ. of British Columbia, Rm. 2H-30, 4500 Oak St., Vancouver, BC, Canada V6H 3V5.

    GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [(3)H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [(3)H]thymidine incorporation, enhanced cell transition from G(0)/G(1) to S and G(2)/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D(1) and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15(INK4B) and p16(INK4A). GDF-9 also activated Rb protein (a critical G(1) to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D(1) and E but not the decrease in p15(INK4B) and p16(INK4A) induced by GDF-9. In contrast, SIS3 reversed the decrease in p15(INK4B) and p16(INK4A) but not the increase in cyclin D(1) and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D(1) and E and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways.

    American journal of physiology. Endocrinology and metabolism 2009;296;6;E1344-53

  • EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway, but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line.

    Kim S, Choi JH, Lim HI, Lee SK, Kim WW, Cho S, Kim JS, Kim JH, Choe JH, Nam SJ, Lee JE and Yang JH

    Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Ilwon-dong 50, Kangnam-gu, Seoul, Republic of Korea.

    The number of epidermal growth factor receptors (EGFRs) and their ligands are highly expressed in malignant tumor cells. The EGF signaling pathway is also activated in up to one-third of patients with breast cancer. In this study, we investigated the novel function of the JAK3 inhibitor, WHI-P131, on EGF-induced MMP-9 expression and the regulatory mechanism of EGF-induced MMP-9 expression in SKBR3 cells. We observed that EGF increased MMP-9 mRNA and protein expression in a dose-dependent manner. EGF also induced the phosphorylation of EGFR, ERK, and STAT-3, and these effects were inhibited by the EGFR inhibitor, AG1478.To investigate the involvement of the STAT-3 pathway on EGF-induced MMP-9 expression, we pretreatedSKBR3 cells with JAK1, JAK2, and JAK3 inhibitors prior to EGF treatment. The results showed that the JAK3 inhibitor, WHI-P131, as well as JAK3 siRNA transfection, but not the JAK1 and JAK2 inhibitors, significantly decreased EGF-induced MMP-9 expression. In addition, EGF-induced STAT-3 phosphorylation was only inhibited by WHI-P131. We then transfected cells with adenoviral STAT-3 (Ad-STAT-3), followed by treatment with EGF. Interestingly, EGF-induced MMP-9 expression was decreased by Ad-STAT-3 overexpression in a dose-dependent manner, while it was significantly increased by STAT-3 siRNA transfection. Our results also showed that basal levels of MMP-9 expression were significantly increased by constitutive active-MEK (CAMEK)overexpression. EGF-induced ERK phosphorylation was prevented by WHI-P131, but not by JAK1 andJAK2 inhibitors. On the other hand, EGF-induced MMP-9 expression was decreased by the MEK1/2 inhibitor,UO126. Therefore, for the first time, we suggest that the JAK3 inhibitor, WHI-P131, inhibits EGF-induced STAT-3 phosphorylation as well as ERK phosphorylation. The JAK3/ERK pathway may play an important role in EGFinduced MMP-9 expression in SKBR3 cells.

    Cellular signalling 2009;21;6;892-8

  • Extracellular signal-regulated kinase 2-dependent phosphorylation induces cytoplasmic localization and degradation of p21Cip1.

    Hwang CY, Lee C and Kwon KS

    Laboratory of Cell Signaling, Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Oun-Dong, Yusong, Daejeon 305-333, South Korea.

    p21(Cip1) is an inhibitor of cell cycle progression that promotes G(1)-phase arrest by direct binding to cyclin-dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21(Cip1) downregulation at the posttranslational level. This downregulation requires the sustained activation of extracellular signal-regulated kinase 2 (ERK2), which directly interacts with and phosphorylates p21(Cip1), promoting p21(Cip1) nucleocytoplasmic translocation and ubiquitin-dependent degradation, thereby resulting in cell cycle progression. ERK1 is not likely involved in this process. Phosphopeptide analysis of in vitro ERK2-phosphorylated p21(Cip1) revealed two phosphorylation sites, Thr57 and Ser130. Double mutation of these sites abolished ERK2-mediated p21(Cip1) translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G(1)/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21(Cip1).

    Molecular and cellular biology 2009;29;12;3379-89

  • Expression of phospho-ERK1/2 and PI3-K in benign and malignant gallbladder lesions and its clinical and pathological correlations.

    Li Q and Yang Z

    Research Laboratory of Hepatobiliary Diseases, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, PR China. liqinglonga@sina.com

    Background: An increasing number of studies have shown that ERK and PI3-K/AKT signaling pathways are involved in various human cancers including hepatocellular carcinoma and cholangiocarcinoma. However, few studies have examined gallbladder cancer specimens, and little is known about the clinical and pathological significance of ERK1/2 and PI3-K/AKT signaling changes in gallbladder adenocarcinoma. In this study, we examined phospho-ERK1/2 (p-ERK1/2) and PI3K expression and analyzed its clinicopathological impact in gallbladder adenocarcinoma.

    Methods: Immunohistochemistry was used to detect and compare the frequency of p-ERK1/2 and PI3-K expression in gallbladder adenocarcinoma, peri-tumor tissues, adenomatous polyps, and chronic cholecystitis specimens.

    Results: The positive staining for p-EKR1/2 and PI3-K were 63/108 (58.3%) and 55/108 (50.9%) in gallbladder adenocarcinoma; 14/46 (30.4%) and 5/46 (10.1%) in peri-tumor tissues; 3/15 (20%) and 3/15 (20%) in adenomatous polyps; and 4/35 (11.4%) and 3/35 (8.6%) in chronic cholecystitis. The positive rate of p-ERK1/2 or PI3-K in gallbladder adenocarcinoma was significantly higher than that in peri-tumor tissue (both, P < 0.01), adenomatous polyps (p-ERK1/2, P < 0.01; PI3-K, P < 0.05), and chronic cholecystitis (both, P < 0.01). The positive staining for p-ERK1/2 or PI3-K was significantly lower in well/highly-differentiated adenocarcinomas with maximal diameter < 2.0 cm, no metastasis to lymph node, and no infiltration of regional tissues or organs compared to poorly-differentiated adenocarcinomas which are characterized by a maximal diameter >or= 2.0 cm, with metastasis to lymph node and infiltration of regional tissues or organs (P < 0.05 or P < 0.01). Moreover, the frequency of p-ERK1/2 expression in gallbladder adenocarcinomas without gallstone was significantly lower than those with gallstones. Increased expression of p-ERK1/2 (P < 0.05) and PI3K (P = 0.062) was associated with decreased overall survival. Multivariate Cox regression analysis showed that increased p-ERK1/2 expression was an independent prognostic predictor in gallbladder carcinoma (P = 0.028).

    Conclusion: Increased expression of p-ERK1/2 and PI3K might contribute to gallbladder carcinogenesis. p-ERK1/2 over-expression is correlated with decreased survival and therefore may serve as an important biological marker in development of gallbladder adenocarcinoma.

    Journal of experimental & clinical cancer research : CR 2009;28;65

  • Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode.

    Lin RY, Wang JH, Lu XM, Zhou XT, Mantion G, Wen H, Vuitton DA and Richert L

    Xinjiang Key Laboratory on Echinococcosis and Liver Surgery, 1st Teaching Hospital of Xinjiang Medical University, No.1 Liyushan Road, Urumqi 830054, China.

    Aim: To explore the effect of Echinococcus multilocularis (E. multilocularis) on the activation of mitogen-activated protein kinase (MAPK) signaling pathways and on liver cell proliferation.

    Methods: Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen (PCNA) expression were measured in the liver of patients with alveolar echinococcosis (AE). MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase (ERK) kinase] and ribosomal S6 kinase (RSK) phosphorylation were detected in primary cultures of rat hepatocytes in contact in vitro with (1) E. multilocularis vesicle fluid (EmF), (2) E. multilocularis-conditioned medium (EmCM).

    Results: In the liver of AE patients, ERK 1/2 and p38 MAPK were activated and PCNA expression was increased, especially in the vicinity of the metacestode. Upon exposure to EmF, p38, c-Jun N-terminal kinase (JNK) and ERK1/2 were also activated in hepatocytes in vitro, as well as MEK1/2 and RSK, in the absence of any toxic effect. Upon exposure to EmCM, only JNK was up-regulated.

    Conclusion: Previous studies have demonstrated an influence of the host on the MAPK cascade in E. multilocularis. Our data suggest that the reverse, i.e. parasite-derived signals efficiently acting on MAPK signaling pathways in host liver cells, is actually operating.

    World journal of gastroenterology 2009;15;17;2116-24

  • Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/Akt pathways.

    Kornasio R, Riederer I, Butler-Browne G, Mouly V, Uni Z and Halevy O

    Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.

    Beta-hydroxy-beta-methylbutyrate (HMB), a leucine catabolite, has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that HMB would directly regulate muscle-cell proliferation and differentiation and would attenuate apoptosis, the latter presumably underlying satellite-cell depletion during muscle degradation or atrophy. Adding various concentrations of HMB to serum-starved myoblasts induced cell proliferation and MyoD expression as well as the phosphorylation of MAPK/ERK. HMB induced differentiation-specific markers, increased IGF-I mRNA levels and accelerated cell fusion. Its inhibition of serum-starvation- or staurosporine-induced apoptosis was reflected by less apoptotic cells, reduced BAX expression and increased levels of Bcl-2 and Bcl-X. Annexin V staining and flow cytometry analysis showed reduced staurosporine-induced apoptosis in human myoblasts in response to HMB. HMB enhanced the association of the p85 subunit of PI3K with tyrosine-phosphorylated proteins. HMB elevated Akt phosphorylation on Thr308 and Ser473 and this was inhibited by Wortmannin, suggesting that HMB acts via Class I PI3K. Blocking of the PI3K/Akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of HMB on muscle cell differentiation and fusion. These direct effects of HMB on myoblast differentiation and survival resembling those of IGF-I, at least in culture, suggest its positive influence in preventing muscle wasting.

    Biochimica et biophysica acta 2009;1793;5;755-63

  • Sphingosine kinase as an oncogene: autocrine sphingosine 1-phosphate modulates ML-1 thyroid carcinoma cell migration by a mechanism dependent on protein kinase C-alpha and ERK1/2.

    Bergelin N, Blom T, Heikkilä J, Löf C, Alam C, Balthasar S, Slotte JP, Hinkkanen A and Törnquist K

    Department of Biology, Abo Akademi University, BioCity, Tykistökatu 6, 20520 Turku, Finland.

    Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

    Endocrinology 2009;150;5;2055-63

  • Mechanical force induces type I collagen expression in human periodontal ligament fibroblasts through activation of ERK/JNK and AP-1.

    Kook SH, Hwang JM, Park JS, Kim EM, Heo JS, Jeon YM and Lee JC

    Division of Biological Sciences, Chonbuk National University, Jeonju 561-756, South Korea.

    Type I collagen (COL I) is the predominant collagen in the extracellular matrix of periodontal ligament (PDL), and its expression in PDL fibroblasts (PLF) is sensitive to mechanical force. However, the mechanism by which PLF induces COL I to respond to mechanical force is unclear. This study examined the nature of human PLF in mediating COL I expression in response to centrifugal force. Signal transduction pathways in the early stages of mechanotransduction involved in the force-driven regulation of COL I expression were also investigated. Centrifugal force up-regulated COL I without cytotoxicity and activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. ERK and JNK inhibitor blocked the expression of COL I but p38 kinase inhibitor had no effect. Centrifugal force activated activator protein-1 (AP-1) through dimerization between c-Fos and c-Jun transcription factors. ERK and JNK inhibitors also inhibited AP-1-DNA binding, c-Fos nuclear translocation, and c-Jun phosphorylation that were increased in the force-exposed PLF. Further, transfecting the cells with c-Jun antisense oligonucleotides almost completely abolished the force-induced increase of c-Jun phosphorylation and COL I induction. Our findings suggest that mechanical signals are transmitted into the nucleus by ERK/JNK signaling pathways and then stimulate COL I expression through AP-1 activation in force-exposed human PLF.

    Journal of cellular biochemistry 2009;106;6;1060-7

  • Mechanical force inhibits osteoclastogenic potential of human periodontal ligament fibroblasts through OPG production and ERK-mediated signaling.

    Kook SH, Son YO, Hwang JM, Kim EM, Lee CB, Jeon YM, Kim JG and Lee JC

    Division of Biological Sciences, Chonbuk National University, Jeonju 561-756, South Korea.

    Periodontal ligament and gingival fibroblasts play important roles in bone remodeling. Periodontal ligament fibroblasts stimulate bone remodeling while gingival fibroblasts protect abnormal bone resorption. However, few studies had examined the differences in stimulation of osteoclast formation between the two fibroblast populations. The precise effect of mechanical forces on osteoclastogenesis of these populations is also unknown. This study revealed that more osteoclast-like cells were induced in the co-cultures of bone marrow cells with periodontal ligament than gingival fibroblasts, and this was considerably increased when anti-osteoprotegerin (OPG) antibody was added to the co-cultures. mRNA levels of receptor activator of nuclear factor-kappaB ligand (RANKL) were increased in both populations when they were cultured with dexamethasone and vitamin D(3). Centrifugal forces inhibited osteoclastogenesis of both populations, and this was likely related to the force-induced OPG up-regulation. Inhibition of extracellular signal-regulated kinase (ERK) signaling by a pharmacological inhibitor (10 microM PD98059) or by siERK transfection suppressed the force-induced OPG up-regulation along with the augmentation of osteoclast-like cells that were decreased by the force. These results suggest that periodontal ligament fibroblasts are naturally better at osteoclast induction than gingival fibroblasts, and that centrifugal force inhibited osteoclastogenesis of the periodontal fibroblasts through OPG production and ERK activation.

    Journal of cellular biochemistry 2009;106;6;1010-9

  • A positive feedback between activated extracellularly regulated kinase and cyclooxygenase/lipoxygenase maintains proliferation and migration of breast cancer cells.

    You J, Mi D, Zhou X, Qiao L, Zhang H, Zhang X and Ye L

    Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin, People's Republic of China.

    Metastasis of breast cancer cells is the leading cause of death in breast cancer patients. Why do breast cancer cells with high metastatic potential always keep in high proliferation and migration? The endogenous signaling pathways associated with tumor metastasis remain unclear. In the present study, we address whether a link between ERK and the enzymes associated with arachidonic acid (AA) metabolism contributes to the proliferation and migration of breast cancer cells. To identify endogenous signaling pathways involved in sustaining proliferation and migration of breast cancer cells, we performed parallel studies of human breast cancer cell lines that differ in their metastatic potential. Our data showed that cell lines with high metastatic potential, including LM-MCF-7 and MDA-MB-231, exhibited significantly high, sustained levels of phosphorylated ERK (pERK) 1/2 relative to MCF-7 cells. Our findings showed that beta-catenin, cyclin D1, and survivin serve downstream effectors of pERK1/2, whereas Gi/o proteins, phospholipase C, and protein kinase C serve upstream activators of pERK1/2. In addition, AA metabolites were able to activate Gi/o proteins, phospholipase C, protein kinase C, and pERK1/2 cascades through cyclooxygenase and lipoxygenase. In contrast, activated ERK1/2 promoted AA metabolism through a positive feedback loop, which conduces to a high proliferative potential and the migration of the breast cancer cells. Together, our data provide new mechanistic insights into possible endogenous signaling metastatic signaling pathways involved in maintaining proliferation and migration of breast cancer cells.

    Endocrinology 2009;150;4;1607-17

  • Cold-inducible RNA-binding protein bypasses replicative senescence in primary cells through extracellular signal-regulated kinase 1 and 2 activation.

    Artero-Castro A, Callejas FB, Castellvi J, Kondoh H, Carnero A, Fernández-Marcos PJ, Serrano M, Ramón y Cajal S and Lleonart ME

    Pathology Department, Fundació Institut de Recerca Hospital Vall d'Hebron, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain.

    Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16(INK4a), p21(WAF1), and p19(ARF) upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research.

    Molecular and cellular biology 2009;29;7;1855-68

  • Effects of MAPK signaling on 1,25-dihydroxyvitamin D-mediated CYP24 gene expression in the enterocyte-like cell line, Caco-2.

    Cui M, Zhao Y, Hance KW, Shao A, Wood RJ and Fleet JC

    Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47906-2059, USA.

    We examined the role of the extracellular signal regulated kinases (ERK) in 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3))-induced gene expression in the differentiated Caco-2 cells. 1,25(OH)(2)D(3)-regulated expression of the 25-hydroxyvitamin D, 24-hydroxylase (CYP24) gene (both natural gene and promoter construct) was strongly modulated by altering ERK activity (i.e., reduced by MEK inhibitors and dominant negative (dn) ERK1 and ERK2, activated by epidermal growth factor) but ERK inhibition had no effect on 1,25(OH)(2)D(3)-regulated expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6). ERK5-mediated phosphorylation of the transcription factor Ets-1 enhanced 1,25(OH)(2)D(3)-mediated CYP24 gene transcription in proliferating but not differentiated Caco-2 cells due to reduced levels of ERK5 and Ets-1 (total and phosphoprotein levels) in differentiated cells. MEK inhibition reduced 1,25(OH)(2)D(3)-induced 3X-VDRE promoter activity but had no impact on the association of vitamin D receptor (VDR) with chromatin suggesting a role for co-activator recruitment in ERK-modulation of vitamin D-regulated CYP24 gene activation. Chromatin immunoprecipitation assays revealed that the ERK1/2 target, mediator 1 (MED1), is recruited to the CYP24, but not the TRPV6, promoter following 1,25(OH)(2)D(3) treatment. MED1 phosphorylation was sensitive to activators and inhibitors of the ERK1/2 signaling and MED1 siRNA reduced 1,25(OH)(2)D(3)-regulated human CYP24 promoter activity. This suggests ERK1/2 signaling enhances 1,25(OH)(2)D(3) effects on the CYP24 promoter by MED1-mediated events. Our data show that there are both promoter-specific and cell stage-specific roles for the ERK signaling pathway on 1,25(OH)(2)D(3)-mediated gene induction in enterocyte-like Caco-2 cells.

    Funded by: NIDDK NIH HHS: DK054111, R01 DK054111, R01 DK054111-12

    Journal of cellular physiology 2009;219;1;132-42

  • Gonadotropin-releasing hormone and protein kinase C signaling to ERK: spatiotemporal regulation of ERK by docking domains and dual-specificity phosphatases.

    Armstrong SP, Caunt CJ and McArdle CA

    Department of Clinical Sciences at South Bristol, University of Bristol, Bristol BS1 3NY, UK.

    Activated ERK translocates to the nucleus to regulate transcription. Spatiotemporal aspects of this response dictate biological consequences and are influenced by dual-specificity phosphatases (DUSPs) that can scaffold and dephosphorylate ERK. In HeLa cells, GnRH causes transient and protein kinase C (PKC)-dependent ERK activation, but termination mechanisms are unknown. We now explore DUSP roles using short inhibitory RNA to knock down endogenous ERK, adenoviruses to express GnRH receptors and add-back ERK2-GFP, and automated microscopy to monitor ERK location and activation. GnRH caused rapid and transient increases in dual phosphorylated ERK2 (ppERK2) and nuclear to cytoplasmic ERK2-green fluorescent protein (GFP) ratio, whereas responses to a PKC-activating phorbol ester were more sustained. In cells expressing D319N ERK2-GFP (D319N mutation impairs docking-domain-dependent binding to DUSPs), GnRH caused more sustained increases in ppERK2 and nuclear to cytoplasmic ERK2-GFP ratio and also had more pronounced effects on Egr-1 luciferase (a transcriptional reporter for ERK activation). Cycloheximide caused more sustained effects of GnRH and phorbol ester on ppERK, suggesting termination by nuclear-inducible DUSPs. GnRH also increased expression of nuclear-inducible DUSP1 and -4, but their knockdown did not alter GnRH-mediated ERK signaling. Screening a short inhibitory RNA library targeting 16 DUSPs (nuclear-inducible DUSPs, cytoplasmic ERK MAPK phosphatases, c-Jun N-terminal kinase/p38 MAPK phosphatases, and atypical DUSPs) revealed GnRH effects to be influenced by DUSPs 5, 9, 10, 16, and 3 (i.e. by each DUSP class). Thus, GnRH-mediated ERK responses (like PKC-mediated ERK responses) are dependent on protein neosynthesis and docking-domain-dependent binding, but for GnRH activation (unlike PKC activation), this does not reflect dependence on nuclear-inducible DUSPs. Termination of these GnRH effects is apparently dependent upon a preexisting rapid turnover protein.

    Funded by: Wellcome Trust: 076557, 078407, 084588

    Molecular endocrinology (Baltimore, Md.) 2009;23;4;510-9

  • Phosphorylation of the human papillomavirus type 16 E1--E4 protein at T57 by ERK triggers a structural change that enhances keratin binding and protein stability.

    Wang Q, Kennedy A, Das P, McIntosh PB, Howell SA, Isaacson ER, Hinz SA, Davy C and Doorbar J

    Division of Virology, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom.

    The E1--E4 protein of human papillomavirus type 16 (HPV16) causes cytokeratin reorganization in the middle and upper epithelial layers and is thought to contribute to multiple facets of the virus life cycle. Although little is known as to how HPV16 E1--E4 (16E1--E4) functions are controlled following the first expression of this protein, the finding that low-risk E1--E4 proteins can be phosphorylated in vivo suggests an important role for kinases. Here, we show that 16E1--E4 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and PKC alpha, with CDK1/2 serine 32 and ERK threonine 57 phosphorylations representing the two primary events seen in cells in cycle. Interestingly, T57 phosphorylation was found to trigger a structural change in the 16E1--E4 protein that compacts the central fold region, leading to an increase in 16E1--E4 stability and overall abundance in the cell. When compared to wild-type 16E1--E4, a T57D phosphomimic was found to have greatly enhanced keratin-binding ability and an ability to modulate the binding of the unphosphorylated form, with keratin binding protecting the T57-phosphorylated form of 16E1--E4 from proteasomal degradation. In HPV16 genome-containing organotypic rafts, the T57-phosphorylated form was specifically detected in the intermediate cell layers, where productive infection occurs, suggesting that T57 phosphorylation may have a functional role at this stage of the viral life cycle. Interestingly, coexpression with 16E5 and ERK activation enhanced T57 phosphorylation, suggesting that E1--E4 and E5 may work together in vivo. Our data suggest a model in which the expression of 16E5 from the major E1--E4-E5 mRNA promotes T57 phosphorylation of E1--E4 and keratin binding, with dephosphorylation occurring following the switch to late poly(A) usage. Other forms of E1--E4, with alternative functional roles, may then increase in prevalence in the upper layers of the epithelium.

    Funded by: Medical Research Council: MC_U117584278

    Journal of virology 2009;83;8;3668-83

  • Proliferative effects of gamma-aminobutyric acid on the gastric cancer cell line are associated with extracellular signal-regulated kinase 1/2 activation.

    Maemura K, Shiraishi N, Sakagami K, Kawakami K, Inoue T, Murano M, Watanabe M and Otsuki Y

    Department of Anatomy and Cell Biology, Division of Basic Medicine I, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan. an2011@art.osaka-med.ac.jp

    Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult mammalian brain. However, GABA is found not only in peripheral neuronal tissue, but also in many peripheral non-neuronal tissues, and is thought to have important physiological functions in addition to neurotransmission. We previously reported that GABA participates in chondrocyte proliferation. In the present study, we investigated the effects of GABA on the proliferation of a gastric cancer cell line, KATO III.

    Methods: Reverse transcription polymerase chain reaction and immunohistochemical analyses were performed to examine the expression of the GABA synthesis enzyme, glutamate decarboxylase (GAD), and that of the GABA(A) and GABA(B) receptor subunits. The production of GABA was confirmed by immunohistochemistry. The proliferative effect of GABA on KATO III cells was analyzed by bromodeoxyuridine incorporation assay, and the activation status of mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase [ERK]-1/2, Jun-N-terminal kinase, and p38) and the expression of cyclin D1 were analyzed by western blotting.

    Results: KATO III cells expressed GAD and GABA. More than five GABA(A) receptor subunits, including the pi subunit, were expressed in KATO III cells; however, GABA(B) receptor subunits were not seen. The addition of GABA to the medium promoted KATO III proliferation, and maximum proliferative effects were observed in the presence of 10 or 1 microM GABA. The addition of 1 microM GABA predominantly activated ERK-1/2 among the three MAP kinases in addition to increasing cyclin D1 expression.

    Conclusion: GABA is able to promote KATO III cell proliferation in an autocrine or a paracrine fashion through GABA(A) receptors followed by MAP kinase activation.

    Journal of gastroenterology and hepatology 2009;24;4;688-96

  • Differentiation impairs low pH-induced Ca2+ signaling and ERK phosphorylation in granule precursor tumour cells.

    Huang WC, Swietach P, Vaughan-Jones RD and Glitsch MD

    Department of Physiology, Anatomy and Genetics, Sherrington Building, Oxford University, Oxford, United Kingdom.

    Extracellular acidification is a hallmark of a number of debilitating pathologies including cancer, ischemia and inflammation. We have recently shown that in human granule precursor tumour cells a fall in extracellular pH triggers increases in intracellular Ca(2+) concentration through activation of G-protein coupled proton-sensing receptors coupling to phospholipase C. This pH-dependent rise in cytosolic Ca(2+) led to activation of the extracellular signal-regulated kinase ERK, providing a mechanistic explanation of how extracellular acidification can promote tumour growth. We now find that differentiation of granule precursor tumour cells profoundly affects their ability to respond to extracellular acidification with gene transcription. Differentiating cells have a lower Ca(2+) release probability from intracellular Ca(2+) stores upon acidification and cells that respond have a significantly smaller and slower Ca(2+) signal than proliferating cells. Importantly, Ca(2+) release in differentiating cells fails to evoke ERK phosphorylation. This altered responsiveness of differentiating cells is not due to reduced proton-sensing receptor expression or diminished Ca(2+) store content. Rather, our results suggest that in differentiating cells, the proton-sensing receptor couples less effectively to phospholipase C activation and IP(3) formation. Hence, the ability of human granule cells to respond to extracellular acidification by generating Ca(2+) signals and ERK activation is state-dependent, being lost upon differentiation.

    Funded by: British Heart Foundation: RG/08/016/26423; Medical Research Council: G0700698

    Cell calcium 2009;45;4;391-9

  • Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells.

    Gao SY, Li EM, Cui L, Lu XF, Meng LY, Yuan HM, Xie JJ, Du ZP, Pang JX and Xu LY

    Department of Biochemistry and Molecular Biology, Shantou University, Shantou, China.

    Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5'-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.

    The Journal of biological chemistry 2009;284;12;7995-8004

  • Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors.

    Griffero F, Daga A, Marubbi D, Capra MC, Melotti A, Pattarozzi A, Gatti M, Bajetto A, Porcile C, Barbieri F, Favoni RE, Lo Casto M, Zona G, Spaziante R, Florio T and Corte G

    Department of Translational Oncology, National Institute for Cancer Research, 16132 Genova, Italy.

    Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

    The Journal of biological chemistry 2009;284;11;7138-48

  • Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer.

    Khandrika L, Lieberman R, Koul S, Kumar B, Maroni P, Chandhoke R, Meacham RB and Koul HK

    Signal Transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology, Department of Surgery, School of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.

    Androgen receptor (AR) signaling is involved in the development and progression of prostate cancer. Tumor microvasculature contributes to continual exposure of prostate cancer cells to hypoxia-reoxygenation, however, the role of hypoxia-reoxygenation in prostate cancer progression and modulation of AR signaling is not understood. In this study, we evaluated the effects of hypoxia-reoxygenation in LNCaP cells, a line of hormone responsive human prostate cancer cells. Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells. Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity. We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness. These results demonstrate a critical role for hypoxia-induced p38 MAP kinase pathway in androgen-independent AR activation in prostate cancer cells, and suggest that hypoxia-reoxygenation may select for aggressive androgen-independent prostate cancer phenotype.

    Funded by: NCI NIH HHS: P20 CA103680, P20 CA103680-01; NIDDK NIH HHS: R01 DK054084, R01 DK054084-12A1

    Oncogene 2009;28;9;1248-60

  • Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

    Watanabe H, Kawaguchi M, Fujishima S, Ogura M, Matsukura S, Takeuchi H, Ohba M, Sueki H, Kokubu F, Hizawa N, Adachi M, Huang SK and Iijima M

    Department of Dermatology, Showa University School of Medicine, Tokyo, Japan. aki2516w@cj8.so-net.ne.jp

    IL-17F is known to be involved in many inflammatory diseases, but its role in skin diseases has not been fully examined. Because IL-8 is involved in many skin diseases such as psoriasis, we investigated the production of IL-8 in normal human epidermal keratinocytes (NHEKs) stimulated by IL-17F, tumor necrosis factor-alpha (TNF-alpha), IL-17A, and control using real-time PCR and ELISA. The results showed that IL-17F induced production of IL-8 in NHEKs in a time-dependent manner. Interestingly, the amounts of IL-8 stimulated by IL-17F were much higher than those stimulated by TNF-alpha or IL-17A. Next, we confirmed that selective mitogen-activated protein kinase kinase inhibitors significantly inhibited IL-17F-induced IL-8 production. Moreover, mouse skin intradermally injected with IL-17F expressed high level of IL-8 mRNA and induced ERK1/2 phosphorylation. Histological examination of mouse skin that was injected with IL-17F revealed marked neutrophilia in dermis and the infiltration was significantly inhibited by anti-IL-8 antibody. Finally, IL-17F expression in skin biopsy samples from psoriasis patients were examined by western blotting and ELISA. IL-17F was upregulated in lesional psoriatic skin compared with nonlesional skin. These results indicate that IL-17F may be involved in psoriasis via, in part, the activation of ERK1/2 and the induction of IL-8 in keratinocytes.

    Funded by: NIAID NIH HHS: R01 AI-052468

    The Journal of investigative dermatology 2009;129;3;650-6

  • ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937 cells.

    Luchetti F, Betti M, Canonico B, Arcangeletti M, Ferri P, Galli F and Papa S

    Institute of Morphological Sciences, University of Urbino Carlo Bo, Urbino, Italy.

    The pineal gland hormone melatonin has been recently described to downregulate the intrinsic (or damage-induced) pathway of apoptosis in human leukocytes. These properties appear to depend on a specific mitochondrial signaling of melatonin which is associated with a lower generation of reactive oxygen species and a better control of redox-sensitive components such as the antiapoptotic protein Bcl-2. Other elements upstream in this signaling are expected to contribute regulatory roles that remain unexplored. The aim of this study was to investigate whether the extracellular signal-regulated kinase (ERK), which controls the balance between survival and death-promoting genes throughout the MAPK pathway, is involved in the antiapoptotic signaling of melatonin. Human monocytic U937 cells irradiated with UVB light were used as a model of stress-induced apoptosis. In this model we found that pharmacological concentrations of melatonin (1 mM) were able to decrease superoxide anion production, mitochondrial damage, and caspase-dependent apoptosis by improved Bcl-2 levels and decreased Cyt c release in the cytoplasm. Moreover, melatonin increased the phosphorylative activation of ERK 1/2 independently from the presence of UVB stress, and decreased the UVB-mediated activation of the stress kinases p38 MAPK and JNK. The ERK 1/2 inhibitor PD98059, but not the p38 MAPK inhibitor SB203580, abolished to different extents the effects that melatonin had on the UVB-induced ROS generation, mitochondrial dysfunction, and apoptosis. Using these inhibitors, a cross-talk effect between stress and survival-promoting kinases was tentatively identified, and confirmed the hierarchical role of ERK MAPK phosphorylation in the signaling of melatonin. In conclusion, melatonin sustains the activation of the survival-promoting pathway ERK MAPK which is required to antagonize UVB-induced apoptosis of U937 cells. This kinase mediates also the antioxidant and mitochondrial protection effects of this hormonal substance that may find therapeutic applications in inflammatory and immune diseases associated with leukocyte oxidative stress and accelerated apoptosis.

    Free radical biology & medicine 2009;46;3;339-51

  • Activation of MAP kinase (ERK1/2) in human neonatal colonic enteric nervous system.

    Rouleau C, Matécki S, Kalfa N, Costes V and de Santa Barbara P

    INSERM, ERI 25, Muscle and Pathologies, Montpellier, France.

    The aim of this study was to examine mitogen-activated protein kinase (ERK1/2) activation in the human neonatal colonic enteric nervous system. For this, we investigated by immunocytochemistry the cellular localization of phosphorylated ERK1/2 (P-ERK) in a series of normal human colon samples removed from newborns and in patients with intestinal obstruction such as Hirschsprung's disease (HSCR), stenosis and atresia. We checked the presence of P-ERK in the three distinct histological layers of normal colon. Phosphorylated ERK was detected in the colonic mucosa, in the enteric nervous system and in endothelial cells. In the mucosa from normal colon, P-ERK was detected at the upper part of the crypt, while P-ERK activation in epithelial cells is altered in HSCR, stenosis and atresia. In the normal colon, strong P-ERK staining was detected in myenteric and submucosal enteric plexuses. Using confocal microscopy analyses, we observed that P-ERK staining was localized in enteric glial cells and not in enteric neurons. Strong P-ERK staining was also observed in plexuses from stenosis and atresia whereas in HSCR, hypertrophic nerve fibres were not stained.

    Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society 2009;21;2;207-14

  • PRL-3 facilitates angiogenesis and metastasis by increasing ERK phosphorylation and up-regulating the levels and activities of Rho-A/C in lung cancer.

    Ming J, Liu N, Gu Y, Qiu X and Wang EH

    Department of Pathology, College of Basic Medical Sciences, China Medical University and First Affiliated Hospital of China Medical University, Shenyang, China.

    Aims: The aim of this study was to investigate the mechanism of PRL-3 in inducing angiogenesis and lymphangiogenesis to promote distant and lymph node metastasis in human lung cancer tissues and cells.

    Methods: We investigated the expression of PRL-3, VEGF, and VEGF-C from 94 patients with non-small cell lung cancer (NSCLC) using immunohistochemical staining. The relationship between PRL-3 expression and microvessel density (MVD), lymphatic vessel density (LVD), clinicopathological factors, and surgical treatment outcome was also studied. Following this, we studied the effect on A549 by blocking PRL-3.

    Results: PRL-3 expression in NSCLC was high, and this over-expression is correlated with advanced clinical stage (p = 0.019), distant metastasis (p = 0.001), lymph node metastasis (p = 0.001), and poor post-operative survival. PRL-3 over-expression was associated with vascular endothelial growth factor (VEGF; p = 0.000) and VEGF-C (p = 0.008) expressions, MVD and LVD (p = 0.000 and p = 0.000). Blocking PRL-3 expression in A549 cell resulted in decreased cellular proliferative, migratory, and invasive abilities, and VEGF, VEGF-C, pERK, Rho-A, and Rho-C expression was inhibited. Following inhibition of either Rho or pERK, VEGF expression was down-regulated.

    Conclusions: PRL-3 induces microvascular and lymphatic vessel formation by facilitating VEGF and VEGF-C expression in lung cancer tissues, thus promoting distant and lymph node metastasis of lung cancer. PRL-3 up-regulates pERK and Rho expression and activity, facilitating VEGF expression, and accelerating angiogenesis and distant metastasis. How to regulate VEGF-C expression needs to be further studied.

    Pathology 2009;41;2;118-26

  • Signal pathway of 17beta-estradiol-induced MUC5B expression in human airway epithelial cells.

    Choi HJ, Chung YS, Kim HJ, Moon UY, Choi YH, Van Seuningen I, Baek SJ, Yoon HG and Yoon JH

    Department of Otorhinolaryngology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, Korea.

    MUC5B is a major mucin of the human respiratory tract, and it is not clear how MUC5B expression is regulated in various airway diseases. The goal of this study was to determine the mechanisms by which 17beta-estradiol induces MUC5B gene expression in airway epithelial cells. It was found that E2, a sex hormone, stimulates MUC5B gene overexpression by interaction with estrogen receptor alpha (ERalpha) and by acting through extracellular signal-regulated kinase 1/2 (ERK1/2)-mitogen-activated protein kinase (MAPK). Pretreatment with ER antagonist ICI 182,780 blocked both E2-induced ERK1/2-MAPK activation and MUC5B gene expression. It was also found that the activation of p90 ribosomal S 6 protein kinase 1 (RSK1), cAMP-response element-binding protein (CREB), and cAMP-response element (CRE) (-956 region of the MUC5B promoter)-responsive signaling cascades via ERK1/2 MAPK are crucial aspects of the intracellular mechanisms that mediate MUC5B gene expression. Taken together, these studies give additional insights into the molecular mechanism of hormone-induced MUC5B gene expression and enhance our understanding of abnormal mucin secretion in response to hormonal imbalances.

    American journal of respiratory cell and molecular biology 2009;40;2;168-78

  • Sphingosine-1-phosphate inhibits high glucose-mediated ERK1/2 action in endothelium through induction of MAP kinase phosphatase-3.

    Whetzel AM, Bolick DT and Hedrick CC

    Cardiovascular Research Center, Univ. of Virginia, P. O. Box 801394, 415 Lane Rd., MR5 Rm. G123, Charlottesville, VA 22908, USA.

    Endothelial activation is a key early event in vascular complications of Type 1 diabetes. The nonobese diabetic (NOD) mouse is a well-characterized model of Type 1 diabetes. We previously reported that Type 1 diabetic NOD mice have increased endothelial activation, with increased production of monocyte chemoattractant protein (MCP)-1 and IL-6, and a 30% increase of surface VCAM-1 expression leading to a fourfold increase in monocyte adhesion to the endothelium. Sphingosine-1-phosphate (S1P) prevents monocyte:endothelial interactions in these diabetic NOD mice. Incubation of diabetic NOD endothelial cells (EC) with S1P (100 nmol/l) reduced ERK1/2 phosphorylation by 90%, with no significant changes in total ERK1/2 protein. In the current study, we investigated the mechanism of S1P action on ERK1/2 to reduce activation of diabetic endothelium. S1P caused a significant threefold increase in mitogen-activated kinase phosphatase-3 (MKP-3) expression in EC. MKP-3 selectively regulates ERK1/2 activity through dephosphorylation. Incubation of diabetic NOD EC with S1P and the S1P(1)-selective agonist SEW2871 significantly increased expression of MKP-3 and reduced ERK1/2 phosphorylation, while incubation with the S1P(1)/S1P(3) antagonist VPC23019 decreased the expression of MKP-3, both results supporting a role for S1P(1) in MKP-3 regulation. To mimic the S1P-mediated induction of MKP-3 diabetic NOD EC, we overexpressed MKP-3 in human aortic endothelial cells (HAEC) cultured in elevated glucose (25 mmol/l). Overexpression of MKP-3 in glucose-cultured HAEC decreased ERK1/2 phosphorylation and resulted in decreased monocyte:endothelial interactions in a static monocyte adhesion assay. Finally, we used small interfering RNA to MKP-3 and observed increased monocyte adhesion. Moreover, S1P was unable to inhibit monocyte adhesion in the absence of MKP-3. Thus, one mechanism for the anti-inflammatory action of S1P in diabetic EC is inhibition of ERK1/2 phosphorylation through induction of MKP-3 expression via the S1P-S1P(1) receptor axis.

    Funded by: NHLBI NIH HHS: R01 HL079621

    American journal of physiology. Cell physiology 2009;296;2;C339-45

  • Silibinin suppresses growth of human prostate carcinoma PC-3 orthotopic xenograft via activation of extracellular signal-regulated kinase 1/2 and inhibition of signal transducers and activators of transcription signaling.

    Singh RP, Raina K, Deep G, Chan D and Agarwal R

    Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado-Denver, 4200 East Ninth Street, Box C238, Denver, CO 80262, USA.

    Purpose: Silibinin is currently under phase II clinical trial in prostate cancer patients; however, its antitumor effects and mechanisms are not completely understood. Herein, we studied the efficacy and associated mechanisms of silibinin against orthotopically growing advanced human prostate carcinoma PC-3 tumors.

    Athymic male mice were orthotopically implanted with PC-3 cells in prostate and 1 week later after surgical recovery were gavaged daily with silibinin (100 mg/kg body weight) for 7 weeks.

    Results: Silibinin treatment reduced the lower urogenital weight (including tumor, prostate, and seminal vesicle) by 40% (P < 0.05) without any toxicity in mice. Silibinin decreased proliferating cell nuclear antigen expression and proliferating cells (P < 0.001) but increased cleaved caspase-3-positive cells (P < 0.01) and apoptotic cells (P < 0.001) and suppressed tumor microvessel density (P < 0.001) and vascular endothelial growth factor expression (P = 0.02). Decreased levels of cyclin-dependent kinases 2, 4, and 6, CDC2, and cyclins D1, D3, E, and A were observed, indicating an inhibitory effect of silibinin on cell cycle progression. Silibinin showed a tremendous increase in extracellular signal-regulated kinase 1/2 phosphorylation but decreased c-Jun NH(2)-terminal kinase 1/2 and p38 mitogen-activated protein kinase phosphorylation. A moderate decrease in phosphorylated and total levels of Akt was also noted. A marked inhibitory effect of silibinin on signal transducers and activators of transcription (STAT) 1 (Tyr(701)), STAT1 (Ser(727)), STAT3 (Tyr(705)), STAT3 (Ser(727)), and STAT5 (Tyr(794)) phosphorylation together with a decrease in their total levels was also observed.

    Conclusions: These findings provide evidence for antitumor efficacy of silibinin against orthotopically growing prostate tumor in mice with multitargeted mechanistic insights and support its clinical investigation in prostate cancer.

    Funded by: NCI NIH HHS: CA102514, R01 CA102514, R01 CA102514-05

    Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15;2;613-21

  • How does arrestin assemble MAPKs into a signaling complex?

    Song X, Coffa S, Fu H and Gurevich VV

    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.

    Arrestins bind active phosphorylated G protein-coupled receptors, precluding G protein activation and channeling signaling to alternative pathways. Arrestins also function as mitogen-activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules. Here we have demonstrated that all four vertebrate arrestins interact with JNK3, MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the functional specificity of arrestins is not determined by differential binding of the kinases. Using receptor binding-impaired mutant, we have shown that free arrestin3 readily promotes JNK3 phosphorylation. We identified key arrestin-binding elements in JNK3 and ASK1 and investigated the molecular interactions of arrestin2 and arrestin3 and their individual domains with the components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We found that both arrestin domains interact with all six kinases. These findings shed new light on the mechanism of arrestin-mediated MAPK activation and the spatial arrangement of the three kinases on arrestin molecule.

    Funded by: NEI NIH HHS: EY011500; NIGMS NIH HHS: GM077561, GM081756

    The Journal of biological chemistry 2009;284;1;685-95

  • A new type of ERK1/2 autophosphorylation causes cardiac hypertrophy.

    Lorenz K, Schmitt JP, Schmitteckert EM and Lohse MJ

    Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, Würzburg, Germany. lorenz@toxi.uni-wuerzburg.de

    The extracellular-regulated kinases ERK1 and ERK2 (commonly referred to as ERK1/2) have a crucial role in cardiac hypertrophy. ERK1/2 is activated by mitogen-activated protein kinase kinase-1 (MEK1) and MEK2 (commonly referred to as MEK1/2)-dependent phosphorylation in the TEY motif of the activation loop, but how ERK1/2 is targeted toward specific substrates is not well understood. Here we show that autophosphorylation of ERK1/2 on Thr188 directs ERK1/2 to phosphorylate nuclear targets known to cause cardiac hypertrophy. Thr188 autophosphorylation requires the activation and assembly of the entire Raf-MEK-ERK kinase cascade, phosphorylation of the TEY motif, dimerization of ERK1/2 and binding to G protein betagamma subunits released from activated G(q). Thr188 phosphorylation of ERK1/2 was observed in isolated cardiomyocytes induced to undergo hypertrophic growth, in mice upon stimulation of G(q)-coupled receptors or after aortic banding and in failing human hearts. Experiments using transgenic mouse models carrying mutations at the Thr188 phosphorylation site of ERK2 suggested a causal relationship to cardiac hypertrophy. We propose that specific phosphorylation events on ERK1/2 integrate differing upstream signals (Raf1-MEK1/2 or G protein-coupled receptor-G(q)) to induce cardiac hypertrophy.

    Nature medicine 2009;15;1;75-83

  • Focal adhesion kinase: important to prostaglandin E2-mediated adhesion, migration and invasion in hepatocellular carcinoma cells.

    Bai XM, Zhang W, Liu NB, Jiang H, Lou KX, Peng T, Ma J, Zhang L, Zhang H and Leng J

    Laboratory of Reproductive Medicine, Cancer Center, Department of Pathology, Nanjing Medical University, Nanjing 210029, PR China.

    Prostaglandin E2 has been implicated in cell growth and metastasis in many types of cancers. However, the effects of PGE2 and its mechanism on cell adhesion, migration, and invasion have not been clarified yet. In this study, we found PGE2 treatment significantly increased the cell adhesion, migration, and invasion in hepatocellular carcinoma (HCC) cells. In addition, the effects of PGE2 were found to be associated with focal adhesion kinase (FAK). PGE2 treatment increased the phosphorylation and synthesis of FAK in a dose-dependent manner. RNA interference targeting FAK suppressed PGE2-mediated cell adhesion and migration. Furthermore, the downstream proteins of FAK, paxillin and Erk2, were also activated by PGE2. PGE2 treatment increased the phosphorylation and synthesis of paxillin in a dose-dependent manner. PGE2 treatment also induced the phosphorylation of Erk2. PD98059, the specific inhibitor of MEK, suppressed PGE2-mediated cell adhesion and migration. However, it had no effect on PGE2-induced activation and synthesis of FAK. These results demonstrated that PGE2 greatly induced HCC cell adhesion, migration, and invasion by activating FAK/paxillin/Erk pathway.

    Oncology reports 2009;21;1;129-36

  • Relationship of activated extracellular signal-regulated kinase 1/2 with lung metastasis in salivary adenoid cystic carcinoma.

    Hu K, Gan YH, Li SL, Gao Y, Wu DC, Wang CY and Yu GY

    Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing 100081, PR China.

    Lung metastasis is one of the important characteristics of salivary adenoid cystic carcinoma (SACC). Although activation of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway may be associated with cancer progression in some malignant tumors, its roles in lung metastasis of SACC remains unclear. We examined the expression of activated ERK1/2 in SACC-LM with high lung-metastatic rate and SACC-83 with low lung-metastatic rate, as well as in the tissues from lung-metastatic and non-metastatic groups of SACC patients. Western blot analysis indicated that SACC-LM exhibited higher expression of activated ERK1/2 than SACC-83. Similarly, immunohistochemistry showed that expression of activated ERK1/2 was detected in 73% (8/11) of the primary tissues from SACC patients with lung metastasis, while only 25% (3/12) of the primary tissues from SACC patients without lung metastasis (P<0.05). Furthermore, we examined the effects of U0126, a specific inhibitor of mitogen activated protein kinase kinase (MEK or MAPKK), on migration and invasion in SACC-LM cells, showing U0126 not only inactivated ERK1/2, but also inhibited migration and invasion of SACC-LM. The present findings suggested that the elevated expression of activated ERK1/2 may play a role in lung metastasis of salivary adenoid cystic carcinoma.

    Oncology reports 2009;21;1;137-43

  • Sustained activation of Akt and Erk1/2 is required for Coxiella burnetii antiapoptotic activity.

    Voth DE and Heinzen RA

    Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, 903 S. 4th Street, Hamilton, MT 59840, USA.

    Coxiella burnetii is an obligate intracellular bacterial pathogen that directs biogenesis of a lysosome-like, parasitophorous vacuole in mammalian cells. We recently reported that C. burnetii inhibits apoptotic cell death in macrophages, presumably as a mechanism to sustain the host for completion of its lengthy infectious cycle. In the current study, we further investigated C. burnetii manipulation of host cell signaling and apoptosis by examining the effect of C. burnetii infection on activation of 15 host proteins involved in stress responses, cytokine production, and apoptosis. C. burnetii infection of THP-1 human macrophage-like cells caused increased levels of phosphorylated c-Jun, Hsp27, Jun N-terminal protein kinase, and p38 at 2 h postinfection (hpi), and this activation rapidly decreased to near basal levels by 24 hpi. The prosurvival kinases Akt and Erk1/2 (extracellular signal-regulated kinases 1 and 2) were also activated at 2 to 6 hpi; however, the phosphorylation of these proteins increased coincident with C. burnetii replication through at least 72 hpi. Sustained phosphorylation of Akt and Erk1/2 was abolished by treatment of infected cells with rifampin, indicating their activation is a C. burnetii-directed event requiring pathogen RNA synthesis. Moreover, pharmacological inhibition of Akt or Erk1/2 significantly decreased C. burnetii antiapoptotic activity. Collectively, these results indicate the importance of C. burnetii modulation of host signaling and demonstrate a critical role for Akt and Erk1/2 in successful intracellular parasitism and maintenance of host cell viability.

    Funded by: Intramural NIH HHS

    Infection and immunity 2009;77;1;205-13

  • Activation of extracellular regulated kinases (ERK1/2) predicts poor prognosis in urothelial bladder carcinoma and is not associated with B-Raf gene mutations.

    Karlou M, Saetta AA, Korkolopoulou P, Levidou G, Papanastasiou P, Boltetsou E, Isaiadis D, Pavlopoulos P, Thymara I, Thomas-Tsagli E and Patsouris E

    Department of Pathology, Medical School, National and Kapodistrian University of Athens, Goudi, Athens, Greece.

    Aim: The analysis of the presence of B-Raf gene mutations in relation to ERK1/2 activation in bladder urothelial carcinoma (UC), in order to determine their potential role in tumour aggressiveness and patients' survival.

    Methods: Polymerase chain reaction-single strand confirmation polymorphism (PCR-SSCP) and sequencing analysis were used for B-Raf gene mutation detection. pERK1/2 and FGFR3 expression were examined by immunohistochemistry in 152 and 116 primary UCs, respectively.

    Results: None of the cases displayed mutations in exon 15 of B-Raf gene. Nuclear or cytoplasmic pERK immunoreactivity was displayed in 99.3% and 96.7% of cases, respectively. pERK nuclear expression increased with histological grade and with T-category. Nuclear and cytoplasmic pERK expression was unrelated to FGFR3 expression. In univariate survival analysis of muscle-invasive carcinomas, advanced T-category and higher pERK nuclear expression (p = 0.018) adversely affected survival. However, multivariate analysis in non-invasive as well as in muscle-invasive carcinomas selected only T-category as a significant prognosticator.

    Conclusions: Our findings suggest that elevated pERK expression occurs in UCs in the absence of B-Raf mutations and is not correlated with FGFR3 over-expression. Moreover, it implicates ERK activation in the acquisition of a more aggressive phenotype. However, the assessment of pERK1/2 expression does not seem to add to the prognostic information provided by classical prognosticators.

    Pathology 2009;41;4;327-34

  • Cysteinyl leukotrienes induce macrophage inflammatory protein-1 in human monocytes/macrophages.

    Ichiyama T, Hasegawa M, Hashimoto K, Matsushige T, Hirano R and Furukawa S

    Department of Pediatrics, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan. ichiyama@yamaguchi-u.ac.jp

    Background: Macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta are known for their chemotactic and proinflammatory effects on monocytes/macrophages which have a cysteinyl leukotriene 1 (CysLT(1)) receptor.

    Methods: We examined MIP-1alpha and MIP-1beta production stimulated by CysLTs (LTC(4), LTD(4), and LTE(4)) in THP-1 cells, a human monocytic leukemia cell line, and peripheral blood mononuclear cells (PBMCs). Moreover, we examined the inhibitory effect of pranlukast, a CysLT(1) receptor antagonist, and inhibitors of three major mitogen-activated protein kinases (MAPK) on the induction of MIP-1alpha and MIP-1beta production by CysLTs.

    Results: ELISA demonstrated that CysLTs induced MIP-1alpha and MIP-1beta production in THP-1 cells and PBMCs. PCR demonstrated that LTD(4) increased MIP-1alpha and MIP-1beta mRNA expressions in THP-1 cells. Pranlukast blocked MIP-1alpha and MIP-1beta production promoted by LTD(4) in THP-1 cells and PBMCs. Moreover, an inhibitor of extracellular signal-regulated kinase (ERK) attenuated the induction of MIP-1alpha and MIP-1beta production by LTD(4) in THP-1 cells whereas the inhibitors of c-Jun NH2-terminal kinase or p38 MAPK did not.

    Conclusion: CysLTs induce MIP-1alpha and MIP-1beta production mediated by ERK via binding to the CysLT(1) receptor in human monocytes/macrophages.

    International archives of allergy and immunology 2009;148;2;147-53

  • ICBP90 mediates the ERK1/2 signaling to regulate the proliferation of Jurkat T cells.

    Fang Z, Xing F, Bronner C, Teng Z and Guo Z

    Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou, PR China.

    ERK1/2 activation leads to the release of E2F that could bind to the E2F binding sites in the inverted CCAAT box binding protein (ICBP90) gene promoter. Therefore, in the present study the relationship between ERK1/2 signaling and ICBP90 in the regulation of Jurkat T cell proliferation was explored. Jurkat T cells were treated with different concentrations of various signal pathway inhibitors. The cell viability and cell cycle were determined. Furthermore, the expression of non-phosphorylated and phosphorylated ERK1/2, and ICBP90 was measured by Western blot analysis. All the inhibitors, including PD98059, LY294002, AG490, genistein and GF109203X, suppressed the cell colony formation and proliferation to different extent in a dose-dependent manner. PD98059 could suppress the cell proliferation remarkably, arrested the cell cycle at G1/G0 stage and blocked its entrance from S phase to G2/M phase. Three or 24h after exposure to the inhibitors, all the inhibitors downregulated the level of the phosphorylated ERK1/2, of which the inhibitory roles of PD98059, LY294002 and AG490 were more significant. All the inhibitors had no effect on the expression of ICBP90 after 3h treatment, but downregulated markedly its expression after 24h treatment, especially PD98059, LY294002 and AG490. The expression of ICBP90 was directly proportioned to the level of ERK1/2 phosphorylation and the cell proliferation. Our results demonstrate that ICBP90 might be a pivotal target for the ERK1/2 signaling pathway to control the proliferation of Jurkat T cells.

    Cellular immunology 2009;257;1-2;80-7

  • Mitochondrial respiration is required for activation of ERK1/2 and caspase-3 in human eosinophils stimulated with hydrogen peroxide.

    Lee YA and Shin MH

    Department of Environmental Medical Biology, Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, Korea.

    Background: Eosinophils are important effector cells in the pathogenesis of allergic diseases such as bronchial asthma. Oxidative stress in the form of cellular reactive oxygen species (ROS) has been implicated in the pathogenesis of several allergic diseases. Recently, it has become evident that mitochondrial-derived ROS are important transducers of apoptosis and intracellular signaling. In this study, we investigated the role of mitochondrial ROS in the activation of extracellular signal-regulated kinases (ERK) 1 and 2-mitogen-activated protein kinase (MAPK) and caspase-3 in human eosinophils stimulated with H2O2.

    Methods: Human eosinophils were purified using immunomagnetic negative selection and then stimulated with H2O. H2O2-induced eosinophil apoptosis was measured by staining cells with annexin V. Activation of ERK1/2 MAPK and caspases was assessed by Western blotting. Eosinophils were pretreated with rotenone, an inhibitor of the mitochondrial electron transport chain, before H2O2 was added.

    Results: Treatment with 1 mM H2O2 induced externalization of phosphatidylserine (PS) and activation of caspases in eosinophils. H2O2-triggered PS externalization and cleavage of caspase-3 were markedly inhibited by pretreatment with the mitochondrial ROS scavenger N-acetyl-L-cysteine. In addition, H2O2 strongly induced phosphorylation of ERK1/2, but not ERK5, in eosinophils. Hydrogen peroxide-triggered activation of caspase-3 and ERK1/2 was attenuated by pretreatment with rotenone.

    Conclusions: These results suggest that mitochondrial respiration is essential for activation of ERK1/2 and caspase-3 in human eosinophils stimulated with H2O2.

    Journal of investigational allergology & clinical immunology 2009;19;3;188-94

  • Neurotrophin receptors TrkA and TrkB in retinoblastoma are differentially expressed depending on cellular differentiation.

    Kim JH, Kim JH, Kim DH, Cho CS, Jun HO, Yu YS and Kim KW

    Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Korea.

    Retinoblastoma is the most common primary intraocular malignancy in children. With the progression of retinoblastoma, retinoblastoma cells lose their ability to differentiate. Regardless of many attempts to identify prognostic factors in retinoblastoma, further investigation for prognostic factors of retinoblastoma progression is still required because of the lack of sensitivity and specificity of these prognostic factors in predicting disease progression. We demonstrated that the differential expression of the neurotrophin receptors TrkA and TrkB is closely related to the differentiation of retinoblastoma cells. While retinoblastoma cells expressed TrkA as well as TrkB, their growth rates were not influenced by the addition of nerve growth factor to the culture medium. In experimental animal models of retinoblastoma, TrkA expression was primarily detected in more differentiated areas with high nm23 immunoreactivity whereas TrkB expression was apparent in more proliferative areas with high Ki67 immunoreactivity. With retinoic-acid-induced differentiation of retinoblastoma cells, TrkA expression significantly increased whereas TrkB significantly decreased. The differential expression of TrkA and TrkB with differentiation of retinoblastoma cells was mediated by extracellular-signal-regulated kinase 1/2 activation, which was confirmed by immunocytochemistry of TrkA. Therefore, our results suggest that the differential expression of TrkA and TrkB could be valuable as a therapeutic target, for instance using specific inhibitors.

    Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2009;30;5-6;233-41

  • PI-3 kinase activity is necessary for ERK1/2-induced disruption of mammary epithelial architecture.

    Pearson GW and Hunter T

    Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, CA 92037, USA. gray.pearson@utsouthwestern.edu

    Introduction: Epithelial tumors, including breast cancer, are being identified and treated at earlier stages of tumor development because of technological advances in screening and detection methods. It is likely that early-stage epithelial tumors, such as mammary ductal carcinoma in situ (DCIS), will be amenable to new and more efficacious diagnostic tests and forms of therapy. However, our limited understanding of the underlying molecular mechanisms of early-stage epithelial tumor growth has hampered the development of new forms treatment and preventative therapy.

    Methods: The Raf-MEK1/2-ERK1/2 mitogen-activated protein kinase module is activated by stimuli complicit in mammary neoplastic progression. We have recently demonstrated that the activation of ERK1/2 induces a non-invasive form of motility, where cells can track along the basement membrane and adjacent epithelial cells, but do not become invasive over time, using real-time imaging of a mammary epithelial organotypic culture model. Using this novel approach combined with traditional biochemical techniques, we have analyzed at the molecular level how ERK1/2 induces this new non-invasive form of motility as well as proliferation and cell survival.

    Results: We find that the activation of Raf:ER in the differentiated epithelium of fully formed acini promotes proliferation and cell survival, which are characteristic features of pre-invasive DCIS lesions. The activation of ERK1/2 correlated with induction of c-Fos, a transcriptional regulator of proliferation and reduced expression of the pro-apoptotic BH3-only protein BIM. Both ERK1/2 and PI-3 kinase-dependent effector pathways were required for activated Raf:ER to reduce expression of p27 and promote proliferation. In addition, PI-3K activity was necessary for the induction of non-invasive motility induced by ERK1/2.

    Conclusions: ERK1/2 activation is sufficient to induce cell behaviors in organotypic culture that could promote recurrent and invasive growth in DCIS patients. Interestingly, PI-3K activity is necessary for two of these behaviors, proliferation and cell motility. Collectively, our results suggest that the relationship between the activity state of the ERK1/2 and PI-3K signaling pathways and recurrent growth in DCIS patients should be investigated.

    Funded by: NCI NIH HHS: CA14195, CA82683, R01 CA082683, T32 CA009370

    Breast cancer research : BCR 2009;11;3;R29

  • Feedback regulation of DUSP6 transcription responding to MAPK1 via ETS2 in human cells.

    Furukawa T, Tanji E, Xu S and Horii A

    International Research and Educational Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Tokyo, Japan. furukawa@imcir.twmu.ac.jp

    DUSP6/MKP-3 is a dual specificity phosphatase exclusively specific to MAPK1/ERK2 for its substrate recognition and dephosphorylating activity. DUSP6 is demonstrated to play a negative regulatory role in MAPK1 in a feedback loop manner; however, the regulation mechanisms of its expression in human cells have been largely unknown. We previously found that human pancreatic cancer cells frequently lost DUSP6 expression, which could induce constitutively active MAPK1, and the loss was associated with hypermethylation of the CpG cluster region of intron 1 of DUSP6. In this study, we investigated the promoter activity of intron 1 of DUSP6 in human cells. We demonstrated that the intron indeed had promoter activity and this activity was associated with MAPK1 activity. Moreover, promoter activity depended on a consensus binding sequence of ETS transcription factors and ETS2 was specifically associated with the intron. Because ETS2 is a direct target of MAPK, these results indicate that intron 1 of DUSP6 plays a crucial role in transcriptional regulation of DUSP6 in a feedback loop manner responding to MAPK1 via ETS2 in human cells.

    Biochemical and biophysical research communications 2008;377;1;317-20

  • Aldosterone induces collagen synthesis via activation of extracellular signal-regulated kinase 1 and 2 in renal proximal tubules.

    Xu G, Liu A and Liu X

    Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi'an, China. xugsh882003@yahoo.com.cn

    Aim: Aldosterone plays a crucial role in renal fibrosis by inducing mesangial cell proliferation and promoting collagen synthesis in renal fibroblasts. However, renal proximal tubule involvement in aldosterone-induced collagen synthesis has not yet been identified. The aim of this study was to examine the potential role of aldosterone in collagen expression and its possible mineralocorticoid receptor (MR)-dependent pathway, mediated by activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in cultured human renal proximal tubular epithelial (HKC) cells.

    Methods: After HKC cells were stimulated by aldosterone with different concentrations for various time and periods, the gene expression and protein synthesis of collagen I, II, III and IV were measured by real-time polymerase chain reaction and western blot, respectively. ERK1/2 activation, alpha-smooth muscle actin (alpha-SMA), and E-cadherin were also detected by western blot.

    Results: Aldosterone can increase ERK1/2 phosphorylation of human renal proximal tubular epithelial cells in a time- and dose-dependent manner. Although aldosterone had no effect on collagen I and II expression, it increased expression of alpha-SMA and collagen III and IV and decreased that of E-cadherin in HKC cells after 48 h. These effects could be prevented by a ERK pathway inhibitor, U0126, or by a selective MR antagonist, spironolactone.

    Conclusion: The results suggest that aldosterone plays a pivotal role in tubulointerstitial fibrosis by promoting tubular epithelial-mesenchymal transition and collagen synthesis in proximal tubular cells. The process is MR-dependent, and mediated by ERK1/2 mitogen-activated protein kinase pathway.

    Nephrology (Carlton, Vic.) 2008;13;8;694-701

  • EGF stimulates uPAR expression and cell invasiveness through ERK, AP-1, and NF-kappaB signaling in human gastric carcinoma cells.

    Baek MK, Kim MH, Jang HJ, Park JS, Chung IJ, Shin BA, Ahn BW and Jung YD

    The Brain Korea 21 Project, Center for Biomedical Human Resources, Chonnam National University Medical School, Kwangju 501-190, Korea.

    Overexpression of epidermal growth factor (EGF) and urokinase plasminogen activator receptor (uPAR) have been observed in human gastric cancers. However, the interaction between EGF and uPAR in gastric cancer has not been well elucidated. In this study, we investigated the effect of EGF on uPAR expression and the underlying signal pathways in human gastric cancer AGS cells. EGF induced uPAR mRNA expression in a time- and concentration-dependent manner. EGF also induced uPAR promoter activity. In addition, EGF induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2) and P38 mitogen-activated protein kinase (MAPK) but not the activation of c-Jun amino terminal kinase. A specific inhibitor of MEK-1 (an upstream effector of ERK-1/2) and a dominant negative MEK-1 were able to suppress the EGF-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays demonstrated that the binding sites of transcription factors, activator protein-1 (AP-1) and nuclear factor (NF)-kappaB, are involved in the EGF-induced uPAR transcription. Suppression of the EGF-induced uPAR promoter activity by the AP-1 decoy oligonuclotide, as well as expression vectors encoding mutated-type NF-kappaB-inducting kinase and I-kappaB, confirmed that the activation of AP-1 and NF-kappaB are essential for the EGF-induced uPAR upregulation. The AGS cells pretreated with EGF showed a remarkably enhanced invasiveness and this effect was partially abrogated by uPAR neutralizing antibodies and by the inhibitors of ERK-1/2, AP-1, and NF-kappaB. The above results suggest that EGF induces uPAR expression via ERK-1/2, AP-1, and NF-kappaB signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

    Oncology reports 2008;20;6;1569-75

  • Identification of a key element for hydrogen-bonding patterns between protein kinases and their inhibitors.

    Katayama N, Orita M, Yamaguchi T, Hisamichi H, Kuromitsu S, Kurihara H, Sakashita H, Matsumoto Y, Fujita S and Niimi T

    Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan.

    In this article, we report crystal structures for inhibitor-kinase complexes in which the inhibitor has different binding orientations and hydrogen-bonding patterns with extracellular-signal regulated kinase 2 and insulin receptor tyrosine kinase. Our crystallographic studies, and sequence and structural analyses of 532 coordinates of kinases held in the Protein Data Bank, suggest that the length of the "specificity linker" described here is a key structural element of the hydrogen-bonding patterns between protein kinases and their inhibitors.

    Proteins 2008;73;4;795-801

  • Role of endogenous RGS proteins on endothelial ERK 1/2 activation.

    Anger T, Grebe N, Osinski D, Stelzer N, Carson W, Daniel WG, Hoeher M and Garlichs CD

    Department for Cardiology, Friedrich-Alexander University of Erlangen, Germany. th.anger@arcor.de

    Endothelial cells are maintaining atherosclerotic signaling mediated by Extracellular Regulated Kinases 1 and 2 (ERK). Signaling gets activated upon stimulation of G protein-coupled receptors mediated by G(q) and G(i/o) proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity of RGS proteins modulating induced ERK phosphorylation. We used stimulated HUVEC, silenced specifically RGS proteins and compared assessed ERK 1/2 activation with immunohistochemical stainings on atherosclerotic plaques. Increased ERK phosphorylation was detected upon stimulation with Phenylephrine (2.6+/-0.1 times over basal), Endothelin-1 (1.8+/-0.2), Dopamine (5.1+/-0.2), TNF (9.8+/-0.7) or IL-4 (3.1+/-0.3). RGS silencing increased activation of ERK 1/2: Phen (RGS3, 5), ET-1 (RGS3, 4), Dopa (RGS3), TNF (RGS2, 3, 4) or IL-4 (RGS2, 3, 4). Immunohistochemically, increased ERK activation was detected on atherosclerotic plaques. This data supports the role of RGS proteins on ERK activation in human atherosclerosis which identifies RGS proteins as new therapeutical targets.

    Experimental and molecular pathology 2008;85;3;165-73

  • Cl- -channel is essential for LDL-induced cell proliferation via the activation of Erk1/2 and PI3k/Akt and the upregulation of Egr-1 in human aortic smooth muscle cells.

    Heo KS, Ryoo SW, Kim L, Nam M, Baek ST, Lee H, Lee AR, Park SK, Park Y, Myung CS, Kim DU and Hoe KL

    Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea.

    Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that Cl- channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of Cl-channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. Cl- channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated Cl- concentration, as judged by flow cytometry analysis using MQAE as a Cl- -detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS (Cl- channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive Cl- channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.

    Molecules and cells 2008;26;5;468-73

  • Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope.

    González JM, Navarro-Puche A, Casar B, Crespo P and Andrés V

    Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (CSIC), Valencia, Spain.

    Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1-dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A-null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C.

    The Journal of cell biology 2008;183;4;653-66

  • Sepsis-induced inhibition of neutrophil chemotaxis is mediated by activation of peroxisome proliferator-activated receptor-{gamma}.

    Reddy RC, Narala VR, Keshamouni VG, Milam JE, Newstead MW and Standiford TJ

    Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, USA. rajuc@umich.edu

    Neutrophils (polymorphonuclear leukocytes [PMNs]) are critical to the immune response, including clearance of infectious pathogens. Sepsis is associated with impaired PMN function, including chemotaxis. PMNs express peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a ligand-activated nuclear transcription factor involved in immune and inflammatory regulation. The role of PPAR-gamma in PMN responses, however, is not well characterized. We report that freshly isolated human PMNs constitutively express PPAR-gamma, which is up-regulated by the sepsis-induced cytokines TNF-alpha and IL-4. PMN chemotactic responses to formylmethionyl-leucyl-phenylalanine (fMLP) and IL-8 were dose-dependently inhibited by treatment with the PPAR-gamma ligands troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and by transfection of PMN-like HL-60 cells with a constitutively active PPAR-gamma construct. Inhibition of chemotaxis by PPAR-gamma ligands correlated with decreases in extracellular signal-regulated kinase-1 and -2 activation, actin polymerization, and adherence to a fibrinogen substrate. Furthermore, PMN expression of PPAR-gamma was increased in sepsis patients and mice with either of 2 models of sepsis. Finally, treatment with the PPAR-gamma antagonist GW9662 significantly reversed the inhibition of PMN chemotaxis and increased peritoneal PMN recruitment in murine sepsis. This study indicates that PPAR-gamma activation is involved in PMN chemotactic responses in vitro and may play a role in the migration of these cells in vivo.

    Funded by: NHLBI NIH HHS: HL70068, K08 HL070068, P50 HL074024, P50 HL60289

    Blood 2008;112;10;4250-8

  • Myosin light-chain kinase contributes to the proliferation and migration of breast cancer cells through cross-talk with activated ERK1/2.

    Zhou X, Liu Y, You J, Zhang H, Zhang X and Ye L

    Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, PR China.

    Myosin light-chain kinase (MLCK) plays a crucial role in the cell migration and tumor metastasis. Herein, we investigated the signaling pathways involved in MLCK using ML-7, a specific inhibitor of MLCK, in breast cancer cell proliferation and migration. Our data showed that reduction of MLCK in breast cancer cells mediated by 20 microM ML-7 was able to depress the cell proliferation and migration using two parallel cell lines (MCF-7 and LM-MCF/MDA-MB-231) with different metastatic abilities through reciprocal cross-talk with activated ERK1/2, in which both phosphorylated myosin light chain (p-MLC) and cascades of beta-catenin, cyclin D1, survivin, and c-Myc serve as essential downstream effectors.

    Cancer letters 2008;270;2;312-27

  • Aspergillus fumigatus-induced interleukin-8 synthesis by respiratory epithelial cells is controlled by the phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2 pathways and not by the toll-like receptor-MyD88 pathway.

    Balloy V, Sallenave JM, Wu Y, Touqui L, Latgé JP, Si-Tahar M and Chignard M

    Unité de Défense Innée et Inflammation, Institut Pasteur, Paris 75015, France.

    Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-kappaB-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-kappaB activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis.

    The Journal of biological chemistry 2008;283;45;30513-21

  • MKP1 regulates the induction of MUC5AC mucin by Streptococcus pneumoniae pneumolysin by inhibiting the PAK4-JNK signaling pathway.

    Ha UH, Lim JH, Kim HJ, Wu W, Jin S, Xu H and Li JD

    Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA.

    Mucosal epithelial cells in the respiratory tract act as the first line of host innate defense against inhaled microbes by producing a range of molecules for clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping the inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in defective mucosal defenses. Thus, tight regulation of mucin production is critical for maintaining an appropriate balance between beneficial and detrimental outcomes. Among various mechanisms, negative regulation plays an important role in tightly regulating mucin production. Here we show that the PAK4-JNK signaling pathway acted as a negative regulator for Streptococcus pneumoniae pneumolysin-induced MUC5AC mucin transcription. Moreover pneumolysin also selectively induced expression of MKP1 via a TLR4-dependent MyD88-TRAF6-ERK signaling pathway, which inhibited the PAK4-JNK signaling pathway, thereby leading to up-regulation of MUC5AC mucin production to maintain effective mucosal protection against S. pneumoniae infection. These studies provide novel insights into the molecular mechanisms underlying the tight regulation of mucin overproduction in the pathogenesis of airway infectious diseases and may lead to development of new therapeutic strategies.

    Funded by: NIDCD NIH HHS: DC004562, DC005843, DC008703

    The Journal of biological chemistry 2008;283;45;30624-31

  • Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.

    Newbern J, Zhong J, Wickramasinghe RS, Li X, Wu Y, Samuels I, Cherosky N, Karlo JC, O'Loughlin B, Wikenheiser J, Gargesha M, Doughman YQ, Charron J, Ginty DD, Watanabe M, Saitta SC, Snider WD and Landreth GE

    Neuroscience Center, University of North Carolina, Chapel Hill, NC 27599, USA.

    Disrupted ERK1/2 (MAPK3/MAPK1) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel approximately 1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2. These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade (B-Raf and C-Raf, MEK1 and MEK2) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: HL074731, HL080637, P50 HL074731, R21 HL080637; NIMH NIH HHS: F31 MH074241, F31-MH074241; NINDS NIH HHS: F32 NS061591, NS031768, NS34814, R01 NS031768, R01 NS034814, R37 NS034814

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;44;17115-20

  • Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3.

    Zeliadt NA, Mauro LJ and Wattenberg EV

    Division of Environmental Health Sciences, School of Public Health, University of Minnesota, Mayo Mail Code #807, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

    Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

    Funded by: NCI NIH HHS: R01 CA104609, R01 CA104609-01A1, R01 CA104609-02, R01 CA104609-03, R01 CA104609-04, R01-CA104609

    Toxicology and applied pharmacology 2008;232;3;408-17

  • Induction of high mobility group box 1 release from serotonin-stimulated human umbilical vein endothelial cells.

    Kawahara K, Hashiguchi T, Kikuchi K, Tancharoen S, Miura N, Ito T, Oyama Y, Nawa Y, Biswas KK, Meng X, Morimoto Y, Shrestha B, Sameshima H and Maruyama I

    Department of Laboratory and Vascular Medicine, Cardiovascular and Respiratory Disorders, Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8520, Japan.

    High mobility group box 1 (HMGB1) is a non-histone nuclear protein which is released from the nucleus of activated macrophages into the extracellular space in response to stimuli such as endotoxin or necrosis. The HMGB1 functions as a potent proinflammatory cytokine in the extracellular spaces. Recently, HMGB1 has been implicated in the progression of atherosclerosis. However, the association between HMGB1 and the development of atherosclerosis is poorly understood. Therefore, we examined whether serotonin (5-HT), a key factor involved in the development of atherosclerosis, induced HMGB1 release in human umbilical vein endothelial cells (HUVECs). We found that 5-HT induced the release of HMGB1 but not of ERK1/2 and JNK from HUVECs via the 5-HT receptor (5-HT1B)/p38 mitogen-activated protein kinase (MAPK) signaling pathway. The p38MAPK inhibitor SB203580 and the 5-HT1B antagonist GR55526 markedly inhibited HMGB1 release from 5-HT-stimulated HUVECs. The vascular endothelial growth factor (VEGF) derived from activated macrophages in atherosclerotic lesions also plays an important role in the progression of atherosclerosis. We found that HMGB1 induced VEGF production in macrophage-like RAW264.7 cells. HMGB1 induced the activation of p38MAPK, ERK1/2 and Akt. The PI3-kinase inhibitor LY294002 significantly inhibited VEGF production in HMGB1-stimulated macrophages, while other kinase inhibitors did not. These results suggest that HMGB1 release may contribute as a risk factor in the development and progression of atherosclerosis.

    International journal of molecular medicine 2008;22;5;639-44

  • Lack of cytoplasmic ERK activation is an independent adverse prognostic factor in primary cutaneous melanoma.

    Jovanovic B, Kröckel D, Linden D, Nilsson B, Egyhazi S and Hansson J

    Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska, Karolinska University Hospital Solna, Stockholm, Sweden.

    The aim of this study was to estimate the impact on survival of NRAS and BRAF mutations and activation of Akt and extracellular signal-regulated kinase (ERK) in primary melanomas. A cohort of 57 primary cutaneous T1-2 melanoma tumors was analyzed. Mutation frequency for both genes was 61% (NRAS 26% and BRAF 39%). In a univariate analysis, shorter overall survival was associated with the presence of ulceration (P=0.001) and BRAF exon 15 mutations (P=0.005) as well as the absence of nuclear activation of Akt (P=0.022) and of cytoplasmic activation of ERK (P=0.003). Unexpectedly, ulceration was a significant adverse prognostic factor only in melanomas with BRAF mutations, whereas there was no effect of ulceration on overall survival in tumors with wild-type BRAF. A multivariate analysis showed that significant independent adverse survival prognostic markers were absence of cytoplasmic activation of ERK (P=0.007) and ulceration (P=0.008), whereas BRAF exon 15 mutation status showed a nonsignificant trend (P=0.066). The absence of cytoplasmic ERK activation in poor prognosis T1-2 melanomas may be associated with activation of some other uncharacterized pathway leading to tumor progression and adverse outcome. Immunohistochemical analysis of cytoplasmic phosphorylated ERK could be used as a prognostic marker in primary melanomas if confirmed in another data set.

    The Journal of investigative dermatology 2008;128;11;2696-704

  • Target gene-specific regulation of androgen receptor activity by p42/p44 mitogen-activated protein kinase.

    Agoulnik IU, Bingman WE, Nakka M, Li W, Wang Q, Liu XS, Brown M and Weigel NL

    Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

    Evidence that the androgen receptor (AR) is not only important in androgen-dependent prostate cancer, but also continues to play a role in tumors that become resistant to androgen deprivation therapies, highlights the need to find alternate means to block AR activity. AR, a hormone-activated transcription factor, and its coactivators are phosphoproteins. Thus, we sought to determine whether inhibition of specific cell signaling pathways would reduce AR function. We found that short-term inhibition of p42/p44 MAPK activity either by a MAPK kinase inhibitor, U0126, or by depletion of kinase with small interfering RNA caused target gene-specific reductions in AR activity. AR enhances histone H3 acetylation of target genes that are sensitive to U0126 including prostate-specific antigen and TMPRSS2, but does not increase histone H3 acetylation of the U0126-resistant PMEPA1 gene. Thus, although AR induces transcription of many target genes, the molecular changes induced by AR at the chromatin level are target gene specific. Long-term treatment (24-48 h) with U0126 causes a G1 cell cycle arrest and reduces AR expression both through a decrease in AR mRNA and a reduction in AR protein stability. Thus, treatments that reduce p42/p44 MAPK activity in prostate cancer have the potential to reduce AR activity through a reduction in expression levels as well as by target gene-selective inhibition of AR function.

    Funded by: NCI NIH HHS: CA58204, P50 CA058204; NHGRI NIH HHS: R01 HG004069, R01 HG004069-02, R01 HG004069-03; NIDDK NIH HHS: DK65262, T32 DK007696, T32DK07696

    Molecular endocrinology (Baltimore, Md.) 2008;22;11;2420-32

  • TC1 (C8orf4) is involved in ERK1/2 pathway-regulated G(1)- to S-phase transition.

    Wang YD, Bian GH, Lv XY, Zheng R, Sun H, Zhang Z, Chen Y, Li QW, Xiao Y, Yang QT, Ai JZ, Wei YQ and Zhou Q

    Core Facility of Gene Engineered Mouse, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, PR China.

    Although previous studies have implicated a role for TC1 (C8orf4) in cancer cell proliferation, the molecular mechanism of its action is still largely unclear. In this study, we showed, for the first time, that the mRNA levels of TC1 were upregulated by mitogens (FBS/thrombin) and at least partially, through the ERK1/2 signaling pathway. Interestingly, the over-expression of TC1 promoted the G(1)- to S-phase transition of the cell cycle, which was delayed by the deficiency of ERK1/2 signaling in fibroblast cells. Furthermore, the luciferase reporter assay indicated that the over-expression of TC1 significantly increased Cyclin D1 promoter-driven luciferase activity. Taken together, our findings revealed that TC1 was involved in the mitogen-activated ERK1/2 signaling pathway and positively regulated G(1)- to S-phase transition of the cell cycle. Our results may provide a novel mechanism of the role of TC1 in the regulation of cell proliferation.

    BMB reports 2008;41;10;733-8

  • The transactivated epidermal growth factor receptor recruits Pyk2 to regulate Src kinase activity.

    Schauwienold D, Sastre AP, Genzel N, Schaefer M and Reusch HP

    Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.

    G protein-coupled receptors such as proteinase-activated receptor 1 induce phosphorylation of mitogen-activated protein kinases through multiple pathways including transactivation of receptor tyrosine kinases. In vascular smooth muscle cells, both matrix-metalloproteinase-dependent extracellular shedding of membrane-bound epidermal growth factor (EGF) receptor ligands and activation of the nonreceptor tyrosine kinases Pyk2 and Src contributed to the thrombin-induced ERK1/2 phosphorylation. Surprisingly, disruption of the HB-EGF-mediated extracellular mode of EGF receptor transactivation also prevented the phosphorylation of the nonreceptor tyrosine kinases Pyk2 and Src, locating these kinases downstream of the transactivated EGF receptor. The ionomycin-induced Pyk2 phosphorylation was partially sensitive to AG1478, heparin, or the matrix-metalloproteinase inhibitor BB2116, and the ionomycin-induced EGF receptor phosphorylation was almost completely blocked by these inhibitors of extracellular transactivation. Coimmunoprecipitation experiments revealed that, upon thrombin stimulation, a signaling complex consisting of Pyk2 and Src assembles at the EGF receptor. Reconstitution of the signaling molecules in HEK293 or vascular smooth muscle cells and subsequent determination of the EGF-induced Src kinase activity applying fluorescent sensor proteins demonstrated that a Ca(2+)-independent mode of Pyk2 activation is critical for the activation of Src downstream of the EGF receptor.

    The Journal of biological chemistry 2008;283;41;27748-56

  • Histamine induces Egr-1 expression in human aortic endothelial cells via the H1 receptor-mediated protein kinase Cdelta-dependent ERK activation pathway.

    Hao F, Tan M, Xu X and Cui MZ

    Department of Pathobiology, The University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37996, USA.

    Histamine, a potent inflammatory mediator, has multiple effects on the pathogenesis of atherosclerosis. This study investigates the effect of histamine on the expression of early growth response factor 1 (Egr-1), a master transcription factor that regulates the expression of an array of atherogenic genes in atherosclerotic lesions. Histamine markedly and rapidly induces Egr-1 mRNA and protein expression in primary human aortic endothelial cells (HAECs). Histamine-induced Egr-1 expression is dependent on the activation of the H1 receptor. Histamine also rapidly and transiently activates protein kinase C-delta (PKCdelta), extracellular signal-regulated kinase (ERK)1/2, p38 kinase, and c-Jun N-terminal kinase (JNK) prior to Egr-1 induction. Using specific pharmacological inhibitors and small interfering RNA technology, we determined that PKCdelta and ERK, but not p38 and JNK, mediate histamine-induced Egr-1 expression. Our data provide the first evidence that histamine regulates expression of Egr-1 in mammalian cells and demonstrate a novel role of PKCdelta in up-regulation of Egr-1 expression. The present study reveals the following regulatory mechanism: histamine up-regulates Egr-1 expression in primary HAECs via the H1 receptor and the PKCdelta-dependent ERK activation pathway. Our data also imply that CREB, a downstream component of the ERK pathway, regulates Egr-1 expression in HAECs. Importantly, these results suggest a central role of Egr-1 in histamine-induced gene expression and in histamine-induced vascular disease.

    Funded by: NHLBI NIH HHS: HL074341, R01 HL074341, R01 HL074341-03; NIA NIH HHS: AG026640

    The Journal of biological chemistry 2008;283;40;26928-36

  • The activation of ERK1/2 MAP kinases in glioblastoma pathobiology and its relationship with EGFR amplification.

    Lopez-Gines C, Gil-Benso R, Benito R, Mata M, Pereda J, Sastre J, Roldan P, Gonzalez-Darder J and Cerdá-Nicolás M

    Department of Pathology, University of Valencia, Valencia, Spain.

    The ERK1/2 activated protein kinase (MAPK) pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation and survival, involved in malignant transformation. The purpose of this study was to determine the activation of ERK1/2 in this tumor, and to determine the relationship of ERK1/2 activation with the amplification/overexpression of EGFR as well as with 9p21 locus gene alterations, both of which are genetic factors frequently associated with glioblastoma. We used immunohistochemistry and Western blot analysis to analyze the activation of ERK1/2 in 22 patients with glioblastoma, and we studied the amplification/overexpression of EGFR; as well as the molecular alterations in 9p21 locus genes. Positive immunostaining ERK1/2 was observed in 86.4% of the tumors, displaying mainly nuclear immunolocalization; and by immunoblotting, ERK1/2 was activated in 68% of the cases. The 70% of cases with EGFR amplification presented activated ERK1/2. The joint presence of amplified EGFR and alterations in the 9p21 genes was observed in 50% of the cases, whereas the simultaneous occurrence of these two phenomena with the activation of ERK1/2 was observed in 40% of the cases. Our results suggest that the activation of ERK1/2 is implicated in the pathobiology of glioblastoma. This activation of ERK1/2 is probably related in part to the amplification of EGFR as well as to alterations in 9p21 locus genes (homozygous deletion and promoter methylation). However, the activation of ERK1/2 also involves pathways that are independent of the EGFR.

    Neuropathology : official journal of the Japanese Society of Neuropathology 2008;28;5;507-15

  • A study of phosphorylated ERK1/2 and COX-2 in early stage (T1-T2) oral squamous cell carcinomas.

    Søland TM, Husvik C, Koppang HS, Boysen M, Sandvik L, Clausen OP, Christoffersen T and Bryne M

    Department of Pathology and Forensic Odontology, Institute of Clinical Dentistry, University of Oslo, Oslo, Norway. tinehe@odont.uio.no

    Background: Histomorphological grading at the invasive front of oral squamous cell carcinomas (OSCCs) may provide useful prognostic information. In the present study, we investigated the presence and prognostic value of activated phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2) and cyclo-oxygenase-2 (COX-2) both at the invasive front and in central/superficial parts of OSCCs.

    Methods: Using immunohistochemistry, we assessed the presence of p-ERK1/2 and COX-2 in 53 early stage OSCCs. Clinical data were recorded prospectively. The end point was disease-free survival.

    Results: p-ERK1/2 staining was present in almost all tumours. The staining was mostly nuclear in the cells of the invasive front and either nuclear or nuclear/cytoplasmic in central/superficial tumour parts. COX-2 was observed in almost all tumours (98%) and the staining was often restricted to focal areas. Most tumours were COX-2 negative at the invasive front. The lowest P-value in survival analyses was P = 0.06 for p-ERK1/2 at the invasive front. COX-2, the histomorphological grading systems and TNM stage were of no prognostic value.

    Conclusion: p-ERK1/2 was present in almost all tumours and p-ERK1/2 may be a prognostic marker at the invasive front of OSCCs. In early stage OSCCs, most tumours did not express COX-2 at the invasive front.

    Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 2008;37;9;535-42

  • C-reactive protein (CRP) increases VEGF-A expression in monocytic cells via a PI3-kinase and ERK 1/2 signaling dependent pathway.

    Bello G, Cailotto F, Hanriot D, Kolopp-Sarda MN, Latger-Cannard V, Hess K, Zannad F, Longrois D and Ropars A

    INSERM, U684, Vandoeuvre les Nancy, F-54500 France.

    C-reactive protein (CRP) is an independent predictor of atherosclerosis and its complications. Monocytes/macrophages are implicated in this complex disease which is, among other mechanisms, characterised by angiogenesis. The aim of this study was to analyse whether CRP plays a role in VEGF-A regulation by monocytic cells. Our findings show that CRP up-regulates VEGF-A mRNA expression and protein excretion in THP-1 cells in a concentration- and time-dependent manner. Furthermore, we studied the signaling pathway underlying this effect. CRP increases VEGF-A expression via a PI3-kinase and an extracellular-signal-regulated kinase (ERK) 1/2 dependent pathway. Our results suggest that CRP could play a role in the angiogenesis process via immune cells such as monocytes.

    Atherosclerosis 2008;200;2;286-93

  • ERK1/2-mediated phosphorylation of small hepatitis delta antigen at serine 177 enhances hepatitis delta virus antigenomic RNA replication.

    Chen YS, Huang WH, Hong SY, Tsay YG and Chen PJ

    Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

    The small hepatitis delta virus (HDV) antigen (SHDAg) plays an essential role in HDV RNA double-rolling-circle replication. Several posttranslational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. Among the PTMs, the serine 177 residue of SHDAg is a phosphorylation site, and its mutation preferentially abolishes HDV RNA replication from antigenomic RNA to genomic RNA. Using coimmunoprecipitation analysis, the cellular kinases extracellular signal-related kinases 1 and 2 (ERK1/2) are found to be associated with the Flag-tagged SHDAg mutant (Ser-177 replaced with Cys-177). In an in vitro kinase assay, serine 177 of SHDAg was phosphorylated directly by either Flag-ERK1 or Flag-ERK2. Activation of endogenous ERK1/2 by a constitutively active MEK1 (hemagglutinin-AcMEK1) increased phosphorylation of SHDAg at Ser-177; this phosphorylation was confirmed by immunoblotting using an antibody against phosphorylated S177 and mass spectrometric analysis. Interestingly, we found an increase in the HDV replication from antigenomic RNA to genomic RNA but not in that from genomic RNA to antigenomic RNA. The Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. The results may shed light on the mechanisms of HDV RNA replication.

    Journal of virology 2008;82;19;9345-58

  • ERK2 CD domain mutation from a human cancer cell line enhanced anchorage-independent cell growth and abnormality in Drosophila.

    Mahalingam M, Arvind R, Ida H, Murugan AK, Yamaguchi M and Tsuchida N

    Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, Tokyo, Japan.

    In a human cancer cell line, we previously found a mutation in codon 322 of the extracellular signal-regulated kinase (ERK2E322K), the protein showed a faster migration when compared to wild-type in SDS-PAGE and constitutive phosphorylation. However, the reason for the faster migration, and the biochemical and biological properties of the mutation is unknown. In this study, we report that the amino acid charge-change mutation in the common docking (CD) domain is important for fast migration. In vitro binding of ERK2E322K to MKP1 and RSK2 was lost, resulting in constitutive activation and possibly contributing to a more efficient colony formation in soft agar. We established transgenic flies by carrying the corresponding CD domain mutation, DERKE335K, which developed smaller and rougher eyes compared with the wild-type. Taken together, these data are consistent with ERK2E322K loss of contact with downstream effectors and its constitutive activation, presenting an oncogenic potential and weak abnormality in differentiation.

    Oncology reports 2008;20;4;957-62

  • G-CSF stimulates Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation.

    Wang L, Xue J, Zadorozny EV and Robinson LJ

    Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.

    Granulocyte colony-stimulating factor (G-CSF), the major cytokine regulator of neutrophilic granulopoiesis, stimulates both the proliferation and differentiation of myeloid precursors. A variety of signaling proteins have been identified as mediators of G-CSF signaling, but understanding of their specific interactions and organization into signaling pathways for particular cellular effects is incomplete. The present study examined the role of the scaffolding protein Grb2-associated binding protein-2 (Gab2) in G-CSF signaling. We found that a chemical inhibitor of Janus kinases inhibited G-CSF-stimulated Gab2 phosphorylation. Transfection with Jak2 antisense and dominant negative constructs also inhibited Gab2 phosphorylation in response to G-CSF. In addition, G-CSF enhanced the association of Jak2 with Gab2. In vitro, activated Jak2 directly phosphorylated specific Gab2 tyrosine residues. Mutagenesis studies revealed that Gab2 tyrosine 643 (Y643) was a major target of Jak2 in vitro, and a key residue for Jak2-dependent phosphorylation in intact cells. Mutation of Gab2 Y643 inhibited G-CSF-stimulated Erk1/2 activation and Shp2 binding to Gab2. Loss of Y643 also inhibited Gab2-mediated G-CSF-stimulated cell proliferation. Together, these results identify a novel signaling pathway involving Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation in response to G-CSF.

    Funded by: NIAMS NIH HHS: K08 AR053566, K08 AR053566-01A1, K08 AR053566-02

    Cellular signalling 2008;20;10;1890-9

  • Genistein induces G2/M cell cycle arrest via stable activation of ERK1/2 pathway in MDA-MB-231 breast cancer cells.

    Li Z, Li J, Mo B, Hu C, Liu H, Qi H, Wang X and Xu J

    The Key Laboratory of Reproductive Medicine of Jiangsu Province, Institute of Toxicology, Nanjing Medical University, NO. 140 Hanzhong Rd, Jiangsu, Nanjing 210029, China. uiuclz@126.com

    Genistein is an isoflavonoid present in soybeans that exhibits anti-carcinogenic effect. Several studies have shown that genistein can trigger G2/M cell cycle arrest and inhibit cell growth in human breast cancer cells. In the present study, we assessed the role of MEK-ERK cascade in regulation of genistein-mediated G2/M cell cycle arrest in the hormone-independent cell line MDA-MB-231. Flow cytometric analysis showed that treatment of MDA-MB-231 cells with genistein induced a concentration-dependent accumulation of cells in the G2/M phase of the cell cycle, with a parallel depletion of the percentage of cells in G0/G1. Genistein-mediated G2/M arrest was associated with a decrease in the protein levels of Cdk1, cyclinB1, and Cdc25C as determined by Western blot analysis. Genistein induced a slow and stable activation of phosphorylated ERK1/2 in a concentration- and time-dependent manner in MDA-MB-231 cells. MEK1/2-specific inhibitor PD98059 blocked genistein-induced activation of ERK1/2 and markedly attenuated genistein-induced G2/M arrest. Furthermore, genistein induced the expression of Ras and Raf-1 protein. Genistein also up-regulated steady-state levels of both c-Jun and c-Fos. PD98059 did not depress genistein-induced up-regulation of Ras and Raf-1 protein. However, it markedly blocked genistein-induced up-regulation of c-Jun and c-Fos. These results suggest that the Ras/MAPK/AP-1 signal pathway may be involved in genistein-induced G2/M cell cycle arrest in MDA-MB-231 breast cancer cells.

    Cell biology and toxicology 2008;24;5;401-9

  • Serum-induced phosphorylation of the serum response factor coactivator MKL1 by the extracellular signal-regulated kinase 1/2 pathway inhibits its nuclear localization.

    Muehlich S, Wang R, Lee SM, Lewis TC, Dai C and Prywes R

    Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

    Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related coactivator of the serum response factor (SRF) transcription factor, which has an integral role in differentiation, migration, and proliferation. Serum induces RhoA-dependent translocation of MKL1 from the cytoplasm to the nucleus and also causes a rapid increase in MKL1 phosphorylation. We have mapped a serum-inducible phosphorylation site and found, surprisingly, that its mutation causes constitutive localization to the nucleus, suggesting that phosphorylation of MKL1 inhibits its serum-induced nuclear localization. The key site, serine 454, resembles a mitogen-activated protein kinase phosphorylation site, and its modification was blocked by the MEK1 inhibitor U0126, implying that extracellular signal-regulated kinase 1/2 (ERK1/2) is the serum-inducible kinase that phosphorylates MKL1. Previous results indicated that G-actin binding to MKL1 promotes its nuclear export, and we found that MKL1 phosphorylation is required for its binding to actin, explaining its effect on localization. We propose a model in which serum induction initially stimulates MKL1 nuclear localization due to a decrease in G-actin levels, but MKL1 is then downregulated by nuclear export due to ERK1/2 phosphorylation.

    Funded by: NCI NIH HHS: CA050329, R01 CA050329; NIGMS NIH HHS: 1F31GM082027-01, F31 GM082027

    Molecular and cellular biology 2008;28;20;6302-13

  • Identification and characterization of a general nuclear translocation signal in signaling proteins.

    Chuderland D, Konson A and Seger R

    Department of Biological Regulation, The Weizmann Institute of Science, 76100 Rehovot, Israel.

    Upon stimulation, many proteins translocate into the nucleus in order to regulate a variety of cellular processes. The mechanism underlying the translocation is not clear since many of these proteins lack a canonical nuclear localization signal (NLS). We searched for an alternative mechanism in extracellular signal-regulated kinase (ERK)-2 and identified a 3 amino acid domain (SPS) that is phosphorylated upon stimulation to induce nuclear translocation of ERK2. A 19 amino acid stretch containing this phosphorylated domain inserts nondiffusible proteins to the nucleus autonomously. The phosphorylated SPS acts by binding to importin7 and the release from nuclear pore proteins. This allows its functioning both in passive and active ERK transports. A similar domain appears in many cytonuclear shuttling proteins, and we found that phosphorylation of similar sequences in SMAD3 or MEK1 also induces their nuclear accumulation. Therefore, our findings show that this phosphorylated domain acts as a general nuclear translocation signal (NTS).

    Molecular cell 2008;31;6;850-61

  • Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in beta-cells.

    Lawrence MC, McGlynn K, Shao C, Duan L, Naziruddin B, Levy MF and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

    MAPK pathways regulate transcription through phosphorylation of transcription factors and other DNA-binding proteins. In pancreatic beta-cells, ERK1/2 are required for transcription of the insulin gene and several other genes in response to glucose. We show that binding of glucose-sensitive transcription activators and repressors to the insulin gene promoter depends on ERK1/2 activity. We also find that glucose and NGF stimulate the binding of ERK1/2 to the insulin gene and other promoters. An ERK1/2 cascade module, including MEK1/2 and Rsk, are found in complexes bound to these promoters. These findings imply that MAPK-containing signaling complexes are positioned on sensitive promoters with their protein substrates to modulate transcription in situ in response to incoming signals.

    Funded by: NIDDK NIH HHS: DK34128, DK55310, R01 DK034128, R01 DK055310, R37 DK034128

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;36;13315-20

  • COX2 expression and Erk1/Erk2 activity mediate Cot-induced cell migration.

    Rodríguez C, López P, Pozo M, Duce AM, López-Pelaéz M, Fernández M and Alemany S

    Instituto de Investigaciones Biomédicas, Dpto. de Bioquímica, UAM, Arturo Duperier 4, 28029 Madrid, Spain.

    The MAPKKK8 Cot/tpl-2, identified as an oncogene (Cot-T), participates in the intracellular signaling activated by members of the TLR and TNFalpha receptor superfamilies. Here we demonstrate that Cot promotes cell migration by regulating different steps involved in this process, such as cell adhesion and metalloproteinase activity. Indeed, Cot also regulates the cytoskeleton and Cot-T overexpression provokes the polarization of microtubules and the loss of stress fibers. Moreover, and in accordance with the increased Rac-GTP levels observed, Cot-T overexpressing cells develop more lamellipodia than control cells. Conversely, depletion of endogenous Cot increases the formation of stress fibers which is correlated with the high levels of Rho-GTP observed in these cells. In addition, the increase in COX2 expression and the activation of Erk1/2 regulated by Cot are essential for the induction of cell migration. Together, these data provide evidence of a new role for both proto-oncogenic and oncogenic Cot.

    Cellular signalling 2008;20;9;1625-31

  • Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus.

    Zhang HY, Zhang X, Chen X, Thomas D, Hormi-Carver K, Elder F, Spechler SJ and Souza RF

    Department of Medcine, Veterans Affairs North tExas Health Care System and the University of Texas Southwestern Medical School, MC# 111B1, Dallas VA Medical Ctr., 4500 South Lancaster Rd., Dallas, TX 75216, USA.

    We hypothesized that, in esophageal squamous epithelial cells, there are differences among individuals in the signal transduction pathways activated by acid reflux that might underlie the development of Barrett's esophagus. To explore that hypothesis, we immortalized nonneoplastic, esophageal squamous cells from patients with gastroesophageal reflux disease (GERD) with (NES-B3T) and without (NES-G2T) Barrett's esophagus and used those cells to study acid effects on MAPK proteins. During endoscopy in patients with GERD with and without Barrett's esophagus, we took biopsy specimens from the distal squamous esophagus to study MAPK proteins before and after esophageal perfusion with 0.1 N HCl. We used immunoblotting and Western blotting to study MEK1/2 phosphorylation at two activating sites (serines 217/221), MEK1 phosphorylation at an inhibitory site (threonine 286), and MEK1/2 activity. After acid exposure, both cell lines exhibited increased MEK1/2 phosphorylation at the activating sites; the NES-B3T cells had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the NES-G2T cells showed an acid-induced increase in MEK1/2 activity. Similarly, in the squamous epithelium of patients with GERD with and without Barrett's esophagus, acid perfusion increased MEK1/2 phosphorylation at the activating sites in both patient groups; the Barrett's patients had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the patients without Barrett's demonstrated an acid-induced increase in ERK1/2 phosphorylation. In esophageal squamous cell lines and biopsies from patients with GERD with and without Barrett's esophagus, we have found differences in MAPK pathways activated by acid exposure. We speculate that these differences might underlie the development of Barrett's metaplasia.

    Funded by: NIDDK NIH HHS: DK63621

    American journal of physiology. Gastrointestinal and liver physiology 2008;295;3;G470-8

  • Dual ERK and phosphatidylinositol 3-kinase pathways control airway smooth muscle proliferation: differences in asthma.

    Burgess JK, Lee JH, Ge Q, Ramsay EE, Poniris MH, Parmentier J, Roth M, Johnson PR, Hunt NH, Black JL and Ammit AJ

    Respiratory Research Group, Discipline of Pharmacology, Faculty of Medicine, University of Sydney, Sydney, NSW, Australia.

    Hyperplasia of airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely due to increased muscle proliferation. We have shown that ASM cells obtained from asthmatic patients proliferate faster than those obtained from non-asthmatic patients. In ASM from non-asthmatics, mitogens act via dual signaling pathways (both ERK- and PI 3-kinase-dependent) to control growth. In this study we are the first to examine whether dual pathways control the enhanced proliferation of ASM from asthmatics. When cells were incubated with 0.1% or 1% FBS, ERK activation was significantly greater in cells from asthmatic subjects (P < 0.05). In contrast, when cells were stimulated with 10% FBS, ERK activity was significantly greater in the non-asthmatic cells. However, cell proliferation in asthmatic cells was still significantly higher in cells stimulated by both 1% and 10% FBS. Pharmacological inhibition revealed that although dual proliferative pathways control ASM growth in cells from non-asthmatics stimulated with 10% FBS to an equal extent ([(3)H]-thymidine incorporation reduced to 57.2 +/- 6.9% by the PI 3-kinase inhibitor LY294002 and 57.8 +/- 1.1% by the ERK-pathway inhibitor U0126); in asthmatics, the presence of a strong proliferative stimulus (10% FBS) reduces ERK activation resulting in a shift to the PI 3-kinase pathway. The underlying mechanism appears to be upregulation of an endogenous MAPK inhibitor--MKP-1--that constrains ERK signaling in asthmatic cells under strong mitogenic stimulation. This study suggests that the PI 3-kinase pathway may be an attractive target for reversing hyperplasia in asthma.

    Journal of cellular physiology 2008;216;3;673-9

  • Effects of small interfering RNAs targeting MAPK1 on gene expression profile in HeLa cells as revealed by microarray analysis.

    Huang C, Liu LY, Li ZF, Wang P, Ni L, Song LP, Xu DH and Song TS

    Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education/Department of Genetics and Molecular Biology, Medical School, Xi'an Jiaotong University, 205 Zhu Que Road, Xi'an, Shaanxi 710061, PR China. hchen@mail.xjtu.edu.cn

    The mitogen activated protein kinases (MAPK) signaling cascade plays an important role in cell life. We proved that small interfering RNAs targeting MAPK1 (siRNA-2) could inhibit HeLa cell growth, but the effects of siRNA-2 on gene expression profile were unclear. Using Affymetrix GeneChip HG-U133A 2.0, we identified the long-term changes for 48 h in gene expression profile in HeLa cell treated by siRNA-2. The results showed that expressions of 181 genes were altered by siRNA-2 and were divided into two groups: (i) one group showed downregulation by siRNA-2, including the proliferation associated genes, small G protein, cytoskeleton associated protein and extracellular matrix associated protein; and (ii) the other group showed upregulation by siRNA-2, including interferon response genes, OAS family, TRIM family and apoptosis associated genes. The results of Real-time quantitative PCR for MAPK1, NUP188, P38, STAT1, STAT2, MPL and OAS1 were consistent with that of gene chip. Two networks were found to react substantially to the downregulation of MAPK1 by siRNA-2. One of the networks regulates cell growth through cell-cycle control, apoptosis and cytoskeleton. The other network is related to interferon-like response. Our findings suggest that siRNA-mediated downregulation of MAPK1 could be an attractive strategy for cancer therapy.

    Cell biology international 2008;32;9;1081-90

  • Extracellularly signal-regulated kinase activity in the human endometrium: possible roles in the pathogenesis of endometriosis.

    Murk W, Atabekoglu CS, Cakmak H, Heper A, Ensari A, Kayisli UA and Arici A

    Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8063, USA.

    Context: Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial tissue outside of the uterine cavity, causing pelvic pain and infertility in 10% of reproductive-aged women. It is unclear why ectopic endometrium remains viable in only a subset of women. ERK1/2 plays key intracellular roles in activating cellular survival and differentiation processes.

    Objective: We sought to determine ERK1/2 activity in patients with endometriosis and its possible roles in regulating endometrial cell survival.

    Design: ERK1/2 phosphorylation and expression throughout the menstrual cycle were evaluated in vivo in normal and endometriotic human endometrium, and in vitro techniques assessed the steroidal regulation of ERK1/2 and its effect on endometrial cell survival.

    Results: Total ERK1/2 remained constant in normal and endometriotic endometrium throughout the menstrual cycle. Phospho-ERK1/2 was high in the late proliferative and secretory phases in normal endometrium (P < 0.05). In endometriotic glandular cells, there was no cyclical variation in phospho-ERK1/2. In endometriotic stromal cells, there was also a reduction in phospho-ERK1/2 variation, with higher levels in the early-mid secretory phase (P < 0.05). In cultured endometrial stromal cells (ESCs), estrogen plus progesterone increased ERK1/2 phosphorylation within 15 min (P < 0.05). Although estrogen alone did not induce ERK1/2 phosphorylation in normal ESCs, there was a significant response to estrogen in ESCs isolated from eutopic endometriotic endometrium (P < 0.05). ERK1/2 inhibition in ESCs reduced proliferation and increased apoptosis (P < 0.05).

    Conclusion: Abnormally high levels of ERK1/2 activity may be involved in endometriosis, possibly by stimulating endometrial cell survival.

    The Journal of clinical endocrinology and metabolism 2008;93;9;3532-40

  • Interaction of scaffolding adaptor protein Gab1 with tyrosine phosphatase SHP2 negatively regulates IGF-I-dependent myogenic differentiation via the ERK1/2 signaling pathway.

    Koyama T, Nakaoka Y, Fujio Y, Hirota H, Nishida K, Sugiyama S, Okamoto K, Yamauchi-Takihara K, Yoshimura M, Mochizuki S, Hori M, Hirano T and Mochizuki N

    Department of Structural Analysis, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka, Japan.

    Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1(WT)), mutated Gab1 that is unable to bind SHP2 (Gab1(DeltaSHP2)), or mutated Gab1 that is unable to bind p85 (Gab1(Deltap85)), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1(DeltaSHP2), but inhibited in those overexpressing either Gab1(WT) or Gab1(Deltap85). Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1(DeltaSHP2) but enhanced in those overexpressing either Gab1(WT) or Gab1(Deltap85). Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1(DeltaSHP2), suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1(WT) overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1(DeltaSHP2). Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.

    The Journal of biological chemistry 2008;283;35;24234-44

  • Constitutive hypophosphorylation of extracellular signal-regulated kinases-1/2 and down-regulation of c-Jun in human gastric adenocarcinoma.

    Wu WK, Sung JJ, Yu L, Li ZJ, Chu KM and Cho CH

    Department of Pharmacology, Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China.

    Hyperphosphorylation of extracellular signal-regulated protein kinases-1/2 (ERK1/2) is known to promote cancer cell proliferation. We therefore investigated the constitutive phosphorylation levels of ERK1/2 and the expression of its downstream targets c-Fos, c-Jun, and cyclooxygenase-2 (COX-2) in biopsied human gastric cancer tissues. Results showed that ERK1/2 phosphorylation and c-Jun expression were significantly lowered in gastric cancer compared with the non-cancer adjacent tissues. The expression of c-Fos, however, was not altered while COX-2 was significantly up-regulated. To conclude, we demonstrate that hypophosphorylation of ERK1/2 may occur in gastric cancer. Such discovery may have implication in the application of pathway-directed therapy for this malignant disease.

    Biochemical and biophysical research communications 2008;373;2;330-4

  • The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation.

    Hansen JL, Aplin M, Hansen JT, Christensen GL, Bonde MM, Schneider M, Haunsø S, Schiffer HH, Burstein ES, Weiner DM and Sheikh SP

    Laboratory for Molecular Cardiology, The Danish National Research Foundation Centre for Cardiac Arrhythmia, The Heart Centre, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark. jlhansen@sund.ku.dk

    The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensin AT(1) receptor signalling is illustrated by the common use of angiotensin AT(1) receptor-inverse agonists in clinical practice. It is well established that rodent orthologues of the angiotensin AT(1) receptor can selectively signal through G protein-dependent and -independent mechanisms in recombinant expression systems, primary cells and in vivo. The in vivo work clearly demonstrates profoundly different cellular consequences of angiotensin AT(1) receptor signalling in the cardiovascular system, suggesting pharmacological potential for drugs which specifically affect a subset of angiotensin AT(1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes.

    European journal of pharmacology 2008;590;1-3;255-63

  • Oncogenic B-RafV600E inhibits apoptosis and promotes ERK-dependent inactivation of Bad and Bim.

    Sheridan C, Brumatti G and Martin SJ

    Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.

    Recent studies have revealed that B-Raf mutations are very common in malignant melanoma and are required for tumor growth and maintenance. The majority of melanoma-associated B-Raf mutations involve a single point mutation, V600E, which results in greatly elevated B-Raf kinase activity and constitutive activation of MAPK/ERK downstream. Here we show that B-Raf(V600E) increases resistance to apoptosis induced by chemotherapeutic drugs and promotes ERK-dependent phosphorylation of the BH3-only proteins Bim and Bad that are involved in setting thresholds for apoptosis. ERK-dependent phosphorylation of Bim resulted in degradation of this BH3-only protein, whereas phosphorylation of Bad has previously been shown to result in its sequestration by 14-3-3 proteins. Consistent with this, inhibition of ERK activity in a panel of melanoma cell lines resulted in stabilization of Bim and dephosphorylation of Bad. Furthermore, apoptosis induced through overexpression of Bad or Bim was efficiently blocked by coexpression of mutant B-Raf(V600E). However, small interfering RNA-mediated silencing of Bim and Bad expression conferred only modest protection against cytotoxic drugs, whereas oncogenic B-Raf strongly protected against the same stimuli. These observations suggest that B-Raf-initiated inactivation of Bad and Bim only partly contributes to the anti-apoptotic activities of this oncogene and that other points within the cell death machinery are also targeted by deregulated ERK signaling.

    The Journal of biological chemistry 2008;283;32;22128-35

  • Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases.

    LaMarca HL, Dash PR, Vishnuthevan K, Harvey E, Sullivan DE, Morris CA and Whitley GS

    Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, LA, USA.

    Background: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy.

    In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3beta as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3beta. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3beta phosphorylation and the subsequent expression of beta-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype.

    Conclusion: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of beta-catenin alone was insufficient to stimulate cell motility.

    Funded by: British Heart Foundation; NICHD NIH HHS: HD045768, R01 HD051998

    Human reproduction (Oxford, England) 2008;23;8;1733-41

  • Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-alpha-induced cell death in MCF-7 cells.

    Sivaprasad U and Basu A

    Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

    The role of autophagy in cell death is under considerable debate. The process of autophagy has been shown to lead to either cell survival or cell death depending on cell type and stimulus. In the present study, we determined the contribution of ERK1/2 signalling to autophagy and cell death induced by tumour necrosis factor-alpha (TNF) in MCF-7 breast cancer cells. Treatment of MCF-7 cells with TNF caused a time-dependent increase in ERK1/2 activity. There was an induction of autophagy and cleavage of caspase-7, -8, -9 and PARP. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 or PD98059 resulted in a decrease in TNF-induced autophagy that was accompanied by an increase in cleavage of caspase-7, -8, -9 and PARP Furthermore, inhibition of ERK1/2 signalling resulted in decreased clonogenic capacity of MCF-7 cells. These data suggest that TNF-induces autophagy through ERK1/2 and that inhibition of autophagy increases cellular sensitivity to TNF.

    Funded by: NCI NIH HHS: CA71727, R01 CA071727

    Journal of cellular and molecular medicine 2008;12;4;1265-71

  • Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson's disease.

    Dagda RK, Zhu J, Kulich SM and Chu CT

    Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    Degenerating neurons of Parkinson's disease (PD) patient brains exhibit granules of phosphorylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) that localize to autophagocytosed mitochondria. Here we show that 6-hydroxydopamine (6-OHDA) elicits activity-related localization of ERK1/2 in mitochondria of SH-SY5Y cells, and these events coincide with induction of autophagy and precede mitochondrial degradation. Transient transfection of wildtype (WT) ERK2 or constitutively active MAPK/ERK Kinase 2 (MEK2-CA) was sufficient to induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.

    Funded by: NIA NIH HHS: AG026389, F32 AG030821, F32 AG030821-01, R01 AG026389, R01 AG026389-01A2, R01 AG026389-02; NIDCD NIH HHS: DC009120, K18 DC009120, K18 DC009120-01; NINDS NIH HHS: T32 NS007495, T32 NS07495

    Autophagy 2008;4;6;770-82

  • Suppression of astrovirus replication by an ERK1/2 inhibitor.

    Moser LA and Schultz-Cherry S

    University of Wisconsin-Madison, Department of Medical Microbiology and Immunology, Madison, WI 53706, USA.

    Human astroviruses are nonenveloped, positive-sense single-strand RNA viruses associated with self-limiting diarrhea. Although they are recognized as a leading cause of disease in young children, the cellular factors involved in astrovirus replication are not well defined. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate many viral infections, but its role during astrovirus infection is unknown. In this report, we show that astrovirus activates ERK1/2 early in infection independently of replication. Inhibition of ERK activation with U0126, a specific ERK inhibitor, significantly reduced viral production. Investigations into the mechanism of ERK1/2 regulation revealed that all steps of the viral life cycle, including early and late protein expression as well as subgenomic and genomic RNA transcription, were diminished during U0126 treatment of monolayers. These data support a role for ERK1/2 in a postattachment step, although the precise mechanism remains under investigation.

    Funded by: PHS HHS: T32 A10055397

    Journal of virology 2008;82;15;7475-82

  • Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling.

    Seomun Y and Joo CK

    Laboratory of Ophthalmology and Visual Science, Korean Eye Tissue and Gene Bank related to Blindness, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-040, Republic of Korea.

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins alpha2 and beta1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin beta1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

    Biochemical and biophysical research communications 2008;372;1;221-5

  • Gamma-irradiation-induced DNA damage checkpoint activation involves feedback regulation between extracellular signal-regulated kinase 1/2 and BRCA1.

    Yan Y, Black CP, Cao PT, Haferbier JL, Kolb RH, Spieker RS, Ristow AM and Cowan KH

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.

    Previous studies from our laboratory have shown that the activation of G(2)-M checkpoint after exposure of MCF-7 breast cancer cells to gamma-irradiation (IR) is dependent on the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Studies presented in this report indicate that IR exposure of MCF-7 cells is associated with a marked increase in expression of breast cancer 1 (BRCA1) tumor suppressor, an effect that requires ERK1/2 activation and involves posttranscriptional control mechanisms. Furthermore, reciprocal coimmunoprecipitation, as well as colocalization studies, indicate an interaction between BRCA1 and ERK1/2 in both nonirradiated and irradiated cells. Studies using short hairpin RNA targeting BRCA1 show that BRCA1 expression is necessary for IR-induced G(2)-M cell cycle arrest, as well as ERK1/2 activation in MCF-7 cells. Although BRCA1 expression is not required for IR-induced phosphorylation of ataxia telangiectasia mutated (ATM)-Ser1981, it is required for ATM-mediated downstream signaling events, including IR-induced phosphorylation of Chk2-Thr68 and p53-Ser20. Moreover, BRCA1 expression is also required for IR-induced ATM and rad3 related activation and Chk1 phosphorylation in MCF-7 cells. These results implicate an important interaction between BRCA1 and ERK1/2 in the regulation of cellular response after IR-induced DNA damage in MCF-7 cells.

    Funded by: NCI NIH HHS: P30 CA036727, T32 CA009476

    Cancer research 2008;68;13;5113-21

  • The ERK-RSK1 activation by growth factors at G2 phase delays cell cycle progression and reduces mitotic aberrations.

    Nam HJ, Kim S, Lee MW, Lee BS, Hara T, Saya H, Cho H and Lee JH

    Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, South Korea.

    Growth factors accelerate G0 to S progression in the cell cycle, however, the roles of growth factors in other cell cycle phases are largely unknown. Here, we show that treatment of HeLa cells with hepatocyte growth factor (HGF) at G2 phase induced the G2/M transition delay as evidenced by FACS analysis as well as by mitotic index and time-lapse analyses. Growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) also induced G2/M transition delay like HGF. HGF treatment at G2 phase causes a delayed activation of cyclin B1-associated kinase and a diminished nuclear translocation of cyclin B1. Either U0126, a MAPK kinase (MEK) inhibitor, or kinase-dead mutant of ribosomal S6 kinase (RSK) abolished the delay. Additionally, knockdown of RSK1, but not RSK2, with siRNA abrogated the delay, indicating that the extracellular-regulated protein kinase (ERK)-RSK1 mediates the HGF-induced delay. We further found that the delay in G2/M transition of cells expressing oncogenic HGF receptor, M1268T, was abolished by RSK1 knockdown. Intriguingly, we observed that HGF induced chromosomal segregation defects, and depletion of RSK1, but not RSK2, aggravated these chromosomal aberrations. Taken together, the ERK-RSK1 activation by growth factors delays G2/M transition and this might be required to maintain genomic integrity during growth factor stimulation.

    Cellular signalling 2008;20;7;1349-58

  • Extracellular ATP activates the PLC/PKC/ERK signaling pathway through the P2Y2 purinergic receptor leading to the induction of early growth response 1 expression and the inhibition of viability in human endometrial stromal cells.

    Chang SJ, Tzeng CR, Lee YH and Tai CJ

    Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taiwan.

    ATP is an extracellular signaling molecule that activates specific G protein-coupled P2Y receptors in most cell types to mediate diverse biological effects. ATP has been shown to activate the phospholipase C (PLC)/diacylglycerol/protein kinase C (PKC) pathway in various systems. However, little is known about the signaling events in human endometrial stromal cells (hESCs). The objective of this study was to examine the presence of the P2Y2 receptor and the effects of exogenous ATP on the intracellular mitogen-activated protein kinases (MAPKs) signaling pathway, immediate early genes expression, and cell viability in hESCs. Western blot analysis, gene array analysis, and MTT assay for cell viability were performed. The current study demonstrated the existence of the P2Y2 purinergic receptor in hESCs. UTP and ATP activated MAPK in a dose- and time-dependent manner. Suramin (a P2-purinoceptor antagonist), neomycin (a PLC inhibitor), staurosporin (a PKC inhibitor), and PD98059 (a MEK inhibitor) significantly attenuated the ATP-induced activation of MAPK. ATP activated ERK1/2 and induced translocation of activated ERK1/2 to the nucleus. The gene array for 23 genes associated with members of the mitogenic pathway cascade and immediate early genes revealed that the expression of early growth response 1 was increased. In addition, MTT assay revealed an inhibition effect of ATP on cell viability. ATP activated MAPKs through the P2Y2 purinoceptor/PLC/PKC/ERK signaling pathway and induced translocation of ERK1/2 into the nucleus. Further, ATP induced the expression of early growth response 1 and inhibited cell viability in hESCs.

    Cellular signalling 2008;20;7;1248-55

  • Redox-regulation of Erk1/2-directed phosphatase by reactive oxygen species: role in signaling TPA-induced growth arrest in ML-1 cells.

    Traore K, Sharma R, Thimmulappa RK, Watson WH, Biswal S and Trush MA

    Department of Environmental Health Sciences, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA. ktraore@jhsph.edu

    Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.

    Funded by: NHLBI NIH HHS: HL081205, R01 HL081205, R01 HL081205-03; NIEHS NIH HHS: P30 ES 03819, P30 ES003819, R01 ES 03760, T32 ES 07141, T32 ES007141; NIGMS NIH HHS: R01 GM079239

    Journal of cellular physiology 2008;216;1;276-85

  • Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha.

    Bove PF, Hristova M, Wesley UV, Olson N, Lounsbury KM and van der Vliet A

    Department of Pathology, College of Medicine, University of Vermont, Burlington, VT 05405, USA.

    Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.

    The Journal of biological chemistry 2008;283;26;17919-28

  • MicroRNA miR-199a* regulates the MET proto-oncogene and the downstream extracellular signal-regulated kinase 2 (ERK2).

    Kim S, Lee UJ, Kim MN, Lee EJ, Kim JY, Lee MY, Choung S, Kim YJ and Choi YC

    Gene2Drug Research Center, Bioneer Corporation, and National Genome Information Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.

    MicroRNAs (miRNAs) constitute a class of small noncoding RNAs that play important roles in a variety of biological processes including development, apoptosis, proliferation, and differentiation. Here we show that the expression of miR-199a and miR-199a* (miR-199a/a*), which are processed from the same precursor, is confined to fibroblast cells among cultured cell lines. The fibroblast-specific expression pattern correlated well with methylation patterns: gene loci on chromosome 1 and 19 were fully methylated in all examined cell lines but unmethylated in fibroblasts. Transfection of miR-199a and/or -199a* mimetics into several cancer cell lines caused prominent apoptosis with miR-199a* being more pro-apoptotic. The mechanism underlying apoptosis induced by miR-199a was caspase-dependent, whereas a caspase-independent pathway was involved in apoptosis induced by miR-199a* in A549 cells. By employing microarray and immunoblotting analyses, we identified the MET proto-oncogene as a target of miR-199a*. Studies with a luciferase reporter fused to the 3'-untranslated region of the MET gene demonstrated miR-199a*-mediated down-regulation of luciferase activity through a binding site of miR-199a*. Interestingly, extracellular signal-regulated kinase 2 (ERK2) was also down-regulated by miR-199a*. Coordinated down-regulation of both MET and its downstream effector ERK2 by miR-199a* may be effective in inhibiting not only cell proliferation but also motility and invasive capabilities of tumor cells.

    The Journal of biological chemistry 2008;283;26;18158-66

  • The functional interaction of 14-3-3 proteins with the ERK1/2 scaffold KSR1 occurs in an isoform-specific manner.

    Jagemann LR, Pérez-Rivas LG, Ruiz EJ, Ranea JA, Sánchez-Jiménez F, Nebreda AR, Alba E and Lozano J

    Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain.

    Identifying 14-3-3 isoform-specific substrates and functions may be of broad relevance to cell signaling research because of the key role played by this family of proteins in many vital processes. A multitude of ligands have been identified, but the extent to which they are isoform-specific is a matter of debate. Herein we demonstrate, both in vitro and in vivo, a specific, functionally relevant interaction of human 14-3-3gamma with the molecular scaffold KSR1, which is mediated by the C-terminal stretch of 14-3-3gamma. Specific binding to 14-3-3gamma protected KSR1 from epidermal growth factor-induced dephosphorylation and impaired its ability to activate ERK2 and facilitate Ras signaling in Xenopus oocytes. Furthermore, RNA interference-mediated inhibition of 14-3-3gamma resulted in the accumulation of KSR1 in the plasma membrane, all in accordance with 14-3-3gamma being the cytosolic anchor that keeps KSR1 inactive. We also provide evidence that KSR1-bound 14-3-3gamma heterodimerized preferentially with selected isoforms and that KSR1 bound monomeric 14-3-3gamma. In sum, we have demonstrated ligand discrimination among 14-3-3 isoforms and shed light on molecular mechanisms of 14-3-3 functional specificity and KSR1 regulation.

    The Journal of biological chemistry 2008;283;25;17450-62

  • Phosphorylation of protein kinase Cdelta on distinct tyrosine residues induces sustained activation of Erk1/2 via down-regulation of MKP-1: role in the apoptotic effect of etoposide.

    Lomonaco SL, Kahana S, Blass M, Brody Y, Okhrimenko H, Xiang C, Finniss S, Blumberg PM, Lee HK and Brodie C

    William and Karen Davidson Laboratory of Cell Signaling and Tumorigenesis, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit, Michigan 48202, USA.

    The mechanism underlying the important role of protein kinase Cdelta (PKCdelta) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCdelta in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCdelta since the phosphorylation of Erk1/2 was inhibited by a PKCdelta-KD mutant and PKCdelta small interfering RNA. We recently reported that phosphorylation of PKCdelta on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCdeltatyrosine mutants, we found that the phosphorylation of PKCdeltaon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCdelta was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCdelta. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCdelta and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCdeltaon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.

    Funded by: NCI NIH HHS: CA109196

    The Journal of biological chemistry 2008;283;25;17731-9

  • NF-kappaB-dependent transcriptional activation in lung carcinoma cells by farnesol involves p65/RelA(Ser276) phosphorylation via the MEK-MSK1 signaling pathway.

    Joo JH and Jetten AM

    Cell Biology Section, LRB, Division of Intramural Research, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

    In this study, we demonstrate that treatment of human lung adenocarcinoma H460 cells with farnesol induces the expression of a number of immune response and inflammatory genes, including IL-6, CXCL3, IL-1alpha, and COX-2. This response was dependent on the activation of the NF-kappaB signaling pathway. Farnesol treatment reduces the level of IkappaBalpha and induces translocation of p65/RelA to the nucleus, its phosphorylation at Ser(276), and transactivation of NF-kappaB-dependent transcription. Moreover, overexpression of IkappaBalpha or treatment with the NF-kappaB inhibitor caffeic acid phenethyl ester greatly diminishes the induction of inflammatory gene expression by farnesol. We provide evidence indicating that the farnesol-induced phosphorylation of p65/RelA at Ser(276) is important for optimal transcriptional activity of NF-kappaB. The MEK1/2 inhibitor U0126 and knockdown of MEK1/2 expression with small interfering RNAs effectively blocked the phosphorylation of p65/RelA(Ser(276)) but not that of Ser(536), suggesting that this phosphorylation is dependent on the activation of the MEK1/2-ERK1/2 pathway. We further show that inhibition of MSK1, a kinase acting downstream of MEK1/2-ERK1/2, by H89 or knockdown of MSK1 expression also inhibited phosphorylation of p65/RelA(Ser(276)), suggesting that this phosphorylation is dependent on MSK1. Knockdown of MEK1/2 or MSK1 expression inhibits farnesol-induced expression of CXCL3, IL-1alpha, and COX-2 mRNA. Our results indicate that the induction of inflammatory genes by farnesol is mediated by the activation of the NF-kappaB pathway and involves MEK1/2-ERK1/2-MSK1-dependent phosphorylation of p65/RelA(Ser(276)). The activation of the NF-kappaB pathway by farnesol might be part of a prosurvival response during farnesol-induced ER stress.

    Funded by: Intramural NIH HHS

    The Journal of biological chemistry 2008;283;24;16391-9

  • Constitutive ERK MAPK activity regulates macrophage ATP production and mitochondrial integrity.

    Monick MM, Powers LS, Barrett CW, Hinde S, Ashare A, Groskreutz DJ, Nyunoya T, Coleman M, Spitz DR and Hunninghake GW

    Department of Medicine, University of Iowa Carver College of Medicine and Veterans Administration Medical Center, Iowa City, IA 52242, USA. martha-monick@uiowa.edu

    A unique feature of human alveolar macrophages is their prolonged survival in the face of a stressful environment. We have shown previously that the ERK MAPK is constitutively active in these cells and is important in prolonging cell survival. This study examines the role of the ERK pathway in maintaining mitochondrial energy production. The data demonstrate that ATP levels in alveolar macrophages depend on intact mitochondria and optimal functioning of the electron transport chain. Significant levels of MEK and ERK localize to the mitochondria and inhibition of ERK activity induces an early and profound depletion in cellular ATP coincident with a loss of mitochondrial transmembrane potential. The effect of ERK suppression on ATP levels was specific, since it did not occur with PI3K/Akt, p38, or JNK suppression. ERK inhibition led to cytosolic release of mitochondrial proteins and caspase activation. Both ERK inhibition and mitochondrial blockers induced loss of plasma membrane permeability and cell death. The cell death induced by ERK inhibition had hallmarks of both apoptotic (caspase activation) and necrotic (ATP loss) cell death. By blocking ERK inhibition-induced reactive oxygen species, caspase activation was prevented, although necrotic pathways continued to induce cell death. This suggests that mitochondrial dysfunction caused by ERK inhibition generates both apoptotic and necrotic cell death-inducing pathways. As a composite, these data demonstrate a novel mitochondrial role for ERK in maintaining mitochondrial membrane potential and ATP production in human alveolar macrophages.

    Funded by: NCI NIH HHS: CA-086862, P30 CA086862; NCRR NIH HHS: M01 RR000059, RR00059, UL1 RR024979; NHLBI NIH HHS: HL-077431, HL-60316, HL079901-01A1, K08 HL089392, R01 HL073967, R01 HL073967-04, R01 HL077431, R01 HL077431-04, R01 HL079901

    Journal of immunology (Baltimore, Md. : 1950) 2008;180;11;7485-96

  • Dual-specificity phosphatase 1 ubiquitination in extracellular signal-regulated kinase-mediated control of growth in human hepatocellular carcinoma.

    Calvisi DF, Pinna F, Meloni F, Ladu S, Pellegrino R, Sini M, Daino L, Simile MM, De Miglio MR, Virdis P, Frau M, Tomasi ML, Seddaiu MA, Muroni MR, Feo F and Pascale RM

    Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, 07100 Sassari, Italy.

    Sustained activation of extracellular signal-regulated kinase (ERK) has been detected previously in numerous tumors in the absence of RAS-activating mutations. However, the molecular mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in human hepatocellular carcinoma (HCC). Levels of DUSP1, as assessed by real-time reverse transcription-PCR and Western blot analysis, were significantly higher in tumors with better prognosis (as defined by the length of patients' survival) when compared with both normal and nontumorous surrounding livers, whereas DUSP1 protein expression sharply declined in all HCC with poorer prognosis. In the latter HCC subtype, DUSP1 inactivation was due to either ERK/SKP2/CKS1-dependent ubiquitination or promoter hypermethylation associated with loss of heterozygosity at the DUSP1 locus. Noticeably, expression levels of DUSP1 inversely correlated with those of activated ERK, as well as with proliferation index and microvessel density, and directly with apoptosis and survival rate. Subsequent functional studies revealed that DUSP1 reactivation led to suppression of ERK, CKS1, and SKP2 activity, inhibition of proliferation and induction of apoptosis in human hepatoma cell lines. Taken together, the present data indicate that ERK achieves unrestrained activity during HCC progression by triggering ubiquitin-mediated proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may represent a valuable prognostic marker and ERK, CKS1, or SKP2 potential therapeutic targets for human HCC.

    Cancer research 2008;68;11;4192-200

  • A functionally significant cross-talk between androgen receptor and ErbB2 pathways in estrogen receptor negative breast cancer.

    Naderi A and Hughes-Davies L

    Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Cancer Biology Program, University of Queensland, Princess Alexandra Hospital, Brisbane Qld 4102, Australia. a.naderi@uq.edu.au

    Recent studies have identified novel subgroups in ER-negative breast cancer based on the expression pattern of androgen receptor (AR). One subtype (molecular apocrine) has an over-expression of steroid-response genes and ErbB2. Using breast cancer cell lines with molecular apocrine features, we demonstrate a functional cross-talk between AR and ErbB2 pathways. We show that stimulation of AR and ErbB2 pathways leads to the cross-regulation of gene expression for AR, ErbB2, FOXA1, XBP1, TFF3, and KLK3. As opposed to the physiologic transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) observed with the testosterone treatment, we demonstrate that the addition of ErbB2 inhibition leads to a persistent phosphorylation of ERK1/2, which negatively regulates the downstream signaling and cell growth. This suggests a mechanism for the cross-talk involving the ERK pathway. Moreover, testosterone stimulates the proliferation of molecular apocrine breast cell lines, and this effect can be reversed using antiandrogen flutamide and anti-ErbB2 AG825. Conversely, the growth stimulatory effect of heregulin can also be inhibited with flutamide, suggesting a cross-talk between the AR and ErbB2 pathways affecting cell proliferation. Importantly, there is a synergy with the combined use of flutamide and AG825 on cell proliferation and apoptosis, which indicates a therapeutic advantage in the combined blockage of AR and ErbB2 pathways.

    Neoplasia (New York, N.Y.) 2008;10;6;542-8

  • Dual roles of the MAPK/ERK1/2 cell signaling pathway after stroke.

    Sawe N, Steinberg G and Zhao H

    Department of Neurosurgery and Stanford Stroke Center, Stanford University, Stanford, California 94305-5327, USA.

    Extracellular signal-regulated kinase 1/2 (ERK1/2), one of the best-characterized members of the mitogen-activated protein kinase (MAPK) family, mediates a range of activity from metabolism, motility, and inflammation to cell death and survival. It is phosphorylated and activated through a three-tiered MEK mode via cell surface receptors stimulated by growth factors or cytokines. The phosphorylated ERK1/2 level is usually increased after cerebral ischemia/reperfusion, but whether an increase in ERK1/2 phosphorylation is protective or detrimental is highly debatable. Much of the support for ERK1/2's role as a neuroprotectant against stroke stems from its apparent involvement in the beneficial effects of growth factors, estrogen, preconditioning, and hypothermia on the ischemic brain. Conversely, evidence supporting the detrimental effects of ERK1/2 activity is derived from its activation promoting inflammation and oxidative stress and its inhibition reducing ischemic damage. The dual potential of ERK1/2 actions in the ischemic brain is likely related to its responses to a diverse array of agonists and cell surface receptors. Plausibly, the ERK1/2 activity generated by cytokines and free radicals or other inflammatory factors after stroke may worsen ischemic damage, whereas the ERK1/2 activity produced by exogenous growth factors, estrogen, and preconditioning favors neuroprotection. Future experiments should be conducted to optimize the protective effect of ERK1/2 while blocking its detrimental actions.

    Funded by: NINDS NIH HHS: P01 NS37520, R01 NS27292

    Journal of neuroscience research 2008;86;8;1659-69

  • Inhibition of ERK and activation of p38 are involved in diallyl disulfide induced apoptosis of leukemia HL-60 cells.

    Tan H, Ling H, He J, Yi L, Zhou J, Lin M and Su Q

    Cancer Research Institute, University of South China, Hengyang City, Hunan Province, 421001, P.R.China. tanhuiy@sohu.com

    We investigated the effects of diallyl disulfide (DADS) on the induction of apoptosis in human leukemia cell line HL-60 and explored the roles of mitogen-activated protein kinase (ERK and p38 MAPK) pathways in the growth inhibition and apoptosis induced by DADS. MTT assay was used to determine the DADS induced cell growth inhibition in HL-60 cells. Flow cytometry and DNA fragmentation were used to examine the roles of apoptosis in DADS-mediated cell death. Western blot analysis of the expression of phospho-MAPKs (ERK and p38) was employed to elucidate the possible mechanisms of DADS induced apoptosis. We found that growth inhibition of HL-60 cells treated with DADS exhibited a dose-dependent response (P<0.05) and DADS induced significant apoptosis. DADS at the concentration of 10 mg/L persistently activated p38 and simultaneously reduced ERK activity. PD98059, an inhibitor of ERK upstream activators MAPK kinase MKK1 and MKK2, promoted cytotoxicity and apoptosis in HL-60 cells treated with DADS. In contrast, SB203580, an inhibitor of p38, decreased cytotoxicity and apoptosis induced by DADS. Therefore, DADS can effectively inhibit the proliferation and induce apoptosis of human leukemia cell line HL-60. Inhibition of ERK signaling pathways and activation of p38 signaling pathways are likely involved in DADS induced apoptosis in HL-60 cells.

    Archives of pharmacal research 2008;31;6;786-93

  • Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells.

    Chaturvedi LS, Gayer CP, Marsh HM and Basson MD

    Surgical Service, John D Dingell Veterans Affairs Medical Center, Detroit, MI 48201, USA.

    Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr 418, FAK-Tyr 397-Tyr 576-Tyr 925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 micromol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr 397 and Tyr 576 but not FAK-Tyr 925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr 925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr 925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr 925-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.

    Funded by: NIDDK NIH HHS: R01-DK-067257

    American journal of physiology. Cell physiology 2008;294;6;C1350-61

  • Sphingosine kinases and sphingosine-1-phosphate are critical for transforming growth factor beta-induced extracellular signal-regulated kinase 1 and 2 activation and promotion of migration and invasion of esophageal cancer cells.

    Miller AV, Alvarez SE, Spiegel S and Lebman DA

    Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298-0678, USA.

    Transforming growth factor beta (TGFbeta) plays a dual role in oncogenesis, acting as both a tumor suppressor and a tumor promoter. These disparate processes of suppression and promotion are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGFbeta in the progression of epithelial cancers is the elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator sphingosine-1-phosphate (S1P) has been shown to transactivate the TGFbeta receptor and activate Smad3, we examined its role in TGFbeta activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and promotion of migration and invasion of esophageal cancer cells. Both S1P and TGFbeta activate ERK1/2, but only TGFbeta activates Smad3. Both ligands promoted ERK1/2-dependent migration and invasion. Furthermore, TGFbeta rapidly increased S1P, which was required for TGFbeta-induced ERK1/2 activation, as well as migration and invasion, since downregulation of sphingosine kinases, the enzymes that produce S1P, inhibited these responses. Finally, our data demonstrate that TGFbeta activation of ERK1/2, as well as induction of migration and invasion, is mediated at least in part by ligation of the S1P receptor, S1PR2. Thus, these studies provide the first evidence that TGFbeta activation of sphingosine kinases and formation of S1P contribute to non-Smad signaling and could be important for progression of esophageal cancer.

    Funded by: NIGMS NIH HHS: R37 GM043880, R37GM043880

    Molecular and cellular biology 2008;28;12;4142-51

  • OspF directly attenuates the activity of extracellular signal-regulated kinase during invasion by Shigella flexneri in human dendritic cells.

    Kim DW, Chu H, Joo DH, Jang MS, Choi JH, Park SM, Choi YJ, Han SH and Yun CH

    International Vaccine Institute, SNU Research Park, Seoul, Republic of Korea.

    Shigella spp., Gram-negative pathogenic bacteria, deliver various effector molecules into the host cell cytoplasm through their type III secretion system to facilitate their invasive process and control the host innate immune responses. Although the function of these effectors is well characterized in epithelial cells during Shigella infection, it has not been elucidated in the dendritic cell (DC), a major antigen presenting cell playing an important role in the initiation of immune responses. In this study, we showed that an invasive Shigella strain (M90T), but not its non-invasive counterpart strain (BS176) induced apoptotic cell death in the human monocyte-derived DCs. Confocal microscopy using a lysosome-associated membrane protein 2 specific antibody demonstrated that the M90T escaped from phagosomes 2h post-DC invasion while BS176 remained in the phagosome. Furthermore, Shigella expressed outer Shigella protein F (OspF), one of the effector proteins that are released through type III secretion system during the invasion, at non-secretion state and further up-regulated OspF expression in the cytoplasm of DC during the invasion. Interestingly, in the host cell, OspF could directly bind to the extracellular signal-regulated kinase (Erk) 1/2 and dephosphorylate phospho-Erk. These results suggest that induction of OspF is enhanced during Shigella invasion of DCs and decreases the phosphorylation level of Erk1/2, which could be at least partially involved in the apoptotic death of DC, eventually resulting in the down-regulation of the host immune response.

    Molecular immunology 2008;45;11;3295-301

  • The hyaluronan receptor for endocytosis mediates hyaluronan-dependent signal transduction via extracellular signal-regulated kinases.

    Kyosseva SV, Harris EN and Weigel PH

    Department of Biochemistry and Molecular Biology, and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

    The hyaluronan (HA) receptor for endocytosis (HARE) mediates the endocytotic clearance of HA and other glycosaminoglycans from lymph and blood. Two isoforms of human HARE, 315- and 190-kDa, are highly expressed in sinusoidal endothelial cells of liver, lymph node, and spleen; HARE is also in specialized cells in the eye, heart, brain, and kidney. Here we determined whether HA binding to HARE initiates intracellular signaling in Flp-In 293 cells stably expressing either the 315- and 190-kDa HARE or the 190-kDa HARE alone. HARE was co-immunoprecipitated with extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 members of the mitogen-activated protein kinase signaling cascade. ERK phosphorylation increased in a dose- and time-dependent manner when HA was added to cells expressing full-length or 190-kDa HARE, but not cells with vector-only or a HARE(DeltaLink) construct with greatly decreased ( approximately 90%) HA uptake. HA did not induce phosphorylation of JNK or p38. A maximum increase in phospho-ERK1/2 occurred within 30 min at 5 mug/ml HA, and the response was dampened at >20 mug/ml HA. HA binding did not increase the level of HARE-ERK complexes, but did increase HARE phosphorylation. These findings demonstrate a novel functional response, when HARE binds HA, that leads to activation of ERK1/2, important mediators of intracellular signal transduction.

    Funded by: NIGMS NIH HHS: GM 69961

    The Journal of biological chemistry 2008;283;22;15047-55

  • Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK as regulators of human sperm motility and acrosome reaction and as predictors of poor spermatozoan quality.

    Almog T, Lazar S, Reiss N, Etkovitz N, Milch E, Rahamim N, Dobkin-Bekman M, Rotem R, Kalina M, Ramon J, Raziel A, Breitbart H, Brietbart H, Seger R and Naor Z

    Department of Biochemistry, George S Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.

    Mature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCbetaI and PKCepsilon). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality.

    The Journal of biological chemistry 2008;283;21;14479-89

  • Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms.

    García S, López E and López-Colomé AM

    Instituto de Fisiología Celular, Departamento de Neurociencias, Universidad Nacional Autónoma de México, Mexico, Mexico.

    The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.

    Journal of cellular biochemistry 2008;104;2;377-90

  • Prostaglandin E1 inhibits IL-6-induced MCP-1 expression by interfering specifically in IL-6-dependent ERK1/2, but not STAT3, activation.

    Sobota RM, Müller PJ, Heinrich PC and Schaper F

    Department of Biochemistry, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany.

    IL (interleukin)-6 exerts pro- as well as anti-inflammatory activities. Beside many other activities, IL-6 is the major inducer of acute phase proteins in the liver, acts as a differentiation factor for blood cells, as migration factor for T-cells and is a potent inducer of the chemokine MCP-1 (monocyte chemoattractant protein-1). Recent studies have focused on the negative regulation of IL-6 signal transduction through the IL-6-induced feedback inhibitors SOCS (suppressor of cytokine signalling) 1 and SOCS3 or the protein tyrosine phosphatases SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and TcPTP (T-cell protein tyrosine phosphatase). Studies on the cross-talk between pro-inflammatory mediators (IL-1, tumour necrosis factor, lipopolysaccharide) and IL-6 elucidated further regulatory mechanisms. Less is known about the regulation of IL-6 signal transduction by hormone/cytokine signalling through G-protein-coupled receptors. This is particularly surprising since many of these hormones (such as prostaglandins and chemokines) play an important role in inflammatory processes. In the present study, we have investigated the inhibitory activity of PGE(1) (prostaglandin E(1)) on IL-6-induced MCP-1 expression and have elucidated the underlying molecular mechanism. Surprisingly, PGE(1) does not affect IL-6-induced STAT (signal transducer and activator of transcription) 3 activation, but does affect ERK (extracellular-signal-regulated kinase) 1/2 activation which is crucial for IL-6-dependent expression of MCP-1. In summary, we have discovered a specific cross-talk between the adenylate cyclase cascade and the IL-6-induced MAPK (mitogen-activated protein kinase) cascade and have investigated its impact on IL-6-dependent gene expression.

    The Biochemical journal 2008;412;1;65-72

  • IMP modulates KSR1-dependent multivalent complex formation to specify ERK1/2 pathway activation and response thresholds.

    Chen C, Lewis RE and White MA

    Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    The Ras effector and ubiquitin-protein isopeptide ligase family member IMP acts as a steady-state resistor within the Raf-MEK-ERK kinase module. IMP concentrations are regulated by Ras through induction of autodegradation and can modulate signal/response thresholds by directly limiting the assembly of functional KSR1-dependent Raf.MEK complexes. Here, we show that the capacity of IMP to inhibit signal propagation through Raf to MEK is a consequence of disrupting KSR1 homooligomerization and B-Raf/c-Raf hetero-oligomerization. This impairs both the recruitment of MEK to activated Raf family members and the contribution of Raf oligomers to c-Raf kinase activation. Our observations indicate that human KSR1 proteins promote assembly of multivalent Raf.MEK complexes that are required for c-Raf kinase activation and functional coupling of active kinases to downstream substrates. This property is engaged by IMP for modulation of signal amplitude.

    Funded by: NCI NIH HHS: CA71443

    The Journal of biological chemistry 2008;283;19;12789-96

  • ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential.

    Cárcamo-Orive I, Tejados N, Delgado J, Gaztelumendi A, Otaegui D, Lang V and Trigueros C

    Fundación Inbiomed, Foundation for Stem Cell Research, Mesenchymal Stem Cell Department, Paseo Mikeletegi, 61 Bajo 20009, San Sebastián, Spain.

    Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes.

    Experimental cell research 2008;314;8;1777-88

  • DUSP6/MKP-3 inactivates ERK1/2 but fails to bind and inactivate ERK5.

    Arkell RS, Dickinson RJ, Squires M, Hayat S, Keyse SM and Cook SJ

    Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

    Extracellular signal-regulated kinase-1 and -2 (ERK1/2) are activated by dual threonine and tyrosine phosphorylation of a TEY motif. The highly related kinase ERK5 is also activated by phosphorylation at a TEY motif. Inactivation of ERK1/2 is achieved by distinct members of the dual-specificity protein phosphatase (DUSP) family, which are responsible for the specific, regulated de-phosphorylation of the TEY motif. These include both nuclear (DUSP5) and cytoplasmic (DUSP6) enzymes. DUSP6, a candidate tumour suppressor gene, is thought to be highly specific for inactivation of ERK1/2 but several reports have suggested that it may also inactivate ERK5. Here we have compared the ability of DUSP6 to regulate the ERK1/2 and ERK5 protein kinases. We find that DUSP6 binds to ERK1/2 in both yeast and human cells but fails to bind to ERK5. Recombinant ERK2 can induce catalytic activation of DUSP6 whereas ERK5 cannot. Ectopic expression of DUSP6 can de-phosphorylate a co-expressed ERK2 construct but does not de-phosphorylate ERK5. Finally, expression of DUSP6 blocks the MEK1-driven activation of GAL4-ELK1, an ERK1/2-regulated transcription factor, but fails to block the MEK5-driven activation of GAL4-MEF2D, an ERK5-regulated transcription factor. These results demonstrate that even upon over-expression DUSP6 fails to inactivate ERK5, confirming that it is indeed an ERK1/2-specific DUSP.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/C509190/1, BBS/E/B/0000C199; Cancer Research UK

    Cellular signalling 2008;20;5;836-43

  • Expression of extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinase in the invasive trophoblasts at the human placental bed.

    Moon KC, Park JS, Norwitz ER, Kim DI, Oh KJ, Park CW, Jun JK and Syn HC

    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea.

    Background: Mitogen-activated protein kinases (MAP kinases) participate in signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. The roles of extracellular signal-regulated kinase1/2 (ERK1/2) and p38 MAP kinase in the differentiation and invasion of human trophoblasts have been studied. However, the in vivo expression and activation of ERK1/2 and p38 at the placental bed have not been elucidated.

    Methods: The study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n=24) and with preeclampsia (n=8) between 31 and 40 weeks of gestation. We evaluated the expressions and phosphorylations of ERK1/2 and p38 MAP kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry.

    Results: p38 and phospho-p38 MAP kinase were not detected in invasive trophoblasts in cases or controls. ERK1/2 and phospho-ERK1/2 were positive in invasive trophoblasts albeit with variable staining. Phosphorylation of ERK1/2 was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls.

    Conclusion: These findings suggest that preeclampsia is associated with decreased activation of ERK1/2 in invasive trophoblasts in vivo.

    Placenta 2008;29;5;391-5

  • Extracellular regulated kinase-2 immunoreactivity increases in parallel with cervical intraepithelial neoplasia grade in cervical neoplasia.

    Kapetanios V, Lazaris AC, Bogris P, Kouneli S, Nonni A, Arvaniti H, Kouri E, Tzavara M, Giannakodimos G, Koutselini H and Patsouris ES

    5th Gynaecological Department, "Elena Venizelou" Maternity Hospital, Athens, Greece.

    The cell cycle control system includes cyclins, cyclin-dependent kinases (CDK), and their inhibitors (CDK1). Extracellular regulated kinase (ERK1/2) (p44 and p42 mitogen-activated protein kinases [MAPKs]) is a component of the MAPK pathway, which is associated with cyclin D1 and CDK. It is a critical signaling system for the induction of cell proliferation, differentiation, and cell survival. The aim of this study was to investigate the usefulness of ERK2 expression as a marker of biological aggressiveness complementary to cervical intraepithelial neoplasia (CIN) grade as well as to compare its expression in preinvasive lesions with that in invasive carcinoma. Paraffin-embedded sections of 146 CIN lesions (32 CIN I, 49 CIN II, and 43 CIN III) and 22 invasive cervical carcinomas (13 squamous and 9 adenocarcinomas) were used for the standard immunohistochemical procedure with the application of the ERK2 monoclonal antibody. ERK2 staining displayed a cytoplasmic and nuclear pattern. The staining intensity was gradually increased according to the severity of the dysplastic lesions; ERK2 immunoreactivity was significantly increased in high-grade dysplastic lesions (CIN II and CIN III) and invasive carcinomas by comparison to low-grade dysplastic lesions (CIN I) (P < 0.001). When high-grade lesions were separately assessed, the differences between each one of them and CIN I retained their statistical significance: CIN II versus CIN I (P < 0.001) and CIN III versus CIN I (P < 0.001). In conclusion, our study found a direct relationship between the increasing grade of the dysplastic cervical lesions and the intensity of ERK2 staining, thus implying a role of ERK2 as an early event in cervical carcinogenesis.

    International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 2008;18;3;540-5

  • Involvement of connexin 43 in angiotensin II-induced migration and proliferation of saphenous vein smooth muscle cells via the MAPK-AP-1 signaling pathway.

    Jia G, Cheng G, Gangahar DM and Agrawal DK

    Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA.

    Proliferation and migration of vascular smooth muscle cells (VSMCs) lead to intimal thickening and influence the long-term patency of venous graft post coronary arterial bypass graft. There is increasing evidence that connexins are involved in the development of intimal hyperplasia and restenosis. We assessed connexin 43 (Cx43) expression and its role in angiotensin II-induced proliferation and migration of smooth muscle cells and the signal pathways involved in human saphenous vein bypass conduits. Angiotensin II significantly increased gap junctional intercellular communication and induced the expression of Cx43 in human saphenous vein SMCs in a dose- and time-dependent manner through angiotensin II type 1 receptor. The effect of angiotensin II was blocked by siRNA of ERK 1/2, p38 MAPK and JNK, respectively. Overexpression of Cx43 markedly increased the proliferation of saphenous vein SMCs. However, siRNA for Cx43 inhibited angiotensin II-induced proliferation, cyclin E expression and migration of human saphenous vein SMCs. In dual-luciferase reporter assay, angiotensin II markedly activated AP-1 transcription factor, which was significantly attenuated by a dominant-negative AP-1 (A-Fos) with subsequent inhibition of angiotensin II-induced transcriptional expression of Cx43. These data demonstrate the role of Cx43 in the proliferation and migration of human saphenous vein SMCs and angiotensin II-induced Cx43 expression via mitogen-activated protein kinases (MAPK)-AP-1 signaling pathway.

    Funded by: NHLBI NIH HHS: R01 HL070885, R01 HL073349, R01 HL090580, R01 HL090580-01A1, R01HL070885, R01HL073349

    Journal of molecular and cellular cardiology 2008;44;5;882-90

  • Mifepristone attenuates human chorionic gonadotropin-induced extracellular signal-regulated kinase 1/2 phosphorylation, cyclooxygenase-2, and prostaglandin E2 production in human granulosa luteal cells.

    Tsai EM, Chan TF, Chen YH, Hsu SC, Chuang CY and Lee JN

    Department of Obstetrics and Gynecology, Graduate Institute of Medicine, College of Medicine, Kaohsiung, Taiwan, Republic of China.

    Objective: To elucidate the role of RU486 in regulating the function of granulosa luteal cells and its possible involvement in ovarian dysfunction.

    Design: An in vitro study.

    Setting: University hospital.

    Our subjects were women under the age of 40 who were unable to get pregnant as a result of male-factor infertility.

    HCG and RU486 were added to cultured granulosa luteal cells; after incubation for 12 hours, the harvested cells were subjected to total mRNA and protein measurements.

    Reverse transcriptase-polymerase chain reaction, immunoblot assay, immunocytochemistry, and enzyme immunoassay were performed.

    RU486 attenuates hCG-induced cyclooxygenase-2 (COX-2) mRNA and protein expression and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and decreases the hCG-induced prostaglandin E2 (PGE2) production in a dose-dependent manner. RU486 treatment had no significant effect on COX-1 mRNA expression.

    Treatments using gonadotropins are able to induce ERK1/2 phosphorylation resulting in increased COX-2 protein expression and prostaglandin synthesis. RU486 attenuates the activation of ERK1/2, decreases the expression of COX-2, and affects PGE2 production by inhibiting hCG-induced COX-2 expression.

    Fertility and sterility 2008;89;5 Suppl;1522-9

  • Potential role of phospholipase D2 in increasing interleukin-2 production by T-lymphocytes through activation of mitogen-activated protein kinases ERK1/ERK2.

    Hamdi SM, Cariven C, Coronas S, Malet N, Chap H, Perret B, Salles JP and Record M

    INSERM U563, CHU Purpan, Toulouse, France. safouane.hamdi@toulouse.inserm.fr

    Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras-MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders.

    Biochimica et biophysica acta 2008;1781;5;263-9

  • C-FLIP promotes the motility of cancer cells by activating FAK and ERK, and increasing MMP-9 expression.

    Park D, Shim E, Kim Y, Kim YM, Lee H, Choe J, Kang D, Lee YS and Jeoung D

    School of Biological Sciences, College of Natural Sciences, Kangwon National University, Chunchon 200-701, Korea.

    We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP (c-FLIPL), but not the short form (c-FLIPS), enhanced adhesion and motility. Downregulation of c-FLIPL with siRNA decreased phosphorylation of FAK and ERK, while overexpression of c-FLIPL increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed c-FLIPL-promoted motility. Inhibition of ROCK by Y27632 suppressed the c-FLIPL-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of c-FLIPL increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced c-FLIPL- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the c-FLIPL-promoted cell motility. We conclude that c-FLIPL promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

    Molecules and cells 2008;25;2;184-95

  • Epidermal growth factor receptor signaling to Erk1/2 and STATs control the intensity of the epithelial inflammatory responses to rhinovirus infection.

    Liu K, Gualano RC, Hibbs ML, Anderson GP and Bozinovski S

    Departments of Pharmacology and Medicine, University of Melbourne, Melbourne, Victoria 3010, Australia.

    Rhinovirus infection is the most common cause of acute exacerbations of inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease, where it provokes steroid refractory and abnormally intense neutrophilic inflammation that can be life threatening. Epidermal growth factor receptor (EGFR) expression correlates with disease severity and neutrophil infiltration in these conditions. However, the role of EGFR signaling in rhinovirus infection is unknown. We measured the key determinants of neutrophilic inflammation interleukin (IL)-8 and ICAM-1 in rhinovirus (RV16 serotype)-infected bronchial epithelial cells, BEAS-2B. RV16 infection stimulated IL-8 and ICAM-1 expression, which was further elevated (2-fold) by transient up-regulation of EGFR levels. Detection of viral RNA by quantitative real time PCR confirmed that enhanced expression was not associated with increased viral replication. EGFR ligands (epiregulin, amphiregulin, and heparin-binding epidermal growth factor) were induced by RV16 infection, and inhibition of metalloproteases responsible for ligand shedding partially suppressed this response. The EGFR inhibitor AG1478, completely blocked IL-8 and ICAM-1 expression to basal levels, as did the specific Erk1/2 inhibitor U0126. The p38 mitogen-activated protein kinase inhibitor SB203580 blocked IL-8 secretion but not ICAM-1 expression, whereas the PI3K inhibitor wortmannin was ineffective in both responses. Kinase inactive K721R EGFR, which is selectively deficient in STAT signaling, reversed RV16 responses associated with EGFR overexpression. In conclusion, RV16 infection rapidly promotes induction of EGFR ligands and utilizes EGFR signaling to increase IL-8 and ICAM-1 levels. These results suggest that targeting EGFR may provide a selective therapy that dampens neutrophil-driven inflammation without compromising essential antiviral pathways mediated by pathogen recognition receptors such as TLR3.

    The Journal of biological chemistry 2008;283;15;9977-85

  • Interaction between ERK and GSK3beta mediates basic fibroblast growth factor-induced apoptosis in SK-N-MC neuroblastoma cells.

    Ma C, Bower KA, Chen G, Shi X, Ke ZJ and Luo J

    Department of Microbiology, Immunology and Cell Biology, West Virginia University School of Medicine, Robert C. Byrd Health Sciences Center, Morgantown, WV 26506, USA.

    The Ewing's sarcoma family of tumors (ESFT) includes Ewing's sarcoma (ES), Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor. Basic fibroblast growth factor (FGF2) suppresses the growth of ESFT cells and causes their apoptosis. The underlying mechanism is unclear. Using a human peripheral primitive neuroectodermal tumor cell line, SK-N-MC, we demonstrated FGF2 stimulated phosphorylation of ERK1 and ERK2 (pERK1/2) and GSK3beta (pGSK3beta(Tyr-216)), all of which were primarily retained in the cytoplasm. FGF2 promoted the association between ERK and pGSK3beta(Tyr-216). Inhibitors for GSK3beta (TDZD and LiCl) and ERK (PD98059) protected cells from FGF2-induced apoptosis. On the other hand, inhibitors of GSK3beta, but not PD98059 decreased ERK/pGSK3beta(Tyr-216) association and caused a nuclear translocation of pERK1/2. Similarly, expression of a kinase-deficient (K85R) GSK3beta or GSK3beta-small interfering RNA inhibited FGF2-regulated ERK/pGSK3beta(Tyr-216) association and translocated pERK to the nucleus. Both K85R GSK3beta and small interfering RNA offered protection against FGF2-induced cell death. In contrast, overexpression of wild-type GSK3beta sensitized cells to FGF2 cytotoxicity. Hydrogen peroxide and ethanol enhanced FGF2-stimulated pGSK3beta(Tyr-216), ERK/pGSK3beta(Tyr-216) association, and cytoplasmic retention of pERK1/2. As a result, they potentiated FGF2-induced cell death. Taken together, our results suggested that FGF2-induced accumulation of pERK1/2 in the cytoplasm is toxic for SK-N-MC cells. The formation of an ERK.GSK3beta complex retained pERK1/2 in the cytoplasm. In contrast, disruption of the ERK.GSK3beta complex resulted in nuclear translocation of pERK1/2 and offered protection.

    Funded by: NIAAA NIH HHS: AA 015407, R01 AA015407, R01 AA015407-03

    The Journal of biological chemistry 2008;283;14;9248-56

  • Mitogen-activated protein kinase ERK1/2 regulates the class II transactivator.

    Voong LN, Slater AR, Kratovac S and Cressman DE

    Department of Biology, Sarah Lawrence College, 1 Mead Way, Bronxville, NY 10708, USA.

    The expression of major histocompatibility class II genes is necessary for proper antigen presentation and induction of an immune response. This expression is initiated by the class II transactivator, CIITA. The establishment of the active form of CIITA is controlled by a series of post-translational events, including GTP binding, ubiquitination, and dimerization. However, the role of phosphorylation is less clearly defined as are the consequences of phosphorylation on CIITA activity and the identity of the kinases involved. In this study we show that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) interact directly with CIITA, targeting serine residues in the amino terminus of the protein, including serine 288. Inhibition of this phosphorylation by dominant-negative forms of ERK or by treatment of cells with the ERK inhibitor PD98059 resulted in the increase in CIITA-mediated gene expression from a class II promoter, enhanced the nuclear concentration of CIITA, and impaired its ability to bind to the nuclear export factor, CRM1. In contrast, inhibition of ERK1/2 activity had little effect on serine-to-alanine mutant forms of CIITA. These data suggest a model whereby ERK1/2-mediated phosphorylation of CIITA down-regulates CIITA activity by priming it for nuclear export, thus providing a means for cells to tightly regulate the extent of antigen presentation.

    The Journal of biological chemistry 2008;283;14;9031-9

  • The antiadipogenic effect of macrophage-conditioned medium depends on ERK1/2 activation.

    Constant VA, Gagnon A, Yarmo M and Sorisky A

    Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

    The proatherogenic state of obesity is associated with hypertrophied adipocytes that may arise because of deficient adipogenesis. Macrophages infiltrate adipose tissue as a function of obesity and may release factors that attenuate adipogenesis. Macrophage-conditioned medium inhibits human and 3T3-L1 adipocyte differentiation in culture, but underlying molecular mechanisms have yet to be defined. Exposure of 3T3-L1 cells throughout the 8-day period of differentiation to medium conditioned by THP-1 macrophages (THP-1-MacCM) blocked adipogenesis. Triacylglycerol (TG) accumulation and induction of peroxisome proliferator-activated receptor gamma and fatty acid synthase protein levels were inhibited by 59% (n = 4, P < .001), 29% (n = 4, P < .01), and 47% (n = 4, P < .01), respectively. THP-1-MacCM had no effect when added after the first 2 days of differentiation, indicating that early exposure of its targets must be needed to inhibit 3T3-L1 adipogenesis. Cell enumeration revealed a 44% decrease in clonal expansion compared with standard differentiation (n = 3, P < .01). Addition of THP-1-MacCM to 3T3-L1 preadipocytes increased ERK1/2 phosphorylation by 6.5-fold (n = 3, P < .01). PD98059 (an inhibitor of the ERK1/2 pathway) impaired the negative effect of THP-1-MacCM on TG accumulation, indicated by an inhibition of 25% vs 69% (n = 3, P < .001), without altering fatty acid synthase or peroxisome proliferator-activated receptor gamma levels. Our data implicate ERK1/2 as an important signaling mediator for the inhibitory effect of THP-1-MacCM on TG accumulation during 3T3-L1 adipogenesis.

    Metabolism: clinical and experimental 2008;57;4;465-72

  • Concordant overexpression of p-FAK and p-ERK1/2 in extramammary Paget's disease.

    Chen SY, Moroi Y, Urabe K, Takeuchi S, Kido M, Hayashida S, Uchi H, Uenotsuchi T, Tu YT and Furue M

    Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka, 812-8582, Japan.

    Focal adhesion kinase (FAK) is a tyrosine kinase which is at the crossroad of extracellular signal-regulated kinase-1/2 (ERK1/2), PI3K/Akt, MAPK and JAK/STAT signaling pathways. We have previously reported that p-ERK1/2, p-Akt, p38MAPK and p-STAT3 are overexpressed in extramammary Paget's diseases (EMPD), this study aimed to examine the expression of phosphorylated (p)-FAK and p-ERK1/2 proteins in EMPD and to evaluate the relationships among them. Paraffin-embedded EMPD specimens (35 tissue samples from 33 patients with primary EMPD, including two samples of metastatic lymph nodes from two of the 33 patients) were subjected to immunohistochemical staining for p-FAK and p-ERK1/2. All of the 35 EMPD specimens, including all of six invasive EMPD and two metastatic lymph node specimens, showed cytoplasmic overexpression of p-FAK and nuclear overexpression of p-ERK1/2. The expression levels (% positive cells) of p-FAK and p-ERK1/2 (88.34 +/- 14.66 and 91.26 +/- 11.21%) in EMPD were significantly higher than those in normal skin (22.38 +/- 2.13 and 29.00 +/- 4.44%), respectively. The expression levels of p-FAK (95.38 +/- 4.57%) and p-ERK1/2 (96.25 +/- 5.01%) in the advanced EMPD showed slightly higher than that in the non-invasive EMPD (86.26 +/- 15.99 and 89.78 +/- 12.15%), respectively. There exhibited a significantly high positive correlation between expression levels of p-ERK1/2 and p-FAK in EMPD. The present study shows that the concordant overexpression of p-FAK and p-ERK1/2 in EMPD which is associated with the grade of malignancy of EMPD, indicating that p-FAK and p-ERK1/2 may play pivotal roles in the tumorigenesis and further malignant transduction of EMPD.

    Archives of dermatological research 2008;300;4;195-201

  • Diallyl sulfide induces apoptosis in Colo 320 DM human colon cancer cells: involvement of caspase-3, NF-kappaB, and ERK-2.

    Sriram N, Kalayarasan S, Ashokkumar P, Sureshkumar A and Sudhandiran G

    Department of Biochemistry, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600 025, India.

    Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Diallyl sulfide (DAS), an organosulfur component of garlic has been known for its chemopreventive activities against various cancers and also in recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. Thus the present study was focused to elucidate the anticancerous effect and the mode of action of DAS against Colo 320 DM colon cancer cells. DAS induced apoptosis in Colo 320 DM cells was revealed by flow cytometer analysis and phosphatidyl serine exposure. DAS also promoted cell cycle arrest substantially at G2/M phase in Colo 320 DM cells. The production of reactive oxygen intermediates, which were examined by 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA), increased with time, after treatment with DAS. The activities of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were decreased upon DAS treatment, which shows the antiproliferative and the cytotoxic effects, respectively. The expression of NF-kappaB was upregulated in DAS treated cells, compared to normal cells. Further, DAS promoted the expression of caspase-3 and suppression of Extracellular Regulatory Kinase-2 (ERK-2) activity in Colo 320 DM cells that was determined by Western blot analysis. In conclusion, DAS increased the production of ROS, caused cell cycle arrest, decreased cell proliferation and induced apoptosis in Colo 320 DM cells. Thus, this study put forward DAS as a drug that can possibly be used to treat cancers.

    Molecular and cellular biochemistry 2008;311;1-2;157-65

  • Interleukin-15 improves cytotoxicity of natural killer cells via up-regulating NKG2D and cytotoxic effector molecule expression as well as STAT1 and ERK1/2 phosphorylation.

    Zhang C, Zhang J, Niu J, Zhang J and Tian Z

    Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, China. caizhangsd@yahoo.com.cn

    NK cells are crucial components of the innate immune system, providing a first line of defense against infectious pathogens and tumors. IL-15 is the major physiologic growth factor responsible for NK cell differentiation, survival and cytolytic activity of mature NK cells. However, the exact regulatory mechanism of IL-15 on NK cell function is still unclear. In this study, we try to investigate the mechanism of IL-15 on NK cytolysis. Our results demonstrate that IL-15 treatment increased NKG2D transcripts and surface expression in NK cells. NKG2D or MICA blockade attenuated the up-regulation of IL-15 on NK cytolysis, demonstrating that the up-regulatory effect of IL-15 on NK cytolysis is at least partly dependent of the interaction of NKG2D and MICA. Furthermore, IL-15 augmented the expression of cytotoxic effector molecules (TRAIL and Perforin) and the phosphorylation of STAT1 and ERK1/2, which may also contribute the NK lysis. These results may have therapeutic implications when designing cytokine immunotherapy against cancer.

    Cytokine 2008;42;1;128-36

  • Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism.

    Seth A, Yan F, Polk DB and Rao RK

    Dept. of Physiology, Univ. of Tennessee Health Science Center, Memphis, TN 38163, USA.

    Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and beta-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCbetaI and PKCepsilon. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

    Funded by: NIAAA NIH HHS: AA12307, R01 AA012307; NIDDK NIH HHS: DK065788, DK54993, DK55532, R01 DK054993, R01 DK054993-05, R01 DK055532, R01 DK056008, R01 DK056008-09, R56 DK055532

    American journal of physiology. Gastrointestinal and liver physiology 2008;294;4;G1060-9

  • The N terminus controls sterol binding while the C terminus regulates the scaffolding function of OSBP.

    Wang PY, Weng J, Lee S and Anderson RG

    Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9039, USA.

    Previously we reported that when cell cholesterol is acutely lowered with beta-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP(408-809), it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway.

    Funded by: NHLBI NIH HHS: HL20948; NIGMS NIH HHS: GM52016, R01 GM052016, R01 GM052016-14

    The Journal of biological chemistry 2008;283;12;8034-45

  • Anandamide regulates keratinocyte differentiation by inducing DNA methylation in a CB1 receptor-dependent manner.

    Paradisi A, Pasquariello N, Barcaroli D and Maccarrone M

    Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, Teramo, Italy.

    Anandamide (arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed "endocannabinoids." Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances.

    The Journal of biological chemistry 2008;283;10;6005-12

  • Epidermal growth factor receptor and protein kinase C signaling to ERK2: spatiotemporal regulation of ERK2 by dual specificity phosphatases.

    Caunt CJ, Rivers CA, Conway-Campbell BL, Norman MR and McArdle CA

    Laboratories for Integrated Neuroscience and Endocrinology, Department of Clinical Sciences at South Bristol, University of Bristol, Whitson Street, Bristol, U.K.

    Spatiotemporal aspects of ERK activation are stimulus-specific and dictate cellular consequences. They are dependent upon dual specificity phosphatases (DUSPs) that bind ERK via docking domains and can both inactivate and anchor ERK in cellular compartments. Using high throughput fluorescence microscopy in combination with a system where endogenous ERKs are removed and replaced with wild-type or mutated ERK2-green fluorescent protein (GFP), we show that ERK2 activation responses to epidermal growth factor (EGF) and protein kinase C (PKC) are transient and sustained, respectively. PKC-mediated ERK2 activation is associated with prolonged nuclear localization in the dephosphorylated form, whereas EGF-stimulated ERK2 activation mediates only transient nuclear accumulation. By using short inhibitory RNAs to nuclear inducible DUSP1, -2, or -4 (alone or in combination), we demonstrate that all three of these enzymes contribute to the dephosphorylation of PKC (but not EGF)-activated ERK2 in the nucleus but that they have opposing effects on localization. DUSP2 and -4 inactivate and anchor ERK2, whereas DUSP1 dephosphorylates ERK in the nucleus but allows its traffic back to the cytoplasm. Overexpression of DUSP1, -2, or -4 prevented ERK2 activation, but only DUSP2 and -4 caused ERK2-GFP nuclear accumulation or could be immunoprecipitated with ERK2. Furthermore, protein synthesis inhibition or replacement of wild-type ERK2-GFP with docking domain mutants selectively increased PKC effects on ERK activity and altered ERK2-GFP localization. These mutations also impaired the ability of ERK2-GFP to bind DUSP2 and -4. Together, our data reveal a novel, stimulus-specific, and phosphatase-specific mechanism of ERK2 regulation in the nucleus by DUSP1, -2, and -4.

    Funded by: Wellcome Trust: 062918, 076557, 078407

    The Journal of biological chemistry 2008;283;10;6241-52

  • Abnormalities of insulin-like growth factor-I signaling and impaired cell proliferation in osteoblasts from subjects with osteoporosis.

    Perrini S, Natalicchio A, Laviola L, Cignarelli A, Melchiorre M, De Stefano F, Caccioppoli C, Leonardini A, Martemucci S, Belsanti G, Miccoli S, Ciampolillo A, Corrado A, Cantatore FP, Giorgino R and Giorgino F

    Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, and Metabolic Diseases, University of Bari, Piazza Giulio Cesare, 11, I-70124 Bari, Italy.

    IGF-I regulates bone acquisition and maintenance, even though the cellular targets and signaling pathways responsible for its action in human bone cells are poorly understood. Whether abnormalities in IGF-I action and signaling occur in human osteoblasts under conditions of net bone loss has not been determined. Herein we carried out a comparative analysis of IGF-I signaling in primary cultures of human osteoblasts from osteoporotic and control donors. In comparison with control cells, osteoporotic osteoblasts showed increased tyrosine phosphorylation of the IGF-I receptor in the basal state and blunted stimulation of receptor phosphorylation by IGF-I. Augmentation of basal IGF-I receptor phosphorylation was associated with coordinate increases in basal tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and activation of Erk, which were also minimally responsive to IGF-I stimulation. By contrast, phosphorylation levels of IRS-1, Akt, and glycogen synthase kinase-3 were similar in the basal state in control and osteoporotic osteoblasts and showed marked increases after IGF-I stimulation in both cell populations, even though these responses were significantly lower in the osteoporotic osteoblasts. The IGF-I signaling abnormalities in osteoporotic osteoblasts were associated with reduced DNA synthesis both under basal conditions and after stimulation with IGF-I. Interestingly, treatment of the osteoporotic osteoblasts with the MAPK kinase inhibitor PD098059 reduced the elevated levels of Erk phosphorylation and increased basal DNA synthesis. Collectively, our data show that altered osteoblast proliferation in human osteoporosis may result from dysregulation of IGF-I receptor signaling, including constitutive activation of the IRS-2/Erk signaling pathway, which becomes unresponsive to IGF-I, and defective induction of the IRS-1/Akt signaling pathway.

    Endocrinology 2008;149;3;1302-13

  • FAK-mediated activation of ERK for eosinophil migration: a novel mechanism for infection-induced allergic inflammation.

    Cheung PF, Wong CK, Ip WK and Lam CW

    Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong.

    Bacterial and viral infections often induce the exacerbation of allergic diseases. In this study, we investigated the activation of human eosinophils by different microbial products via Toll-like receptors (TLRs). The underlying intracellular mechanism involving activation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK), an integrin-associated focal adhesion molecule, was also examined. Seven TLR ligands were studied for their abilities in promoting survival, modulating the expression of adhesion molecules and facilitating chemotactic migration of eosinophils. While peptidoglycan (PGN) (TLR2 ligand) showed the most prominent effects, flagellin (TLR5 ligand) and imiquimod R837 (TLR7 ligand) were also effective in activating eosinophils. However, little or no effect was observed for double-stranded polyinosinic-polycytidylic acid (TLR3 ligand), ultra-purified LPS (TLR4 ligand), single-stranded RNA (ssRNA) (TLR8 ligand) and CpG-DNA (TLR9 ligand). Further investigation confirmed that PGN, flagellin and R837 commonly transmitted signals through ERK activation that required prior phosphorylation of tyrosine 925, but not tyrosine 577, on FAK. Moreover, the inhibition of ERK activation by selective inhibitor PD98059 and FAK expression by FAK-specific RNA interference could significantly abolish the stimulatory effects induced by PGN, flagellin and R837. Taken together, our findings indicate the involvement of FAK-dependent activation of ERK1 in TLR-mediated eosinophil stimulation. A potential role of eosinophils was also suggested in exacerbating allergic inflammation in response to microbial infections.

    International immunology 2008;20;3;353-63

  • Human chorionic gonadotropin stimulates trophoblast invasion through extracellularly regulated kinase and AKT signaling.

    Prast J, Saleh L, Husslein H, Sonderegger S, Helmer H and Knöfler M

    Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

    Chorionic gonadotropin (CG) is indispensable for human pregnancy because it controls implantation, decidualization, and placental development. However, its particular role in the differentiation process of invasive trophoblasts has not been fully unraveled. Here we demonstrate that the hormone promotes trophoblast invasion and migration in different trophoblast model systems. RT-PCR and Western blot analyses revealed expression of the LH/CG receptor in trophoblast cell lines and different trophoblast primary cultures. In vitro, CG increased migration and invasion of trophoblastic SGHPL-5 cells through uncoated and Matrigel-coated transwells, respectively. The hormone also increased migration of first-trimester villous explant cultures on collagen I. Proliferation of the trophoblast cell line and villous explant cultures measured by cumulative cell numbers and in situ 5-bromo-2'-deoxyuridine labeling, respectively, was unaffected by CG. Addition of the hormone activated ERK-1/2 and AKT in SGHPL-5 cells and pure, extravillous trophoblasts. Inhibition of MAPK kinase/ERK and phosphatidylinositide 3-kinase/AKT blocked phosphorylation of the kinases and attenuated CG-dependent invasion of SGHPL-5 cells. Similarly, the inhibitors decreased hormone-stimulated migration in villous explant cultures. Western blot analyses and gelatin zymography suggested that CG increased matrix metalloproteinase (MMP)-2 protein levels and activity in both culture systems. Inhibition of ERK or AKT diminished CG-induced MMP-2 expression. In summary, the data demonstrate that CG promotes trophoblast invasion and migration through activation of ERK and AKT signaling involving their downstream effector MMP-2. Because the increase of CG during the first trimester of pregnancy correlates with rising trophoblast motility, the hormone could be a critical regulator of the early invasion process.

    Funded by: Austrian Science Fund FWF: P 17894

    Endocrinology 2008;149;3;979-87

  • Induction of ERK-kinase signalling triggers morphotype-specific killing of Candida albicans filaments by human neutrophils.

    Wozniok I, Hornbach A, Schmitt C, Frosch M, Einsele H, Hube B, Löffler J and Kurzai O

    University of Würzburg, Department of Internal Medicine II, Germany.

    Candida albicans is among the most important fungal pathogens in humans. Morphological plasticity has been linked to its pathogenic potential as filamentous forms are associated with tissue invasion and infection. Here we show that human neutrophils discriminate between yeasts and filaments of C. albicans. Whereas filaments induced targeted motility, resulting in the establishment of close contact between neutrophils and fungal cells, yeast forms were largely ignored during coincubation. In transwell assays, C. albicans filaments induced significantly higher migratory activity in neutrophils than yeasts. Neutrophil motility based on actin rearrangement was essential for killing of C. albicans filaments but not involved in killing of yeast forms. Using inhibitors for MAP-kinase cascades, it was shown that recognition of C. albicans filaments by neutrophils is mediated via the MEK/ERK cascade and independent of JNK or p38 activation. Inhibition of the ERK signalling pathway abolished neutrophil migration induced by C. albicans filaments and selectively impaired the ability to kill this morphotype. These data show that invasive filamentous forms of C. albicans trigger a morphotype-specific activation of neutrophils, which is strongly dependent on neutrophil motility. Therefore, human neutrophils are capable of sensing C. albicans invasion and initiating an appropriate early immune response.

    Cellular microbiology 2008;10;3;807-20

  • Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide.

    Feldt S, Batenburg WW, Mazak I, Maschke U, Wellner M, Kvakan H, Dechend R, Fiebeler A, Burckle C, Contrepas A, Jan Danser AH, Bader M, Nguyen G, Luft FC and Muller DN

    Medical Faculty of the Charité, Experimental and Clinical Research Center, Franz Volhard Clinic and HELIOS Klinikum, Berlin-Buch, Germany.

    The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the "handle-region" peptide (HRP). We tested whether human prorenin and renin induce extracellular signal-regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involve the epidermal growth factor receptor. A mitogen-activated protein kinase kinase 1/2 inhibitor inhibited both renin and prorenin-induced ERK 1/2 phosphorylation. Neither aliskiren nor HRP inhibited binding of (125)I-renin or (125)I-prorenin to (P)RR. Aliskiren did not inhibit renin and prorenin-induced ERK 1/2 phosphorylation and kinase activity. Fluorescence-activated cell sorter analysis showed that, although fluorescein isothiocyanate-labeled HRP bound to U937 cells, HRP did not inhibit renin or prorenin-induced ERK 1/2 activation. In conclusion, prorenin and renin-induced ERK 1/2 activation are independent of angiotensin II. The signal transduction is different from that evoked by angiotensin II. Aliskiren has no (P)RR blocking effect and did not inhibit ERK 1/2 phosphorylation or kinase activity. Finally, we found no evidence that HRP affects renin or prorenin binding and signaling.

    Hypertension (Dallas, Tex. : 1979) 2008;51;3;682-8

  • RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells.

    Flynn JM, Lannigan DA, Clark DE, Garner MH and Cammarata PR

    Dept. of Cell Biology and Genetics, Univ. of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.

    17beta-Estradiol (E(2)) reduces oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3). The mechanism by which the nongenomic effects of E(2) contributed to the protection against mitochondrial membrane depolarization was investigated. Mitochondrial membrane integrity is regulated by phosphorylation of BAD, and it is known that phosphorylation of Ser(112) inactivates BAD and prevents its participation in the mitochondrial death pathway. We found that E(2) rapidly increased both the phosphorylation of ERK2 and Ser(112) in BAD. Ser(112) is phosphorylated by p90 ribosomal S6 kinase (RSK), a Ser/Thr kinase, which is a downstream effector of ERK1/2. Inhibition of RSK by the RSK-specific inhibitor SL0101 did not reduce the level of E(2)-induced phosphorylation of Ser(112). Silencing BAD using small interfering RNA did not alter mitochondrial membrane depolarization elicited by peroxide insult. However, under the same conditions, silencing ERK2 dramatically increased membrane depolarization compared with the control small interfering RNA. Therefore, ERK2, functioning through a BAD-independent mechanism regulates MMP in humans lens epithelial cells. We propose that estrogen-induced activation of ERK2 acts to protect cells from acute oxidative stress. Moreover, despite the fact that ERK2 plays a regulatory role in mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism.

    Funded by: NEI NIH HHS: EY05570

    American journal of physiology. Endocrinology and metabolism 2008;294;3;E589-99

  • Roles of Rho guanosine 5'-triphosphatase A, Rho kinases, and extracellular signal regulated kinase (1/2) in prostaglandin E2-mediated migration of first-trimester human extravillous trophoblast.

    Nicola C, Chirpac A, Lala PK and Chakraborty C

    Department of Anatomy, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1.

    Prostaglandin (PG) E(2) may regulate invasiveness of human placenta because we previously reported stimulation of migration of placental trophoblasts by PGE(2) acting through PGE receptor (EP)-1 and activating calpain. RhoA GTPase and its important effector Rho kinase (ROCK) have also been previously shown to regulate trophoblast migration. Using immortalized HTR-8/SVneo trophoblast cells and first-trimester human chorionic villus explant cultures on matrigel, we further examined the role of RhoA/ROCK and MAPK (ERK1/2) pathways on PGE(2)-mediated stimulation of trophoblast migration. Migration of cytotrophoblasts was shown to be inhibited by treatment of the trophoblast cell line and chorionic villus explants with either cell-permeable C3 transferase or selective RhoA small interfering RNA. These inhibitions were significantly mitigated by the addition of PGE(2), an EP1/EP3 agonist or an EP3/EP4 agonist, suggesting that RhoA plays an important role in trophoblast migration but may not be obligatory for PGE(2) action. Treatment of HTR-8/SVneo cells with nonselective ROCK inhibitor Y27632 or ROCK small interfering RNAs inhibited migration of these cells, which could not be rescued with PGE(2) or the other two EP agonists, suggesting the obligatory role of ROCK in PGE(2)-induced migratory response. Furthermore, U0126, an inhibitor of MAPK kinases MEK1 and MEK2, abrogated PGE(2)-induced migration of trophoblasts, and PGE(2) or the other two EP agonists stimulated ERK1/2 activation in trophoblasts, which was not abrogated by pretreatment with C3 transferase, indicating that ERK signaling pathway is an efficient alternate pathway for RhoA in PGE(2)-mediated migration of trophoblasts. These results suggest that ROCK and ERK1/2 play more important roles than RhoA in PGE(2)-mediated migration stimulation of first-trimester trophoblasts.

    Endocrinology 2008;149;3;1243-51

  • Tissue factor pathway inhibitor (TFPI) interferes with endothelial cell migration by inhibition of both the Erk pathway and focal adhesion proteins.

    Provençal M, Michaud M, Beaulieu E, Ratel D, Rivard GE, Gingras D and Béliveau R

    Laboratoire de médecine moléculaire Sainte-Justine-UQAM, Centre de cancérologie Charles-Bruneau, 3175, Chemin Côte-Sainte-Catherine, Montréal, Québec, Canada.

    Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glioblastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion, suggesting an alteration of focal adhesion complex integrity. Accordingly, we observed that TFPI inhibited the phosphorylation of focal adhesion kinase and paxillin, two key proteins involved in the scaffolding of these complexes, and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells.

    Thrombosis and haemostasis 2008;99;3;576-85

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Suppression of v-Src transformation by andrographolide via degradation of the v-Src protein and attenuation of the Erk signaling pathway.

    Liang FP, Lin CH, Kuo CD, Chao HP and Fu SL

    Institute of Traditional Medicine, Faculty of Life Sciences, National Yang-Ming University, 155, Sec. 2, Li-Nong St., Taipei 11221, and Department of Research and Education, Taipei City Hospital, Taiwan.

    Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.

    The Journal of biological chemistry 2008;283;8;5023-33

  • The docking interaction of caspase-9 with ERK2 provides a mechanism for the selective inhibitory phosphorylation of caspase-9 at threonine 125.

    Martin MC, Allan LA, Mancini EJ and Clarke PR

    Biomedical Research Centre, Ninewells Hospital and Medical School, Level 5, University of Dundee, Dundee DD1 9SY, United Kingdom.

    Caspase-9 plays a critical role in the initiation of apoptosis by the mitochondrial pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr(125) by ERK1/2 MAPKs in response to growth factors. Here, we show that phosphorylation of this site is specific for these classical MAPKs and is not strongly induced when JNK and p38alpha/beta MAPKs are activated by anisomycin. By deletion and mutagenic analysis, we identify domains in caspase-9 and ERK2 that mediate their interaction. Binding of ERK2 to caspase-9 and subsequent phosphorylation of caspase-9 requires a basic docking domain (D domain) in the N-terminal prodomain of the caspase. Mutational analysis of ERK2 reveals a (157)TTCD(160) motif required for recognition of caspase-9 that acts independently of the putative common docking domain. Molecular modeling supports the conclusion that Arg(10) in the D domain of caspase-9 interacts with Asp(160) in the TTCD motif of ERK2. Differences in the TTCD motif in other MAPK family members could account for the selective recognition of caspase-9 by ERK1/2. This selectivity may be important for the antiapoptotic role of classical MAPKs in contrast to the proapoptotic roles of stress-activated MAPKs.

    Funded by: Cancer Research UK; Medical Research Council

    The Journal of biological chemistry 2008;283;7;3854-65

  • IL-1beta-stimulated activation of ERK1/2 and p38alpha MAPK mediates the transcriptional up-regulation of IL-6, IL-8 and GRO-alpha in HeLa cells.

    Yang HT, Cohen P and Rousseau S

    MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Sir James Black Centre, Dow Street, Dundee, DD1 5EH, United Kingdom.

    Epithelial cells represent the first line of defense against infection. Here we have studied the production of inflammatory mediators induced by IL-1beta in the HeLa epithelial cell line. We found that GRO-alpha, IL-6 and IL-8 were the only three inflammatory mediators elevated out of 36 tested. Specific inhibition of p38alpha MAP kinase or preventing the activation of ERK1/ERK2 partially reduced the production of these substances, while the combined blockade of both pathways almost abolished secretion. The suppression of these signaling pathways mainly reduced transcription of the genes encoding GRO-alpha, IL-6 and IL-8, rather than affecting mRNA stability, translation or secretion. The production of these three inflammatory mediators was shown to account for the ability of the HeLa cell culture medium to stimulate the migration of monocytes/macrophages, suggesting a key role for p38 MAPK and ERK1/ERK2 in orchestrating the epithelial cell response to infection.

    Funded by: Medical Research Council: MC_U127084348

    Cellular signalling 2008;20;2;375-80

  • Mesothelin promotes anchorage-independent growth and prevents anoikis via extracellular signal-regulated kinase signaling pathway in human breast cancer cells.

    Uehara N, Matsuoka Y and Tsubura A

    Second Department of Pathology, Kansai Medical University, Moriguchi-Shi, Osaka, Japan. ueharan@takii.kmu.ac.jp

    Mesothelin (MSLN) is a glycoprotein that is overexpressed in various tumors. MSLN is present on the cell surface and is also released into body fluids or culture supernatants from MSLN-positive tumor cells. Despite intensive study of MSLN as a diagnostic marker or target for immunotherapy, its biological function is largely unknown. In the present study, we examined the effects of ectopic expression of MSLN in human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). We found that overexpression of MSLN promoted anchorage-independent growth in soft agar. In addition, MDA-MB-231 cells expressing high levels of MSLN exhibited resistance to anoikis (a type of apoptosis induced by detachment from substratum), as indicated by decreased DNA fragmentation and down-regulation of the proapoptotic protein Bim. Incubating MSLN-expressing MDA-MB-231 cells in the presence of U0126, an inhibitor of mitogen-activated protein/extracellular-signal-regulated kinase kinase, induced accumulation of Bim and restored susceptibility to anoikis. Western blot analysis also revealed that overexpression of MSLN resulted in sustained activation of extracellular signal-regulated kinase 1/2 and suppression of Bim. The present results constitute novel evidence that MSLN enables cells to survive under anchorage-independent conditions by suppressing Bim induction via the extracellular signal-regulated kinase signaling pathway.

    Molecular cancer research : MCR 2008;6;2;186-93

  • Pyk2/ERK 1/2 mediate Sp1- and c-Myc-dependent induction of telomerase activity by epidermal growth factor.

    Bermudez Y, Yang H, Cheng JQ and Kruk PA

    Department of Pathology and Cell Biology, University of South Florida, Tampa, Florida 33612, USA.

    Epidermal growth factor (EGF) promotes growth of normal ovarian surface as well as malignant ovarian epithelial cells. Further, EGF receptors are present on both normal and malignant ovarian surface epithelial cells and they are often constitutively activated in many cancers. Since telomerase confers cellular immortalization and survival through increased cellular proliferation, we sought to investigate the potential role of EGF to regulate telomerase activity in normal and ovarian cancer cells. While exogenous EGF failed to activate telomerase in normal ovarian surface epithelial cells, in cancer cells we herein report that: exogenous EGF activates telomerase activity and human telomerase reverse transcriptase gene (hTERT) transcription; EGF-induced telomerase activity is ERK 1/2-dependent; EGF targets Sp1 and c-Myc binding sites within the core region of the hTERT promoter; and proline-rich tyrosine kinase 2 (Pyk2) is a key mediator of EGF-mediated telomerase activity. Together, these data show that dysregulation of EGF signaling may promote cancer cell survival through up-regulation of telomerase activity.

    Growth factors (Chur, Switzerland) 2008;26;1;1-11

  • Resveratrol reverses hydrogen peroxide-induced proliferative effects in human coronary smooth muscle cells: a novel signaling mechanism.

    El-Mowafy AM, Alkhalaf M and El-Kashef HA

    Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. aelmowafy@yahoo.com

    Background: In human coronary smooth muscle cells (HCSMCs), we tested the proatherogenic/proliferative potential of the reactive oxygen species (ROS), hydrogen peroxide (HP), and the ability of the polyphenol stilbene resveratrol (RSVL) to protect against such effects.

    Methods: Activity for ERK1/2 and the kinase-G cascade were determined and correlated with HCSMC count before and after treatment with HP and/or RSVL.

    Results: HP evoked concentration-dependent cell proliferation and stimulated ERK1/2 phosphorylation at active sites. Pretreatment with the MEK-ERK inhibitor (PD98059) reversed these effects of HP. RSVL (1-100 microM) elicited more prominent inhibition of HP-evoked cell proliferation and ERK1/2 activation. In addition, RSVL markedly enhanced cGMP formation, a response that was insensitive to the soluble guanylyl-cyclase (sGC) inhibitor (ODQ, 10 microM) but was obliterated with the phorbol ester, (PMA, 0.1 microM), a desensitizer of the pGC enzyme. Likewise, the RSVL-evoked cytostatic and ERK inhibitory effects were significantly reversed by the kinase-G-inhibitor, KT-5823 (10 microM).

    Conclusions: Collectively, RSVL activates the kinase-G system to counteract HP-induced ERK1/2 activation and coronary arterial proliferation. These effects for RSVL remain functional in endothelium-disrupted arteries, scenarios that commonly occur in advanced coronary heart disease.

    Archives of medical research 2008;39;2;155-61

  • Three-dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway.

    Damianova R, Stefanova N, Cukierman E, Momchilova A and Pankov R

    Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University St. Kliment Ohridski, 1164 Sofia, Bulgaria.

    Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion-mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three-dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady-state levels of ERK1/2 activity in primary human fibroblasts, induced by cell-derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5-fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo-like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.

    Cell biology international 2008;32;2;229-34

  • Role of beta-arrestin-mediated desensitization and signaling in the control of angiotensin AT1a receptor-stimulated transcription.

    Lee MH, El-Shewy HM, Luttrell DK and Luttrell LM

    Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    Heptahelical G protein-coupled receptors employ several mechanisms to activate the ERK1/2 cascade and control gene transcription. Previous work with the angiotensin AT1a receptor has shown that G(q/11) activation leads to a rapid and transient rise in ERK1/2 activity, whereas beta-arrestin binding supports sustained ERK1/2 activation by scaffolding a Raf.MEK.ERK complex associated with the internalized receptor. In this study, we compared the role of the two beta-arrestin isoforms in AT1a receptor desensitization, ERK1/2 activation and transcription using selective RNA interference. In HEK293 cells, both the native AT1a receptor and a G protein-coupling deficient DRY/AAY mutant recruited beta-arrestin1 and beta-arrestin2 upon angiotensin binding and internalized with the receptor. In contrast, only beta-arrestin2 supported protein kinase C-independent ERK1/2 activation by both the AT1a and DRY/AAY receptors. Using focused gene expression filter arrays to screen for endogenous transcriptional responses, we found that silencing beta-arrestin1 or beta-arrestin2 individually did not alter the response pattern but that silencing both caused a marked increase in the number of transcripts that were significantly up-regulated in response to AT1a receptor activation. The DRY/AAY receptor failed to elicit any detectable transcriptional response despite its ability to stimulate beta-arrestin2-dependent ERK1/2 activation. These results indicate that the transcriptional response to AT1a receptor activation primarily reflects heterotrimeric G protein activation. Although beta-arrestin1 and beta-arrestin2 are functionally specialized with respect to supporting G protein-independent ERK1/2 activation, their common effect is to dampen the transcriptional response by promoting receptor desensitization.

    Funded by: NIDDK NIH HHS: DK55524

    The Journal of biological chemistry 2008;283;4;2088-97

  • Blockade of tumor growth due to matrix metalloproteinase-9 inhibition is mediated by sequential activation of beta1-integrin, ERK, and NF-kappaB.

    Bhoopathi P, Chetty C, Kunigal S, Vanamala SK, Rao JS and Lakka SS

    Program of Cancer Biology, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605, USA.

    We previously showed that matrix metalloproteinase (MMP)-9 inhibition using an adenovirus-mediated delivery of MMP-9 small interfering RNA (Ad-MMP-9), caused senescence in medulloblastoma cells. Regardless of whether or not Ad-MMP-9 would induce apoptosis, the possible signaling mechanism is still obscure. In this report, we demonstrate that Ad-MMP-9 induced apoptosis in DAOY cells as determined by propidium iodide and terminal deoxynucleotidyltransferase-mediated nick end labeling staining. Ad-MMP-9 infection induced the release of cytochrome c, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase. Ad-MMP-9 infection stimulated ERK, and electrophoretic mobility shift assay indicated an increase in NF-kappaB activation. ERK inhibition, using a kinase-dead mutant for ERK, ameliorated NF-kappaB activation and caspase-mediated apoptosis in Ad-MMP-9-infected cells. beta1-Integrin expression in Ad-MMP-9-infected cells also increased, and this increase was reversed by the reintroduction of MMP-9. We found that the addition of beta1 blocking antibodies inhibited Ad-MMP-9-induced ERK activation. Taken together, our results indicate that MMP-9 inhibition induces apoptosis due to altered beta1-integrin expression in medulloblastoma. In addition, ERK activation plays an active role in this process and functions upstream of NF-kappaB activation to initiate the apoptotic signal.

    Funded by: NCI NIH HHS: CA 116708, CA 75557, CA 92393, CA 95058, R01 CA075557, R01 CA075557-09, R01 CA092393, R01 CA092393-04, R01 CA095058, R01 CA095058-04, R01 CA116708, R01 CA116708-02; NINDS NIH HHS: NS 47699, NS 57529, R01 NS047699, R01 NS047699-03, R01 NS057529, R01 NS057529-01

    The Journal of biological chemistry 2008;283;3;1545-52

  • Coupling of Grb2 to Gab1 mediates hepatocyte growth factor-induced high intensity ERK signal required for inhibition of HepG2 hepatoma cell proliferation.

    Kondo A, Hirayama N, Sugito Y, Shono M, Tanaka T and Kitamura N

    Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.

    Activation of the extracellular signal-regulated kinase (ERK) pathway is a key factor in the regulation of cell proliferation by growth factors. Hepatocyte growth factor (HGF)-induced cell cycle arrest in the human hepatocellular carcinoma cell line HepG2 requires strong activation of the ERK pathway. In this study, we investigated the molecular mechanism of the activation. We constructed a chimeric receptor composed of the extracellular domain of the NGF receptor and the cytoplasmic domain of the HGF receptor (c-Met) and introduced a point mutation (N1358H) into the chimeric receptor, which specifically abrogates the direct binding of Grb2 to c-Met. The mutant chimeric receptor failed to mediate the strong activation of ERK, up-regulation of the expression of a Cdk inhibitor p16(INK4a) and inhibition of HepG2 cell proliferation by ligand stimulation. Moreover, the mutant receptor did not induce tyrosine phosphorylation of the docking protein Gab1. Knockdown of Gab1 using siRNA suppressed the HGF-induced strong activation of ERK and inhibition of HepG2 cell proliferation. These results suggest that coupling of Grb2 to Gab1 mediates the HGF-induced strong activation of the ERK pathway, which is required for the inhibition of HepG2 cell proliferation.

    The Journal of biological chemistry 2008;283;3;1428-36

  • Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling.

    Chandramouli S, Yu CY, Yusoff P, Lao DH, Leong HF, Mizuno K and Guy GR

    Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.

    The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of Spry2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by Spry2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of Spry2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of Spry2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of Spry2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.

    The Journal of biological chemistry 2008;283;3;1679-91

  • The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways.

    de Totero D, Meazza R, Capaia M, Fabbi M, Azzarone B, Balleari E, Gobbi M, Cutrona G, Ferrarini M and Ferrini S

    Department of Translational Oncology and Medical Oncology C, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.

    The clonal expansion of chronic lymphocytic leukemia (CLL) cells requires the interaction with the microenvironment and is under the control of several cytokines. Here, we investigated the effect of IL-15 and IL-21, which are closely related to IL-2 and share the usage of the common gamma chain and of its JAK3-associated pathway. We found remarkable differences in the signal transduction pathways activated by these cytokines, which determined different responses in CLL cells. IL-15 caused cell proliferation and prevented apoptosis induced by surface IgM cross-linking. These effects were more evident in cells stimulated via surface CD40, which exhibited increased cell expression of IL-15Ralpha chain and, in some of the cases, also of IL-2Rbeta. IL-21 failed to induce CLL cell proliferation and instead promoted apoptosis. Following cell exposure to IL-15, phosphorylation of STAT5 was predominantly observed, whereas, following stimulation with IL-21, there was predominant STAT1 and STAT3 activation. Moreover, IL-15 but not IL-21 caused an increased phosphorylation of Shc and ERK1/2. Pharmacological inhibition of JAK3 or of MEK, which phosphorylates ERK1/2, efficiently blocked IL-15-induced CLL cell proliferation and the antiapoptotic effect of this cytokine. The knowledge of the signaling pathways regulating CLL cell survival and proliferation may provide new molecular targets for therapeutic intervention.

    Blood 2008;111;2;517-24

  • TPL2-mediated activation of ERK1 and ERK2 regulates the processing of pre-TNF alpha in LPS-stimulated macrophages.

    Rousseau S, Papoutsopoulou M, Symons A, Cook D, Lucocq JM, Prescott AR, O'Garra A, Ley SC and Cohen P

    MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK. s.rousseau@dundee.ac.uk

    Activation of the TPL2-MKK1/2-ERK1/2 signalling pathway is essential for lipopolysaccharide (LPS)-stimulated production of TNF alpha in macrophages. Here, we demonstrate that, unexpectedly, TPL2-deficient or MKK1-inhibited macrophages produce near normal levels of pre-TNF alpha when TLR2, TLR4 and TLR6 are activated by their respective agonists, but fail to secrete TNFalpha. We show that LPS stimulates the appearance of pre-TNFalpha at the cell surface and that this is prevented by inhibition of MAPK kinases 1 and 2 (MKK1/2) or in TPL2-deficient macrophages. However, the transport of pre-TNF alpha from the Golgi to the plasma membrane is unaffected by inhibition of the TPL2-MKK1/2-ERK1/2 pathway. Finally, we show that TACE, the protease that cleaves pre-TNF alpha to secreted TNFalpha, is phosphorylated by ERK1 and ERK2 (ERK1/2) at Thr735 in LPS-stimulated macrophages. Therefore, although TACE activity per se is not required for the LPS-stimulated cell surface expression of pre-TNF alpha, the phosphorylation of this protease might contribute to, or be required for, the cell surface expression of the pre-TNF alpha-TACE complex.

    Funded by: Medical Research Council: MC_U117565642, MC_U127084348

    Journal of cell science 2008;121;Pt 2;149-54

  • Cyclophilin A is required for CXCR4-mediated nuclear export of heterogeneous nuclear ribonucleoprotein A2, activation and nuclear translocation of ERK1/2, and chemotactic cell migration.

    Pan H, Luo C, Li R, Qiao A, Zhang L, Mines M, Nyanda AM, Zhang J and Fan GH

    Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

    The chemokine receptor CXCR4-mediated signaling cascades play an important role in cell proliferation and migration, but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood. Here, we demonstrate that CXCR4 was co-immunoprecipitated with cyclophilin A (CyPA) from the lysate of HEK293 cells stably expressing CXCR4. Although both the glutathione S-transferase-CXCR4 N- and C-terminal fusion proteins were associated with the purified CyPA, truncation of the C-terminal domain of CXCR4 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells, thereby suggesting a critical role of the receptor C terminus in this interaction. Ligand stimulation of CXCR4 induced CyPA phosphorylation and nuclear translocation, both of which were inhibited by truncation of the C-terminal domain of CXCR4. CyPA was associated with transportin 1, and knockdown of transportin 1 by RNA interference (RNAi) blocked CXCL12-induced nuclear translocation of CyPA, thereby suggesting a transportin 1-mediated nuclear import of CyPA. CyPA formed a complex with heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which underwent nuclear export in response to activation of CXCR4. Interestingly, the CXCR4-mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA. Moreover, CXCR4-evoked activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was attenuated by CyPA RNAi, by overexpression of a PPIase-deficient mutant of CyPA (CyPA-R55A), and by pretreatment of the immunosuppressive drugs, cyclosporine A and sanglifehrin A. Finally, CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 or Jurkat T cells was inhibited by CyPA RNAi or CsA treatment.

    Funded by: NCRR NIH HHS: RR 03032-19

    The Journal of biological chemistry 2008;283;1;623-37

  • Angiotensin II induces vascular endothelial growth factor in pancreatic cancer cells through an angiotensin II type 1 receptor and ERK1/2 signaling.

    Anandanadesan R, Gong Q, Chipitsyna G, Witkiewicz A, Yeo CJ and Arafat HA

    Department of Surgery, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA, USA.

    Vascular endothelial growth factor (VEGF) is a crucial pro-angiogenic component in pancreatic ductal adenocarcinoma (PDA), and its high expression levels have been correlated with poor prognosis and early postoperative recurrence. We have recently shown that high levels of angiotensin II (AngII) type 1 receptor (AT1R) correlate and colocalize with VEGF in invasive PDA and that AngII induces VEGF expression in PDA cell lines. In this study, we explored the signaling mechanisms involved in the AngII-mediated VEGF induction and correlated AT1R and VEGF expression in noninvasive precursor lesions. An AT1R antagonist significantly (p<0.05) inhibited the AngII-mediated induction of VEGF messenger RNA and protein in all PDA cell lines. AngII-VEGF induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a mitogen-activated protein kinase signaling mechanism. AngII activated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the AngII-induced VEGF synthesis. Immunohistochemical analysis of precursor lesions showed increased expression of AT1R in most ductal cells undergoing metaplasia. Pancreatic intraepithelial neoplasms showed more intense AT1R staining when compared to intraductal papillary mucinous neoplasms, which showed heterogeneous immunoreactivity. VEGF followed the same distribution pattern of AT1R in both lesions. AT1R expression in the premalignant pancreatic lesions suggests its involvement in tumor progression and angiogenesis. Our mechanistic findings provide the first insight into an AngII-initiated signaling pathway that regulates PDA angiogenesis. An AT1R-mediated VEGF induction suggests the possibility of AT1R blockade as a novel therapeutic strategy to control angiogenesis in PDA.

    Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract 2008;12;1;57-66

  • CCDC134, a novel secretory protein, inhibits activation of ERK and JNK, but not p38 MAPK.

    Huang J, Shi T, Ma T, Zhang Y, Ma X, Lu Y, Song Q, Liu W, Ma D and Qiu X

    School of Basic Medical Science, Peking University Health Science Center, Beijing, People's Republic of China.

    In this study, we report a novel gene, CCDC134 (coiled-coil domain containing 134), that encodes a secretory protein that can inhibit the MAPK pathway as a novel human MAPK-regulating protein. The CCDC134 mRNA contains 1280 nucleotides, encoding a protein of 229 amino acids. CCDC134 is a classical secretory protein. Expression profile analysis by Northern blot, RT-PCR, immunohistochemistry and Western blot reveals that CCDC134 is widely expressed in normal adult tissues, tumor tissues and cell lines. Functional investigation reveals that overexpression of CCDC134 and its purified protein significantly inhibit transcriptional activity of Elk1 and phosphorylation of Erk and JNK/SAPK but not p38 MAPK. Conversely, specific siRNA against CCDC134 activates Elk1 transcriptional activity and promotes Erk and JNK/SAPK phosphorylation. These results clearly indicate that CCDC134 is a novel member of the secretory family and down-regulates the Raf-1/MEK/ERK and JNK/ SAPK pathways.

    Cellular and molecular life sciences : CMLS 2008;65;2;338-49

  • Gastrin transactivates the chromogranin A gene through MEK-1/ERK- and PKC-dependent phosphorylation of Sp1 and CREB.

    Cramer T, Jüttner S, Plath T, Mergler S, Seufferlein T, Wang TC, Merchant J and Höcker M

    Laboratory for Angiogenesis and Tumor Metastasis, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Germany.

    Our previous work revealed that gastrin regulates chromogranin A (CgA) transcription through enhanced binding of Sp1, CREB and Egr-1 to a proximal gastrin-responsive promoter element (Gas-RE). Here, we provide a detailed characterization of the signalling pathways transmitting the effect of gastrin on the CgA promoter. Gastrin treatment of gastric AGS-B cells potently stimulated MEK-1 as well as MAP kinases ERK-1/-2, JNK and p38 in a time-dependent manner. Interruption of ERK-1/-2/MEK-1 pathways abolished the transactivating effect of gastrin, whereas blockade of JNK or p38 activity was without effect. Functional promoter analysis revealed that the minimal element CgA-85/-64 was sufficient and necessary to confer MEK-1/ERK responsiveness. Analysis of proximal signalling pathways showed that activation of the MEK-1/ERK-1/2 module by gastrin does not require Ras, PI3-kinase or intracellular calcium signals, but depends on activation of kinases of the PKC family. This report demonstrates that a pathway comprising PKCs>Raf-1>MEK-1>ERK-1/-2 mediates the effect of gastrin on the CgA promoter, and strongly suggests that enhanced phosphorylation of Sp1 and CREB is crucial for CgA transactivation through the G protein-coupled CCK-B/gastrin receptor.

    Funded by: NIDDK NIH HHS: R0 1 DK 48077, R01 DK045729, R37 DK045729

    Cellular signalling 2008;20;1;60-72

  • Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways.

    Liu D, Yumoto H, Hirota K, Murakami K, Takahashi K, Hirao K, Matsuo T, Ohkura K, Nagamune H and Miyake Y

    Department of Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan.

    Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.

    Cellular microbiology 2008;10;1;262-76

  • Interferon alpha induces nucleus-independent apoptosis by activating extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase downstream of phosphatidylinositol 3-kinase and mammalian target of rapamycin.

    Panaretakis T, Hjortsberg L, Tamm KP, Björklund AC, Joseph B and Grandér D

    Department of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden.

    Interferon (IFN)alpha induces apoptosis via Bak and Bax and the mitochondrial pathway. Here, we investigated the role of known IFNalpha-induced signaling cascades upstream of Bak activation. By pharmacological and genetic inhibition of the kinases protein kinase C (PKC)delta, extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) in U266-1984 and RHEK-1 cells, we could demonstrate that all three enzymes are critical for the apoptosis-associated mitochondrial events and apoptotic cell death induced by IFNalpha, at a step downstream of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR). Furthermore, the activation of JNK was found to occur in a PKCdelta/ERK-dependent manner. Inhibition of these kinases did not affect the canonical IFNalpha-stimulated Janus tyrosine kinase-signal transducer and activator of transcription signaling or expression of IFN-responsive genes. Therefore, enucleated cells (cytoplasts) were examined for IFNalpha-induced apoptosis, to test directly whether this process depends on gene transcription. Cytoplasts were found to undergo apoptosis after IFNalpha treatment, as analyzed by several apoptosis markers by using flow cytometry, live cell imaging, and biochemical analysis of flow-sorted cytoplasts. Furthermore, inhibition of mTOR, ERK, and JNK blocked IFNalpha-induced apoptosis in cytoplasts. In conclusion, IFNalpha-induced apoptosis requires activation of ERK1/2, PKCdelta, and JNK downstream of PI3K and mTOR, and it can occur in a nucleus-independent manner, thus demonstrating for the first time that IFNalpha induces apoptosis in the absence of de novo transcription.

    Molecular biology of the cell 2008;19;1;41-50

  • Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro.

    Fan Y, Ye J, Shen F, Zhu Y, Yeghiazarians Y, Zhu W, Chen Y, Lawton MT, Young WL and Yang GY

    Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, CA 94110, USA.

    Circulating blood endothelial progenitor cells (EPCs) contribute to postnatal vasculogenesis, providing a novel therapeutic target for vascular diseases. However, the molecular mechanism of EPC-induced vasculogenesis is unknown. Interleukin-6 plays multiple functions in angiogenesis and vascular remodeling. Our previous study demonstrated that the polymorphism (174G>C) in IL-6 gene promoter was associated with brain vascular disease. In this study, we investigated if IL-6 receptor is expressed in human EPCs derived from circulating mononuclear cells, and if interleukin-6 (IL-6) stimulates EPC angiogenesis in vitro. First, we isolated and cultured mononuclear cells from adult human circulating blood. We obtained EPC clones that were further cultured and expended for the angiogenesis study. We found that the EPCs possessed human mature endothelial cell phenotypes; however, they proliferated much faster than mature endothelial cells (P<0.05). We then found that IL-6 receptor (gp-80) was expressed in the EPCs, and that administration of IL-6 could activate receptor gp80/gp130 signaling pathways including downstream extracellular signal-regulated kinase 1/2 and STAT3 phosphorylation in EPCs. Furthermore, IL-6 stimulated EPC proliferation, migration, and matrigel tube formation in a dose-dependent manner (P<0.05); anti-IL-6 antibodies or IL-6 receptor could abolish these effects (P<0.05). These results suggest that IL-6 plays a crucial role in the biologic behavior of blood-derived EPCs, which may help clarify the mechanism of IL-6 inflammatory-related diseases.

    Funded by: NINDS NIH HHS: P01 NS044145, P01 NS044155-05, R01 NS027713-17, R01 NS27713, R21 NS50668

    Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 2008;28;1;90-8

  • Reverse signaling initiated from GITRL induces NF-kappaB activation through ERK in the inflammatory activation of macrophages.

    Bae EM, Kim WJ, Suk K, Kang YM, Park JE, Kim WY, Choi EM, Choi BK, Kwon BS and Lee WH

    Department of Genetic Engineering, School of Life Sciences and Biotechology, Kyungpook National University, Taegu 702-701, Republic of Korea.

    Glucocorticoid-induced TNF receptor family related protein ligand (GITRL) is known to interact with its cognate receptor GITR. In order to investigate the potential role of GITRL in the pro-inflammatory activation of macrophages and the signaling pathway induced by GITRL, we stimulated the macrophage cell line, THP-1, and primary macrophages with an anti-GITRL monoclonal antibody or a GITR:Fc fusion protein and analyzed the cellular responses. The stimulation of GITRL induced the expression of pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9 and up-regulated ICAM-1 expression levels, which was responsible for enhanced cellular aggregation and adhesion to extracellular matrix proteins. The activation of these pro-inflammatory mediators required the activation of ERK1/2 mitogen-activated protein kinase (MAPK) and negatively regulated by p38 MAPK and JNK. Immunofluorescence analysis detected nuclear translocation of the NF-kappaB p50 subunit and this was blocked by ERK inhibitor, indicating that GITRL stimulation induced ERK1/2 phosphorylation and subsequent activation of NF-kappaB. Furthermore, the expression of GITRL and GITR was detected in macrophages in inflammatory disease specimens such as atherosclerotic plaques and synovial tissues of rheumatoid arthritis. These observations raise the possibility that the GITRL-mediated inflammatory activation of macrophages is involved in the pathogenesis of inflammatory diseases.

    Molecular immunology 2008;45;2;523-33

  • SKAP55 modulates T cell antigen receptor-induced activation of the Ras-Erk-AP1 pathway by binding RasGRP1.

    Kosco KA, Cerignoli F, Williams S, Abraham RT and Mustelin T

    Program on Inflammatory Disease Research, Infectious and Inflammatory Disease Center, The Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. karenakosco@yahoo.com

    Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is an adapter protein with an N-terminal region, a pleckstrin homology domain, a linker with tyrosine phosphorylation sites, and a C-terminal Src homology 3 domain. We report that overexpression of SKAP55 disrupts signaling from the TCR to the Ras-Erk-AP-1 pathway and transcription of the IL-2 gene in primary human T cells and in Jurkat T leukemia cells. In contrast, moderate overexpression of SKAP55 increased TCR-dependent AP-1 transcriptional activity, suggesting that high-level SKAP55 overexpression interfered with the assembly of functional signaling complexes required for TCR coupling to the Ras pathway. In support of this view, knock-down of SKAP55 by RNA interference resulted in decreased reporter gene activation and decreased ERK phosphorylation. In contrast, TCR-induced NF-kappaB activation was not affected. Since constitutively active forms of Ras or Raf-1 overcame the inhibitory effects of SKAP55 overexpression, we searched for a mechanism upstream of Ras and found that SKAP55 co-immunoprecipitated with the Ras activator RasGRP1. The binding of RasGRP1 to SKAP55 required the C-terminus of SKAP55 and was enhanced by tyrosine phosphorylation of SKAP55. These results suggest that SKAP55 modulates signal transduction from the TCR to Ras by binding to RasGRP1.

    Funded by: NIAID NIH HHS: F32 AI056895, R01 AI035603, R01 AI053585, R01 AI35603, R01 AI53585

    Molecular immunology 2008;45;2;510-22

  • High intracellular Zn2+ ions modulate the VHR, ZAP-70 and ERK activities of LNCaP prostate cancer cells.

    Wong PF and Abubakar S

    Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

    Malignant prostate tissues have markedly reduced zinc (Zn(2+)) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn(2+) level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn(2+) (10 microg/ml) over 5 weeks. The intracellular Zn(2+) level increased in the Zn(2+)-treated cells, and there was a marked increase in the presence of zincosomes, a Zn(2+)-specific intracellular organelle. The proliferation rate of the Zn(2+)-treated cells was markedly reduced. There was also a significant increase (36.6% +/- 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn(2+), reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn(2+)-treated LNCaP cell proliferation to a rate comparable to that of the non Zn(2+)-treated cells. These results highlight the importance of a high intracellular Zn(2+) content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.

    Cellular & molecular biology letters 2008;13;3;375-90

  • Implication of active Erk in the classic type of human medulloblastoma.

    Wlodarski PK, Boszczyk A, Grajkowska W, Roszkowski M and Jozwiak J

    Department of Histology and Embryology, Centre for Biostructure Research, Medical University of Warsaw, Warsaw, Poland.

    Molecular pathways underlying medulloblastoma (MB), the most common malignant brain tumour in children, are still under scrutiny. The mammalian target of the rapamycin (mTOR) pathway is one of the kinases that was recently found to be implicated in a number of human tumours. Also in the case of MB it is suspected that mTOR dysregulation may play an important role in pathogenesis. Active mTOR leads to translation of several proteins, some of which affect cellular proliferation. On the other hand, Akt/PKB (protein kinase B) and Erk (extracellular signal-regulated kinase, also called mitogen-activated protein kinase, MAPK) are two protein kinases whose hyperactivity leads to a number of downstream effects, including activation of mTOR. In our previous report we found that indeed Akt and Erk are variably activated in human MBs. However, because MBs are a highly heterogeneous group of tumours, we were unable to associate Akt or Erk activation with all the cases of MB. In this paper we evaluated six cases of MB, only of the classic subtype. We found that elements of the Erk pathway are hyperactive in all six tumours. Thus, we postulate that in classic type of MB, growth factor stimulation may lead to Erk upregulation and mTOR-dependent protein translation, causing malignant growth.

    Folia neuropathologica 2008;46;2;117-22

  • Morphoproteomic profile of mTOR, Ras/Raf kinase/ERK, and NF-kappaB pathways in human gastric adenocarcinoma.

    Feng W, Brown RE, Trung CD, Li W, Wang L, Khoury T, Alrawi S, Yao J, Xia K and Tan D

    Department of Pathology, MD Anderson Cancer Center, Houston, TX 77030, USA.

    Preclinical studies using human gastric adenocarcinoma (GAC) cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, can inhibit tumor growth and that the extracellular signal-regulated kinase (ERK) of the Ras/Raf kinase/ERK pathway is related to chemoresistance and apoptosis. We examined the state of activation of components of mTOR, Ras/Raf kinase/ERK, and nuclear factor (NF)-kappaB signal transduction pathways, as well as cell cycle protein analyte correlates in GAC cases. Formalin-fixed paraffin-embedded tissue microarray blocks containing samples from 210 cases of GAC were examined. Immunohistochemistry was utilized to detect the following antigens: S100P, upstream stimulator of ERK, and NF-kappaB pathways; phosphorylated (p)-mTOR (Ser 2448), p-ERK-1/2 (Thr 202/Tyr 204), and one of their common down-stream effectors, p-p70S6K(Thr 389); p-NF-kappaBp65(Ser 536); and cell cycle associated proteins, Ki-67, and S phase kinase-associated protein (Skp)2. Immunoreactivity (0 to 4+) of protein expression and compartmentalization were assessed by bright-field microscopy. The majority of cases showed positive (1+ to 4+) cytoplasmic/plasmalemmal p-mTOR (88%), and moderate-strong (2+ to 4+) nuclear p-p70S6K (93%) and nuclear S100P (81%) expression. A subset of cases exhibited moderate-strong nuclear p-ERK-1/2 (15%) and p-NF-kappaBp65 (36%) expression. The majority of cases showed concomitant moderate-strong (2+ to 4+) nuclear Ki-67 (71%) and Skp2 (68%). Nuclear expression levels of p-ERK-1/2 and p-NF-kappaBp65, of p-p70S6K and p-NF-kappaB, and of Ki-67 and Skp2, respectively, showed significant linear correlations in GAC (p <0.001). Additionally, there were statistically significant differences in the mean expression levels of p-ERK-1/2 and p-NF-kappaBp65 in diffuse vs intestinal types of GAC, with higher levels of both in the diffuse type ( p = 0.001 and p <0.0001, respectively). In summary, morphoproteomic analysis reveals constitutive activation of mTOR and to some extent, Ras/Raf kinase and NF-kappaB pathways in GAC, as evidenced by increased cytoplasmic p-mTOR, nuclear translocation of p-p70S6K and p-ERK-1/2 phosphorylated at putative sites of activation (Ser 2448, Thr 389, and Thr 202/Tyr 204, respectively), as well as correlative expression of cell cycle analytes, Ki-67, and Skp2. These results suggest that a prospective study is warranted to evaluate the use of morphoproteomic profiling of individual patients with GAC in order to design combinatorial treatment strategies that target the mTOR, Ras/Raf kinase/ERK, and/or NF-kappaB pathways.

    Annals of clinical and laboratory science 2008;38;3;195-209

  • Oxidative stress induces extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase in cystic fibrosis lung epithelial cells: Potential mechanism for excessive IL-8 expression.

    Boncoeur E, Criq VS, Bonvin E, Roque T, Henrion-Caude A, Gruenert DC, Clement A, Jacquot J and Tabary O

    Inserm, U719, Paris F-75012, France.

    Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the CF transmembrane conductance regulator (CFTR) that leads to oxidative damage and excessive inflammatory response in lungs of CF patients. We here report the effects of oxidative stress (hyperoxia, 95% O(2)) on the expression of pro-inflammatory interleukin (IL)-8 and CXCR1/2 receptors in two human CF lung epithelial cell lines (IB3-1, with the heterozygous F508del/W1282X mutation and CFBE41o- with the homozygous F508del/F508del mutation) and two control non-CF lung epithelial cell lines (S9 cell line derived from IB3-1 after correction with wtCFTR and the normal bronchial cell line 16HBE14o-). Under oxidative stress, the expression of IL-8 and CXCR1/2 receptors was increased in CF, corrected and normal lung cell lines. The effects of oxidative stress were also investigated by measuring the transcription nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activities. Under oxidative stress, no increase of NF-kappaB activation was observed in CF lung cells in contrast to that observed in normal and corrected CF lung cells. The signalling of mitogen-activated protein (MAP) kinases was further studied. We demonstrated that extracellular signal-regulated kinase (ERK1/2) and AP-1 activity was markedly enhanced in CF but not non-CF lung cells under oxidative stress. Consistently, inhibition of ERK1/2 in oxidative stress-exposed CF lung cells strongly decreased both the IL-8 production and CXCR1/2 expression. Therefore, targeting of ERK1/2 MAP kinase may be critical to reduce oxidative stress-mediated inflammation in lungs of CF patients.

    The international journal of biochemistry & cell biology 2008;40;3;432-46

  • Taurine increases cell proliferation and generates an increase in [Mg2+]i accompanied by ERK 1/2 activation in human osteoblast cells.

    Jeon SH, Lee MY, Kim SJ, Joe SG, Kim GB, Kim IS, Kim NS, Hong CU, Kim SZ, Kim JS and Kang HS

    Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Republic of Korea.

    Taurine has been reported to influence bone metabolism, and its specific transport system, the taurine transporter, is expressed in osteoblasts. The mean [Mg2+]i was 0.51+/-0.01 mM in normal culture media. Taurine caused an increase in [Mg(2+)]i by 0.72+/-0.04 mM in human osteoblast (HOB) cells. This increment in [Mg2+]i was inhibited significantly by PD98059, nifedipine, lidocaine, and imipramine. Taurine was also shown to stimulate the activation of ERK 1/2. This taurine-stimulated ERK 1/2 activation was inhibited by PD98059. In the present study, taurine was shown to increase cell proliferation and generate an increase in [Mg2+]i accompanied by ERK 1/2 activation in HOB cells.

    FEBS letters 2007;581;30;5929-34

  • Shp2E76K mutant confers cytokine-independent survival of TF-1 myeloid cells by up-regulating Bcl-XL.

    Ren Y, Chen Z, Chen L, Woods NT, Reuther GW, Cheng JQ, Wang HG and Wu J

    Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Interdisciplinary Oncology, University of South Florida, Tampa 33612, USA.

    Shp2 has been known to mediate growth factor-stimulated cell proliferation, but its role in cell survival is less clear. Gain-of-function Shp2 mutants such as Shp2E76K are associated with myeloid leukemias. We found that Shp2E76K could transform cytokine-dependent human TF-1 myeloid cells into cytokine independence and further characterized the Shp2E76K-induced cell survival mechanism in this study. Expression of Shp2E76K suppressed the cytokine withdrawal-induced intrinsic/mitochondrial apoptosis pathway, which is controlled by the Bcl-2 family proteins. Analysis of Bcl-2 family proteins showed that Bcl-XL and Mcl-1 were up-regulated in Shp2E76K-transformed TF-1 (TF-1/Shp2E76K) cells. Knockdown of Bcl-XL but not Mcl-1 with short hairpin RNAs prevented Shp2E76K-induced cytokine-independent survival. Roscovitine, which down-regulated Mcl-1, also did not prevent cytokine-independent survival of TF-1/Shp2E76K cells, whereas the Bcl-XL inhibitor HA14-1 did. Ras and mitogen-activated protein kinases Erk1 and Erk2 (Erk1/2) were constitutively activated in TF-1/Shp2E76K cells, whereas little active Akt was detected under cytokine-free conditions. Shp2E76K-induced Bcl-XL expression was suppressed by Mek inhibitors and by a dominant-negative Mek1 mutant but not by the phosphoinositide 3-phosphate inhibitor LY294002 and the Akt inhibitor API-2. Inhibition of Erk1/2 blocked cytokine-independent survival of TF-1/Shp2E76K cells, whereas inhibition of Akt had a minimal effect on cytokine-independent survival of TF-1/Shp2E76K cells. These results show that Shp2E76K can evoke constitutive Erk1/2 activation in TF-1 cells. Furthermore, Shp2E76K induces cytokine-independent survival of TF-1 cells by a novel mechanism involving up-regulation of Bcl-XL through the Erk1/2 pathway.

    Funded by: NCI NIH HHS: P01 CA118210, P01CA118210, R01 CA077467, R01 CA077467-09, R01CA077467

    The Journal of biological chemistry 2007;282;50;36463-73

  • ERK signaling is a central regulator for BMP-4 dependent capillary sprouting.

    Zhou Q, Heinke J, Vargas A, Winnik S, Krauss T, Bode C, Patterson C and Moser M

    Department of Cardiology, University of Freiburg, Freiburg, Germany.

    Objective: Bone Morphogenetic Protein-4 (BMP-4) and Extracellular-Signal Regulated Kinases (ERK) play crucial roles in vascular diseases. Here, we demonstrate that BMP-4 not only signals through the classical Smad cascade but also activates ERK phosphorylation as an alternative pathway in human umbilical vein endothelial cells (HUVEC) and that Smad and ERK pathways communicate through signal crosstalk.

    Methods: HUVECs were treated with BMP-4 and/or MEK inhibitors. Smad 6 and constitutively active (ca) MEK1 were overexpressed. Loss of function of Smad 4 and Smad 6 was achieved by specific siRNA transfection. Cell lysates were analyzed by western blotting for Smad and ERK phosphorylation. HUVEC spheroids were generated for angiogenesis quantification.

    Results: Treatment with BMP-4 results in a dose- and time-dependent activation of the MEK-ERK 1/2 pathway in addition to activation of the Smad pathway and is blocked by MEK inhibitors. Quantitative in-gel angiogenesis assays in the presence or absence of MEK inhibitors demonstrate that ERK signals are necessary for BMP-4 induced capillary sprouting. Furthermore sprouting is not blocked by inhibition of the Smad signaling pathway. Overexpression of the inhibitory Smad 6 inhibits ERK phosphorylation and ERK-induced capillary sprouting, whereas loss of function of Smad 4 has no effect.

    Conclusions: We demonstrate that ERK1/2 functions as an alternative pathway in BMP-4 signaling in HUVECs. Capillary sprouting induced by BMP-4 is dependent on ERK phosphorylation. ERK is essential for efficient transduction of BMP signals and serves as a positive feedback mechanism. On the other hand, stimulation of Smad 6 inhibits ERK activation and thus results in a negative feedback loop to fine-tune BMP signaling in HUVECs.

    Funded by: NHLBI NIH HHS: HL072347, HL61656, HL65619; NIA NIH HHS: AG024282

    Cardiovascular research 2007;76;3;390-9

  • An altered fibronectin matrix induces anoikis of human squamous cell carcinoma cells by suppressing integrin alpha v levels and phosphorylation of FAK and ERK.

    Kamarajan P and Kapila YL

    Department of Periodontics and Oral Medicine, University of Michigan, School of Dentistry, 1011 N. University Ave, Room 5223, Ann Arbor, MI 48109-1078, USA.

    Fibronectin regulates many cellular processes, including migration, proliferation, differentiation, and survival. Previously, we showed that squamous cell carcinoma (SCC) cell aggregates escape suspension-induced, p53-mediated anoikis by engaging in fibronectin-mediated survival signals through focal adhesion kinase (FAK). Here we report that an altered matrix, consisting of a mutated, nonfunctional high-affinity heparin-binding domain and the V region of fibronectin (V+H-), induced anoikis in human SCC cells; this response was blocked by inhibitors of caspase-8 and caspase-3. Anoikis was mediated by downregulation of integrin alpha v in a panel of SCC cells and was shown to be proteasome-dependent. Overexpression of integrin alpha v or FAK inhibited the increase in caspase-3 activation and apoptosis, whereas suppression of alpha v or FAK triggered a further significant increase in apoptosis, indicating that the apoptosis was mediated by suppression of integrin alpha v levels and dephosphorylation of FAK. Treatment with V+H- decreased the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, and direct activation of ERK by constitutively active MEK1, an ERK kinase, increased ERK1 and ERK2 phosphorylation and inhibited the increase in apoptosis induced by V+H-. ERK acted downstream from alpha v and FAK signals, since alpha v and FAK overexpression inhibited both the decrease in ERK phosphorylation and the increase in anoikis triggered by V+H-. These findings provide evidence that mutations in the high-affinity heparin-binding domain in association with the V region of fibronectin, or altered fibronectin matrices, induce anoikis in human SCC cells by modulating integrin alpha v-mediated phosphorylation of FAK and ERK.

    Funded by: NIDCR NIH HHS: R01 DE014429

    Apoptosis : an international journal on programmed cell death 2007;12;12;2221-31

  • Activated protein C decreases tumor necrosis factor related apoptosis-inducing ligand by an EPCR- independent mechanism involving Egr-1/Erk-1/2 activation.

    O'Brien LA, Richardson MA, Mehrbod SF, Berg DT, Gerlitz B, Gupta A and Grinnell BW

    Biotechnology Discovery Research, Lilly Research Laboratories, Indianapolis, IN 46285, USA.

    Unlabelled: Background- APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism.

    APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase.

    Conclusions: Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.

    Arteriosclerosis, thrombosis, and vascular biology 2007;27;12;2634-41

  • c-Cbl is not required for ERK1/2-dependent degradation of BimEL.

    Wiggins CM, Band H and Cook SJ

    Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

    Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim(EL) is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim(EL) turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim(EL) is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim(EL) does not involve its tyrosine phosphorylation; indeed, Bim(EL) is not a tyrosine phosphoprotein. Furthermore, Bim(EL) fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim(EL) turnover proceeds normally in Cbl-containing or Cbl-/- fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim(EL) turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/E02162X/1, BBS/E/B/0000C199, BBS/E/B/0000H151; NCI NIH HHS: CA 76118, CA 87986, CA 99163, CA 99900, R01 CA076118-02, R01 CA087986, R01 CA087986-01, R01 CA099163, R01 CA099163-01, R01 CA099900, R01 CA099900-01

    Cellular signalling 2007;19;12;2605-11

  • Calcitonin targets extracellular signal-regulated kinase signaling pathway in human cancers.

    Nakamura M, Han B, Nishishita T, Bai Y and Kakudo K

    Department of Pathology, Wakayama Medical University, Kimiidera 811-1, Wakayama 641-0012, Japan. nakamuram@kawasakigakuen.ac.jp

    The mitogen-activated protein kinases (MAPKs) signaling pathway is a potential target in cancer therapy. Constitutive phosphorylated extracellular signal-regulated kinase (ERK1/2), which is one of the MAPKs has been detected in a variety of tumors. Calcitonin (CT) is a polypeptide hormone secreted by the thyroid gland and has been used to treat the osteoporosis and humoral hypercalcemia of malignancy. We report that CT decreases ERK1/2 phosphorylation in cancer cells showing constitutive phosphorylated ERK1/2. In MDA-MB-231 cells, a breast cancer cell line showing constitutive phosphorylated ERK1/2, CT phosphorylated c-Raf at Ser(259) via the protein kinase A pathway, resulting in suppression of ERK1/2 phosphorylation. CT significantly reduced the tumor volume of MDA-MB-231 cells showing constitutive phosphorylated ERK1/2 compared with saline buffer. However, CT did not exert any significant effects on the proliferation of MCF-7 cells, a breast cancer cell line, showing non-constitutive phosphorylated ERK1/2. These novel findings indicate that CT may be used to target ERK in the treatment of cancer.

    Journal of molecular endocrinology 2007;39;6;375-84

  • IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-beta , NF-kappaB, and AP-1 activation.

    Cortez DM, Feldman MD, Mummidi S, Valente AJ, Steffensen B, Vincenti M, Barnes JL and Chandrasekar B

    Department of Veterans Affairs South Texas Veterans Health Care System, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

    Matrix metalloproteinases (MMPs) degrade collagen and mediate tissue remodeling. The novel cytokine IL-17 is expressed during various inflammatory conditions and modulates MMP expression. We investigated the effect of IL-17 on MMP-1 expression in primary human cardiac fibroblasts (HCF) and delineated the signaling pathways involved. HCF were treated with recombinant human IL-17. MMP-1 expression was analyzed by Northern blotting, RT-quantitative PCR, Western blotting, and ELISA; transcriptional induction and transcription factor binding by EMSA, ELISA, and reporter assay; and p38 MAPK and ERK1/2 activation by protein kinase assays and Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA), and adenoviral dominant-negative expression vectors. IL-17 stimulated MMP-1 gene transcription, net mRNA levels, protein, and promoter-reporter activity in HCF. This response was blocked by IL-17 receptor-Fc chimera and IL-17 receptor antibodies, but not by IL-6, TNF-alpha, or IL-1beta antibodies. IL-17-stimulated type I collagenase activity was inhibited by the MMP inhibitor GM-6001 and by siRNA-mediated MMP-1 knockdown. IL-17 stimulated activator protein-1 [AP-1 (c-Fos, c-Jun, and Fra-1)], NF-kappaB (p50 and p65), and CCAAT enhancer-binding protein (C/EBP)-beta DNA binding and reporter gene activities, effects attenuated by antisense oligonucleotides, siRNA-mediated knockdown, or expression of dominant-negative signaling proteins. Inhibition of AP-1, NF-kappaB, or C/EBP activation attenuated IL-17-stimulated MMP-1 expression. IL-17 induced p38 MAPK and ERK1/2 activation, and inhibition by SB-203580 and PD-98059 blunted IL-17-mediated transcription factor activation and MMP-1 expression. Our data indicate that IL-17 induces MMP-1 in human cardiac fibroblasts directly via p38 MAPK- and ERK-dependent AP-1, NF-kappaB, and C/EBP-beta activation and suggest that IL-17 may play a critical role in myocardial remodeling.

    Funded by: NIDCR NIH HHS: DE 017139, K02 DE 016312

    American journal of physiology. Heart and circulatory physiology 2007;293;6;H3356-65

  • LIV-1 suppression inhibits HeLa cell invasion by targeting ERK1/2-Snail/Slug pathway.

    Zhao L, Chen W, Taylor KM, Cai B and Li X

    Center for Laboratory Medicine, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.

    It was reported that expression of the estrogen-regulated zinc transporter LIV-1 was particularly high in human cervical cancer cell line HeLa. This result prompted us to study the role that LIV-1 played in human cervical cancer. The results of real-time PCR showed that LIV-1 mRNA was significantly higher in cervical cancer in situ than in normal tissues. RNAi mediated suppression of LIV-1 in HeLa cells significantly inhibited cell proliferation, colony formation, migration, and invasive ability, but had no effect on cell apoptosis. Furthermore, LIV-1 suppression is accompanied by down-regulation of p44/42 MAPK, phospho-p44/42 MAPK, Snail and Slug expression levels. Hence, our data provide the first evidence that LIV-1 mRNA is overexpressed in cervical cancer in situ and is involved in invasion of cervical cancer cells through targeting MAPK-mediated Snail and Slug expression.

    Biochemical and biophysical research communications 2007;363;1;82-8

  • [The role of ERK1/2 in leptin promoting the proliferation of human endometrial cancer cell line Ishikawa].

    Gong C, Liu Y, Xiao W, Yin J, Wang DH and Sheng H

    Department of Gynecology and Obstetrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, PR China.

    Epidemiologic studies showed that leptin is closely related to the tumorigenesis of endometrial cancer, but the mechanism is unclear. As a mitogenic agent, leptin can promote the proliferation of many kinds of cells. This study was to explore the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in leptin promoting the proliferation of human endometrial cancer cell line Ishikawa.

    Methods: The expression of leptin receptor OB-Rb in Ishikawa cells was detected by fluoroimmunoassay. Ishikawa cells were treated by leptin at various concentrations (0, 10, 50, 100, and 150 ng/ml) for different time (6, 12, and 24 h). Cell proliferation was examined by MTT assay. Meanwhile, the effect of PD98059, selective inhibitor of ERK1/2, on the proliferation of Ishikawa cells induced by leptin was also studied. Ishikawa cells were treated with 100 ng/ml leptin for different time (20, 40, and 60 min), then the levels of phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 were examined by Western blot.

    Results: Fluoroimmunoassay showed the presence of OB-Rb in Ishikawa cells. Leptin stimulated the proliferation of Ishikawa cells. This effect was maximal at 100 ng/ml after 24-hour treatment, and there was no significant difference between 100 ng/ml group and 150 ng/ml group (P=0.129). Blocking ERK1/2 phosphorylation by PD98059 significantly reduced the proliferation of Ishikawa cells stimulated by leptin. When treated with 100 ng/ml Leptin and 100 micromol/L PD98059 for 24 h, cell proliferation rate was (6.88+/-0.86)%. ERK1/2 phosphorylation was enhanced significantly in Ishikawa cells after treatment of 100 ng/ml leptin.

    Conclusion: Leptin may promote the proliferation of endometrial cancer Ishikawa cells by activating ERK1/2 signaling pathway.

    Ai zheng = Aizheng = Chinese journal of cancer 2007;26;11;1211-4

  • The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells.

    Gomes E, Papa L, Hao T and Rockwell P

    Department of Biological Sciences, Hunter College of The City University of New York, 695 Park Ave, New York, NY 10021, USA.

    Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli. Whether a relationship exists between VEGFR2 and PKA in mediating neuroprotection under stressful conditions is unknown. Using SK-N-SH neuronal cells as a model system, we show that serum deprivation induces an upregulation in VEGF and VEGFR2 that concomitantly serves as a prosurvival signaling pathway. Inhibitor studies revealed that PKA functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and a subsequent cell death. The loss in cell viability induced by VEGFR2 and PKA inhibition was prevented by caspase inhibition or overexpression of ERK1. Overexpression of the antiapoptotic protein Bcl-xL also promoted survival when VEGFR2 function was blocked. However, the protection elicited by all three treatments were prevented by the inclusion of a selective inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase that activates ERK1/2. Taken together, these findings suggested that PKA and VEGFR2 converge at the MEK/ERK1/2 pathway to protect serum starved neuronal cells from a caspase-dependent cell death.

    Funded by: NCRR NIH HHS: RR03037

    Molecular and cellular biochemistry 2007;305;1-2;179-90

  • Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK-->ERK1/2 pathway.

    Kodiha M, Rassi JG, Brown CM and Stochaj U

    Department of Physiology, McGill University, Montreal H3G 1Y6, Canada.

    5'-AMP-activated protein kinase (AMPK) serves as an energy sensor and is at the center of control for a large number of metabolic reactions, thereby playing a crucial role in Type 2 diabetes and other human diseases. AMPK is present in the nucleus and cytoplasm; however, the mechanisms that regulate the intracellular localization of AMPK are poorly understood. We have now identified several factors that control the distribution of AMPK. Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)-->extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEK-->ERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease.

    American journal of physiology. Cell physiology 2007;293;5;C1427-36

  • Oncostatin M-induced effects on EMT in human proximal tubular cells: differential role of ERK signaling.

    Pollack V, Sarközi R, Banki Z, Feifel E, Wehn S, Gstraunthaler G, Stoiber H, Mayer G, Montesano R, Strutz F and Schramek H

    Division of Nephrology, Department of Internal Medicine, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria.

    Growing evidence suggests that a proportion of interstitial myofibroblasts detected during renal tubulointerstitial fibrosis originates from tubular epithelial cells by a process called epithelial-mesenchymal transition (EMT). The IL-6-type cytokine oncostatin M (OSM) has been recently implicated in the induction of EMT. We investigated OSM effects on the expression of both cell-cell contact proteins and mesenchymal markers and studied OSM-induced intracellular signaling mechanisms associated with these events in human proximal tubular cells. Human recombinant OSM attenuated the expression of N-cadherin, E-cadherin, and claudin-2 in human kidney-2 (HK-2) cells associated with the induction of HK-2 cell scattering in 3D collagen matrices. Conversely, expression of collagen type I, vimentin, and S100A4 was induced by OSM. OSM-stimulated cell scattering was inhibited by antibodies against gp130. Besides inducing phosphorylation of Stat1 and Stat3, OSM led to a strong concentration- and time-dependent phosphorylation of the mitogen-activated protein kinases ERK1, ERK2, and ERK5. MEK1/2 inhibitor U0126 (10 muM) blocked basal and OSM-induced ERK1/2 phosphorylation but not phosphorylation of either ERK5 or Stat1/3. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at concentrations which inhibit ERK1/2 phosphorylation but not ERK5 phosphorylation, restored N-cadherin expression in the presence of OSM, inhibited basal claudin-2 expression, but did not affect either basal or OSM-inhibited E-cadherin expression or OSM-induced expression of collagen type I and vimentin. These results suggest that in human proximal tubular cells ERK1/2 signaling represents an important component of OSM's inhibitory effect on N-cadherin expression. Furthermore, functional ERK1/2 signaling is necessary for basal claudin-2 expression.

    American journal of physiology. Renal physiology 2007;293;5;F1714-26

  • Retinol-binding protein-4 attenuates insulin-induced phosphorylation of IRS1 and ERK1/2 in primary human adipocytes.

    Ost A, Danielsson A, Lidén M, Eriksson U, Nystrom FH and Strålfors P

    Department of Cell Biology and Diabetes Research Centre, Linköping University, SE58185 Linköping, Sweden.

    Reduced sensitivity to insulin in adipose, muscle, and liver tissues is a hallmark of type 2 diabetes. Animal models and patients with type 2 diabetes exhibit elevated levels of circulating retinol-binding protein (RBP4), and RBP4 can induce insulin resistance in mice. However, little is known about how RBP4 affects insulin signaling. We examined the mechanisms of action of RBP4 in primary human adipocytes. RBP4-treated adipocytes exhibited the same molecular defects in insulin signaling, via IRS1 to MAP kinase, as in adipocytes from patients with type 2 diabetes. Without affecting autophosphorylation of the insulin receptor, RBP4 blocked the insulin-stimulated phosphorylation of IRS1 at serine (307) [corresponding to serine (302) in the murine sequence] and concomitantly increased the EC50 (from 0.5 to 2 nM) for insulin stimulation of IRS1 phosphorylation at tyrosine. The phosphorylation of IRS1 at serine (312) [corresponding to serine (307) in the murine sequence] was not affected in cells from diabetic patients and was also not affected by RBP4. The EC50 for insulin stimulation of downstream phosphorylation of MAP kinase ERK1/2 was increased (from 0.2 to 0.8 nM) by RBP4. We show that ERK1/2 phosphorylation is similarly impaired in adipocytes from patients with type 2 diabetes. However, the sensitivity to insulin for downstream signaling to control of protein kinase B and glucose uptake was not affected by RBP4. When insulin-resistant adipocytes from patients with type 2 diabetes were incubated with antibodies against RBP4, insulin-induced phosphorylation of IRS1 at serine (307) was normalized and the EC50 for insulin stimulation of ERK1/2 phosphorylation was reduced. Endogenous levels of RBP4 were markedly reduced in adipocytes from obese or type 2 diabetic subjects, whereas expression levels of RBP4 mRNA were unaffected. These findings indicate that RBP4 may be released from diabetic adipocytes and act locally to inhibit phosphorylation of IRS1 at serine (307), a phosphorylation site that may integrate nutrient sensing with insulin signaling.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;13;3696-704

  • Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction.

    Martínez N, García-Domínguez CA, Domingo B, Oliva JL, Zarich N, Sánchez A, Gutiérrez-Eisman S, Llopis J and Rojas JM

    Unidad de Biología Celular, Centro Nacional de Microbiología, Instituto de Salud Carlos III, carretera Majadahonda-Pozuelo, Km 2, Majadahonda-28220, Madrid, Spain.

    Sprouty2 has been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. Sprouty2 directly interacts with the adapter protein Grb2, member of the receptor tyrosine kinase-induced signaling pathways. In considering the functional role of Grb2, we investigated whether the interaction with this protein was responsible for ERK pathway inhibition. We found that the binding between Sprouty2 and Grb2 is constitutive, independent of Sprouty2 tyrosine phosphorylation, although it is increased when fibroblast growth factor receptor is activated. This connection is mediated by the N-terminal SH3 domain of Grb2 and two Sprouty2 proline-rich stretches (residues 59-64 and 303-307). Most importantly, a double Sprouty2 mutant (hSpry2 P59AP304A), which is unable to bind Grb2, developed at a similar inhibition level of fibroblast growth factor receptor-ERK pathway than that which originated from Sprouty2 wt. These results are evidence that the Sprouty2 mechanism of ERK inhibition is independent of Grb2 binding.

    Cellular signalling 2007;19;11;2277-85

  • Glycogen synthase kinase-3beta and p38 phosphorylate cyclin D2 on Thr280 to trigger its ubiquitin/proteasome-dependent degradation in hematopoietic cells.

    Kida A, Kakihana K, Kotani S, Kurosu T and Miura O

    Department of Hematology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan.

    Cyclin D2 plays an important role in regulation of hematopoietic cell proliferation by cytokines and is implicated in oncogenesis of various hematopoietic malignancies. However, mechanisms regulating cyclin D2 stability and its expression level have remained to be known. Here, we demonstrate that interleukin-3 signaling stabilizes cyclin D2 by inhibition of glycogen synthase kinase-3beta (GSK3beta) through Janus kinase2-dependent activation of phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathway in hematopoietic 32Dcl3 cells. On the other hand, osmotic stress was shown to induce a rapid proteasomal degradation of cyclin D2, which was mediated by activation of p38. GSK3beta and p38 was demonstrated to phosphorylate cyclin D2 on Thr280 in vitro, while a cyclin D2 mutant with this residue substituted with Ala was found to be resistant to ubiquitination and proteasome-dependent degradation in 32Dcl3 cells. Inhibition of the PI3K pathway or induction of osmotic stress also caused a rapid proteasomal degradation of cyclin D2 in primary leukemic or myeloma cells. These results indicate that cyclin D2 expression in normal and malignant hematopoietic cells is regulated by ubiquitin/proteasome-dependent degradation that is triggered by Thr280 phosphorylation by GSK3beta or p38, which is induced by inhibition of the PI3K pathway or by osmotic stress, respectively.

    Oncogene 2007;26;46;6630-40

  • Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP): identification of sites targeted by different kinases.

    Hlavanda E, Klement E, Kókai E, Kovács J, Vincze O, Tökési N, Orosz F, Medzihradszky KF, Dombrádi V and Ovádi J

    Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, H-1113, Hungary.

    Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 +/- 0.3, 2.2 +/- 0.3, and 0.9 +/- 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr(14), Ser(18), Ser(160) for Cdk5; Ser(18), Ser(160) for ERK2, and Ser(32) for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (K(d) = 2.5-2.7 microM) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr(14) and/or Ser(18) in its unfolded N-terminal tail.

    The Journal of biological chemistry 2007;282;40;29531-9

  • The hepatitis B virus protein MHBs(t) sensitizes hepatoma cells to TRAIL-induced apoptosis through ERK2.

    Liang X, Du J, Liu Y, Cui M, Ma C, Han L, Qu Z, Zhang Z, Sun Z, Zhang L, Chen YH and Sun W

    Institute of Immunology, School of Medicine, Shandong University, Jinan, 250012, People's Republic of China.

    The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Infectious agents, such as hepatitis B virus (HBV), may play important roles in regulating the sensitivity of hepatocytes to TRAIL. Our previous studies showed that HBx, a protein encoded by the HBV genome, enhanced TRAIL-induced apoptosis through upregulating Bax. We report here that another HBV protein called MHBs(t) (C-terminally truncated middle hepatitis B surface protein) is also a potent regulator of TRAIL-induced apoptosis. Overexpressing MHBs(t) in hepatoma cells enhanced TRAIL-induced apoptosis. Mechanistic studies reveal that MHBs(t) had no effect on Bax or TRAIL receptor expression or procaspase-8 activation, but selectively enhanced the activation of ERK2 (extracellular signal-regulated kinase 2) and the degradation of procaspases-3 and 9. ERK2 activation is required for the MHBs(t) effect because ERK2 inhibition by its inhibitor PD98059 significantly reversed TRAIL-induced apoptosis of MHBs(t)-transfected cells. These results establish that unlike HBx, MHBs(t) enhances TRAIL-induced hepatocyte apoptosis through a novel mechanism that involves ERK2. Therefore, manipulating the ERK2 signaling pathway may provide new therapeutic opportunities to contain hepatic cell death during HBV infection.

    Apoptosis : an international journal on programmed cell death 2007;12;10;1827-36

  • Significance of the expression of phosphorylated-STAT3, -Akt, and -ERK1/2 in several tumors of the epidermis.

    Lin N, Moroi Y, Uchi H, Fukiwake N, Dainichi T, Takeuchi S, Takahara M, Tu Y, Furue M and Urabe K

    Journal of dermatological science 2007;48;1;71-3

  • The complete pathway for ERK2-catalyzed reaction. Evidence for an iso random Bi Bi mechanism.

    Wang ZX and Wu JW

    Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. zxwang@sun5.ibp.ac.cn

    In the present study, the enzymatic mechanism of ERK2 is re-examined by a combination of steady-state kinetic studies in the absence and presence of viscosogenic agents. Kinetic studies carried out in various concentrations of sucrose revealed that both k(cat) and k(cat)/K(m) for either ATP or EtsDelta138 were highly sensitive to solvent viscosity, suggesting that the rapid equilibrium assumption is not valid for the phosphorylation of protein substrate by ERK2. Furthermore, the kinetic analysis with the minimal random Bi Bi reaction mechanism is shown to be inconsistent with the principle of the detailed balance. This inconsistent calculation strongly suggests that there is isomerization of the enzyme-substrate ternary complex. The viscosity-dependent steady-state kinetic data are combined to establish a kinetic mechanism for the ERK2-catalyzed reaction that predicts initial reaction velocities under varying concentrations of ATP and substrate. These results complement previous structure-function studies of mitogen-activated protein kinases and provide important insight for mechanistic interpretation of the kinase functions.

    The Journal of biological chemistry 2007;282;38;27678-84

  • ERK2-mediated C-terminal serine phosphorylation of p300 is vital to the regulation of epidermal growth factor-induced keratin 16 gene expression.

    Chen YJ, Wang YN and Chang WC

    Department of Pharmacology, Institute of Basic Medical Sciences, College of Medicine, Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan 701, Taiwan.

    We previously reported that the epidermal growth factor (EGF) regulates the gene expression of keratin 16 by activating the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling which in turn enhances the recruitment of p300 to the keratin 16 promoter. The recruited p300 functionally cooperates with Sp1 and c-Jun to regulate the gene expression of keratin 16. This study investigated in detail the molecular events incurred upon p300 whereby EGF caused an enhanced interaction between p300 and Sp1. EGF apparently induced time- and dose-dependent phosphorylation of p300, both in vitro and in vivo, through the activation of ERK2. The six potential ERK2 phosphorylation sites, including three threonine and three serine residues as revealed by sequential analysis, were first identified in vitro. Confirmation of these six sites in vivo indicated that these three serine residues (Ser-2279, Ser-2315, and Ser-2366) on the C terminus of p300 were the major signaling targets of EGF. Furthermore, the C-terminal serine phosphorylation of p300 stimulated its histone acetyltransferase activity and enhanced its interaction with Sp1. These serine phosphorylation sites on p300 controlled the p300 recruitment to the keratin 16 promoter. When all three serine residues on p300 were replaced by alanine, EGF could no longer induce the gene expression of keratin 16. Taken together, these results strongly suggested that the ERK2-mediated C-terminal serine phosphorylation of p300 was a key event in the regulation of EGF-induced keratin 16 expression. These results also constituted the first report identifying the unique p300 phosphorylation sites induced by ERK2 in vivo.

    The Journal of biological chemistry 2007;282;37;27215-28

  • Interleukin-17 stimulates C-reactive protein expression in hepatocytes and smooth muscle cells via p38 MAPK and ERK1/2-dependent NF-kappaB and C/EBPbeta activation.

    Patel DN, King CA, Bailey SR, Holt JW, Venkatachalam K, Agrawal A, Valente AJ and Chandrasekar B

    Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, TX 78229-4404, USA.

    Elevated systemic levels of the acute phase C-reactive protein (CRP) are predictors of future cardiovascular events. There is evidence that CRP may also play a direct role in atherogenesis. Here we determined whether the proinflammatory interleukin (IL)-17 stimulates CRP expression in hepatocytes (Hep3B cell line and primary hepatocytes) and coronary artery smooth muscle cells (CASMC). Our results demonstrate that IL-17 potently induces CRP expression in Hep3B cells independent of IL-1beta and IL-6. IL-17 induced CRP promoter-driven reporter gene activity that could be attenuated by dominant negative IkappaBalpha or C/EBPbeta knockdown and stimulated both NF-kappaB and C/EBP DNA binding and reporter gene activities. Targeting NF-kappaB and C/EBPbeta activation by pharmacological inhibitors, small interfering RNA interference and adenoviral transduction of dominant negative expression vectors blocked IL-17-mediated CRP induction. Overexpression of wild type p50, p65, and C/EBPbeta stimulated CRP transcription. IL-17 stimulated p38 MAPK and ERK1/2 activation, and SB203580 and PD98059 blunted IL-17-mediated NF-kappaB and C/EBP activation and CRP transcription. These results, confirmed in primary human hepatocytes and CASMC, demonstrate for the first time that IL-17 is a potent inducer of CRP expression via p38 MAPK and ERK1/2-dependent NF-kappaB and C/EBPbeta activation and suggest that IL-17 may mediate chronic inflammation, atherosclerosis, and thrombosis.

    Funded by: NHLBI NIH HHS: HL 68020, R01 HL068020, R01 HL071233

    The Journal of biological chemistry 2007;282;37;27229-38

  • The insulin-like growth factor type 1 and insulin-like growth factor type 2/mannose-6-phosphate receptors independently regulate ERK1/2 activity in HEK293 cells.

    El-Shewy HM, Lee MH, Obeid LM, Jaffa AA and Luttrell LM

    Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    Insulin-like growth factor types 1 and 2 (IGF-1; IGF-2) and insulin-like peptides are all members of the insulin superfamily of peptide hormones but bind to several distinct classes of membrane receptor. Like the insulin receptor, the IGF-1 receptor is a heterotetrameric receptor tyrosine kinase, whereas the IGF-2/ mannose 6-phosphate receptor is a single transmembrane domain protein that is thought to function primarily as clearance receptors. We recently reported that IGF-1 and IGF-2 stimulate the ERK1/2 cascade by triggering sphingosine kinase-dependent "transactivation" of G protein-coupled sphingosine-1-phosphate receptors. To determine which IGF receptors mediate this effect, we tested seven insulin family peptides, IGF-1, IGF-2, insulin, and insulin-like peptides 3, 4, 6, and 7, for the ability to activate ERK1/2 in HEK293 cells. Only IGF-1 and IGF-2 potently activated ERK1/2. Although IGF-2 was predictably less potent than IGF-1 in activating the IGF-1 receptor, they were equipotent stimulators of ERK1/2. Knockdown of IGF-1 receptor expression by RNA interference reduced the IGF-1 response to a greater extent than the IGF-2 response, suggesting that IGF-2 did not signal exclusively via the IGF-1 receptor. In contrast, IGF-2 receptor knockdown markedly reduced IGF-2-stimulated ERK1/2 phosphorylation, with no effect on the IGF-1 response. As observed previously, both the IGF-1 and the IGF-2 responses were sensitive to pertussis toxin and the sphingosine kinase inhibitor, dimethylsphingosine. These data indicate that endogenous IGF-1 and IGF-2 receptors can independently initiate ERK1/2 signaling and point to a potential physiologic role for IGF-2 receptors in the cellular response to IGF-2.

    Funded by: NIDDK NIH HHS: DK58283; NIGMS NIH HHS: GM62887

    The Journal of biological chemistry 2007;282;36;26150-7

  • Protein kinase WNK2 inhibits cell proliferation by negatively modulating the activation of MEK1/ERK1/2.

    Moniz S, Veríssimo F, Matos P, Brazão R, Silva E, Kotelevets L, Kotevelets L, Chastre E, Gespach C and Jordan P

    Centre of Human Genetics, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisbon, Portugal.

    The recently identified subfamily of WNK protein kinases is characterized by a unique sequence variation in the catalytic domain and four related human WNK genes were identified. Here, we describe the cloning and functional analysis of the human family member WNK2. We show that the depletion of endogenous WNK2 expression by RNA interference in human cervical HeLa cancer cells led to the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases but, in contrast to the depletion of WNK1, had no effect on ERK5. Furthermore, expression of a kinase-dead WNK2-K207M mutant also activated ERK1/2 suggesting that WNK2 catalytic activity is required. Depletion of WNK2 expression increased G1/S progression and potentiated the cellular response to low epidermal growth factor concentrations. The molecular mechanism of ERK1/2 activation in WNK2-depleted cells lies downstream of the Raf kinases and involves MEK1 phosphorylation at serine 298 in both HeLa and HT29 colon cancer cells. This modification is linked to the upregulation of MEK1 activity toward ERK1/2. Together, these results provide evidence that WNK2 is involved in the modulation of growth factor-induced cancer cell proliferation through the MEK1/ERK1/2 pathway. The data identify WNK2 as a candidate tumor suppressor gene and suggest a coordinated activity of WNK kinases in the regulation of cell proliferation.

    Oncogene 2007;26;41;6071-81

  • AKT and MAPK signaling in KGF-treated and UVB-exposed human epidermal cells.

    Lotti LV, Rotolo S, Francescangeli F, Frati L, Torrisi MR and Marchese C

    Department of Experimental Medicine, University Sapienza, Viale Regina Elena, Rome, Italy.

    Regulation of proliferation and differentiation in keratinocyte is a complex and dynamic process that involves activation of multiple signaling pathways triggered by different growth factors. Keratinocyte growth factor (KGF) is not only a potent mitogen, but differently from other growth factors, is a potent inducer of differentiation. The MAP kinase and AKT pathways are involved in proliferation and differentiation of many cell types including keratinocytes. We investigated here the role of KGF in modulating AKT and MAPK activity during differentiation of human keratinocytes. Our results show that the mechanisms of action of KGF are dose-dependent and that a sustained activation of the MAPK signaling cascade causes a negative regulation of AKT. We also demostrated increasing expression of KGFR substrates, such as PAK4 during keratinocyte differentiation parallel to the receptor upregulation.

    Journal of cellular physiology 2007;212;3;633-42

  • Angiotensin II and IGF-1 regulate connexin43 expression via ERK and p38 signaling pathways in vascular smooth muscle cells of coronary artery bypass conduits.

    Jia G, Mitra AK, Cheng G, Gangahar DM and Agrawal DK

    Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska 68178, USA.

    Background: Changes in connexin expression have been found in vascular smooth muscle cells (VSMCs) during the progression of atherosclerotic lesion and intimal hyperplasia. It is our hypothesis that increased connexin43 expression following stimulation of VSMCs with Ang II and IGF-1 contributes to more proliferation in saphenous vein (SV) than in the internal mammary artery (IMA).

    Using immunohistochemistry, Western blot, and reverse-transcription polymerase chain reaction, we assessed the effect of Ang II and IGF-1 stimulation on connexin43 expression and the signaling pathways involved in VSMCs of SV and IMA.

    Results: Immunostaining demonstrated strong expression of connexin43 in SV compared with IMA after stimulation with Ang II and IGF-1. Ang II up-regulated the expression of connexin43 in VSMCs of SV in a dose- and time-dependent manner. This was inhibited by p38 and ERK MAP kinase inhibitors, SB203580 and PD98059, respectively. In the VSMCs of IMA, the connexin43 expression was markedly low and maintained at a reduced level even after 3 h stimulation. IGF-1 dose-dependently induced mRNA expression of connexin43 in the VSMCs of SV, which was blocked by PD98059. However, in VSMCs of IMA there was no significant effect of IGF-1 on the connexin43 mRNA expression.

    Conclusion: These data suggest that connexin43 expression can be influenced by Ang II and IGF-1 through ERK and p38 pathways and may contribute to the pathogenesis of vein graft disease following coronary artery bypass grafting.

    Funded by: NHLBI NIH HHS: R01HL070885, R01HL073349

    The Journal of surgical research 2007;142;1;137-42

  • D-glucose stimulation of L-arginine transport and nitric oxide synthesis results from activation of mitogen-activated protein kinases p42/44 and Smad2 requiring functional type II TGF-beta receptors in human umbilical vein endothelium.

    Vásquez R, Farías M, Vega JL, Martin RS, Vecchiola A, Casanello P and Sobrevia L

    Cellular and Molecular Physiology Laboratory, Department of Obstetrics and Gynaecology, Medical Research Centre, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.

    Elevated extracellular D-glucose increases transforming growth factor beta1 (TGF-beta1) release from human umbilical vein endothelium (HUVEC). TGF-beta1, via TGF-beta receptors I (TbetaRI) and TbetaRII, activates Smad2 and mitogen-activated protein kinases p44 and p42 (p42/44(mapk)). We studied whether D-glucose-stimulation of L-arginine transport and nitric oxide synthesis involves TGF-beta1 in primary cultures of HUVEC. TGF-beta1 release was higher ( approximately 1.6-fold) in 25 mM (high) compared with 5 mM (normal) D-glucose. TGF-beta1 increases L-arginine transport (half maximal effect approximately 1.6 ng/ml) in normal D-glucose, but did not alter high D-glucose-increased L-arginine transport. TGF-beta1 and high D-glucose increased hCAT-1 mRNA expression ( approximately 8-fold) and maximal transport velocity (V(max)), L-[(3)H]citrulline formation from L-[(3)H]arginine (index of NO synthesis) and endothelial NO synthase (eNOS) protein abundance, but did not alter eNOS phosphorylation. TGF-beta1 and high D-glucose increased p42/44(mapk) and Smad2 phosphorylation, an effect blocked by PD-98059 (MEK1/2 inhibitor). However, TGF-beta1 and high D-glucose were ineffective in cells expressing a truncated, negative dominant TbetaRII. High D-glucose increases L-arginine transport and eNOS expression following TbetaRII activation by TGF-beta1 involving p42/44(mapk) and Smad2 in HUVEC. Thus, TGF-beta1 could play a crucial role under conditions of hyperglycemia, such as gestational diabetes mellitus, which is associated with fetal endothelial dysfunction.

    Journal of cellular physiology 2007;212;3;626-32

  • Evidence for the regulation of contraction-induced fatty acid oxidation via extracellular signal-regulated kinase 1/2 activation independent of changes in fatty acid uptake.

    Raney MA and Turcotte LP

    Department of Kinesiology, College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, CA 90089-0652, USA.

    Data show that extracellular signal-regulated kinase 1/2 (ERK1/2) may be involved in the regulation of fatty acid (FA) uptake during muscle contraction via stimulation of CD36 translocation to the plasma membrane. The perfused hind limb model was used to determine (1) the importance of ERK1/2 signaling on contraction-induced FA uptake and (2) the effect of ERK1/2-mediated FA uptake on contraction-induced FA oxidation. We perfused rat hind limbs with 8 mmol/L glucose, 550 micromol/L palmitate, and no insulin at rest in the absence of inhibitor and during moderate-intensity electrical stimulation and dose-dependent pharmacologic inhibition of ERK1/2 using increasing concentrations of PD98059 (P1 = none, P2 = 10 micromol/L, P3 = 20 micromol/L, P4 = 50 micromol/L). Increasing PD98059 concentration resulted in a gradual decrease in contraction-induced ERK1/2 phosphorylation, and this was accompanied by a decrease in contraction-induced FA uptake (concentration required for 50% inhibition [IC(50)] = 15.8 +/- 1.6 mumol/L) and in plasma membrane CD36 content (IC(50) = 8.7 +/- 0.3 micromol/L) (P < .05). Percent FA oxidation was significantly lower in P3 and P4 compared with P1 and P2. Based on IC(50) values, FA oxidation demonstrated a greater sensitivity than FA uptake to changes in ERK1/2 phosphorylation (IC(50) = 5.4 +/- 0.3 micromol/L) (P < .05). A positive correlation was found between FA uptake and plasma membrane CD36 content (R(2) = 0.85, P < .05). Plasma membrane CD36 content, FA uptake, and FA oxidation each shared a positive correlation with ERK1/2 phosphorylation (R(2) = 0.64, 0.66, and 0.71, respectively; P < .05). These results suggest that during moderate-intensity muscle contraction, ERK1/2 phosphorylation is required for translocation of CD36 to the plasma membrane and the subsequent increase in FA uptake. In addition, these data suggest that ERK1/2 signaling may be involved in the regulation of FA oxidation independently of its effects on FA uptake.

    Metabolism: clinical and experimental 2007;56;9;1192-200

  • Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways.

    Bouchard V, Demers MJ, Thibodeau S, Laquerre V, Fujita N, Tsuruo T, Beaulieu JF, Gauthier R, Vézina A, Villeneuve L and Vachon PH

    Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

    Human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. In the present study, we analyzed the roles of focal adhesion kinase (Fak)/Src signaling to the PI3-K/Akt-1 and mitogen-activated protein kinase (MEK)/extracellular regulated kinases (Erk) pathways, within the context of such differentiation-state distinctions. Anoikis was induced by inhibition of beta1 integrins (antibody blocking), inhibition of Fak (pharmacologic inhibition or overexpression of dominant negative mutants), or by maintaining cells in suspension. Activation parameters of Fak, Src, Akt-1, and Erk1/2 were analyzed. Activities of Src, Akt-1, or Erk1/2 were also blocked by pharmacological inhibition or by overexpression of dominant-negative mutants. We report that: (1) the loss or inhibition of beta1 integrin binding activity causes anoikis and results in a down-activation of Fak, Src, Akt-1, and Erk1/2 in both undifferentiated, and differentiated cells; (2) the inhibition of Fak likewise causes anoikis and a down-activation of Src, Akt-1, and Erk1/2, regardless of the differentiation state; (3) Src, PI3-K/Akt-1, and MEK/Erk contribute to the survival of differentiated cells, whereas MEK/Erk does not play a role in the survival of undifferentiated ones; (4) the inhibition/loss of beta1 integrin binding and/or Fak activity results in a loss of Src engagement with Fak, regardless of the state of differentiation; and (5) Src contributes to the activation of both the PI3-K/Akt-1 and MEK/Erk pathways in undifferentiated cells, but does not influence PI3-K/Akt-1 in differentiated ones. Hence, Fak/Src signaling to the PI3-K/Akt-1 and MEK/Erk pathways undergoes a differentiation state-specific uncoupling which ultimately reflects upon the selective engagement of these same pathways in the mediation of intestinal epithelial cell survival.

    Journal of cellular physiology 2007;212;3;717-28

  • High-dose acetaminophen inhibits the lethal effect of doxorubicin in HepG2 cells: the role of P-glycoprotein and mitogen-activated protein kinase p44/42 pathway.

    Manov I, Bashenko Y, Eliaz-Wolkowicz A, Mizrahi M, Liran O and Iancu TC

    Pediatric Research and Electron Microscopy Unit, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096, Israel. irmanov@tx.technion.ac.il

    Doxorubicin (DOX) is a widely used chemotherapeutic drug for human hepatocellular carcinoma (HCC). A major limitation to its effectiveness is the development of multidrug resistance of cancer cells. In clinical trials, patients with advanced HCC were treated with high-dose acetaminophen (HAAP) in an effort to improve the antitumor activity of chemotherapeutics. In this study, we investigated the effect of concomitant treatment of DOX and HAAP on hepatoma-derived HepG2 cells. Viability, cell cycle distribution, and ultrastructure were examined. Unexpectedly, HAAP, when added to DOX-exposed cells, increased cell viability, released cell cycle arrest, and decreased apoptosis. To elucidate the mechanisms by which HAAP reduces the DOX lethal effect to HepG2 cells, we investigated the multidrug resistance P-glycoprotein (P-gp) and p44/42-mitogen-activated protein kinase (MAPK) pathways. The P-gp function was enhanced by DOX and HAAP, and it was further stimulated during combined treatment, leading to decreased DOX retention. Verapamil (VRP), when added to DOX + HAAP exposure, increased DOX accumulation and restored DOX-induced toxicity. The increased phospho-p44/42-MAPK level in DOX-exposed cells was inhibited by HAAP. In addition, suppression of p44/42 activation by the p44/42-MAPK inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked DOX-induced apoptosis. These findings suggest that the antagonistic effect of concomitant DOX + HAAP treatment occurs as a result of interactive stimulation of P-gp, generating decreased intracellular drug concentrations. Furthermore, inhibition of the p44/42-MAPK phosphorylation by HAAP could abolish the DOX-induced cell death pathway. Thus, combined treatment by DOX + HAAP, intended to improve chemotherapeutic efficacy, could have an opposite effect facilitating cancer cell survival.

    The Journal of pharmacology and experimental therapeutics 2007;322;3;1013-22

  • Leptin induces C-reactive protein expression in vascular endothelial cells.

    Singh P, Hoffmann M, Wolk R, Shamsuzzaman AS and Somers VK

    Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, MN 55905, USA.

    Objective: There is increasing evidence of an association between leptin and increased cardiovascular risk. Higher leptin levels are associated with increased levels of C-reactive protein (CRP), which itself elicits proatherogenic effects in the vascular endothelium. We tested the hypothesis that leptin induces CRP expression in human coronary artery endothelial cells (HCAECs).

    We confirmed the presence of both long and short isoforms of the leptin receptor in cultured HCAECs. Leptin but not IFNalphaA/D nor tumor necrosis factor (TNF) alpha, induced expression of CRP. A dose dependent increase of CRP mRNA and protein was observed with increasing concentration of leptin (0 to 400 ng/mL). This increased CRP expression was attenuated in the presence of anti-leptin receptor antibodies and also by inhibition of ERK1/2 by PD98059 (20 to 40 micromol/L). Time (0 to 60 minutes) and leptin concentration (0 to 200 ng/mL)-dependence of ERK1/2 phosphorylation were evident in response to leptin treatment. Leptin also elicited ROS generation. Inhibition of ROS by catalase (200 microg/mL) prevented ERK1/2 phosphorylation and CRP mRNA transcription.

    Conclusion: Leptin induces CRP expression in HCAECs via activation of the leptin receptor, increased ROS production, and phosphorylation of ERK1/2. These studies suggest a mechanism for the proatherogenic effects of leptin.

    Funded by: NCRR NIH HHS: M01-RR00585; NHLBI NIH HHS: R01HL65176, R01HL70302, R01HL73211

    Arteriosclerosis, thrombosis, and vascular biology 2007;27;9;e302-7

  • Stem cell factor/c-kit receptor signaling enhances the proliferation and invasion of colorectal cancer cells through the PI3K/Akt pathway.

    Yasuda A, Sawai H, Takahashi H, Ochi N, Matsuo Y, Funahashi H, Sato M, Okada Y, Takeyama H and Manabe T

    Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 4678601, Japan. a-yasuda@med.nagoya-cu.ac.jp

    In this study, we examined the role of c-kit receptor (KIT) signal transduction on the proliferation and invasion of colorectal cancer cells. We found that c-kit was expressed in 2 colorectal cancer cell lines as determined by RT-PCR, Western blot, and flow cytometry. In KIT-positive lines, KIT was activated by stem cell factor (SCF). SCF enhanced cellular proliferation of positive lines as demonstrated by the WST-1 proliferation assay. Furthermore, SCF enhanced the invasive ability of KIT-positive cell lines. SCF stimulation upregulated p44/42 mitogen-activated protein kinase (MAPK) and Akt as shown by Western blot. We examined the roles played by p44/42 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways in proliferation and invasion. PI3K/Akt activity strongly correlated with proliferation and invasion and p44/42 MAPK was correlated with only invasion. In conclusion, the SCF-enhanced proliferation and invasion of KIT-positive colorectal cancer cells is achieved mainly through the PI3K/Akt pathway.

    Digestive diseases and sciences 2007;52;9;2292-300

  • Rosiglitazone attenuates NF-kappaB-dependent ICAM-1 and TNF-alpha production caused by homocysteine via inhibiting ERK1/2/p38MAPK activation.

    Bai YP, Liu YH, Chen J, Song T, You Y, Tang ZY, Li YJ and Zhang GG

    Department of Cardiovascular Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410078, China.

    Previous studies demonstrated an important interaction between nuclear factor-kappaB (NF-kappaB) activation and homocysteine (Hcy)-induced cytokines expression in endothelial cells and vascular smooth muscle cells. However, the underlying mechanism remains illusive. In this study, we investigated the effects of Hcy on NF-kappaB-mediated sICAM-1, TNF-alpha production and the possible involvement of ERK(1/2)/p38MAPK pathway. The effects of rosiglitazone intervention were also examined. Our results show that Hcy increased the levels of sICAM-1 and TNF-alpha in cultured human umbilical vein endothelial cells (HUVECs) in a time- and concentration-dependent manner. This effect was significantly depressed by rosiglitazone and different inhibitors (PDTC, NF-kappaB inhibitor; PD98059, MEK inhibitor; SB203580, p38MAPK specific inhibitor; and staurosporine, PKC inhibitor). Next, we investigated the effect of Hcy on ERK(1/2)/p38MAPK pathway and NF-kappaB activity in HUVECs. The results show that Hcy activated both ERK(1/2)/p38MAPK pathway and NF-kappaB-DNA-binding activity. These effects were markedly inhibited by rosiglitazone as well as other inhibitors (SB203580, PD98059, and PDTC). Further, the pretreatment of staurosporine abrogated ERK(1/2)/p38MAPK phosphorylation, suggesting that Hcy-induced ERK(1/2)/p38MAPK activation is associated with PKC activity. Our results provide evidence that Hcy-induced NF-kappaB activation was mediated by activation of ERK(1/2)/p38MAPK pathway involving PKC activity. Rosiglitazone reduces the NF-kappaB-mediated sICAM-1 and TNF-alpha production induced by Hcy via inhibition of ERK(1/2)/p38MAPK pathway.

    Biochemical and biophysical research communications 2007;360;1;20-6

  • Radiation-induced PARP activation is enhanced through EGFR-ERK signaling.

    Hagan MP, Yacoub A and Dent P

    Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298, USA. mhagan@mcvh-vcu.edu

    We examined the impact of EGFR-ERK signaling on poly (ADP-ribose) polymerase (PARP) activation following ionizing irradiation of human prostate cancer (PCa) cell lines displaying marked differences in ERK dependence. PARP activation was indicated by the appearance of polyADP-ribose, the incorporation of P32-labelled NADH, and by cellular NADH. EGFR-ERK signaling was manipulated through ligand activation or signal interruption using the tyrphostin AG1478, or MEK inhibitor PD 184352. EGF activation of ERK prior to irradiation was associated with a marked increase in PARP activation and decreased survival in both cell lines. Prior inactivation of PARP protected both cell lines from the initial decrease in NAD+ and improved the survival of LNCaP cells following combined EGF and IR treatment. MEK inhibitor PD 184352 also reduced PARP activation and improved LNCaP survival following EGF and IR treatment. These data imply that PARP activation following exposure to ionizing radiation is enhanced through EGFR-ERK signaling.

    Funded by: PHS HHS: P01-72955-07

    Journal of cellular biochemistry 2007;101;6;1384-93

  • De-phosphorylation of TRalpha-1 by p44/42 MAPK inhibition enhances T(3)-mediated GLUT5 gene expression in the intestinal cell line Caco-2 cells.

    Mochizuki K, Sakaguchi N, Takabe S and Goda T

    Laboratory of Nutritional Physiology, Graduate School of Nutritional and Environmental Sciences, COE Program in the 21st Century, The University of Shizuoka, Shizuoka 422-8526, Japan.

    Thyroid hormone and p44/42 MAPK inactivation are important in intestinal differentiation. We demonstrated not only that treatment with p44/42 MAPK inhibitor U0126 in intestinal cell line Caco-2 cells reduced the phosphorylation of serine and threonine residues of TRalpha-1, but also that T(3) and U0126 synergistically induced GLUT5 gene expression. EMSA demonstrated that the binding activity of TRalpha-1-RXR heterodimer on GLUT5-TRE in nuclear proteins of Caco-2 cells was synergistically enhanced by co-incubation in vitro with T(3) and CIAP, which strongly de-phosphorylates proteins. ChIP and transfection assays revealed that co-treatment of T(3) and U0126 induces TRalpha-1-RXR binding to GLUT5-TRE on the human GLUT5 enhancer region, and recruitment of the transcriptional complex in cells. These results suggest that inactivation of p44/42 MAPK enhances T(3)-induced GLUT5 gene expression in Caco-2 cells through increasing TRalpha-1 transactivity and binding activity to the GLUT5-TRE, probably due to de-phosphorylation of TRalpha-1.

    Biochemical and biophysical research communications 2007;359;4;979-84

  • Extracellular signal-regulated protein kinase in human intractable epilepsy.

    Xi ZQ, Wang XF, He RQ, Li MW, Liu XZ, Wang LY, Zhu X, Xiao F, Sun JJ, Li JM, Gong Y and Guan LF

    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.

    Extracellular signal-regulated kinases (ERK) such as ERK1 [p44 mitogen-activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the central nervous system under physiological and pathological conditions such as ischemia and epilepsy. Our aim is to investigate ERK1, ERK2, and phosphorylated ERK (p-ERK) (Thr202/Tyr 204) expression in the temporal lobe of patients with intractable epilepsy (IE) and to explore its possible role of ERK in it. Tissue samples from temporal neocortices of 40 patients who had surgery for IE were used to detect ERK1, ERK2, and p-ERK (Thr 202/Tyr 204) expression through immunohistochemistry and western blot. We compared these tissues against 17 histological normal temporal lobes from head-trauma patients. ERK1, ERK2, and p-ERK in IE were significantly higher than those in the controls. They were mainly expressed in the cytoplasm of neurons and glial cells. There was also increased detection of p-ERK in the gliotic cortex of IE compared with the non-gliotic cortex. These findings were consistently observed in western blot and immunohistochemistry techniques. ERK expression in patients with IE was significantly increased compared with the controls. This suggested a probable role of ERK in the pathogenesis of IE.

    Funded by: PHS HHS: 98-1-3457

    European journal of neurology 2007;14;8;865-72

  • Irradiation-induced G2/M checkpoint response requires ERK1/2 activation.

    Yan Y, Black CP and Cowan KH

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.

    Following DNA damage, cells undergo G2/M cell cycle arrest, allowing time for DNA repair. G2/M checkpoint activation involves activation of Wee1 and Chk1 kinases and inhibition of Cdc25A and Cdc25C phosphatases, which results in inhibition of Cdc2 kinase. Results presented in this report indicate that gamma-irradiation (IR) exposure of MCF-7 cells resulted in extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation and induction of G2/M arrest. Furthermore, inhibition of ERK1/2 signaling resulted in >or=85% attenuation in IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ataxia telangiectasia mutated- and rad3-related (ATR), Chk1 and Wee1 kinases as well as phosphorylation of Cdc25A-Thr506, Cdc25C-Ser216 and Cdc2-Tyr15. Moreover, incubation of cells with caffeine, which inhibits ataxia telangiectasia mutated (ATM)/ATR, or transfection of cells with short interfering RNA targeting ATR abrogated IR-induced Chk1 phosphorylation and G2/M arrest but had no effect on IR-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling resulted in marked attenuation in IR-induced ATR activity with little, if any, effect on IR-induced ATM activation. These results implicate IR-induced ERK1/2 activation as an important regulator of G2/M checkpoint response to IR in MCF-7 cells.

    Oncogene 2007;26;32;4689-98

  • Extracellular signal-regulated kinase-2 phosphorylates RORalpha4 in vitro.

    Lechtken A, Hörnig M, Werz O, Corvey N, Zündorf I, Dingermann T, Brandes R and Steinhilber D

    Institute of Pharmaceutical Chemistry/ZAFES, Johann Wolfgang Goethe University Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.

    The retinoic acid related orphan receptor RORalpha activates transcription of genes that play an important role in cerebellar development, the protection against age-related degenerative processes, the regulation of inflammatory responses, and is one of the pivotal participants that control the circadian rhythmicity in the core-clock of mammals. We identified the extracellular signal-regulated kinase 2 (ERK-2) as RORalpha4 phosphorylating kinase in vitro. The primary sequence of RORalpha4 contains an ERK-2 recognition motif (P-L-T(128)-P) within the hinge domain, and mutation of Thr-128 to Ala prevents RORalpha4 phosphorylation by ERK. The RORalpha4-T128A mutant exhibits an increased DNA-binding affinity, an increased transcriptional activity and, in the interplay with the opponent RevErbalpha, acts as a stronger competitor at ROR response elements than RORalpha4-WT.

    Biochemical and biophysical research communications 2007;358;3;890-6

  • Cholesterol level of lipid raft microdomains regulates apoptotic cell death in prostate cancer cells through EGFR-mediated Akt and ERK signal transduction.

    Oh HY, Lee EJ, Yoon S, Chung BH, Cho KS and Hong SJ

    Department of Urology, Urological Science Institute, Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea.

    Background: Lipid rafts are cholesterol-enriched microdomains in cell membranes that have been shown to regulate signal transduction. We investigated whether membrane cholesterol could regulate apoptosis and attempted to elucidate the mechanism by which apoptosis is induced in prostate cancer cells.

    Methods: LNCaP cells were exposed to 2-hydroxyprophyl-beta-cyclodextrin (HPCD) to deplete membrane cholesterol. Cell viability and apoptosis were evaluated by Celltiter Bluetrade mark Cell Viability assay and ethidium bromide/acridine orange staining. Signal transduction was investigated by immunoblot analysis of cell lysates.

    Results: Cell viability was dose dependent inhibited by HPCD and restored by replenishment of cholesterol. HPCD induced apoptotic cell death through down-regulation of Bcl-xL and up-regulation of caspase-3 and PARP cleavages. HPCD inhibited both EGFR/Akt and EGFR/ERK signal transduction.

    Conclusions: Lipid raft cholesterol regulates apoptotic cell death in prostate cancer cells through EGFR-mediated Akt and ERK pathways.

    The Prostate 2007;67;10;1061-9

  • Bidirectional signals transduced by TOPK-ERK interaction increase tumorigenesis of HCT116 colorectal cancer cells.

    Zhu F, Zykova TA, Kang BS, Wang Z, Ebeling MC, Abe Y, Ma WY, Bode AM and Dong Z

    Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.

    Aberrant activation of Ras and Raf in mitogen-activated protein kinase (MAPK) signaling has been linked with cancer. However, the role of MAPK kinases (MAPKKs or MEKs) in cancer is unclear, although constitutively activated MEK1, which does not exist in nature, is "oncogenic." Herein, we found that T-cell-originated protein kinase (TOPK), a member of the MAPKK protein family, is highly expressed in human colorectal cancer tissues and cell lines and plays an important role in the transformation of colorectal cancer.

    Methods: The biologic consequences of overexpression or knockdown of TOPK in JB6 Cl41 and HCT116 colorectal cancer cells were studied in vitro and in vivo, respectively. Kinase assay or transient transfection experiments were performed to study the bidirectional signaling pathway between TOPK and extracellular signal-regulated kinase (ERK).

    Results: TOPK was shown to promote transformation in vitro and in vivo, and knockdown of TOPK in HCT116 colorectal cancer cells reduced this cell lines' tumorigenic properties in vitro and in vivo. Furthermore, a positive feedback loop between TOPK and ERK2 was identified. With epidermal growth factor treatment, knockdown of either TOPK or ERK2 in HCT116 cells resulted in a decreased phosphorylation of ERK2 or TOPK, respectively, and knockdown of TOPK in HCT116 colorectal cancer cells blocked the phosphorylation of downstream substrates of ERK2.

    Conclusions: The positive feedback loop between TOPK and ERK2 increases tumorigenesis properties of HCT116 colorectal cancer cells, and TOPK-regulated signaling may serve as a potential therapeutic target in colorectal cancer.

    Funded by: NCI NIH HHS: CA077646, CA111356, CA111536

    Gastroenterology 2007;133;1;219-31

  • [Expressions and significance of STAT3 and p38 in the carcinogenesis of sporadic colorectal tubular adenoma].

    Liu HM, Wu WX, Zhang XH, Lü GH and Zhang JY

    Department of Pathology, the Second Hospital of Hebei Medical University, Shijiazhuang, China.

    Objective: To investigate the possible role of STAT3 and p38 in the carcinogenesis of sporadic colorectal tubular adenoma.

    Methods: The expression of STAT3 and p38 at protein level was studied in 107 sporadic colorectal tubular adenomas with different dysplasia (SCTA-D) or with cancerous changes (SCTA-Ca) by immunohistochemical staining method, meanwhile the expression of STAT3 at mRNA level was detected by in situ hybridization.

    Results: Immunohistochemical staining results showed that the positive expression rate of STAT3 and p38 was 12.0%, 59.0%, 91.7% and 8.0%, 47.0%, 91.7% in normal colorectal mucosa (NCM), SCTA-D and SCTA-Ca, respectively, with a statistically significant difference of STAT3 and p38 expression among the SCTA-D, SCTA-Ca and NCM (P < 0.05). The expression of STAT3 and p38 was positively correlated with the degree of dysplasia from mild to severe SCTA-D (P < 0.05). In situ hybridization results showed that the positive expression rate of STAT3 at mRNA level in NCM, SCTA-D and SCTA-Ca was 8.00%, 51.8% and 100.0%, respectively, with a statistically significant difference among these either (P < 0.05). The positive expression of STAT3 at mRNA level was not only positively correlated with the degree of dysplasia (P < 0.05), but also with the expression of p38 (P < 0.05).

    Conclusion: STAT3 and p38 may be involved in the carcinogenesis of sporadic colorectal tubular adenoma.

    Zhonghua zhong liu za zhi [Chinese journal of oncology] 2007;29;7;514-7

  • Induction of urokinase-type plasminogen activator, interleukin-8 and early growth response-1 by STI571 through activating mitogen activated protein kinase in human small cell lung cancer cells.

    Yoshida C, Niiya K, Niiya M, Shibakura M, Asaumi N and Tanimoto M

    Department of Hematology, Oncology and Respiratory Medicine, Okayama University Medical School, Okayama, Japan. c-yoshi@md.okayama-u.ac.jp

    We previously demonstrated the simultaneous induction of urokinase-type plasminogen activator and interleukin-8, a CXC chemokine, in doxorubicin-treated human NCI-H69 small cell lung cancer cells in which extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase might be involved. NCI-H69 cells expressed one of the receptor tyrosine kinases, c-Kit, and STI571 inhibited the cell growth and stem cell factor-induced phosphorylation of c-Kit. We therefore investigated the effects of STI571 on the expression of urokinase-type plasminogen activator and interleukin-8 in NCI-H69 cells. Microarray analysis revealed the gene induction of not only urokinase-type plasminogen activator and interleukin-8, but also early growth response-1 in STI571-treated cells. Treatment with STI571 resulted in the induction of phosphorylation of all three mitogen-activated protein kinases, such as extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and stress-activated protein kinase/c-jun N-terminal protein kinase. U0126, an inhibitor against extracellular signal-regulated kinase 1/2, however, only inhibited the STI571-induced interleukin-8 accumulation. Urokinase-type plasminogen activator and interleukin-8 are important biological factors in tumor cell regulation; STI571 may therefore influence many aspects of tumor cell biology through inducing urokinase-type plasminogen activator and interleukin-8, in which the induction of early growth response-1 expression and extracellular signal-regulated kinase 1/2 phosphorylation might be involved.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2007;18;5;425-33

  • Insulin-like growth factor-I induces cyclooxygenase-2 expression via PI3K, MAPK and PKC signaling pathways in human ovarian cancer cells.

    Cao Z, Liu LZ, Dixon DA, Zheng JZ, Chandran B and Jiang BH

    Mary Babb Randolph Cancer Center, Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA.

    Elevated levels of insulin-like growth factor-I (IGF-I) are associated with ovarian carcinogenesis and progression. However, the molecular mechanisms by which IGF-I contributes to ovarian cancer development remain to be elucidated. Cyclooxygenase-2 (COX-2) is a crucial player in the pathogenesis of human malignancies. Herein we showed that IGF-I efficiently induced COX-2 expression and PGE(2) biosynthesis at physiologically relevant concentrations in human ovarian cancer cells. IGF-I treatment significantly increased COX-2 transcriptional activation. IGF-I also stabilized COX-2 mRNA through the COX-2 3'-untranslated region (3'-UTR), which appeared independent of the conserved AU-rich elements. We next investigated the signaling pathways involved in IGF-I-induced COX-2 expression. We found that PI3K inhibitor wortmannin or LY294002 blocked COX-2 expression induced by IGF-I. Wortmannin treatment or a dominant negative PI3K mutant significantly inhibited IGF-I-induced COX-2 mRNA stabilization, but only slightly decreased COX-2 transcriptional activation. We showed that ERK1/2 and p38 MAPKs were required for IGF-I-induced COX-2 expression and that activation of both pathways by IGF-I increased COX-2 transcriptional activation and its mRNA stability. IGF-I stimulated PKC activation in the cells and pretreatment with PKC inhibitor bisindolylmaleimide prevented IGF-I-induced COX-2 transcriptional activation and mRNA stabilization, and inhibited COX-2 mRNA and protein expression. Taken together, our data demonstrate that IGF-I induces COX-2 expression in human ovarian cancer cells, which is mediated by three parallel signaling cascades--PI3K, MAPK, and PKC pathways that differentially regulate COX-2 expression at transcriptional and post-transcriptional levels.

    Funded by: NCI NIH HHS: CA 099925, CA 109460, CA 123675, CA 75911, R01 CA075911, R01 CA099925, R01 CA109460, R01 CA109460-02, R03 CA123675, R03 CA123675-02; NCRR NIH HHS: P20 RR016440, P20 RR016440-050001

    Cellular signalling 2007;19;7;1542-53

  • Lysophosphatidic acid induces prostate cancer PC3 cell migration via activation of LPA(1), p42 and p38alpha.

    Hao F, Tan M, Xu X, Han J, Miller DD, Tigyi G and Cui MZ

    Department of Pathobiology, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN 37996, USA.

    Prostate cancer cell migration is an essential event both in the progression of prostate cancer and in the steps leading to metastasis. We report here that lysophosphatidic acid (LPA), a potent bioactive phospholipid, induces prostate cancer PC3 cell migration via the activation of the LPA(1) receptor, which is linked to a PTX-sensitive activation mechanism of the mitogen-activated protein kinases (MAPK). Our results demonstrate that parallel activation of ERK1/2 and p38, but not JNK, is responsible for LPA-stimulated PC3 cell migration. Furthermore, using small interfering RNA (siRNA) technology, and overexpressing dominant-negative mutants of p38 MAPK isotypes of alpha, beta, gamma and delta, we have identified that the activation of ERK2 (p42) and p38alpha, but not of ERK1 and the other isoforms of p38 MAPK, is required for LPA-induced migration. Our study provides the first evidence for a functional role of p42 and p38alpha in LPA-induced mammalian cell migration, and also demonstrates, for the first time, that the receptor LPA(1) mediates prostate cancer cell migration. The results of the present study suggest that LPA, the receptor LPA(1), ERK2 and p38alpha are important regulators for prostate cancer cell invasion and thus could play a significant role in the development of metastasis.

    Funded by: NCI NIH HHS: CA92160, R01 CA092160, R01 CA092160-08; NHLBI NIH HHS: HL074341, R01 HL074341, R01 HL074341-03

    Biochimica et biophysica acta 2007;1771;7;883-92

  • Short waves-induced enhancement of proliferation of human chondrocytes: involvement of extracellular signal-regulated map-kinase (erk).

    Wang JL, Chan RC, Cheng HH, Huang CJ, Lu YC, Chen IS, Liu SI, Hsu SS, Chang HT, Huang JK, Chen JS, Ho CM and Jan CR

    Department of Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.

    1. Short-wave diathermy (SWD) is a form of radiofrequency radiation that is used therapeutically by physiotherapists. The cellular mechanisms of SWD are unclear. The present study was performed to explore the effect of different conditions of short-wave exposure on the proliferation of cultured human chondrocytes. 2. Cells exposed to short waves once per day for seven consecutive days exhibited a significant increase in proliferation by 42% compared with the control cells. In cells that were treated with short waves twice per day for seven consecutive days, or only once on Day 1 and then examined for proliferation on Day 7, cell proliferation was greater than the control cells by 40% and 30%, respectively. 3. Given the importance of mitogen-activated protein kinases (MAPK) in the proliferation of different cell types, efforts were extended to explore the role of three major types of MAPK; that is, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal protein kinase (JNK) and p38. 4. It was found that the level of phosphorylated ERK (phospho-ERK 1 and ERK 2) increased significantly within 5-120 min following consecutive exposure to short waves for 7 days. Exposure to short waves failed to alter the intensity of phosphorylated JNK and p38 within 0-240 min. 5. Cells were exposed to short waves once for seven consecutive days in the presence of 0, 10 micromol/L, 20 micromol/L or 50 micromol/L PD98059 (an ERK inhibitor). PD98059 totally inhibited short waves-induced enhancement of proliferation without altering normal control viability. In the presence of short waves and PD98059, the cell viability was lower than the normal control. Together, the data suggest that short waves could increase proliferation in human chondrocytes through activation of the ERK pathway, which is also involved in maintaining normal cell proliferation under physiological conditions.

    Clinical and experimental pharmacology & physiology 2007;34;7;581-5

  • ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL.

    Ewings KE, Hadfield-Moorhouse K, Wiggins CM, Wickenden JA, Balmanno K, Gilley R, Degenhardt K, White E and Cook SJ

    Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, UK.

    The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/E02162X/1, BBS/E/B/0000C199, BBS/E/B/0000H151, BBS/E/B/0000H457

    The EMBO journal 2007;26;12;2856-67

  • Actin dysfunction activates ERK1/2 and delays entry into mitosis in mammalian cells.

    Lee K and Song K

    Department of Biochemistry and Institute of Life Science and Biotechnology, College of Science, Yonsei University, Seoul, South Korea.

    Investigations of actin function during the cell cycle have focused primarily on cytokinesis. Here, we describe the role of actin at the entry into mitosis in primary mammalian cells. Depolymerization of actin with cytochalasin D or inhibition of myosin ATPase with butanedione-2-monoxime (BDM) at G(2) blocked the mitotic spindle formation and central positioning of the nucleus in synchronized MEF and IMR90 cells. Time-lapse microscopy confirmed that these treatments inhibit both spindle formation and separation of duplicated centrosomes to the opposite poles. Concurrent with actin dysfunction, activation of Cdc2 and nuclear localization of cyclin B1 were delayed. Furthermore, cyclin A degradation that is necessary for nuclear envelope breakdown (NEBD) in early mitosis was deferred, supporting the conclusion that mitotic onset was delayed. The activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was sustained in these cells, and the use of a specific ERK inhibitor or a dominant negative form of ERK2 abrogated this delay of entry into mitosis. This delay of mitotic entry and the sustained ERK1/2 activity by actin dysfunction was observed only in primary cells and not in transformed cancer cell lines. These observations demonstrate that an intact actin cytoskeleton is necessary for entry into mitosis and that ERK1/2 is involved in monitoring actin dysfunction to control the onset of mitosis, suggesting the presence of an actin checkpoint at the G(2)/M transition in primary mammalian cells.

    Cell cycle (Georgetown, Tex.) 2007;6;12;1487-95

  • CTGF enhances the motility of breast cancer cells via an integrin-alphavbeta3-ERK1/2-dependent S100A4-upregulated pathway.

    Chen PS, Wang MY, Wu SN, Su JL, Hong CC, Chuang SE, Chen MW, Hua KT, Wu YL, Cha ST, Babu MS, Chen CN, Lee PH, Chang KJ and Kuo ML

    Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, and Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan.

    Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. Presently, overexpression of CTGF in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal-adhesion-complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody against integrin alphavbeta3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin-alphavbeta3-ERK1/2 signaling pathway is crucial in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, whereas overexpression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF-mediated S100A4 upregulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a crucial role in migratory/invasive processes in human breast cancer by a mechanism involving activation of the integrin-alphavbeta3-ERK1/2-S100A4 pathway.

    Journal of cell science 2007;120;Pt 12;2053-65

  • ERK1/2 phosphorylation is an independent predictor of complete remission in newly diagnosed adult acute lymphoblastic leukemia.

    Gregorj C, Ricciardi MR, Petrucci MT, Scerpa MC, De Cave F, Fazi P, Vignetti M, Vitale A, Mancini M, Cimino G, Palmieri S, Di Raimondo F, Specchia G, Fabbiano F, Cantore N, Mosna F, Camera A, Luppi M, Annino L, Miraglia E, Fioritoni G, Ronco F, Meloni G, Mandelli F, Andreeff M, Milella M, Foà R, Tafuri A and GIMEMA Acute Leukemia Working Party

    Division of Hematology, Department of Cellular Biotechnologies and Hematology, University La Sapienza of Rome, Italy.

    Extracellular signal-regulated kinase-1/2 (ERK1/2) is frequently found constitutively activated (p-ERK1/2) in hematopoietic diseases, suggesting a role in leukemogenesis. The aim of this study was to assess the expression and clinical role of p-ERK1/2 in adult acute lymphoblastic leukemia (ALL). In 131 primary samples from adult de novo ALL patients enrolled in the Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA) Leucemia Acute Linfoide (LAL) 2000 protocol and evaluated by flow cytometry, constitutive ERK1/2 activation was found in 34.5% of cases; these results were significantly associated with higher white blood cell (WBC) values (P=.013). In a multivariate analysis, p-ERK1/2 expression was an independent predictor of complete remission achievement (P=.027). Effective approaches toward MEK inhibition need to be explored in order to evaluate whether this may represent a new therapeutic strategy for adult ALL patients.

    Blood 2007;109;12;5473-6

  • Hypoxia induces apoptosis of HUVECs in an in vitro capillary model by activating proapoptotic signal p38 through suppression of ERK1/2.

    Eguchi R, Suzuki A, Miyakaze S, Kaji K and Ohta T

    Laboratory of Cell and Molecular Biology of Aging and COE Program in the 21st Century, Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Japan.

    We recently reported that hypoxia induces chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, to tube-forming HUVECs in an in vitro blood vessel model by activating p38 MAPK. In this report, we further examined what role p38 plays and how it is activated during hypoxia-induced apoptosis. First, in order to confirm that p38 can indeed induce apoptosis, the cells were treated with anisomycin, a p38 activator, during normoxia. The activator treatment induced apoptosis and activation of p38 and caspase-3 in a very short time, which indicated that p38 activation alone was sufficient to trigger apoptosis in tube-forming HUVECs. We then observed hypoxia-induced changes in intracellular signals, ERK1/2 and Akt. ERK1/2 inactivation was shown to occur prior to p38 activation and caspase-3 cleavage during hypoxia. On the other hand, anisomycin had no inhibitory effect on ERK1/2 activation during normoxia. It was also shown that the amount of Akt protein slightly decreased by either hypoxia or anisomycin treatment. We then investigated how these two survival signals, ERK1/2 and Akt, are involved in p38 activation by using MEK inhibitor U0126 and PI3K inhibitor LY294002. When tube-forming HUVECs were treated with U0126 or LY294002 during normoxia, the two inhibitors were able to induce apoptosis and activation of p38 and caspase-3 in a relatively short time. U0126 was able to inhibit ERK1/2 activation, but had almost no effect on Akt activation. In contrast, LY294002 was able to inhibit Akt activation, but had very little effect on ERK1/2 activation. These results indicate that ERK1/2 inactivation, rather than Akt decrease, is responsible for hypoxia-induced p38 activation. Taken together, our results strongly suggest that hypoxia-induced apoptosis is regulated through signal transduction in which inactivation of ERK1/2 leads to activation of p38, which then triggers caspase cascade as an execution mechanism of apoptosis.

    Cellular signalling 2007;19;6;1121-31

  • Impaired apoptosis in lymphoblasts from Alzheimer's disease patients: cross-talk of Ca2+/calmodulin and ERK1/2 signaling pathways.

    Bartolomé F, de Las Cuevas N, Muñoz U, Bermejo F and Martín-Requero A

    Department of Cellular and Molecular Pathophysiology, Centro de Investigaciones Biológicas, Ramiro de Maéztu 9, Madrid, Spain.

    We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer's disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca(2+)/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca(2+)/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawal.

    Cellular and molecular life sciences : CMLS 2007;64;11;1437-48

  • Involvement of MAPKs and NF-kappaB in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells.

    Lin WN, Luo SF, Lee CW, Wang CC, Wang JS and Yang CM

    Department of Physiology and Pharmacology, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.

    Lipopolysaccharide (LPS) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for LPS-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in HTSMCs. LPS-induced expression of VCAM-1 protein and mRNA in a time-dependent manner, was significantly inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and c-Jun-N-terminal kinase (JNK; SP600125). The involvement of p42/p44 MAPK and p38 in these responses was further confirmed by that transfection with small interference RNAs (siRNA) direct against MEK, p42, and p38 significantly attenuated LPS-induced VCAM-1 expression. Consistently, LPS-stimulated phosphorylation of p42/p44 MAPK and p38 was attenuated by pretreatment with U0126 or SB202190, and transfection with these siRNAs, respectively. In addition, LPS-induced VCAM-1 expression was significantly blocked by a specific NF-kappaB inhibitor helenalin. LPS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by helenalin, U0126, SB202190, or SP600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to monolayer of HTSMCs which was blocked by pretreatment with helenalin, U0126, or SP600125 prior to LPS exposure. Taken together, these results suggest that in HTSMCs, activation of p42/p44 MAPK, p38, and JNK pathways, at least in part, mediated through NF-kappaB, is essential for LPS-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of LPS action that bacterial toxins may promote inflammatory responses in the airway disease.

    Cellular signalling 2007;19;6;1258-67

  • Mitogen-activated protein kinases, inhibitory-kappaB kinase, and insulin signaling in human omental versus subcutaneous adipose tissue in obesity.

    Bashan N, Dorfman K, Tarnovscki T, Harman-Boehm I, Liberty IF, Blüher M, Ovadia S, Maymon-Zilberstein T, Potashnik R, Stumvoll M, Avinoach E and Rudich A

    Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel.

    MAPKs and inhibitory-kappaB kinase (IKK) were suggested to link various conditions thought to develop in adipose tissue in obesity (oxidative, endoplasmic reticulum stress, inflammation) with insulin resistance. Yet whether in obesity these kinases are affected in a fat-depot-differential manner is unknown. We assessed the expression and phosphorylation of these kinases in paired omental and abdominal-sc fat biopsies from 48 severely obese women (body mass index > 32 kg/m(2)). Protein and mRNAs of p38MAPK, ERK, c-Jun kinase-1, and IKKbeta were increased 1.5-2.5-fold in omental vs. sc fat. The phosphorylated (activated) forms of these kinases were also increased to similar magnitudes as the total expression. However, phosphorylation of insulin receptor substrate-1 on Ser312 (equivalent of murine Ser307) was not increased in omental, compared with sc, fat. Consistently, fat tissue fragments stimulated with insulin demonstrated that tyrosine phosphorylation and signal transduction to Akt/protein kinase B in omental fat was not inferior to that observable in sc fat. Comparison with lean women (body mass index 23.2 +/- 2.9 kg/m(2)) revealed similar ERK2 and IKKbeta expression and phosphorylation in both fat depots. However, as compared with lean controls, obese women exhibited 480 and 270% higher amount of the phosphorylated forms of p38MAPK and c-Jun kinase, respectively, in omental, but not sc, fat, and this expression level correlated with clinical parameters of glycemia and insulin sensitivity. Increased expression of stress-activated kinases and IKK and their phosphorylated forms in omental fat occurs in obesity, potentially contributing to differential roles of omental and sc fat in the pathophysiology of obesity.

    Endocrinology 2007;148;6;2955-62

  • Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation.

    Lee HJ, Mun HC, Lewis NC, Crouch MF, Culverston EL, Mason RS and Conigrave AD

    School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia.

    The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.

    The Biochemical journal 2007;404;1;141-9

  • The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition.

    Meloche S and Pouysségur J

    Departments of Pharmacology and Molecular Biology, Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec, Canada. sylvain.meloche@umontreal.ca

    The Ras-dependent extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway plays a central role in cell proliferation control. In normal cells, sustained activation of ERK1/ERK2 is necessary for G1- to S-phase progression and is associated with induction of positive regulators of the cell cycle and inactivation of antiproliferative genes. In cells expressing activated Ras or Raf mutants, hyperactivation of the ERK1/2 pathway elicits cell cycle arrest by inducing the accumulation of cyclin-dependent kinase inhibitors. In this review, we discuss the mechanisms by which activated ERK1/ERK2 regulate growth and cell cycle progression of mammalian somatic cells. We also highlight the findings obtained from gene disruption studies.

    Oncogene 2007;26;22;3227-39

  • Activation of ERK1/2 occurs independently of KRAS or BRAF status in endometrial cancer and is associated with favorable prognosis.

    Mizumoto Y, Kyo S, Mori N, Sakaguchi J, Ohno S, Maida Y, Hashimoto M, Takakura M and Inoue M

    Department of Obstetrics and Gynecology, Graduate School of Medical Science, Kanazawa University, 13-1, Takaramachi, Kanazawa, Ishikawa 920-8641, Japan.

    The extracellular-regulated kinase (ERK) signaling pathway plays important roles in regulating the malignant potential of cancer cells in vitro. However, the effect of ERK signaling on the prognosis of human tumors is not clearly understood. The present study examined the expression of phosphorylated ERK1/2 (p-ERK1/2) as a hallmark of ERK activation, in relation to KRAS and BRAF mutations, in 63 endometrial cancer specimens with endometrioid-subtype, in order to clarify the prognostic value of p-ERK1/2 expression. Immmunohistochemical analysis revealed that 40 tumors (63%) expressed p-ERK1/2, with varying levels of expression. Total ERK1/2 expression was also evaluated in a subset of tumors; most cases expressed ERK1/2 constitutively but no correlation was observed with p-ERK expression, indicating that p-ERK1/2 staining was not due to ERK overexpression but to hyperactivation of ERK1/2. There was no statistically significant correlation between p-ERK1/2 expression and clinicopathological features, including patient age, International Federation of Gynecology and Obstetrics stage, pathological grade, myometrial invasion and lymph node metastasis. Sequencing analysis indicated that 23% of patients had a mutation in exon 1 of KRAS, whereas none of the patients had a mutation in exons 11 or 15 of BRAF, which are reportedly hot spots for mutation in many tumor types. There was no significant correlation between KRAS or BRAF status and p-ERK1/2 expression. Unexpectedly, patients with low p-ERK1/2 expression had significantly lower relapse-free survival (P = 0.041) and overall survival (P = 0.020). Multivariate Cox regression analysis indicated that p-ERK1/2 expression was an independent prognostic indicator for overall survival (P = 0.047). These findings suggest that ERK activation occurs in a KRAS- and BRAF-independent manner in endometrial cancer, and is associated with favorable prognosis.

    Cancer science 2007;98;5;652-8

  • CXCR4 expression is associated with pelvic lymph node metastasis in cervical adenocarcinoma.

    Yang YC, Lee ZY, Wu CC, Chen TC, Chang CL and Chen CP

    Departments of Obstetrics and Gynecology and Medical Research, Mackay Memorial Hospital, Taipei, Taiwan.

    The aim of this study is to investigate the expression of CXCR4 receptor in cervical adenocarcinoma and related mechanisms involved in pelvic lymph node metastasis. Immunohistochemistry was used to evaluate the expression of CXCR4 and the association of pelvic lymph node metastasis in archived tissue from clinical stage IB cervical adenocarcinomas (n = 37) and from benign specimens obtained at hysterectomy for other causes (n = 48). The HeLa cell (cervical adenocarcinoma-derived cell) line that expresses CXCR4 was used to study the interaction between the CXCR4 receptor and stromal cell-derived factor 1alpha (SDF-1alpha). Cell migration assays, cell numbers, flow cytometry, cell proliferation assay, and western blot were used to study the function of CXCR4 and its downstream signal transduction. The positive cases were semiquantitatively divided into three score classes according to their staining. Tumors with strong CXCR4 stainings were more likely to have pelvic lymph node metastasis than those with weak or negative stainings (87.5% vs 34.5%; P = 0.014). Only 25% of the benign specimens had weak or negative staining for CXCR4. Functioning CXCR4 receptor was expressed on HeLa cells. SDF-1alpha provoked significant signal transduction events, including chemotaxis and rescue from apoptosis. These actions were apparently mediated by the activation and phosphorylation of the extracellular signal-regulated kinase 1/2 and AKT pathways. We conclude CXCR4 expression is associated with cervical adenocarcinoma cell migration and proliferation, and primary cervical adenocarcinoma cells expressing CXCR4 are significantly more likely to metastasize to pelvic lymph nodes.

    International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 2007;17;3;676-86

  • D2 receptors regulate dopamine transporter function via an extracellular signal-regulated kinases 1 and 2-dependent and phosphoinositide 3 kinase-independent mechanism.

    Bolan EA, Kivell B, Jaligam V, Oz M, Jayanthi LD, Han Y, Sen N, Urizar E, Gomes I, Devi LA, Ramamoorthy S, Javitch JA, Zapata A and Shippenberg TS

    Integrative Neuroscience Section, National Institute on Drug Abuse Intramural Research Program/National Institutes of Health/Department of Health and Human Services, Baltimore, MD 21224, USA.

    The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D(2) dopamine receptors (D(2)R) has been reported. The high affinity of DA and other DAT substrates for the D(2)R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP(+)) with live cell imaging techniques to identify the role of two D(2)R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D(2)R regulation of DAT. Addition of the D(2)/D(3) receptor agonist quinpirole (0.1-10 muM) to human embryonic kidney cells coexpressing human DAT and D(2) receptor (short splice variant, D(2S)R) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP(+) accumulation. The D(2)/D(3) agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP(+) accumulation. D(2S)R activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP(+) accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D(2)R stimulation. These experiments demonstrate that D(2S)R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D(2)R. Together, these results suggest a novel mechanism by which D(2S)R autoreceptors may regulate DAT in the central nervous system.

    Funded by: Intramural NIH HHS; NIDA NIH HHS: DA0019521, DA08863, DA11495, K05 DA022413, P50DA015369; NIMH NIH HHS: MH062612, MH54137, MH57324

    Molecular pharmacology 2007;71;5;1222-32

  • Down-regulation of Sprouty2 in non-small cell lung cancer contributes to tumor malignancy via extracellular signal-regulated kinase pathway-dependent and -independent mechanisms.

    Sutterlüty H, Mayer CE, Setinek U, Attems J, Ovtcharov S, Mikula M, Mikulits W, Micksche M and Berger W

    Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, 1090 Vienna, Austria. hedwig.sutterluety@meduniwien.ac.at

    Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non-small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase-induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC.

    Molecular cancer research : MCR 2007;5;5;509-20

  • Expression of human glutathione S-transferase P1 mediates the chemosensitivity of osteosarcoma cells.

    Huang G, Mills L and Worth LL

    Division of Pediatrics, Unit 087, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

    Chemoresistance is a major reason that patients with osteosarcoma fail to achieve a lasting chemotherapy response, and it contributes to disease relapse, progression, and death. Human glutathione S-transferase P1 (GSTP1), a phase II detoxification enzyme, contributes to chemoresistance in many cancers. However, the role of GSTP1 in osteosarcoma chemoresistance is ill defined. We hypothesized that GSTP1 has cytoprotective effects in human osteosarcoma. To assess this possibility, we used GSTP1 cDNA transfection or RNA interference to overexpress or suppress GSTP1 in osteosarcoma cells, and assessed the cytotoxic effect of chemotherapeutic agents on these cells. Our results showed that GSTP1 expression was up-regulated in osteosarcoma cells when they were treated with doxorubicin or cisplatin. GSTP1 overexpression in SAOS-2 osteosarcoma cells caused the cells to be more resistant to doxorubicin and cisplatin. In contrast, GSTP1 suppression in HOS cells caused more apoptosis and extensive DNA damage in response to doxorubicin and cisplatin. The cytotoxicity assay also showed that GSTP1 suppression caused a 2.5-fold increase in cell growth inhibition resulting from doxorubicin and cisplatin treatments [the IC(50)s are approximately 0.16 micromol/L (doxorubicin) and 1.8 micromol/L (cisplatin) for parental HOS versus 0.06 micromol/L (doxorubicin) and 0.75 micromol/L (cisplatin) for GSTP1-silenced HOS]. Moreover, GSTP1 suppression decreased the activation of extracellular signal-regulated kinase 1/2, which is induced by cisplatin and doxorubicin. Taken together, these findings show that GSTP1 contributes to doxorubicin and cisplatin resistance in osteosarcoma, which may be mediated in part by the activation of extracellular signal-regulated kinase 1/2. Targeting of GSTP1 combined with chemotherapy may have synergistic therapeutic effects on osteosarcoma.

    Funded by: NCI NIH HHS: CA 16672

    Molecular cancer therapeutics 2007;6;5;1610-9

  • p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle.

    Gosens R, Dueck G, Gerthoffer WT, Unruh H, Zaagsma J, Meurs H and Halayko AJ

    Department of Physiology, University of Manitoba, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada. r.gosens@rug.nl

    Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.

    American journal of physiology. Lung cellular and molecular physiology 2007;292;5;L1163-72

  • p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells.

    Ko J, Yun CY, Lee JS, Kim JH and Kim IS

    School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.

    9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.

    Experimental & molecular medicine 2007;39;2;129-38

  • Phosphorylation of Parkin by the cyclin-dependent kinase 5 at the linker region modulates its ubiquitin-ligase activity and aggregation.

    Avraham E, Rott R, Liani E, Szargel R and Engelender S

    Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096, Israel.

    Mutations in Parkin are responsible for a large percentage of autosomal recessive juvenile parkinsonism cases. Parkin displays ubiquitin-ligase activity and protects against cell death promoted by several insults. Therefore, regulation of Parkin activities is important for understanding the dopaminergic cell death observed in Parkinson disease. We now report that cyclin-dependent kinase 5 (Cdk5) phosphorylates Parkin both in vitro and in vivo. We found that highly specific Cdk5 inhibitors and a dominant negative Cdk5 construct inhibited Parkin phosphorylation, suggesting that a significant portion of Parkin is phosphorylated by Cdk5. Parkin interacts with Cdk5 as observed by co-immunoprecipitation experiments of transfected cells and rat brains. Phosphorylation by Cdk5 decreased the auto-ubiquitylation of Parkin both in vitro and in vivo. We identified Ser-131 located at the linker region of Parkin as the major Cdk5 phosphorylation site. The Cdk5 phosphorylation-deficient S131A Parkin mutant displayed a higher auto-ubiquitylation level and increased ubiquitylation activity toward its substrates synphilin-1 and p38. Additionally, the S131A Parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared with the wild-type Parkin. Furthermore, S131A Parkin mutant increased the formation of synphilin-1/alpha-synuclein inclusions, suggesting that the levels of Parkin phosphorylation and ubiquitylation may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of the Parkin ubiquitin-ligase activity and modulates its ability to accumulate into and modify inclusions. Phosphorylation by Cdk5 may contribute to the accumulation of toxic Parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in Parkinson disease.

    The Journal of biological chemistry 2007;282;17;12842-50

  • Antiproliferative autoantigen CDA1 transcriptionally up-regulates p21(Waf1/Cip1) by activating p53 and MEK/ERK1/2 MAPK pathways.

    Tu Y, Wu W, Wu T, Cao Z, Wilkins R, Toh BH, Cooper ME and Chai Z

    Diabetes and Metabolism Division, Baker Heart Research Institute, Melbourne, Victoria 3004, Australia.

    We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells arrests cell growth and inhibits DNA synthesis at S-phase. Here we show that CDA1-induced arrest of cell growth is accompanied by increases in protein and mRNA levels of the cyclin-dependent kinase (Cdk) inhibitor protein, p21(Waf1/Cip1) (p21). Both p21 induction and cell growth arrest are reversed when CDA1 expression is inhibited. CDA1 also increases p53 protein, but not its mRNA, in a time- and dose-dependent manner. MDM2, a ubiquitin ligase regulating p53 degradation, is inactivated by CDA1, suggesting that p53 protein accumulation is due to decreased protein degradation. Knockdown of p53, using siRNA targeting two sites of p53 mRNA, abrogates transcriptional induction of p21 by CDA1. Deletion of the p53 responsive element in the distal region of p21 promoter attenuates promoter activity in response to CDA1. DNA damage caused by camptothecin treatment increases mRNA and protein levels of CDA1, accompanied by induction of p53. The DNA damage-induced p53 induction is markedly attenuated by CDA1 knockdown. CDA1 induces phosphorylation of ERK1/2(p44/42), an activity blocked by PD98059 and U0126, inhibitors of the upstream kinase MEK1/2. The MEK inhibitors also block induction of p21 mRNA and abrogate p21 promoter activity stimulated by CDA1. Cell cycle kinases, Cdk1, -2, -4, and -6 are inhibited by CDA1 overexpression. We conclude that CDA1 induces p53- and MEK/ERK1/2 MAPK-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.

    The Journal of biological chemistry 2007;282;16;11722-31

  • Phosphorylation of MCT-1 by p44/42 MAPK is required for its stabilization in response to DNA damage.

    Nandi S, Reinert LS, Hachem A, Mazan-Mamczarz K, Hagner P, He H and Gartenhaus RB

    Department of Medicine, University of California, San Diego, La Jolla, CA, USA.

    We discovered a novel oncogene in a T-cell lymphoma cell line, multiple copies in T-cell lymphoma-1 (MCT-1), that has been shown to decrease cell-doubling time, shorten the duration of G(1) transit time and/or G(1)-S transition, and transform NIH3T3 fibroblasts. We subsequently demonstrated that there were significantly increased levels of MCT-1 protein in a subset of primary diffuse large B-cell lymphomas. Levels of MCT-1 protein were shown to be increased after exposure to DNA damaging agents. This increase did not require new protein synthesis, suggesting that post-translational mechanisms were involved. Phosphorylation is one potential mechanism by which the activity of molecules involved in cell cycle/survival is rapidly modulated. The RAS/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal-regulated kinases (ERK) pathway plays a prominent role in the regulation of cell growth and proliferation through phosphorylation-dependent regulation of several substrates. The MCT-1 protein is predicted to have numerous putative phosphorylation sites. Using a combination of genetic and pharmacological approaches, we established that phosphorylation of MCT-1 protein by p44/p42 mitogen-activated protein kinases is critical for stabilization of MCT-1 protein and for its ability to promote cell proliferation. Our data suggests that targeting the RAS/MEK/ERK signal transduction cascade may provide a potential therapeutic approach in lymphomas and related malignancies that exhibit high levels of MCT-1 protein.

    Oncogene 2007;26;16;2283-9

  • A module of negative feedback regulators defines growth factor signaling.

    Amit I, Citri A, Shay T, Lu Y, Katz M, Zhang F, Tarcic G, Siwak D, Lahad J, Jacob-Hirsch J, Amariglio N, Vaisman N, Segal E, Rechavi G, Alon U, Mills GB, Domany E and Yarden Y

    Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel.

    Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.

    Funded by: NCI NIH HHS: CA099031, CA102537, CA64602, CA65930, CA72981

    Nature genetics 2007;39;4;503-12

  • Dual blockade of the Hedgehog and ERK1/2 pathways coordinately decreases proliferation and survival of cholangiocarcinoma cells.

    Jinawath A, Akiyama Y, Sripa B and Yuasa Y

    Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45, Yushima, Tokyo 113-8519, Japan.

    Purpose: The Hedgehog (Hh) and pERK1/2 pathways participate in the tumorigenesis of various tissues, but there has been no report on the involvement of these two pathways in cholangiocarcinoma (CCA). The aim of this study was to evaluate the effects of the Hh pathway inhibitor, cyclopamine, and MEK inhibitor, U0126, as a single agent or in combination on CCA cell proliferation and survival.

    Methods: Seven CCA cell lines were treated with cyclopamine and/or U0126, and cell proliferation was determined by WST-1 assay. The cell cycle was investigated by fluorescence-activated cell sorter analysis. The expression levels of several cell cycle-related genes were determined by western blot analyses.

    Results: Cyclopamine decreased cell proliferation and arrested the cell cycle at the G1 phase, while U0126 decreased the proliferation of CCA cells with KRAS mutation stronger than with wild-type KRAS. The combination of both inhibitors had an additive antiproliferative effect, particularly in cells with KRAS mutation, and induced caspase-dependent apoptosis in the CCA cells. The expression levels of cell cycle-related proteins that are targets of the two pathways, such as cyclin D1 and cyclin B1, were strongly decreased in some CCA cell lines after combined inhibitor treatment.

    Conclusion: Our results suggest that the Hedgehog and ERK1/2 pathways are important for CCA cell proliferation, and simultaneous inhibition of the two pathways may lead to stronger decreases in cell growth and viability in a subset of CCA cases.

    Journal of cancer research and clinical oncology 2007;133;4;271-8

  • ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells.

    Sarközi R, Miller B, Pollack V, Feifel E, Mayer G, Sorokin A and Schramek H

    Clinical Division of Nephrology, Department of Internal Medicine, Medical University of Innsbruck, Innsbruck, Austria.

    The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.

    Funded by: NHLBI NIH HHS: HL22563

    Journal of cellular physiology 2007;211;1;88-100

  • MEK-ERK is involved in SUMO-1 foci formation on apoptosis.

    Utsubo-Kuniyoshi R, Terui Y, Mishima Y, Rokudai A, Mishima Y, Sugimura N, Kojima K, Sonoda Y, Kasahara T and Hatake K

    Division of Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan.

    Small ubiquitin-related modifier (SUMO) modification appears to regulate the activity, intracellular localization, and stability of the targeted proteins. To explore the relationship among sumoylation, antitumor reagent, and apoptosis, we treated green fluorescence protein (GFP)-SUMO-1-overexpressed K562 cells (K562/GFP-SUMO-1) with mitoxantrone (MIT) as an antitumor reagent. By the treatment with MIT, GFP-SUMO-1 formed foci in nuclei. While by the treatment with a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), GFP-SUMO-1 located homogeneously in nuclei. When K562/GFP-SUMO-1 cells were treated with TPA plus MIT, GFP-SUMO-1 foci became larger and apoptosis was induced more than with MIT alone. The apoptosis induced by TPA plus MIT was prevented by blockage of GFP-SUMO-1 foci by small interfering RNA (siRNA) against SUMO-1. The formation of GFP-SUMO-1 foci was reduced by a MEK inhibitor U0126 or a nuclear export inhibitor leptomycin B, and endogenous SUMO-1 foci were reduced in K562 cells expressing the dominant-negative MEK1 mutant. These results suggest that the formation of SUMO-1 foci is regulated by the MEK-ERK pathway and may induce apoptosis.

    Cancer science 2007;98;4;569-76

  • Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition.

    Wang R, He G, Nelman-Gonzalez M, Ashorn CL, Gallick GE, Stukenberg PT, Kirschner MW and Kuang J

    Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

    Induction of G(2)/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. Here we demonstrate that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G(2)/M transition.

    Funded by: NCI NIH HHS: CA-16672, R01 CA93941

    Cell 2007;128;6;1119-32

  • In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon.

    Mangino G, Percario ZA, Fiorucci G, Vaccari G, Manrique S, Romeo G, Federico M, Geyer M and Affabris E

    Department of Biology-University Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy.

    The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.

    Journal of virology 2007;81;6;2777-91

  • Inhibition of Flt3-activating mutations does not prevent constitutive activation of ERK/Akt/STAT pathways in some AML cells: a possible cause for the limited effectiveness of monotherapy with small-molecule inhibitors.

    Siendones E, Barbarroja N, Torres LA, Buendía P, Velasco F, Dorado G, Torres A and López-Pedrera C

    Unidad de Investigación, Hospital Universitario Reina Sofía, Córdoba, Spain. esiecas@upo.es

    The Flt3 receptor tyrosine kinase is a critical mediator in the pathogenesis of acute myeloid leukaemia (AML). Flt3-activating mutations have been associated with poor prognosis and decreased overall survival of AML patients, thus Flt3 constitutes an ideal target for drug treatment of such disease. Unfortunately, the monotherapy with small-molecule tyrosine kinase inhibitors in clinical trials shows that remission is not permanent, presumably by resistance of Flt3 mutants to inhibitors. An alternative approach for treatment is based on the cooperation between Flt3 and additional intracellular pathways for AML transformation in some patients. Thus, the inhibition of both Flt3 and such pathways may be exploited for successful treatment of the disease. We investigated the importance of Flt3-activating mutations for the constitutive activation of intracellular pathways in primary AML cells, and their effect on cell survival. We found that the main compounds involved in the differentiation, proliferation and survival of AML (MAPK/AKT/STAT) were constitutively activated. However, only four samples showed internal tandem duplications (ITDs) for Flt3. Surprisingly, contrary to previous reports, we found that inhibition of ITD/Flt3 activity did not prevent the phosphorylation of ERK, STAT5 or Akt in some primary AML cells. In parallel, we found that in these cells, Flt3 and ERK or Akt cooperate to regulate cell survival. Our results support the hypothesis that the optimal therapeutic treatment of AML may require not only the oncogenic tyrosine kinase, but also the appropriate combination of different specific inhibitors, thus providing a more effective approach to reverse leukaemogenesis. Thus, we propose that each AML patient should have an individually tailored combination treatment.

    Hematological oncology 2007;25;1;30-7

  • Leptin augments myofibroblastic conversion and fibrogenic activity of human peritoneal mesothelial cells: a functional implication for peritoneal fibrosis.

    Yang AH, Huang SW, Chen JY, Lin JK and Chen CY

    Division of Ultrastructural and Molecular Pathology, Department of Pathology, Taipei Veterans General Hospital, Taipei 112, Taiwan. ahyang@vghtpe.gov.tw

    Background: Myofibroblastic conversion of mesothelial cells is proposed to play an important role in pathological changes following serosal membrane injury.

    Methods: Human peritoneal mesothelial cells (HPMCs) were isolated and maintained in culture. The gene expression was assessed by RT-PCR. Activation of signal transduction was determined by western blot and densitometry. Morphological changes were observed by phase-contrast and electron microscopy.

    Results: In vitro study showed that TGF-beta1-induced myofibroblastic growth of HPMCs was significantly enhanced in the presence of leptin. Augmented expression of alpha-smooth muscle actin, fibronectin and type I collagen mRNA in HPMCs induced by leptin were TGF-beta1-dependent, suggesting that leptin promoted peritoneal fibrogenesis through synergistic activation of the TGF-beta1 signaling system. Leptin and TGF-beta1 synergistically augmented activation of signalling components of mitogen-activated protein kinase (MAPK), STAT3 and Smad but did not modulate the expression of LEPR-B.

    Conclusion: Leptin may act as a profibrogenic TGF-beta1 activated cytokine in peritoneal bioenvironment associated with TGF-beta1 activated pathogenic processes.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2007;22;3;756-62

  • Overexpression of the wip1 gene abrogates the p38 MAPK/p53/Wip1 pathway and silences p16 expression in human breast cancers.

    Yu E, Ahn YS, Jang SJ, Kim MJ, Yoon HS, Gong G and Choi J

    Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-2 dong, Songpa-gu, Seoul 138-736, Korea.

    Wild-type p53-induced phosphatase (Wip1 or PPM1D) is a serine/threonine protein phosphatase expressed under various stress conditions, which selectively inactivates p38 MAPK. The finding that this gene is amplified in association with frequent gain of 17q21-24 in breast cancers supports its role as a driver oncogene. However, the pathogenetic mechanism of the wip1 gene expression in breast carcinogenesis remains to be elucidated. In this study, we examine Wip1 mRNA and protein expression in 20 breast cancer tissues and six cell lines. We additionally investigate the relationship among Wip1, active p38 MAPK, p53, and p16 proteins. In our experiments, Wip1 mRNA was significantly upregulated in 7 of 20 (35%) invasive breast cancer samples. Overexpression of Wip1 was inversely correlated with that of active (phosphor-) p38 MAPK (P = 0.007). Furthermore, Wip1-overexpressing tumors exhibited no or low levels of p16, which normally accumulates upon p38 MAPK activation (P = 0.057). Loss of p16 expression was not associated with hypermethylation of its promoter or loss of heterozygosity on 9p21. Among the 135 primary breast carcinomas further examined, a significant association was found between the Wip1 overexpression and negative staining for p53 (P value = 0.057), indicating that the tumors are wild-type for p53. This is first report showing that Wip1 overexpression abrogates the homeostatic balance maintained through the p38-p53-Wip1 pathway, and contributes to malignant progression by inactivating wild-type p53 and p38 MAPK as well as decreasing p16 protein levels in human breast tissues.

    Breast cancer research and treatment 2007;101;3;269-78

  • Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

    Wissing J, Jänsch L, Nimtz M, Dieterich G, Hornberger R, Kéri G, Wehland J and Daub H

    Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany.

    Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.

    Molecular & cellular proteomics : MCP 2007;6;3;537-47

  • Systematic identification of cellular signals reactivating Kaposi sarcoma-associated herpesvirus.

    Yu F, Harada JN, Brown HJ, Deng H, Song MJ, Wu TT, Kato-Stankiewicz J, Nelson CG, Vieira J, Tamanoi F, Chanda SK and Sun R

    Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.

    The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.

    Funded by: NCI NIH HHS: CA 32737, CA83525, CA91791, P01 CA032737, R01 CA091791; NIDCR NIH HHS: DE14153, R01 DE014153

    PLoS pathogens 2007;3;3;e44

  • Death effector domain DEDa, a self-cleaved product of caspase-8/Mch5, translocates to the nucleus by binding to ERK1/2 and upregulates procaspase-8 expression via a p53-dependent mechanism.

    Yao Z, Duan S, Hou D, Heese K and Wu M

    Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, People's Republic of China.

    Activation of the apical caspase-8 is crucial to the extrinsic apoptotic pathway. Although the death effector domain (DED) of caspase-8 has been reported to be involved in death-inducing signaling complex formation, the detailed mechanism of how DED functions in regulating apoptosis remains largely unknown. Here, we demonstrate that the prodomain of the caspase-8/Mch5 can be further cleaved between two tandemly repeated DEDs (DEDa-DEDb) at the amino-acid residue Asp129 by caspase-8 itself. The DEDa fragment generated from the endogenous caspase-8 was detected in isolated nucleoli upon treatment with TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). Cleaved DEDa appears to translocate into the nucleus by association with extracellular signal-regulated protein kinases-1/2 (ERK1/2). Elimination of ERK1/2 expression by RNA interference resulted in a significant attenuation of nuclear entry of DEDa and reduced caspase-8-dependent apoptosis. In the nucleus, DEDa interacts with TOPORS, a p53 and topoisomerase I binding protein, and possibly displaces p53 from TOPORS, allowing p53 to stimulate caspase-8 gene expression. In summary, we postulate a positive feedback loop involving DEDa, which enables the continual replenishment of procaspase-8 during apoptosis.

    The EMBO journal 2007;26;4;1068-80

  • Activation of JNK-dependent pathway is required for HIV viral protein R-induced apoptosis in human monocytic cells: involvement of antiapoptotic BCL2 and c-IAP1 genes.

    Mishra S, Mishra JP and Kumar A

    Department of Pathology and Laboratory Medicine and Biochemistry, Microbiology, and Immunology, University of Ottawa K1H 8M5, Ottawa, Ontario, Canada.

    Human immunodeficiency virus (HIV) accessory protein viral protein R (Vpr) plays a key role in virus replication and induces cell cycle arrest and apoptosis in various cell types including T cells and neuronal and tumor cells following infection with Vpr-expressing HIV isolates or exposure to the extracellular Vpr protein. The C-terminal Vpr peptide encompassing amino acids 52-96 (Vpr-(52-96)) is required for exerting the apoptotic effects, whereas the N-terminal Vpr-(1-45) peptide is responsible for virus transcription. We demonstrate that Vpr-(52-96) induced apoptosis in human promonocytic THP-1 cells and primary monocytes through the mitochondrial pathway in a caspase-dependent manner. To understand the regulation of Vpr-induced apoptosis, we investigated the signaling pathways, particularly the MAPKs, and the transcription factors involved. Although both Vpr-(52-96) and Vpr-(1-45) peptides induced phosphorylation of all the three members of the MAPKs, Vpr-(52-96)-activated JNK selectively induced apoptosis in monocytic cells through the mitochondrial pathway as determined by using JNK inhibitors SP60025, dexamethasone, curcumin, and JNK-specific small interfering RNAs. Furthermore Vpr-(52-96)-induced apoptosis was mediated by inhibition of downstream antiapoptotic Bcl2 and c-IAP1 genes whose expression could be restored following pretreatment with JNK-specific inhibitors. Overall the results suggest that Vpr-(52-96)-activated JNK plays a key role in inducing apoptosis through the down-regulation of antiapoptotic Bcl2 and c-IAP1 genes.

    The Journal of biological chemistry 2007;282;7;4288-300

  • Mxi2 promotes stimulus-independent ERK nuclear translocation.

    Casar B, Sanz-Moreno V, Yazicioglu MN, Rodríguez J, Berciano MT, Lafarga M, Cobb MH and Crespo P

    Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas (CSIC), Departamento de Biología Molecular, Unidad de Biomedicina CSIC--Universidad de Cantabria, Santander, Spain.

    Spatial regulation of ERK1/2 MAP kinases is an essential yet largely unveiled mechanism for ensuring the fidelity and specificity of their signals. Mxi2 is a p38alpha isoform with the ability to bind ERK1/2. Herein we show that Mxi2 has profound effects on ERK1/2 nucleocytoplasmic distribution, promoting their accumulation in the nucleus. Downregulation of endogenous Mxi2 by RNAi causes a marked reduction of ERK1/2 in the nucleus, accompanied by a pronounced decline in cellular proliferation. We demonstrate that Mxi2 functions in nuclear shuttling of ERK1/2 by enhancing the nuclear accumulation of both phosphorylated and unphosphorylated forms in the absence of stimulation. This process requires the direct interaction of both proteins and a high-affinity binding of Mxi2 to ERK-binding sites in nucleoporins, In this respect, Mxi2 acts antagonistically to PEA15, displacing it from ERK1/2 complexes. These results point to Mxi2 as a key spatial regulator for ERK1/2 and disclose an unprecedented stimulus-independent mechanism for ERK nuclear import.

    Funded by: NIDDK NIH HHS: DK34128, R01 DK034128, R37 DK034128

    The EMBO journal 2007;26;3;635-46

  • Activation of extracellular regulated kinases (ERK1/2) but not AKT predicts poor prognosis in colorectal carcinoma and is associated with k-ras mutations.

    Schmitz KJ, Wohlschlaeger J, Alakus H, Bohr J, Stauder MA, Worm K, Winde G, Schmid KW and Baba HA

    Institute of Pathology and Neuropathology, University Hospital of Essen, Hufelandstr. 55, 45122 Essen, Germany.

    Signal transduction and modulation represent central mechanisms in cellular processes such as cell-cycle regulation, oncogenesis, and apoptosis. The aim of this study was to determine the prognostic relevance of two kinases important in the regulation of cell proliferation and apoptosis in 135 colorectal cancer cases: AKT and extracellular regulated kinases (ERK1/2). We investigated the relationship of phospho-ERK1/2 (pERK1/2) and phospho-AKT (pAKT) with associated parameters (EGFR, COX-2, cyclin-D1), proliferative activity (Ki-67), and apoptosis (TUNEL) using immunohistochemistry. Additionally, the k-ras gene was screened for mutations to determine its putative association with ERK1/2 activation. Activation of ERK1/2 but not AKT correlated statistically with the presence of k-ras mutations (P = 0.015). Survival analysis of phospho-ERK1/2 immunoexpression showed a significant correlation with decreased overall survival (OS). The multivariate Cox regression analysis identified pERK1/2 as an independent prognostic parameter (P = 0.005). Activation of ERK1/2 in colorectal cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor. Furthermore, our data suggest that mutations of the k-ras oncogene may induce activation of ERK1/2. We propose immunohistochemical determination of pERK1/2 status as a promising candidate for the identification of high-risk patients who would benefit from new anticancer drugs targeting the ERK pathway.

    Virchows Archiv : an international journal of pathology 2007;450;2;151-9

  • Angiotensin II activates p44/42 MAP kinase partly through PKCepsilon in H295R cells.

    Lehoux JG and Lefebvre A

    Department of Biochemistry, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4. jean-guy.lehoux@usherbrooke.ca

    Using pharmaceutical and overexpression approaches we have previously reported that in H295R cells, (a) angiotensin II (AII) activates PKCepsilon, PKCalpha and p44/42 MAPK pathway, (b) PKCepsilon, PKCalpha and p44/42 MAPK overexpression inhibits AII-induced CYP11B2 gene transcription and (c) overexpression of PKCepsilon inhibits CYP11B2 gene transcription through p44/42 MAPK activation [LeHoux, J.G., Dupuis, G., Lefebvre, A., 2001. Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms. J. Biol. Chem. 276 (11), 8021-8028; LeHoux, J.G., Lefebvre, A., 2006. Novel protein kinase C-epsilon inhibits human CYP11B2 gene expression through ERK1/2 signalling pathway and JunB. J. Mol. Endocrinol. 36 (1), 51-64]. The aim of the present work was to evaluate the physiological role of endogenous PKCepsilon and PKCalpha isoforms in the activation of p44/42 MAPK by AII. A 50% reduction of PKCepsilon protein by siRNA-PKCepsilon resulted in 35% inhibition of AII-induced p44/42 MAPK activation. Knockdown of PKCepsilon stimulated AII-induced CYP11B2 transcription indicating that the PKCepsilon is not involved in the activation of CYP11B2 gene expression by AII. Furthermore, knockdown of PKCalpha enhanced AII-stimulated CYP11B2 transcription without altering p44/42 MAPK indicating that inhibition of AII-stimulated CYP11B2 gene by PKCalpha does not involve the p44/42 MAPK signalling pathway. These results thus establish that physiologically, PKCepsilon and PKCalpha act through different signalling pathways to inhibit AII-stimulated CYP11B2 gene expression.

    Molecular and cellular endocrinology 2007;265-266;121-5

  • ERK1/2 regulates two sequential steps promoting monocyte survival to peroxynitrite.

    Cerioni L and Cantoni O

    Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino "Carlo Bo", Via Santa Chiara, Urbino (PU), Italy.

    Previous studies from our laboratory indicate that cytosolic phospholipase A(2) (cPLA(2))-released arachidonic acid promotes monocyte/macrophage survival in the presence of peroxynitrite. In particular, the lipid messenger is metabolised by 5-lipoxygenase (5-LO) to 5-hydroxyeicosatetraenoic acid and causes the mitochondrial translocation of protein kinase Calpha (PKCalpha), an event associated with the cytosolic accumulation of Bad and Bax. Here we show that phosphorylation reactions driven by extracellular regulated kinase 1/2 (ERK1/2) critically regulate the activation/nuclear translocation of 5-LO. Inhibition of ERK1/2 was invariably associated with the cytosolic localisation of PKCalpha, the mitochondrial accumulation of Bad and Bax and with a rapid mitochondrial permeability transition-dependent necrosis. All these events were prevented by nanomolar concentrations of 5-hydroxyeicosatetraenoic acid. Hence, in addition to the previously characterised effects on cPLA(2), ERK1/2 critically regulates 5-LO activity in the absence of additional downstream targets in the survival signalling preventing peroxynitrite toxicity.

    Journal of cellular physiology 2007;210;1;177-82

  • MEK-ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-ApoB100 particle assembly.

    Tsai J, Qiu W, Kohen-Avramoglu R and Adeli K

    Division of Clinical Biochemistry, Hospital for Sick Children, University of Toronto, Ontario, Canada M5G 1X8.

    Objective: Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway.

    HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins.

    Conclusions: Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.

    Arteriosclerosis, thrombosis, and vascular biology 2007;27;1;211-8

  • ERK1/2 inactivation and p38 MAPK-dependent caspase activation during guanosine 5'-triphosphate-mediated terminal erythroid differentiation of K562 cells.

    Moosavi MA, Yazdanparast R and Lotfi A

    Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran.

    Since differentiation therapy is one of the promising strategies for treatment of leukemia, universal efforts have been focused on finding new differentiating agents. In that respect, it was recently shown that guanosine 5'-triphosphate (GTP) induced the differentiation of K562 cells, suggesting its possible efficiency in treatment of chronic myelogenous leukemia (CML). However, further investigations are required to verify this possibility. Here, the effects of GTP on activation of mitogen-activated protein kinases (MAPKs) and caspases in K562 cells were examined. Exposure of K562 cells to 100muM GTP markedly inhibited growth (4-70%) and increased percent glycophorin A positive cells after 1-6 days. GTP-induced terminal erythroid differentiation of K562 cells was accompanied with activation of three key caspases, i.e., caspase-3, -6 and -9. More detailed studies revealed that mitochondrial pathway is activated along with down-regulation of Bcl-xL and releasing of cytochrome c into cytosol. Among MAPKs, ERK1/2and p38 were modulated after GTP treatment. Western blot analyses showed that sustained phosphorylation of p38 MAPK was accompanied by a decrease in ERK1/2 activation. These modulatory effects of GTP were observed at early exposure times before the onset of differentiation (3h), and followed for 24-96h. Interestingly, inhibition of p38 MAPK pathway by SB202190 impeded GTP-mediated caspases activation and differentiation in K562 cells, suggesting that p38 MAPK may act upstream of caspases in our system. These results point to a pivotal role for p38 MAPK pathway during GTP-mediated erythroid differentiation of K562 cells and will hopefully have important impact on pharmaceutical evaluation of GTP for CML treatment in differentiation therapy approaches.

    The international journal of biochemistry & cell biology 2007;39;9;1685-97

  • Keratinocyte apoptosis on type I collagen fibrils is prevented by Erk1/2 activation under high calcium condition.

    Fujisaki H, Ebihara T, Irie S, Kobayashi T, Adachi E, Mochitate K and Hattori S

    Nippi Research Institute of Biomatrix, Toride-shi, Ibaraki, Japan.

    Keratinocytes adhere and proliferate well on collagen-coated surfaces, but they undergo apoptosis without differentiation on collagen gels according to our past research. In the current studies, we investigated the necessary conditions for keratinocyte survival on fibrous collagen gels. We found that keratinocytes survived on collagen gels when the medium contains elevated levels (1.8 mM) of calcium. Under this high calcium condition, cells formed multicellular colonies and differentiated. Akt was not activated in cells cultured on collagen gels regardless of the calcium concentration, whereas it was activated in cells cultured on nonfibrous collagen. On the other hand, Erk1/2, key kinases of MAPK pathway, were phosphorylated in cells cultured under high calcium condition but not in cells cultured on collagen gels under low calcium condition. The necessity of Erk1/2 activation for keratinocyte survival on collagen gel was confirmed with experiment using U0126, an inhibitor for Erk1/2. These studies show that activation of Akt depends on collagen assembly, whereas activation of Erk1/2 is induced by increased extracellular calcium concentration. Thus, activation of the Erk1/2 by increasing calcium concentration in the incubation medium may compensate for the loss of Akt activation, allowing keratinocyte survival on collagen gels.

    Connective tissue research 2007;48;3;159-69

  • Modulatory actions of neuropeptide Y on prostate cancer growth: role of MAP kinase/ERK 1/2 activation.

    Ruscica M, Dozio E, Motta M and Magni P

    Center for Endocrinological Oncology, Department of Endocrinology, University of Milan, Milan, Italy.

    Neuroendocrine molecules play a significant role in the progression of human prostate cancer (PCa) and its neuroendocrine differentiation has been associated to a worse prognosis. Evidence exists that, among these molecules, the pleiotropic neuropeptide Y (NPY) and the related receptors may play a role in the normal prostate as well as in the progression of human PCa, which represents one of the most common malignant diseases among men in the Western world. The role of NPY in PCa biology appears to vary in different in vitro human PCa cell systems, since it has been found to reduce the proliferation of LNCaP and DU145 cells, but to stimulate the growth of PC3 cells. These effects are mediated mainly by the NPY Y1 receptor and are associated with a clone-specific pattern of intracellular signaling activation, including a peculiar time-course of MAPK/ERK1/2 phosphorylation (long-lasting in DU145 and transient in PC3 cells). In conclusion, several studies support the concept that NPY and the related receptors are overexpressed in PCa and may play a relevant role in PCa progression. The diagnostic and therapeutical value of targeting the NPY system in PCa will be evaluated in future studies.

    Advances in experimental medicine and biology 2007;604;96-100

  • Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells.

    Carlisle DL, Liu X, Hopkins TM, Swick MC, Dhir R and Siegfried JM

    Department of Pharmacology, University of Pittsburgh, E1340 Biomedical Science Tower, Pittsburgh, PA 15213, USA.

    Nicotinic acetylcholine receptors (nAChR) are expressed on non-neuronal cell types, including normal bronchial epithelial cells, and nicotine has been reported to cause Akt activation in cultured normal airway cells. This study documents mRNA and protein expression of subunits known to form a muscle-type nAChR in non-small cell lung cancer (NSCLC) cell lines. In one NSCLC examined, mRNA and protein for a heteropentamer neuronal-type alpha3beta2 nAChR was detected in addition to a muscle-type receptor. Protein for the alpha5 nAChR was also detected in NSCLC cells. Although, mRNA for the alpha7 nAChR subunit was observed in all cell lines, alpha7 protein was not detectable by immunoblot in NSCLC cell extracts. Immunohistochemistry (IHC) of NSCLC primary tissues from 18 patients demonstrated protein expression of nAChR alpha1 and beta1 subunits, but not alpha7 subunit, in lung tumors, indicating preferential expression of the muscle-type receptor. In addition, the beta1 subunit showed significantly increased expression in lung tumors as compared to non-tumor bronchial tissue. The alpha1 subunit also showed evidence of high expression in lung tumors. Nicotine at a concentration of 10 microM caused phosphorylation of mitogen-activated protein kinase (MAPK) (p44/42) that could be inhibited using nAChR antagonists. Inhibition was observed at 100 nM alpha-bungarotoxin (alpha-BTX) or 10 microM hexamethonium (HEX); maximal inhibition was achieved using a combination of alpha-BTX and HEX. Akt was also phosphorylated in NSCLC cells after exposure to nicotine; this effect was inhibited by the PI3K inhibitor LY294002 and antagonists to the neuronal-type nAChR, but not to the muscle-type receptor. Nicotine triggered influx of calcium in the 273T NSCLC cell line, suggesting that L-type calcium channels were activated. 273T cells also showed greater activation of p44/42 MAPK than of Akt in response to nicotine. Cultures treated with nicotine and the EGFR tyrosine kinase inhibitor gefitinib showed a significant increase in the number of surviving cells compared to gefitinib alone. These data indicate that the muscle-type nAChR, rather than the alpha7 type, is highly expressed in NSCLC and leads to downstream activation of the p44/42 MAPK pathway. Neuronal-type receptors are also present and functional, as evidenced by antagonist studies, although, the expression levels are lower than muscle-type nAChR. They also lead to downstream activation of MAPK and Akt. Nicotine may play a role in regulating survival of NSCLC cells and endogenous acetylcholine released locally in the lung and/or chronic nicotine exposure might play a role in NSCLC development. In addition, exposure of NSCLC patients to nicotine through use of nicotine replacement products or use of tobacco products may alter the efficacy of therapy with EGFR inhibitors.

    Funded by: NCI NIH HHS: P50 CA090440

    Pulmonary pharmacology & therapeutics 2007;20;6;629-41

  • Role of receptor and nonreceptor protein tyrosine kinases in H2O2-induced PKB and ERK1/2 signaling.

    Mehdi MZ, Azar ZM and Srivastava AK

    Laboratory of Cell Signaling, Research Centre, Centre hospitalier de l'Université de Montréal-Technopole Angus, Université de Montréal, Montreal (Quebec) Canada.

    Excessive generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases, including atherosclerosis, hypertension, and vascular complications of diabetes. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully characterized. Hydrogen peroxide (H2O2), a ROS, has been shown to activate several signaling protein kinases, such as extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (PKB) in different cell types, notably in vascular smooth muscle cells. Because these pathways regulate cellular mitogenesis, migration, proliferation, survival, and death responses, their aberrant activation has been suggested to be a potential mechanism of ROS-induced pathologies. The upstream elements responsible for H2O2-induced ERK1/2 and PKB activation remain poorly characterized, but a potential role of receptor and nonreceptor protein tyrosine kinases (PTKs) as triggers that initiate such events has been postulated. Therefore, the aim of this review is to highlight the involvement of receptor and nonreceptor PTKs in modulating H2O2-induced ERK1/2 and PKB signaling.

    Cell biochemistry and biophysics 2007;47;1;1-10

  • Cannabinoid receptor agonist-induced apoptosis of human prostate cancer cells LNCaP proceeds through sustained activation of ERK1/2 leading to G1 cell cycle arrest.

    Sarfaraz S, Afaq F, Adhami VM, Malik A and Mukhtar H

    Department of Dermatology, Medical Sciences Center, University of Wisconsin, Madison, Wisconsin 53706, USA.

    We have recently shown that the expression levels of both cannabinoid receptors CB(1) and CB(2) are higher in human prostate cancer cells than in normal prostate epithelial cells, and treatment of LNCaP cells with WIN-55,212-2 (a mixed CB(1)/CB(2) agonist) resulted in inhibition of cell growth and induction of apoptosis (Sarfaraz, S., Afaq, F., Adhami, V. M., and Mukhtar, H. (2005) Cancer Res. 65, 1635-1641). This study was conducted to understand the mechanistic basis of these effects. Treatment of LNCaP cells with WIN-55,212-2 (1-10 microm; 24 h) resulted in: (i) an arrest of the cells in the G(0)/G(1) phase of the cell cycle; (ii) an induction of p53 and p27/KIP1; (iii) down-regulation of cyclins D1, D2, E; (iii) decrease in the expression of cdk-2, -4, and -6; (iv) decrease in protein expression of pRb; (v) down-regulation of E2F (1-4); and (vi) decrease in the protein expression of DP1 and DP2. Similar effects were also observed when androgen-independent PC3 cells were treated with WIN-55,212-2 (5-30 microm). We further observed sustained up-regulation of ERK1/2 and inhibition of PI3k/Akt pathways in WIN-55,212-2-treated cells. Inhibition of ERK1/2 abrogated WIN-55,212-2-indued cell death suggesting that sustained activation of ERK1/2 leads to cell cycle dysregulation and arrest of cells in G(0)/G(1) phase subsequently leading to an induction of apoptosis. Further, WIN-55,212-2 treatment of cells resulted in a dose-dependent increase in Bax/Bcl-2 ratio in such a way that favors apoptosis. The induction of apoptosis proceeded through down-regulation of caspases 3, 6, 7, and 9 and cleavage of poly (ADP-ribose) polymerases. Based on these data we suggest that cannabinoid receptor agonists should be considered as novel agents for the management of prostate cancer.

    The Journal of biological chemistry 2006;281;51;39480-91

  • Dephosphorylation by default, a potential mechanism for regulation of insulin receptor substrate-1/2, Akt, and ERK1/2.

    Zhande R, Zhang W, Zheng Y, Pendleton E, Li Y, Polakiewicz RD and Sun XJ

    Section of Endocrinology, The University of Chicago, Chicago, Illinois 60637, USA.

    Protein phosphorylation is an important mechanism that controls many cellular activities. Phosphorylation of a given protein is precisely controlled by two opposing biochemical reactions catalyzed by protein kinases and protein phosphatases. How these two opposing processes are coordinated to achieve regulation of protein phosphorylation is unresolved. We have developed a novel experimental approach to directly study protein dephosphorylation in cells. We determined the kinetics of dephosphorylation of insulin receptor substrate-1/2, Akt, and ERK1/2, phosphoproteins involved in insulin receptor signaling. We found that insulin-induced ERK1/2 and Akt kinase activities were completely abolished 10 min after inhibition of the corresponding upstream kinases with PD98059 and LY294002, respectively. In parallel experiments, insulin-induced phosphorylation of Akt, ERK1/2, and insulin receptor substrate-1/2 was decreased and followed similar kinetics. Our findings suggest that these proteins are dephosphorylated by a default mechanism, presumably via constitutively active phosphatases. However, dephosphorylation of these proteins is overcome by activation of protein kinases following stimulation of the insulin receptor. We propose that, during acute insulin stimulation, the kinetics of protein phosphorylation is determined by the interplay between upstream kinase activity and dephosphorylation by default.

    Funded by: NIDDK NIH HHS: R01 DK060128

    The Journal of biological chemistry 2006;281;51;39071-80

  • Expression of phosphorylated ERK1/2 and homeodomain protein CDX2 in cholangiocarcinoma.

    Jinawath A, Akiyama Y, Yuasa Y and Pairojkul C

    Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.

    Purpose: The extracellular signal-regulated kinase (ERK) 1/2 pathway plays important roles in the regulation of cell proliferation, differentiation and cell survival. The caudal-related homeobox protein CDX2 is essential for the development of the intestine, and is related to gastric and gallbladder cancers with the intestinal phenotype. However, the roles of ERK1/2 phosphorylation (pERK1/2) and CDX2 in cholangiocarcinogenesis remain unknown.

    Methods: We investigated the expression of pERK1/2, CDX2 and MUC2 in Thai cholangiocarcinoma (CCA) specimens by means of immunohistochemical staining, and compared the expression of these proteins with clinicopathological factors.

    Results: The pERK1/2 protein was expressed in 29 of 59 (49.2%) CCA cases. Interestingly, in tubular-type CCA, the frequency of pERK1/2 expression was associated with a higher grade of differentiation (P = 0.001). CDX2 expression was observed in 22 of the 59 (37.3%) CCA cases, showed a relationship with MUC2 expression (P = 0.001), and was much higher in papillary-type than tubular-type CCA (P = 0.002).

    Conclusion: These results imply that pERK1/2 may be important for the differentiation of tubular-type CCA, while CDX2 is related to the intestinal phenotype of papillary-type CCA.

    Journal of cancer research and clinical oncology 2006;132;12;805-10

  • Extracellular signal-regulated kinase 1/2 activity is not required in mammalian cells during late G2 for timely entry into or exit from mitosis.

    Shinohara M, Mikhailov AV, Aguirre-Ghiso JA and Rieder CL

    Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

    Extracellular signal-regulated kinase (ERK)1/2 activity is reported to be required in mammalian cells for timely entry into and exit from mitosis (i.e., the G2-mitosis [G2/M] and metaphase-anaphase [M/A] transitions). However, it is unclear whether this involvement reflects a direct requirement for ERK1/2 activity during these transitions or for activating gene transcription programs at earlier stages of the cell cycle. To examine these possibilities, we followed live cells in which ERK1/2 activity was inhibited through late G2 and mitosis. We find that acute inhibition of ERK1/2 during late G2 and through mitosis does not affect the timing of the G2/M or M/A transitions in normal or transformed human cells, nor does it impede spindle assembly, inactivate the p38 stress-activated checkpoint during late G2 or the spindle assembly checkpoint during mitosis. Using CENP-F as a marker for progress through G2, we also show that sustained inhibition of ERK1/2 transiently delays the cell cycle in early/mid-G2 via a p53-dependent mechanism. Together, our data reveal that ERK1/2 activity is required in early G2 for a timely entry into mitosis but that it does not directly regulate cell cycle progression from late G2 through mitosis in normal or transformed mammalian cells.

    Funded by: NCI NIH HHS: CA109182, R01 CA109182, R01 CA109182-01A1, R01 CA109182-02, R01 CA109182-03, R01 CA109182-04; NIGMS NIH HHS: R37 GM040198, R37 GM040198-23; PHS HHS: GMS-40198

    Molecular biology of the cell 2006;17;12;5227-40

  • p38MAPK mediates benzyl isothiocyanate-induced p21WAF1 expression in vascular smooth muscle cells via the regulation of Sp1.

    Moon SK, Choi YH, Kim CH and Choi WS

    Department of Food and Biotechnology, Chungju National University, Chungju, Chungbuk 380-702, South Korea. sumoon66@dreamwiz.com

    It has recently been reported that the transcription factors involved in p21WAF1 activation by certain signaling factors may vary in different cell types. However, the role and importance of the signaling pathway in the transcriptional regulation of p21WAF1 on vascular smooth muscle cells (VSMC) in response to benzyl isothiocyanate (BITC) has been unclear. In this report, we demonstrate that BITC induces the p21WAF1 expression at the transcriptional level. This increase in p21WAF1 gene expression was due to p38MAPK-dependent activation of the p21WAF1 promoter by BITC. Transcription factor Sp1 binding site was identified as the cis-element for the activation of p21WAF1 promoter by BITC, as determined by deletion and mutation analysis. In addition, gel shift and supershift assays demonstrated that this BITC-responsive element binds specifically to the transcription factor Sp1. Treatment with SB203580, an inhibitor of the p38MAPK, significantly downregulated transactivation of BITC-induced Sp1. Finally, the transient expression of VSMC with dominant negative p38MAPK plasmid suppressed BITC-stimulated Sp1 activity. In conclusion, we report that the transcription factor Sp1 involved in the p38MAPK-mediated control of p21WAF1 regulation on VSMC in response to BITC has now been identified.

    Biochemical and biophysical research communications 2006;350;3;662-8

  • Properties and regulation of a transiently assembled ERK2.Ets-1 signaling complex.

    Callaway KA, Rainey MA, Riggs AF, Abramczyk O and Dalby KN

    Graduate Program in Biochemistry, University of Texas, Austin, Texas 78712-0252, USA.

    ERK2 is a proline-directed protein kinase that displays a high specificity for a single threonine (Thr-38) on the substrate Ets-1, which lies within the consensus sequence 36phi-chi-Thr-Pro39 (where phi is typically a small hydrophobic residue and chi appears to be unrestricted). Thr-38 lies in a long flexible N-terminal tail (residues 1-52), which also contains a second potential phosphorylation site, Ser-26. How Ets-1 binds ERK2 to promote the phosphorylation of Thr-38 while simultaneously discriminating against the phosphorylation of Ser-26 is unclear. To delineate the details of the molecular recognition of Ets-1 by ERK2, the binding of various mutants and truncations of Ets-1 were analyzed by fluorescence anisotropy. The data that were obtained support the notion that the N-terminal tail contains a previously unrecognized docking site that promotes the phosphorylation of Thr-38. This new docking site helps assemble the complex of Ets-1 and ERK2 and makes a similar contribution to the stabilization of the complex as does the pointed domain of Ets-1. The in vitro activation of ERK2 by MKK1 induces a large conformational transition of the activation segment (DFG-APE), but neither induces self-association of ERK2 nor destabilizes the stability of the ERK2.Ets-1 complex. This latter observation suggests that interactions intrinsic to the active site are not important for complex assembly, a notion further supported by the observation that the substitution of a number of different amino acids for Pro-39 does not destabilize the complex. Mutagenesis of ERK2 within loop 13 suggests that Ets-1 binds the substrate-binding groove. These data suggest that ERK2 uses two weak docking interactions to specifically assemble the complex, perhaps in doing so denying Ser-26 access to the active site. Displacement of residues 1-138 of Ets-1 (EtsDelta138) from ERK2 by the peptide N-QKGKPRDLELPLSPSL-C, derived from Elk-1, suggests that Ets-1 engages the D-recruitment site (beta7-beta8 reverse turn and the alphaD-alphaE helix) of ERK2. Displacement of EtsDelta138 from ERK2 by the peptide N-AKLSFQFPS-C derived from Elk-1 shows that EtsDelta138 communicates with the F-recruitment site of ERK2 also.

    Funded by: NCRR NIH HHS: P41 RR-01081; NIEHS NIH HHS: ES07247, ES07784; NIGMS NIH HHS: GM59802

    Biochemistry 2006;45;46;13719-33

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Identification of MAPK phosphorylation sites and their role in the localization and activity of hypoxia-inducible factor-1alpha.

    Mylonis I, Chachami G, Samiotaki M, Panayotou G, Paraskeva E, Kalousi A, Georgatsou E, Bonanou S and Simos G

    Laboratory of Biochemistry, Department of Medicine, University of Thessaly, Larissa 41222, Greece.

    Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1alpha, involves several post-translational modifications. To study HIF-1alpha phosphorylation, we have used human full-length recombinant HIF-1alpha as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1alpha. Analysis of in vitro phosphorylated HIF-1alpha by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1alpha. When these mutant forms of HIF-1alpha were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1alpha mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1alpha. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1alpha mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1alpha. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1alpha by blocking its CRM1-dependent nuclear export.

    The Journal of biological chemistry 2006;281;44;33095-106

  • Neuroprotective role of ERK1/2 and ERK5 in a dopaminergic cell line under basal conditions and in response to oxidative stress.

    Cavanaugh JE, Jaumotte JD, Lakoski JM and Zigmond MJ

    Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15221, USA. jec21@pitt.edu

    Loss of motor function in Parkinson's disease is due in part to degeneration of dopamine (DA) neurons. Pharmacological evidence suggests that the mitogen-activated protein kinase signaling pathways involving extracellular signal-regulated kinases (ERKs) play important roles in neuroprotection of DA neurons. However, the relative roles of the several ERK isoforms in the viability of DA neurons have not yet been determined. In the present study, we investigated the contributions of ERK5, as well as ERK1/2, to MN9D cell survival under basal conditions and in response to 6-hydroxydopamine (6-OHDA). We observed that U0126, an inhibitor of ERK activation, decreased basal survival of these cells. To differentiate between ERK1/2 and ERK5, cells were transfected with a dominant negative form of either ERK5 or MEK1, the upstream activator of ERK1/2. Transfection of MN9D cells with either dominant negative construct mimicked U0126, reducing cell survival. Moreover, transfection of the cells in such a way as to increase ERK5 or ERK1/2 activity inhibited 6-OHDA-induced cell death, although this effect was significant only in the case of ERK1/2 activation. These studies suggest that activations of ERK5 and ERK1/2 both promote basal DA cell survival and that ERK1/2 also protects DA cells from oxidative stress. These are the first studies to demonstrate a role for ERK5 in DA neuronal survival and to investigate the relative roles of ERK1/2 and ERK5 in basal DA survival and neuroprotection from oxidative stress.

    Funded by: NIA NIH HHS: AG25848, AG7476; NINDS NIH HHS: NS19608

    Journal of neuroscience research 2006;84;6;1367-75

  • Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells.

    Chang SH, Yu KN, Lee YS, An GH, Beck GR, Colburn NH, Lee KH and Cho MH

    Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, 56-1, Sillim-dong, Seoul 151-742, Korea.

    Inorganic phosphate (Pi) plays a critical role in diverse cellular functions. Among three classes of sodium/phosphate co-transporters (NPTs), two types have been identified in mammalian lung. The potential importance of Pi as a novel signaling molecule and pulmonary expression of NPTs with poor prognosis of diverse lung diseases including cancer have prompted us to begin to define the pathways by which Pi regulates nontumorigenic human bronchial epithelial cells. Pi activates Akt phosphorylation on Thr308 specifically, and activated signal transmits on the Raf/MEK/ERK signaling. Here, we report that Pi controls cell growth by activating ERK cascades and by facilitating the translocation of Mnk1 from cytosol into nucleus through an Akt-mediated MEK pathway. Sequentially, translocated Mnk1 increases eIF4E-BP1 phosphorylation. As a result, Pi stimulates cap-dependent protein translation. Such Akt-mediated signaling of inorganic phosphate may provide critical clues for treatment as well as prevention of diverse lung diseases.

    Funded by: NCI NIH HHS: CA84573

    American journal of respiratory cell and molecular biology 2006;35;5;528-39

  • Expression and activation of mitogen-activated protein kinase in the human endometrium during the menstrual cycle.

    Ozaki T, Takahashi K, Kanasaki H and Miyazaki K

    Department of Obstetrics and Gynecology, Seirei Hamamatsu General Hospital, Hamamatsu, Japan.

    Objective: The purpose of this study was to investigate the fluctuation of expression and activation of mitogen-activated protein kinase in normal human endometrium throughout the menstrual cycle.

    Thirty-three normal endometrial tissues were obtained from fertile women who had undergone hysterectomies for reasons other than endometrial disease. Extracellular signal-regulated kinase, -1, and -2 expression were studied by immunohistochemistry. Moreover, extracellular signal-regulated kinase activity was analyzed by gel kinase assay.

    Results: Western blotting analysis with anti-pan-extracellular signal-regulated kinase antibody mainly demonstrated an immunoreactive band of 42 kd that corresponded to extracellular signal-regulated kinase 2 in the endometrium. The expression of extracellular signal-regulated kinase 2 tended to increase in the secretory phase. Immunohistochemical analysis for extracellular signal-regulated kinase 1 in endometrial sections revealed a weak staining of glands and almost no staining of stromal cells. Immunohistochemical analysis for extracellular signal-regulated kinase 2 in endometrial sections revealed a distinct staining of glands in both proliferative and secretory phases and a weak staining of stromal cells. Although the intensity of staining for extracellular signal-regulated kinase 2 in stromal cells did not change during the secretory phase, in the glands the extracellular signal-regulated kinase 2 was highly stained in the mid-to-late secretory phase. In gel kinase assay revealed that extracellular signal-regulated kinase activity was increased significantly in the mid-to-late secretory phase.

    Conclusion: Expression and activation of extracellular signal-regulated kinase in the human endometrium was increased particularly during the secretory phase. We suggest that fluctuation of extracellular signal-regulated kinase in the human endometrium may be induced by ovarian steroid hormones.

    American journal of obstetrics and gynecology 2006;195;5;1343-50

  • Insulin-like growth factors mediate heterotrimeric G protein-dependent ERK1/2 activation by transactivating sphingosine 1-phosphate receptors.

    El-Shewy HM, Johnson KR, Lee MH, Jaffa AA, Obeid LM and Luttrell LM

    Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Galpha(q/11)-(305-359) peptide that inhibits G(q/11) signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.

    Funded by: NIDDK NIH HHS: DK58283; NIGMS NIH HHS: GM62887

    The Journal of biological chemistry 2006;281;42;31399-407

  • Intracellular HIV-Tat expression induces IL-10 synthesis by the CREB-1 transcription factor through Ser133 phosphorylation and its regulation by the ERK1/2 MAPK in human monocytic cells.

    Gee K, Angel JB, Ma W, Mishra S, Gajanayaka N, Parato K and Kumar A

    Department of Pathology, Division of Virology, Research Institute, Children's Hospital of Eastern Ontario, Ottawa K1H 8L1, Canada.

    Human immunodeficiency virus (HIV)-Tat plays an important role in virus replication and in various aspects of host immune responses, including dysregulation of cytokine production. IL-10, an anti-inflammatory cytokine, is up-regulated during the course of HIV infection representing an important pathway by which HIV may induce immunodeficiency. Here we show that extracellular as well as intracellular Tat induced IL-10 expression in normal human monocytes and promonocytic THP-1 cells. The signaling pathways involved in the regulation of IL-10 production by endogenous Tat remain unknown. To understand the molecular mechanism underlying intracellular Tat-induced IL-10 transcription, we employed a retroviral expression system to investigate the role of MAPKs and the transcription factor(s) involved. Our results suggest that an inhibitor specific for the ERK1/2, PD98059, selectively blocked intracellular Tat-induced IL-10 expression in THP-1 cells. Furthermore, intracellular Tat activated the CREB-1 transcription factor through Ser(133) phosphorylation that was regulated by ERK MAPK as determined by IL-10 promoter analysis and gel shift assays. Overall, our results suggest that intracellular HIV-Tat induces IL-10 transcription by ERK MAPK-dependent CREB-1 transcription factor activation through Ser(133) phosphorylation.

    The Journal of biological chemistry 2006;281;42;31647-58

  • Phosphorylation of the ARE-binding protein DAZAP1 by ERK2 induces its dissociation from DAZ.

    Morton S, Yang HT, Moleleki N, Campbell DG, Cohen P and Rousseau S

    MRC Protein Phosphorylation Unit, Faculty of Life Sciences, University of Dundee, CIR Building, Dow Street, Dundee DD1 5EH, UK.

    A protein in RAW 264.7 macrophages, which became phosphorylated in response to LPS (lipopolysaccharide), was identified as the RNA-binding protein called DAZAP1 [DAZ (deleted in azoospermia)-associated protein 1]. The phosphorylation of this protein was prevented by specific inhibition of MKK1 [MAPK (mitogen-activated protein kinase) kinase 1], indicating that it was phosphorylated via the classical MAPK cascade. Further experiments showed that DAZAP1 was phosphorylated stoichiometrically in vitro by ERK2 (extracellular-signal-regulated protein kinase 2) at two Thr-Pro sequences (Thr269 and Thr315), and that both sites became phosphorylated in HEK-293 (human embryonic kidney 293) cells in response to PMA or EGF (epidermal growth factor), or RAW 264.7 macrophages in response to LPS. Phosphorylation induced by each stimulus was prevented by two structurally distinct inhibitors of MKK1 (PD184352 and U0126), demonstrating that DAZAP1 is a physiological substrate for ERK1/ERK2. The mutation of Thr269 and Thr315 to aspartate or the phosphorylation of these residues caused DAZAP1 to dissociate from its binding partner DAZ. DAZ interacts with PABP [poly(A)-binding protein] and thereby stimulates the translation of mRNAs containing short poly(A) tails [Collier, Gorgoni, Loveridge, Cooke and Gray (2005) EMBO J. 24, 2656-2666]. In the present study we have shown that DAZ cannot bind simultaneously to DAZAP1 and PABP, and suggest that the phosphorylation-induced dissociation of DAZ and DAZAP1 may allow the former to stimulate translation by interacting with PABP.

    Funded by: Medical Research Council: MC_U127084348

    The Biochemical journal 2006;399;2;265-73

  • A bidirectional "alpha(v)beta(3) integrin-ERK1/ERK2 MAPK" connection regulates the proliferation of breast cancer cells.

    Vellon L, Menendez JA and Lupu R

    Department of Medicine, Breast Cancer Translational Research Laboratory, Evanston Northwestern Healthcare Research Institute, Evanston, Illinois 60201, USA.

    In addition to their role in cell migration and adhesion, integrins elicit a series of transduction events that regulate cell-cycle progression and apoptosis in a process known as "outside-in" signaling. A second mode of integrin regulation known as "inside-out" signaling, in which the activation of major cell transduction cascades can influence the activation status of some integrins, has also been described. Here, we have assessed the role of the extracellular signal-regulated kinase (ERK1)/ERK2, mitogen-activated protein kinase (MAPK), and phospoinositide 3-kinase (PI-3'K) signaling pathways in the expression and function of alpha(v)beta(3) integrin in breast cancer models. Pharmacological inhibition of MEK1 and MEK2 with U0126 drastically increased the levels of alpha(v)beta(3) in Heregulin (HRG)-overexpressing MDA-MB-231 cells (231/WT, 231/VEC) and derivatives transfected with the antisense orientation of the HRG-beta2 full length cDNA (231/ASPOOL, 231/AS31). Interestingly, this was related to a significant decrease of viability and of the S- and G2/M subcompartment of the cell cycle in MDA MB 231 cells in response to U0126. Furthermore, specific inhibition of the PI-3'K pathway with LY294002 also induced an increase of alpha(v)beta(3) levels but to a lesser extent. Moreover, pretreatment of MDA-MB-231 cells with U0126 antagonized the effects of small peptidomimetic alpha(v)beta(3) antagonists. Remarkably, inhibition of the PI-3'K/AKT pathway did not exert the same effects, thus suggesting that the "outside-in" as well as the "inside-out" alpha(v)beta(3)-mediated signaling goes primarily through the ERK1/ERK2 MAPK pathway in MDA MB 231 breast cancer cells. Collectively, these results strongly suggest the existence of a bidirectional molecular connection alpha(v)beta(3)-ERK1/ERK2 MAPK that would regulate breast cancer cells survival and proliferation.

    Molecular carcinogenesis 2006;45;10;795-804

  • Mitogen-activated protein kinase-mediated disruption of enhancer-promoter communication inhibits hepatocyte nuclear factor 4alpha expression.

    Hatzis P, Kyrmizi I and Talianidis I

    Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Herakleion, Crete, Greece.

    Hepatocyte nuclear factor 4 (HNF-4) is a key member of the transcription factor network regulating hepatocyte differentiation and function. Activation of the HNF-4 gene involves physical interaction between a distant enhancer and the proximal promoter region, bound by distinct sets of transcription factors. Here we report that, upon mitogen-activated protein (MAP) kinase activation, HNF-4 expression is downregulated in human hepatoma cells. This effect is mediated by the loss of CEBPalpha expression. During MAP kinase signaling, the recruitment of HNF-3beta and HNF-1alpha to the HNF-4 enhancer and RNA polymerase II to the proximal HNF-4 promoter was compromised. CBP, Brg1, and TFIIB were also dissociated from the HNF-4 regulatory regions, and the enhancer-promoter complex was disrupted. Interestingly, the extent of nucleosome acetylation did not decrease at either regulatory region, and HNF-6 and HNF-1alpha, as well as components of the TFIID, remained associated with the proximal promoter during the repressed state. The results point to an absolute requirement of enhancer-promoter communication for maintaining the active state of the HNF-4 gene and provide evidence for a molecular bookmarking mechanism, which may contribute to the prevention of permanent silencing of the locus during the repressed state.

    Molecular and cellular biology 2006;26;19;7017-29

  • Adenosine downregulates DPPIV on HT-29 colon cancer cells by stimulating protein tyrosine phosphatase(s) and reducing ERK1/2 activity via a novel pathway.

    Tan EY, Richard CL, Zhang H, Hoskin DW and Blay J

    Department of Pharmacology, Faculty of Medicine, Sir Charles Tupper Medical Bldg., Dalhousie University, 1459 Oxford St., Halifax, Nova Scotia, Canada B3H 1X5.

    The multifunctional cell-surface protein dipeptidyl peptidase IV (DPPIV/CD26) is aberrantly expressed in many cancers and plays a key role in tumorigenesis and metastasis. Its diverse cellular roles include modulation of chemokine activity by cleaving dipeptides from the chemokine NH(2)-terminus, perturbation of extracellular nucleoside metabolism by binding the ecto-enzyme adenosine deaminase, and interaction with the extracellular matrix by binding proteins such as collagen and fibronectin. We have recently shown that DPPIV can be downregulated from the cell surface of HT-29 colorectal carcinoma cells by adenosine, which is a metabolite that becomes concentrated in the extracellular fluid of hypoxic solid tumors. Most of the known responses to adenosine are mediated through four different subtypes of G protein-coupled adenosine receptors: A(1), A(2A), A(2B), and A(3). We report here that adenosine downregulation of DPPIV from the surface of HT-29 cells occurs independently of these classic receptor subtypes, and is mediated by a novel cell-surface mechanism that induces an increase in protein tyrosine phosphatase activity. The increase in protein tyrosine phosphatase activity leads to a decrease in the tyrosine phosphorylation of ERK1/2 MAP kinase that in turn links to the decline in DPPIV mRNA and protein. The downregulation of DPPIV occurs independently of changes in the activities of protein kinases A or C, phosphatidylinositol 3-kinase, other serine/threonine phosphatases, or the p38 or JNK MAP kinases. This novel action of adenosine has implications for our ability to manipulate adenosine-dependent events within the solid tumor microenvironment.

    American journal of physiology. Cell physiology 2006;291;3;C433-44

  • C-reactive protein induced activation of MAP-K and RANTES in human renal distal tubular epithelial cells in vitro.

    Baer PC, Gauer S, Wegner B, Schubert R and Geiger H

    Department of Internal Medicine III, Division of Nephrology, J.W. Goethe University, Frankfurt/Main, Germany. P.Baer@em.uni-frankfurt.de

    Aims: C-reactive protein (CRP) is a component of the acute-phase reaction to inflammation, severe tissue injury, and infection. Investigations have shown that CRP concentration is highly increased in the urine during acute renal graft dysfunction and, therefore, may affect tubular cell metabolism. Nevertheless, no data about the effects of CRP on human renal tubular epithelial cells are available.

    Methods: Human renal distal tubular cells (DTC) were isolated immunomagnetically and cultured. Cells were stimulated with affinity chromatography pure native CRP from human ascites (10 - 0.001 microg/ml). Phosphorylation of MAP-K was assessed by Westernblot analysis. Release of RANTES and interleukin-6 was evaluated with an enzyme immunoassay. Cytotoxic effects of CRP were determined by a commercially available Live/Dead assay and MTT assay. Effects on cell proliferation were analyzed by a fluorimetric assay.

    Results: Westernblot analysis clearly showed that CRP activates the MAP-K pathway of DTC. CRP upregulated RANTES expression of DTC in a significant and dose-dependent manner. CRP (10 microg/ml) induced a 12.3-fold upregulation, CRP 1 or 0.1 microg/ml induced a 6.3-/2.8-fold RANTES upregulation, respectively. Interleukin-6 synthesis was not influenced. Cytotoxic, proliferative or apoptotic effects were not observed at the concentrations used.

    Conclusions: We demonstrated an activating effect of CRP on DTC in vitro. In vivo, this effect of CRP might be part of the immune activation cascade during episodes of renal graft rejection or bacterial infections.

    Clinical nephrology 2006;66;3;177-83

  • Dysregulation of NGF-signaling and Egr-1 expression by Tat in neuronal cell culture.

    Darbinian-Sarkissian N, Czernik M, Peruzzi F, Gordon J, Rappaport J, Reiss K, Khalili K and Amini S

    Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, Pennsylvania 19122, USA.

    Examination of signal transduction pathways that modulate neuronal cell differentiation and protection against apoptosis has revealed a central role for the MAPK/Erk cascade. The activation of MAPK/Erk through the TrkA NGF signaling pathway is critical for growth and survival of neuronal cells. Here, we investigate the impact of HIV-1 Tat on the NGF-signaling pathway in SK-N-MC neuroblastoma cells. Expression of Tat decreased cell growth and induced apoptosis. Our results revealed dysregulation of various steps involved in the NGF pathway including suppression of MAPK, and inhibition of the promoter activity of Egr-1, a key pleiotropic mediator of the expression of genes involved in cell growth upon expression of Tat in SK-N-MC cells. Similarly, exposure of SK-N-MC to conditioned media derived from cells expressing Tat decreased phosphorylation of MAPK and reduced the level of Egr-1 protein expression in SK-N-MC cells. Furthermore, MAPK was able to phosphorylate Puralpha, a cellular protein that plays an important role in neuronal cell function and differentiation, and this was inhibited by Tat. The ability of Puralpha to interact with a GA/GC-rich sequence positioned upstream from the transcription start site of the Egr-1 promoter provided a rationale to examine Egr-1 expression. Expression of Tat decreased NGF-induced Egr-1 levels in SK-N-MC cells and reduced binding of Puralpha to the Egr-1 promoter. All of these observations support a model where the interplay between Tat and Puralpha dysregulates the NGF pathway including the MAPK/Erk network, resulting in reduced expression and activity of Egr-1 in neuronal cells.

    Funded by: NINDS NIH HHS: P01 NS43980, R01 NS43970

    Journal of cellular physiology 2006;208;3;506-15

  • Glial cell-derived neurotrophic factor (GDNF) promotes low-grade Hs683 glioma cell migration through JNK, ERK-1/2 and p38 MAPK signaling pathways.

    Song H and Moon A

    College of Pharmacy, Duksung Women's University, 419 Ssangmun-Dong, Dobong-Gu, Seoul, Republic of Korea.

    Invasion of tumor cells is the primary cause of therapeutic failure in the treatment of malignant gliomas. In an attempt to investigate the properties of the malignant progression of glioma cells, we examined the correlation between cell migration and glial cell-derived neurotrophic factor (GDNF) secretion of two glioma cell lines which differ in their invasive phenotypes. Here, we show that the high-grade C6 cells are more migrative and secrete more GDNF than the low-grade Hs683 cells. GDNF signaling is more highly activated in C6 cells than in Hs683 cells. Treatment of the Hs683 cells with GDNF significantly increased migration comparable to the C6 cells, revealing the autocrine and/or paracrine effect of GDNF on promotion of the glioma cell migration. We then examined the involvement of mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinases (ERKs) and p38 MAPK in Hs683 cell migration induced by GDNF. A prominent activation of JNK, ERKs and p38 MAPK was observed in the GDNF-treated cells. Functional studies showed that the activation of these MAPKs was critical for Hs683 cell migration induced by GDNF. Our findings revealing molecular mechanisms for the promoting effect of GDNF on glioma cell migration may provide an insight into a better understanding to the malignant progression of human gliomas.

    Neuroscience research 2006;56;1;29-38

  • Poxviral regulation of the host NF-kappaB response: the vaccinia virus M2L protein inhibits induction of NF-kappaB activation via an ERK2 pathway in virus-infected human embryonic kidney cells.

    Gedey R, Jin XL, Hinthong O and Shisler JL

    Department of Microbiology, College of Medicine, University of Illnois, Urbana, 61801, USA.

    Exposure of eukaryotic cells to viruses will activate the host NF-kappaB transcription factor, resulting in proinflammatory and immune protein production. Vaccinia virus (VV), the prototypic orthopoxvirus, expresses products that inhibit this antiviral event. To identify novel mechanisms responsible for this effect, we made use of a VV deletion mutant (MVA) that stimulates NF-kappaBeta activation in infected 293T cells. In this virus-host system, the extents of NF-kappaBeta-regulated gene expression and nuclear translocation were reduced in the presence of either PD 98059 or U0126, two compounds capable of blocking ERK1 and ERK2 phosphorylation. A similar repression was also observed in cells that contained a dominant, nonactive form of ERK2 but not in cells where ERK1 phosphorylation was inhibited via overexpression of a dominant-negative mutant MEK1 protein. Presumably, proteins expressed from a wild-type VV that block ERK2 activity would also inhibit MVA-induced NF-kappaB activation. Indeed, the expression of one such open reading frame, M2L, supported this prediction. First, ectopic M2L expression hampered ERK2 phosphorylation induced by exposure to phorbol myristate acetate. Second, viral M2L expression via infection of cells with a recombinant MVA construct that stably expressed M2L decreased the phosphorylation of ERK2 compared to that in cells infected with the parental MVA strain. Finally, the recombinant M2L-expressing virus restored the "wild-type" NF-kappaB-inhibitory phenotype, as indicated by decreased NF-kappaB migration to infected cell nuclei and interference in transcription. Thus, in 293T cells, VV apparently utilizes its M2L protein to interfere with a step(s) that would otherwise enable ERK2 phosphorylation and the consequential activation of an NF-kappaBeta response.

    Funded by: NIAID NIH HHS: AI055530, AI57153, R01 AI055530, U54 AI057153

    Journal of virology 2006;80;17;8676-85

  • Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle.

    Gosens R, Stelmack GL, Dueck G, McNeill KD, Yamasaki A, Gerthoffer WT, Unruh H, Gounni AS, Zaagsma J and Halayko AJ

    Department of Physiology, University of Manitoba, Winnipeg, Canada. rgosens@mich.ca

    Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.

    American journal of physiology. Lung cellular and molecular physiology 2006;291;3;L523-34

  • Selective reduction of extracellular signal-regulated protein kinase (ERK) phosphorylation in squamous cell carcinoma of the larynx.

    Garavello W, Nicolini G, Aguzzi A, Maggioni D, Leone BE, Viganò P, Gaini RM and Tredici G

    Department of Otorhinolaryngology - Head and Neck Surgery, 20052 Monza (MI), Italy. werner.garavello@unimib.it

    Mitogen-activated protein kinase (MAPK) cascades transmit and amplify signals involved in cell proliferation as well as in cell death. In this study, the potential derangement of MAPK pathways has been evaluated in human squamous cell carcinomas (SCC) of the larynx. The expression and activity of the MAPK p38, ERK1/2p44/p42 and JNK/SAPKp46/p54 have been investigated by immunoblot analysis of tissue homogenates in 27 samples of primary laryngeal cancer and in 27 paired non-neoplastic laryngeal mucosa. On the same tissues, the activation of MAPK JNK/SAPKp46/p54 was also analyzed by an ELISA assay. The results obtained showed that both total and phosphorylated levels of JNK/SAPKp46/p54 and p38 were not different between tumor and normal samples. Conversely, while total protein levels for both ERK1p44 and ERK2p42 were not statistically different between tumor and normal samples, the analysis of the level of the activated forms of ERK1/2 showed a statistically significant decreased phosphorylation of both isoforms in the tumor samples compared to the control tissues. The rate of reduction was similar for both isoforms. Immunohistochemical analysis of all the activated MAPK (p38, JNK/SAPKp46/p54 and ERK1/2p44/p42) in both laryngeal SCC and normal mucosa demonstrated no difference of cellular localization. Activated ERK1/2p44/p42 and activated p38 demonstrated a nucleo-cytoplasmic distribution whereas activated JNK/SAPKp46/p54 were localized into the cytoplasmic membrane. The decreased activity of ERK1/2p44/42 in laryngeal SCC might reflect alterations in tumor suppressing activity or might derive from the interplay among various transduction pathways.

    Oncology reports 2006;16;3;479-84

  • TGF-beta1 induces COX-2 expression and PGE2 synthesis through MAPK and PI3K pathways in human mesangial cells.

    Rodríguez-Barbero A, Dorado F, Velasco S, Pandiella A, Banas B and López-Novoa JM

    Departamento de Fisiología y Farmacología, Instituto Reina Sofía de Investigación Nefrológica, Universidad de Salamanca, Campus Miguel de Unamuno, Edificio Departamental, Salamanca, Spain.

    Transforming growth factor-beta1 (TGF-beta1) plays a fundamental role in the progression of renal diseases. Accumulating evidence has suggested that eicosanoids derived from cyclooxygenase-2 (COX-2) participate in a number of pathological processes in immune-mediated renal diseases. Mesangial cells (MC) play a major role in physiological and pathophysiological renal processes. MC express receptors for TGF-beta1, and COX-2 expression can be induced in MC. However, to date, there are no published data on the possible role of TGF-beta1 in COX-2 expression in human mesangial cells (HMC). We designed studies to determine (1) whether TGF-beta1 stimulates COX-2 expression in primary HMC, (2) whether mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades are involved in TGF-beta1-induced COX-2 expression, and (3) whether prostaglandin (PG)E2 synthesis is affected by TGF-beta1 and MAP kinases and PI3K activation. Studies were performed in primary cultures of HMC and in an immortalized line of HMC. TGF-beta1 induces COX-2 promoter activity and COX-2 mRNA and protein expression in HMC. COX-2 induction is accompanied by increased PGE2 synthesis. Extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and PI3K pathway inhibition blunted TGF-beta1-induced COX-2 overexpression. We demonstrate that TGF-beta1 regulates COX-2 expression in HMC through the activation of ERK1/2, p38 MAPK, and PI3K. These results can help to elucidate the molecular mechanisms underlying the regulation of COX-2 and open up specific strategies for the treatment of glomerular disease.

    Kidney international 2006;70;5;901-9

  • The Src kinase Lyn is required for CCR5 signaling in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages.

    Tomkowicz B, Lee C, Ravyn V, Cheung R, Ptasznik A and Collman RG

    Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    CCR5 is a receptor for several beta chemokines and the entry coreceptor used by macrophage-tropic (R5) strains of HIV-1. In addition to supporting viral entry, CCR5 ligation by the HIV-1 envelope glycoprotein 120 (gp120) can activate intracellular signals in macrophages and trigger inflammatory mediator release. Using a combination of in vitro kinase assay, Western blotting for phospho-specific proteins, pharmacologic inhibition, CCR5 knockout (CCR5Delta32) cells, and kinase-specific blocking peptide, we show for the first time that signaling through CCR5 in primary human macrophages is linked to the Src kinase Lyn. Stimulation of human monocyte-derived macrophages with either HIV-1 gp120 or MIP-1beta results in the CCR5-mediated activation of Lyn and the concomitant Lyn-dependent activation of the mitogen-activated protein (MAP) kinase ERK-1/2. Furthermore, activation of the CCR5/Lyn/ERK-1/2 pathway is responsible for gp120-triggered production of TNF-alpha by macrophages, which is believed to contribute to HIV-1 pathogenesis. Thus, Lyn kinase may play an important role both in normal CCR5 function in macrophages and in AIDS pathogenesis in syndromes such as AIDS dementia where HIV-1 gp120 contributes to inappropriate macrophage activation, mediator production, and secondary injury.

    Funded by: NCI NIH HHS: CA108552; NIAID NIH HHS: AI07324, P30 AI045008; NIMH NIH HHS: MH061139; NINDS NIH HHS: NS027405

    Blood 2006;108;4;1145-50

  • Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase.

    Uchida M, Enomoto A, Fukuda T, Kurokawa K, Maeda K, Kodama Y, Asai N, Hasegawa T, Shimono Y, Jijiwa M, Ichihara M, Murakumo Y and Takahashi M

    Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

    During development of the central and peripheral nervous systems, neurite extension mediated via glial-cell-line-derived neurotrophic factor (GDNF) and its receptor RET is critical for neuronal differentiation. In the present study, we investigated the role of the RET substrate Dok-4 in neurite outgrowth induced by the GDNF/RET signaling pathway. In TGW neuroblastoma cells, which endogenously express both RET and Dok-4, depletion of Dok-4 through treatment with small interfering RNA resulted in a marked decrease in GDNF-stimulated neurite outgrowth. By contrast, exogenous expression of wild-type Dok-4 induced sustained p44/42 mitogen-activated protein kinase (ERK1/2) activation and enhanced neurite outgrowth. Expression of Dok-4 mutants in which the tyrosine residues at codons 187, 220 and 270, conserved between Dok-4, -5, and -6, were each replaced with a phenylalanine inhibited sustained ERK1/2 activation and neurite outgrowth. We also found that Dok-4 induced a significant activation of the small G protein Rap1 and that expression of a dominant active Rap1 mutant restored neurite outgrowth in Dok-4-depleted cells. By contrast, expression of a dominant negative Rap1 mutant impaired GDNF-stimulated neurite outgrowth from TGW cells. Finally, we found that neurite formation in cultured rat hippocampal neurons was enhanced by the expression of Dok-4. Together, our results suggest that Dok-4, through activation of the Rap1-ERK1/2 pathway, regulates GDNF-mediated neurite outgrowth during neuronal development.

    Journal of cell science 2006;119;Pt 15;3067-77

  • Effects of GM-CSF and M-CSF on tumor progression of lung cancer: roles of MEK1/ERK and AKT/PKB pathways.

    Uemura Y, Kobayashi M, Nakata H, Kubota T, Bandobashi K, Saito T and Taguchi H

    Department of Internal Medicine, Kochi Medical School, Kochi 783-8505, Japan. uemuray@med.kochi-u.ac.jp

    Several studies have demonstrated that colony-stimulating factors (CSFs) are closely associated with tumor progression, metastasis and invasion through autocrine or paracrine mechanism in lung cancer. However, biologic roles of CSFs are still unknown. Elucidating the biologic roles of CSFs and the regulatory mechanisms of tumor-specific behavior by CSFs raises the possibility of having a new therapeutic approach for lung cancer. We previously established two adenocarcinoma cell lines, A924 and A964 and a large cell carcinoma cell line MI-4. MI-4 and A924 constitutively produced an abundant dose of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). We examined the effects of GM-CSF and M-CSF on tumor growth, death, and invasion in CSF-producing (A924 and MI-4) and non-producing lung cancer cells (A549 and A964). These cell lines demonstrated both GM-CSF and M-CSF receptor mRNA expression. In our study, GM-CSF seemed to have advantage for tumor proliferation and invasion in lung cancer cells. M-CSF seemed to have advantage for tumor invasion, but not proliferation. The tumor-specific phenotypes (proliferation, invasion and survival) up-regulated by GM-CSF and M-CSF were mediated through MEK/ERK and PI3k/Akt pathways. However, when MEK/ERK was activated by transfection of active form of MEK1 cDNA, the tumor-specific behavior was promoted in CSF-non-producing cells, whereas inhibited in CSF-producing cells though MEK/ERK activation increased constitutive GM-CSF production. MEK/ERK signaling regulated differently tumor-specific behavior between CSF-producing cells and CSF-non-producing cells.

    International journal of molecular medicine 2006;18;2;365-73

  • HIV-TAT protein upregulates expression of multidrug resistance protein 1 in the blood-brain barrier.

    Hayashi K, Pu H, Andras IE, Eum SY, Yamauchi A, Hennig B and Toborek M

    Department of Surgery, University of Kentucky, Lexington, Kentucky 40536, USA.

    Central nervous system (CNS) complications of human immunodeficiency virus (HIV) infection remain a serious health risk in HIV/acquired immunodeficiency syndrome despite significant advances in highly active antiretroviral therapy (HAART). Specific drugs used for HAART are substrates for the efflux transport systems, such as the multidrug resistance-associated proteins (MRPs), which are present on brain microvascular endothelial cells (BMEC) and astrocytes, that is, the main cell types that form the blood-brain barrier (BBB). Thus, drugs employed in HAART are actively removed from the CNS and do not efficiently inhibit HIV replication in the brain. To study the potential mechanisms of this process, the aim of the present research was to address the hypothesis that HIV Tat protein can contribute to upregulation of MRP expression at the BBB level. Tat is a protein produced and released by HIV-infected cells, which may play an important role in brain vascular pathology in the course of HIV infection. Among the family of MRPs, exposure to Tat specifically induced MRP1 messenger ribonucleic acid and protein expression both in BMEC and astrocytes. These alterations were accompanied by enhanced MRP1-mediated efflux functions. Furthermore, activation of the mitogen-activated protein kinase signaling cascade was identified as the mechanism involved in Tat-mediated overexpression of MRP1. These results indicate that Tat exposure can lead to alterations of the BBB functions and decrease HAART efficacy in the CNS through overexpression of drug efflux transporters.

    Funded by: NIAAA NIH HHS: AA013843; NIEHS NIH HHS: P42 ES007380; NIMH NIH HHS: MH072567, MH63022; NINDS NIH HHS: NS39254

    Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 2006;26;8;1052-65

  • LRRC4, a putative tumor suppressor gene, requires a functional leucine-rich repeat cassette domain to inhibit proliferation of glioma cells in vitro by modulating the extracellular signal-regulated kinase/protein kinase B/nuclear factor-kappaB pathway.

    Wu M, Huang C, Gan K, Huang H, Chen Q, Ouyang J, Tang Y, Li X, Yang Y, Zhou H, Zhou Y, Zeng Z, Xiao L, Li D, Tang K, Shen S and Li G

    Cancer Research Institute, Central South University, Changsha, 410078 Hunan, People's Republic of China.

    We have previously reported that the LRRC4 gene, which contains a conserved leucine-rich repeat (LRR) cassette and an immunoglobulin (Ig) IgC2 domain, is associated with glioma suppression both in vitro and in vivo. The present study provides evidence that the conspicuous absence of LRRC4 in high-grade gliomas directly contributes to the increasing tumor grade. The loss of LRRC4 in U251 cells is caused by the loss of homozygosity at chromosome 7q32-ter. It was also found that LRRC4 requires a functional LRR cassette domain to suppress U251 cell proliferation. In the LRR cassette domain, the third LRR motif of the core LRR is found to be indispensable for the function of LRRC4. The inhibitory effect of LRRC4 is accompanied by a decrease in the expression of pERK, pAkt, pNF-kappaBp65, signal transducer and activator of transcription protein-3 (STAT3), and mutant p53, and an increase in the expression of c-Jun NH2-terminal kinase (JNK)2 and p-c-Jun, suggesting that LRRC4 plays a major role in suppressing U251 cell proliferation by regulating the extracellular signal-regulated kinase (ERK)/Akt/NF-kappaBp65, STAT3, and JNK2/c-Jun pathways. In conclusion, LRRC4 may act as a novel candidate of tumor suppressor gene. Therefore, the loss of LRRC4 function may be an important event in the progression of gliomas.

    Molecular biology of the cell 2006;17;8;3534-42

  • Global gene expression analysis of ERK5 and ERK1/2 signaling reveals a role for HIF-1 in ERK5-mediated responses.

    Schweppe RE, Cheung TH and Ahn NG

    Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, 80309, USA.

    ERK5 is a recently characterized MAPK, which is most similar to the well studied ERK1/2 subfamily but uses distinct mechanisms to elicit responses. To understand the specificity of signaling through ERK5 versus ERK1/2, we examined global gene expression changes in response to each pathway. Microarray measurements in retinal pigment epithelial cells revealed 36 genes regulated by ERK5, all which were novel targets for this pathway. 39 genes were regulated by ERK1/2, which included 11 known genes. Of these genes, 19 were regulated by both pathways. Inspection of the 17 genes uniquely regulated by ERK5 revealed that 14 genes (82%) were previously associated with hypoxia via regulation by HIF-1. In contrast, 16 genes (84%) regulated by either ERK5 or ERK1/2 were implicated in hypoxia, most through mechanisms independent of HIF-1. Of the 20 genes regulated by ERK1/2, only 9 were implicated in hypoxia and were not well characterized hypoxia targets. Thus, unlike ERK5, a mechanistic link between ERK1/2 and HIF-1/HRE could not be established on the basis of gene regulation. Activation of both pathways enhanced transcription from a hypoxia-response element and increased HIF-1alpha protein expression. In contrast, ERK5 but not ERK1/2 elevated transcription through GAL4-HIF-1. Most interestingly, ERK5 is not significantly activated by hypoxia in retinal pigment epithelial cells, indicating that ERK5 regulation of these genes is relevant in normoxia rather than hypoxia. Thus, ERK5 and ERK1/2 differ in their mechanisms of gene regulation, and indicate that ERK5 may control hypoxia-responsive genes by a mechanism independent of HIF-1alpha expression control.

    The Journal of biological chemistry 2006;281;30;20993-1003

  • The adaptor molecule Lnk negatively regulates tumor necrosis factor-alpha-dependent VCAM-1 expression in endothelial cells through inhibition of the ERK1 and -2 pathways.

    Fitau J, Boulday G, Coulon F, Quillard T and Charreau B

    INSERM, U643, Nantes, France.

    Lnk, with APS and SH2-B (Src homology 2-B), belongs to a family of SH2-containing proteins with potential adaptor functions. Lnk regulates growth factor and cytokine receptor-mediated pathways implicated in lymphoid, myeloid, and platelet homeostasis. We have previously shown that Lnk is expressed and up-regulated in vascular endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNFalpha). In this study, we have shown that, in ECs, Lnk down-regulates the expression, at both mRNA and protein levels, of the proinflammatory molecules VCAM-1 and E-selectin induced by TNFalpha. Mechanistically, our data indicated that, in response to TNFalpha, NFkappaB/p65 phosphorylation and translocation as well as IkappaBalpha phosphorylation and degradation were unchanged, suggesting that Lnk does not modulate NFkappaB activity. However, Lnk activates phosphatidylinositol 3-kinase (PI3K) as reflected by Akt phosphorylation. Our results identify endothelial nitric-oxide synthase as a downstream target of Lnk-mediated activation of the PI3K/Akt pathway and HO-1 as a new substrate of Akt. We found that sustained Lnk-mediated activation of PI3K in TNFalpha-activated ECs correlated with the inhibition of ERK1/2 phosphorylation, whereas phosphorylation of p38 and c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was unchanged. ERK1/2 inhibition decreases VCAM-1 expression in TNFalpha-treated ECs. Collectively, our results identify the adaptor Lnk as a negative regulator in the TNFalpha-signaling pathway mediating ERK inhibition and suggest a role for Lnk in the interplay between PI3K and ERK triggered by TNFalpha in ECs.

    The Journal of biological chemistry 2006;281;29;20148-59

  • Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity.

    Raines KW, Cao GL, Lee EK, Rosen GM and Shapiro P

    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.

    nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.

    Funded by: NCI NIH HHS: CA-10529; NIBIB NIH HHS: EB-2034, P41 EB002034; NIGMS NIH HHS: R25 GM055036, R25-GM55036

    The Biochemical journal 2006;397;2;329-36

  • Constitutive ERK1/2 activation by a chimeric neurokinin 1 receptor-beta-arrestin1 fusion protein. Probing the composition and function of the G protein-coupled receptor "signalsome".

    Jafri F, El-Shewy HM, Lee MH, Kelly M, Luttrell DK and Luttrell LM

    Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    The beta-arrestins, a small family of G protein-coupled receptor (GPCR)-binding proteins involved in receptor desensitization, have been shown to bind extracellular signal-regulated kinases 1 and 2 (ERK1/2) and function as scaffolds for GPCR-stimulated ERK1/2 activation. To better understand the mechanism of beta-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having beta-arrestin1 fused to the receptor C terminus (NK1-betaArr1). The NK1 receptor couples to both G(s) and G(q/11), resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-betaArr1 is a G protein-uncoupled "constitutively desensitized" receptor that resides almost entirely in an intracellular endosomal compartment. Despite its inability to respond to neurokinin A, we found that NK1-betaArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-betaArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-betaArr1-mediated ERK1/2 activation was completely inhibited when basal PKA and PKC activity were blocked. In addition, the rate of ERK1/2 dephosphorylation was slowed in NK1-betaArr1-expressing cells, suggesting that beta-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that beta-arrestin binding to GPCRs nucleates the formation of a stable "signalsome" that functions as a passive scaffold for the ERK1/2 cascade while confining ERK1/2 activity to an extranuclear compartment.

    Funded by: NIDDK NIH HHS: DK55524

    The Journal of biological chemistry 2006;281;28;19346-57

  • Bluetongue virus and double-stranded RNA increase human vascular permeability: role of p38 MAPK.

    Chiang ET, Persaud-Sawin DA, Kulkarni S, Garcia JG and Imani F

    University of Chicago, Pritzker School of Medicine, Chicago, Illinois 60637, USA.

    Endothelial cell (EC) involvement in viral hemorrhagic fevers has been clearly established. However, virally activated mechanisms leading to endothelial activation and dysfunction are not well understood. Several different potential mechanisms such as direct viral infection, alterations in procoagulant/anticoagulant balance, and increased cytokine production have been suggested. We utilized a model of EC barrier dysfunction and vascular endothelial leakage to explore the effect of bluetongue virus (BTV), a hemorrhagic fever virus of ruminants, on human lung endothelial cell barrier properties. Infection of human lung EC with BTV induced a significant and dose-dependent decrease in trans-endothelial electrical resistance (TER). Furthermore, decreases in TER occurred in conjunction with cytoskeletal rearrangement, suggesting a direct mechanism for viral infection-mediated endothelial barrier disruption. Interestingly, double-stranded RNA (dsRNA) mimicked the effects of BTV on endothelial barrier properties. Both BTV- and dsRNA-induced endothelial barrier dysfunction was blocked by treatment with a pharmacological inhibitor of p38 MAPK. The induction of vascular permeability by dsRNA treatment or BTV infection was concomitent with induction of inflammatory cytokines. Taken together, our data suggest that the presence of dsRNA during viral infections and subsequent activation of p38 MAPK is a potential molecular pathway for viral induction of hemorrhagic fevers. Collectively, our data suggest that inhibition of p38 MAPK may be a possible therapeutic approach to alter viral-induced acute hemorrhagic diseases.

    Journal of clinical immunology 2006;26;4;406-16

  • Pathogenic role of connective tissue growth factor (CTGF/CCN2) in osteolytic metastasis of breast cancer.

    Shimo T, Kubota S, Yoshioka N, Ibaragi S, Isowa S, Eguchi T, Sasaki A and Takigawa M

    Department of Oral and Maxillofacial Surgery and Biopathological Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan.

    Unlabelled: The role of CTGF/CCN2 in osteolytic metastasis by breast cancer cells and its mechanism of action were studied. Osteolytic metastasis accompanied by CCN2 and PTHrP overproduction was efficiently inhibited by an anti-CCN2 antibody. Furthermore, we found that CCN2 was induced by PTHrP through PKA-, PKC-, and ERK-mediated pathways therein.

    Introduction: Connective tissue growth factor (CTGF/CCN2) is a mediator of local angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. PTH-related peptide (PTHrP) is another critical factor in the development of the osteolytic metastasis. Using both in vivo and in vitro approaches, we studied whether/how neutralization of CCN2 prevented bone metastasis and how PTHrP signaling is related.

    A mouse model of bone metastasis by human breast cancer cell line MDA231 was treated with a CCN2-neutralizing antibody, and osteolytic bone metastases were assessed on radiographs and immunohistochemistry. Ccn2 gene expression and transcription were examined by Northern blot and luciferase analysis. Immunoblot analysis and kinase inhibitors were used to identify the signaling pathways implicated. Anti-angiogenic/osteoclastogenic effects of ccn2 downregulation were also evaluated.

    Results: Treatment of mice with a CCN2-neutralizing antibody greatly decreased osteolytic bone metastasis, microvasculature, and osteoclasts involved. The antibody also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Downregulation of ccn2 also repressed osteoclastogenesis. CCN2 expression was specifically observed in cancer cells producing PTHrP and type I PTH/PTHrP receptor (PTH1R) invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Activation of protein kinase C (PKC) and protein kinase A (PKA) was necessary and sufficient for the stimulation of ccn2 by PTHrP. Indeed, inhibition of the extracellular signal-regulated kinase (ERK1/2), PKC, or PKA by specific inhibitors counteracted the stimulation of ccn2 expression. Incubation of MDA231 cells with PTHrP induced the activation of ERK1/2. Consistent with these findings, inhibition of PKC prevented PTHrP-induced ERK1/2 activation, whereas 12-O-tetradecanoylphorbol13-acetate (TPA), a stimulator of PKC, upregulated it.

    Conclusions: CCN2 was critically involved in osteolytic metastasis and was induced by PKA- and PKC-dependent activation of ERK1/2 signaling by PTHrP. Thus, CCN2 may be a new molecular target for anti-osteolytic therapy to shut off the PTHrP-CCN2 signaling pathway.

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2006;21;7;1045-59

  • Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells.

    Herrema H, Czajkowska D, Théard D, van der Wouden JM, Kalicharan D, Zolghadr B, Hoekstra D and van Ijzendoorn SC

    Membrane Cell Biology and Electron Microscopy, Department of Cell Biology, University Medical Center Groningen, University of Groningen, 9713 AV Groningen, The Netherlands.

    The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.

    Molecular biology of the cell 2006;17;7;3291-303

  • Seminal plasma promotes the expression of tumorigenic and angiogenic genes in cervical adenocarcinoma cells via the E-series prostanoid 4 receptor.

    Muller M, Sales KJ, Katz AA and Jabbour HN

    Medical Research Council Human Reproductive Sciences Unit, The Queen's Institute for Medical Research, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom.

    E-series prostanoid (EP)4 receptor is up-regulated in numerous cancers, including cervical carcinomas, and has been implicated in mediating the effects of prostaglandin (PG)E(2) in tumorigenesis. In addition to regulation by endogenously biosynthesized PGE(2), neoplastic cervical epithelial cells in sexually active women may also be regulated by PGs present in seminal plasma. In this study, we investigated the signal transduction pathways mediating the role of seminal plasma and PGE(2) in the regulation of tumorigenic and angiogenic genes via the EP4 receptor in cervical adenocarcinoma (HeLa) cells. HeLa cells were stably transfected with EP4 receptor in the sense orientation. Seminal plasma and PGE(2) signaling via the EP4 receptor induced the activation of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) promoters, expression of COX-2 and VEGF mRNA and protein, and secretion of VEGF protein into the culture medium. Treatment of HeLa cells with seminal plasma or PGE(2) also rapidly induced the phosphorylation of ERK1/2 via the EP4 receptor. Preincubation of cells with a specific EP4 receptor antagonist (ONO-AE2-227) or chemical inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase or MAPK kinase or cotransfection of cells with dominant-negative mutant cDNA targeted against the EGFR, serine/threonine kinase Raf, or MAPK kinase abolished the EP4-induced activation of COX-2, VEGF, and ERK1/2. Therefore, we have demonstrated that seminal plasma and PGE(2) can promote the expression of tumorigenic and angiogenic factors, in cervical adenocarcinoma cells via the EP4 receptor, EGFR, and ERK1/2 signaling pathways.

    Funded by: Medical Research Council: MC_U127684438, U.1276.00.004.00002.01/2(61014)

    Endocrinology 2006;147;7;3356-65

  • Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation.

    Seo HR, Chung DY, Lee YJ, Lee DH, Kim JI, Bae S, Chung HY, Lee SJ, Jeoung D and Lee YS

    Laboratory of Radiation Effect and Laboratory of Radiation Experimental Therapeutics, Korea Institute of Radiological and Medical Sciences, Seoul 139-706.

    The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.

    The Journal of biological chemistry 2006;281;25;17220-7

  • p38 MAPK turns hepatocyte growth factor to a death signal that commits ovarian cancer cells to chemotherapy-induced apoptosis.

    Coltella N, Rasola A, Nano E, Bardella C, Fassetta M, Filigheddu N, Graziani A, Comoglio PM and Di Renzo MF

    Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment, University of Turin Medical School, Candiolo, Italy.

    We recently showed that Hepatocyte Growth Factor (HGF), known as a survival factor, unexpectedly enhances apoptosis in human ovarian cancer cells treated with the front-line chemotherapeutics cisplatin (CDDP) and paclitaxel (PTX). Here we demonstrate that this effect depends on the p38 mitogen-activated kinase (MAPK). In fact, p38 MAPK activity is stimulated by HGF and further increased by the combined treatment with HGF and either CDDP or PTX. The expression of a dominant negative form of p38 MAPK abrogates apoptosis elicited by drugs, alone or in combination with HGF. HGF and drugs also activate the ERK1/2 MAPKs, the PI3K/AKT and the AKT substrate mTOR. However, activation of these survival pathways does not hinder the ability of HGF to enhance drug-dependent apoptosis. Altogether data show that p38 MAPK is necessary for HGF sensitization of ovarian cancer cells to low-doses of CDDP and PTX and might be sufficient to overcome activation of survival pathways. Therefore, the p38 MAPK pathway might be a suitable target to improve response to conventional chemotherapy in human ovarian cancer.

    International journal of cancer 2006;118;12;2981-90

  • Phosphorylation of the HTLV-1 matrix L-domain-containing protein by virus-associated ERK-2 kinase.

    Hémonnot B, Molle D, Bardy M, Gay B, Laune D, Devaux C and Briant L

    Laboratoire Infections Rétrovirales et Signalisation Cellulaire, Centre National pour la Recherche Scientifique UMR5121-Université Montpellier 1, Institut de Biologie, 4 Boulevard Henri IV, CS89508, 34960 Montpellier cedex 2, France.

    L-domain-containing proteins from animal retroviruses play a critical role in the recruitment of the host cell endocytic machinery that is required for retroviruses budding. We recently demonstrated that phosphorylation of the p6(gag) protein containing the L-domain of the human immunodeficiency virus type 1 regulates viral assembly and budding. Here, we investigated whether or not the L-domain-containing protein from another human retrovirus, namely the matrix protein of the human T-cell leukemia virus type 1, that contains the canonical PTAP and PPPY L-domain motifs, shares similar functional properties. We found that MA is phosphorylated at several sites. We identified one phosphorylated amino acid in the HTLV-1 MA protein as being S105, located in the close vicinity to the L-domain sequence. S105 phosphorylation was found to be mediated by the cellular kinase ERK-2 that is incorporated within HTLV-1 virus particles in an active form. Mutation of the ERK-2 target S105 residue into an alanine was found to decrease viral release and budding efficiency of the HTLV-1(ACH) molecular clone from transfected cells. Our data thus support the postulate that phosphorylation of retroviral L-domain proteins is a common feature to retroviruses that participates in the regulation of viral budding.

    Virology 2006;349;2;430-9

  • Dysregulation of the BMP-p38 MAPK signaling pathway in cells from patients with fibrodysplasia ossificans progressiva (FOP).

    Fiori JL, Billings PC, de la Peña LS, Kaplan FS and Shore EM

    Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6081, USA.

    Unlabelled: FOP is a disabling disorder in which skeletal muscle is progressively replaced with bone. Lymphocytes, our model system for examining BMP signaling, cannot signal through the canonical Smad pathway unless exogenous Smad1 is supplied, providing a unique cell type in which the BMP-p38 MAPK pathway can be examined. FOP lymphocytes exhibit defects in the BMP-p38 MAPK pathway, suggesting that altered BMP signaling underlies ectopic bone formation in this disease.

    Introduction: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the primary genetic defect in this condition is unknown, BMP4 mRNA and protein and BMP receptor type IA (BMPRIA) protein are overexpressed in cultured lymphocytes from FOP patients, supporting that altered BMP signaling is involved in this disease. In this study, we examined downstream signaling targets to study the BMP-Smad and BMP-p38 mitogen-activated protein kinase (MAPK) pathways in FOP.

    Protein phosphorylation was assayed by immunoblots, and p38 MAPK activity was measured by kinase assays. To examine BMP target genes, the mRNA expression of ID1, ID3, and MSX2 was determined by quantitative real-time PCR. Statistical analysis was performed using Student's t-test or ANOVA.

    Results: FOP lymphocytes exhibited increased levels of p38 phosphorylation and p38 MAPK activity in response to BMP4 stimulation. Furthermore, in response to BMP4, FOP cells overexpressed the downstream signaling targets ID1 by 5-fold and ID3 by 3-fold compared with controls. ID1 and ID3 mRNA induction was specifically blocked with a p38 MAPK inhibitor, but not extracellular signal-related kinase (ERK) or c-Jun N-terminal kinase (JNK) inhibitors. MSX2, a known Smad pathway target gene, is not upregulated in control or FOP cells in response to BMP, suggesting that lymphocytes do not use this limb of the BMP pathway. However, introduction of Smad1 into lymphocytes made the cells competent to regulate MSX2 mRNA after BMP4 treatment.

    Conclusions: Lymphocytes are a cell system that signals primarily through the BMP-p38 MAPK pathway rather than the BMP-Smad pathway in response to BMP4. The p38 MAPK pathway is dysregulated in FOP lymphocytes, which may play a role in the pathogenesis of FOP.

    Funded by: NIAMS NIH HHS: R01-AR41916

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2006;21;6;902-9

  • Differential regulation of interleukin 5-stimulated signaling pathways by dynamin.

    Gorska MM, Cen O, Liang Q, Stafford SJ and Alam R

    Division of Allergy and Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

    Through the yeast two-hybrid screen we have identified dynamin-2 as a molecule that interacts with the alpha subunit of the interleukin (IL) 5 receptor. Dynamin-2 is a GTPase that is critical for endocytosis. We have shown that dynamin-2 interacts with the IL-5 receptor-associated tyrosine kinases, Lyn and JAK2, in eosinophils. Tyrosine phosphorylation of dynamin is markedly enhanced upon IL-5 stimulation. The inhibition of tyrosine kinases results in complete abolition of ligand-induced receptor endocytosis. Inhibition of dynamin by a dominant-negative mutant or by small interfering RNA results in enhancement of IL-5-stimulated ERK1/2 signaling and cell proliferation. In contrast, the absence of a functional dynamin does not affect STAT5 or AKT phosphorylation or cell survival. Thus, we have identified specific functions for dynamin in the IL-5 signaling pathway and demonstrated its role in receptor endocytosis and termination of the ERK1/2 signaling pathway.

    Funded by: NIAID NIH HHS: AI059719, AI68088, R01 AI50179

    The Journal of biological chemistry 2006;281;20;14429-39

  • The MAP kinase ERK5 binds to and phosphorylates p90 RSK.

    Ranganathan A, Pearson GW, Chrestensen CA, Sturgill TW and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9041, USA.

    We showed previously that p90 RSK was activated in cells expressing an activated mutant of MEK5, the activator of the MAP kinase ERK5. Based on the following evidence, we suggest that ERK5 can directly activate RSK in cells. ERK5 binds to RSK in vitro and co-immunoprecipitates from cell extracts; activation of ERK5 weakens its binding to RSK, suggesting that RSK is released upon activation. Phosphorylation of RSK by ERK5 in vitro causes its activation, indicating that RSK is a substrate of ERK5. In cells activation of ERK5 but not p38 or the c-Jun N-terminal kinase is associated with RSK activation. The large C-terminal domain of ERK5 is not required for binding or activation of RSK by ERK5; however, the common docking or CD domain of ERK5 and the docking or D domain of RSK are important for their association.

    Funded by: NIDDK NIH HHS: DK34128; NIGMS NIH HHS: GM62890

    Archives of biochemistry and biophysics 2006;449;1-2;8-16

  • A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK).

    Liu N, Qiao Y, Cai C, Lin W, Zhang J, Miao S, Zong S, Koide SS and Wang L

    National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing, 100005, People Republic of China.

    HSD-3.8 cDNA (accession number AF311312) encodes a human sperm component. A 0.7 kb fragment (HSD-0.7) containing three immunological epitopes of HSD-3.8 cDNA was prepared and expressed in E. coli. Immunization of female rats with the recombinant HSD-0.7 proteins induced infertility. A cDNA fragment encoding the C-terminal 144 amino acids of human G-protein beta l subunit (Gbeta1-C144) was screened by yeast two-hybrid, when HSD-0.7 segment was used as a bait. Recombinant His6-tagged-Gbeta1-C144 protein was expressed in E. coli BL21 and Anti-Gbeta1 serum was raised with purified Gbeta1-C144. HA-tagged HSD-0.7 and FLAG-tagged Gbeta1 plasmids were constructed and co-transfected into human embryonal kidney 293 cells. Two proteins were localized at superimposable sites in the cytoplasm, and they formed a complex when 500 micromol/L GDP existed. Overexpression of HSD-0.7 activated the G-protein-mediated extracellular signal-regulated kinases (ERK1/2); however, the truncated fragments of HSD-0.7, which lacked either TPR domain or P-loop, lost the ability to activate the ERK1/2 pathway. Further study revealed that the activation of ERK1/2 was protein kinase C (PKC) rather than Ras dependent. These results provide evidence that HSD-3.8 present in spermatocytes and sperm may participate in spermatogenesis and fertilization process by activating the PKC-dependent ERK1/2 signal transduction pathway.

    Frontiers in bioscience : a journal and virtual library 2006;11;1679-89

  • PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation.

    Liu L, Xie Y and Lou L

    Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.

    The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.

    European journal of cell biology 2006;85;5;367-74

  • Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation.

    Gesty-Palmer D, Chen M, Reiter E, Ahn S, Nelson CD, Wang S, Eckhardt AE, Cowan CL, Spurney RF, Luttrell LM and Lefkowitz RJ

    Department of Medicine, Howard Hughes Medical Institute, Duke University, Medical Center, Durham, North Carolina 27710, USA.

    Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

    Funded by: NHLBI NIH HHS: HL16037, HL70631; NICHD NIH HHS: HD043446; NIDDK NIH HHS: DK64353

    The Journal of biological chemistry 2006;281;16;10856-64

  • Association and regulation of heat shock transcription factor 4b with both extracellular signal-regulated kinase mitogen-activated protein kinase and dual-specificity tyrosine phosphatase DUSP26.

    Hu Y and Mivechi NF

    Molecular Chaperone Biology/Radiobiology Program, Medical College of Georgia, 1120 15th Street, CB2803, Augusta, GA 30912, USA.

    The heat shock transcription factors (Hsfs) activate the stress-inducible expression of heat shock proteins (Hsps) and other molecular chaperones in response to stress and, therefore, play an essential role in protein disaggregation and protein folding. In humans, missense mutation in the hsf4 gene causes cataract, and mice bearing a targeted disruption of the hsf4 gene exhibit defects in lens fiber cell differentiation and early cataract formation. Here, we show that Hsf4b is a direct target of the mitogen-activated protein (MAP) kinase extracellular signal-related kinase (ERK) and that phosphorylation of Hsf4b by ERK leads to increased ability of Hsf4b to bind DNA. Surprisingly, Hsf4b also interacts with an ERK-specific dual-specificity tyrosine phosphatase named DUSP26 identified from a yeast two-hybrid screen. While activated ERK phosphorylates Hsf4b, DUSP26 controls the activity of ERK, leading to phosphorylation/dephosphorylation of Hsf4b, altering its ability to bind DNA. Therefore, DUSP26 interaction with Hsf4b places this transcription factor within a regulatory circuit in the MAP kinase signaling pathway.

    Funded by: NCI NIH HHS: CA 62130, R01 CA062130; NIGMS NIH HHS: GM 0707451

    Molecular and cellular biology 2006;26;8;3282-94

  • HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway.

    Toschi E, Bacigalupo I, Strippoli R, Chiozzini C, Cereseto A, Falchi M, Nappi F, Sgadari C, Barillari G, Mainiero F and Ensoli B

    AIDS National Center, Istituto Superiore di Sanità, 00161 Rome, Italy.

    Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.

    Molecular biology of the cell 2006;17;4;1985-94

  • Prostacyclin receptor up-regulates the expression of angiogenic genes in human endometrium via cross talk with epidermal growth factor Receptor and the extracellular signaling receptor kinase 1/2 pathway.

    Smith OP, Battersby S, Sales KJ, Critchley HO and Jabbour HN

    Medical Research Council Human Reproductive Science Unit, Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh, Scotland, UK.

    Prostacyclin (PGI) is a member of the prostanoid family of lipid mediators that mediates its effects through a seven-transmembrane G protein-coupled receptor (IP receptor). Recent studies have ascertained a role for prostanoid-receptor signaling in angiogenesis. In this study we examined the temporal-spatial expression of the IP receptor within normal human endometrium and additionally explored the signaling pathways mediating the role of IP receptor in activation of target angiogenic genes. Quantitative RT-PCR analysis demonstrated the highest endometrial expression of the IP receptor during the menstrual phase compared with all other stages of the menstrual cycle. Immunohistochemical analysis localized the site of IP receptor expression to the glandular epithelial compartment with stromal and perivascular cell immunoreactivity. Expression of the immunoreactive IP receptor protein was greatest during the proliferative and early secretory phases of the menstrual cycle. To explore the role of the IP receptor in glandular epithelial cells, we used the Ishikawa endometrial epithelial cell line. Stimulation of Ishikawa cells and human endometrial biopsy explants with 100 nm iloprost (a PGI analog) rapidly activated ERK1/2 signaling and induced the expression of proangiogenic genes, basic fibroblast growth factor, angiopoietin-1, and angiopoietin-2, in an epidermal growth factor receptor (EGFR)-dependent manner. Furthermore, EGFR colocalized with IP receptor in the glandular epithelial compartment. These data suggest that PGI-IP interaction within glandular epithelial cells can promote the expression of proangiogenic genes in human endometrium via cross talk with the EGFR.

    Funded by: Medical Research Council: G0500047, MC_U127684438, U.1276.00.004.00002.01/2(61014)

    Endocrinology 2006;147;4;1697-705

  • Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation.

    Li Z, Hosoi Y, Cai K, Tanno Y, Matsumoto Y, Enomoto A, Morita A, Nakagawa K and Miyagawa K

    Department of Radiation Research, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan.

    Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.

    Biochemical and biophysical research communications 2006;341;2;363-8

  • Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death.

    Cagnol S, Van Obberghen-Schilling E and Chambard JC

    Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR6543, Université de Nice Sophia-Antipolis, Centre Antoine Lacassagne, 33 Ave Valombrose, 06189, Nice, France.

    Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death.

    Apoptosis : an international journal on programmed cell death 2006;11;3;337-46

  • B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK.

    Letourneux C, Rocher G and Porteu F

    Department of Hematology, Institut Cochin, INSERM U567, Paris, France.

    The protein phosphatase 2A (PP2A) acts on several kinases in the extracellular signal-regulated kinase (ERK) signaling pathway but whether a specific holoenzyme dephosphorylates ERK and whether this activity is controlled during mitogenic stimulation is unknown. By using both RNA interference and overexpression of PP2A B regulatory subunits, we show that B56, but not B, family members of PP2A increase ERK dephosphorylation, without affecting its activation by MEK. Induction of the early gene product and ERK substrate IEX-1 (ier3) by growth factors leads to opposite effects and reverses B56-PP2A-mediated ERK dephosphorylation. IEX-1 binds to B56 subunits and pERK independently, enhances B56 phosphorylation by ERK at a conserved Ser/Pro site in this complex and triggers dissociation from the catalytic subunit. This is the first demonstration of the involvement of B56-containing PP2A in ERK dephosphorylation and of a B56-specific cellular protein inhibitor regulating its activity in an ERK-dependent fashion. In addition, our results raise a new paradigm in ERK signaling in which ERK associated to a substrate can transphosphorylate nearby proteins.

    The EMBO journal 2006;25;4;727-38

  • Reduced stability of mitogen-activated protein kinase kinase-2 mRNA and phosphorylation of poly(A)-binding protein (PABP) in cells overexpressing PABP.

    Ma S, Musa T and Bag J

    Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.

    The poly(A)-binding protein (PABP) is an important regulator of mRNA translation and stability. The cellular level of PABP is controlled by regulating its mRNA translation by a feedback mechanism. The important aspect of this mechanism is that PABP binds to an adenosine-rich cis-element at the 5'-untranslated region of its own mRNA and inhibits its translation. To assess the importance of controlling the PABP level, we studied the effect of PABP overexpression on the transcription profile using the microarray technique. In PABP-overexpressing cells, 19 mRNAs showed a reduction in cellular levels due to reduced mRNA stability, and one showed an increase due to increased mRNA stability. Among these mRNAs, the MKK-2 mRNA encodes the protein kinase activator of ERK1/2 kinase, which is involved in the phosphorylation of eukaryotic initiation factor (eIF) 4E. As a result, mRNA translation may be regulated by the cellular level of MKK-2. In this study, we show that the abundance of the MKK-2 polypeptide is reduced in PABP-overexpressing cells. In these cells, the levels of phosphorylated PABP, eIF4E, and ERK2 are also reduced. Treatment of HeLa cells with the MKK-2 inhibitor U0126 reduced PABP phosphorylation, suggesting that the phosphorylation of PABP is mediated by the MKK-2/ERK signaling pathway. Thus, a novel signaling pathway involving MKK-2 and ERK1/2 may down-regulate the activity of PABP and eIF4E by controlling their phosphorylation and compensates for the effect of excess cellular PABP.

    The Journal of biological chemistry 2006;281;6;3145-56

  • Helicobacter pylori encoding the pathogenicity island activates matrix metalloproteinase 1 in gastric epithelial cells via JNK and ERK.

    Krueger S, Hundertmark T, Kalinski T, Peitz U, Wex T, Malfertheiner P, Naumann M and Roessner A

    Institute of Pathology, Department of Gastroenterology, Hepatology and Infectious Diseases, Magdeburg, Germany. sabine.krueger@medizin.uni-magdeburg.de

    Helicobacter pylori colonizes the human gastric epithelium and induces an inflammatory response that is a trigger for gastric carcinogenesis. Matrix metalloproteinases (MMPs) have recently been shown to be up-regulated in gastric epithelial cells infected with H. pylori and might contribute to the pathogenesis of peptic ulcer. The aim of this study was to extend the knowledge about the effect of H. pylori infection on MMP-1 expression by gastric epithelial cells, the kinetics of induction, the pathogenetic properties of the bacterium, and the intracellular signaling pathways required for MMP-1 up-regulation. Expression of MMP-1 was induced more than 10-fold by co-culture of AGS+cells with H. pylori strains carrying the pathogenicity island (PAI). H. pylori strains with mutations in the PAI and a defective type IV secretion system had no effect on MMP-1. Double immunofluorescence revealed strong MMP-1 staining in epithelial cells of gastric biopsies at sites of bacterial attachment. In vitro, MMP-1 is up-regulated by interleukin-1beta and tumor necrosis factor-alpha, but these regulatory mechanisms are not operating in H. pylori infection as shown by inhibitory antibodies. Specific inhibitors of JNK kinase and ERK1/2 kinase were found to suppress the H. pylori-induced MMP-1 expression and activity. AGS cells treated with antisense MMP-1 showed a significantly reduced potential to degrade reconstituted basement membrane. Our results suggest that in gastric epithelial cells, H. pylori up-regulates MMP-1 in a type IV secretion system-dependent manner via JNK and ERK1/2. Induction of MMP-1 is further implicated in complex processes induced by H. pylori, resulting in tissue degradation and remodeling of the gastric mucosa.

    The Journal of biological chemistry 2006;281;5;2868-75

  • The tyrosine kinase Syk regulates TPL2 activation signals.

    Eliopoulos AG, Das S and Tsichlis PN

    Laboratory of Molecular and Cellular Biology, Division of Basic Sciences, the University of Crete Medical School, Heraklion 71003, Crete, Greece. eliopag@med.uoc.gr

    Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.

    Funded by: NCI NIH HHS: R01 CA38047

    The Journal of biological chemistry 2006;281;3;1371-80

  • Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases.

    Hayashi H, Matsuzaki O, Muramatsu S, Tsuchiya Y, Harada T, Suzuki Y, Sugano S, Matsuda A and Nishida E

    Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

    Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.

    The Journal of biological chemistry 2006;281;3;1332-7

  • beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor.

    Shenoy SK, Drake MT, Nelson CD, Houtz DA, Xiao K, Madabushi S, Reiter E, Premont RT, Lichtarge O and Lefkowitz RJ

    Howard Hughes Medical Institute at Duke University Medical Center, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

    Physiological effects of beta adrenergic receptor (beta2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G(i) inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous beta-arrestins by siRNA. beta-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either beta-arrestin1 or -2 by small interfering RNA. In G(s) knock-out mouse embryonic fibroblasts, wild-type beta2AR recruited beta-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel beta2AR mutant (beta2AR(T68F,Y132G,Y219A) or beta2AR(TYY)), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit beta-arrestins, undergo beta-arrestin-dependent internalization, and activate beta-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and beta-arrestin recruitment, led to the formation of stable receptor-beta-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gbetagamma release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the beta2AR can signal to ERK via a GRK5/6-beta-arrestin-dependent pathway, which is independent of G protein coupling.

    Funded by: NHLBI NIH HHS: HL16037, HL70631; NIDDK NIH HHS: T32-DK-00701; NIGMS NIH HHS: GM66099

    The Journal of biological chemistry 2006;281;2;1261-73

  • Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling.

    Lin YW and Yang JL

    Molecular Carcinogenesis Laboratory, Institute of Biotechnology, Hsinchu, Taiwan.

    The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339-342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser(296)/Ser(323) domain, which was not affected by substituting Ala for Ser at Ser(359)/Ser(364). The DEF and Ser(296)/Ser(323) sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser(359)/Ser(364) sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser(296) MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser(296) MKP-1 bound to SCF(Skp2) ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCF(Skp2) cooperation, thereby sustaining ERK activation.

    The Journal of biological chemistry 2006;281;2;915-26

  • Glycogen synthase kinase-3 is a negative regulator of extracellular signal-regulated kinase.

    Wang Q, Zhou Y, Wang X and Evers BM

    Department of Surgery, The University of Texas Medical Branch, Galveston, TX 77555-0536, USA.

    Glycogen-synthase kinase-3 (GSK-3) and extracellular signal-regulated kinase (ERK) are critical downstream signaling proteins for the PI3-kinase/Akt and Ras/Raf/MEK-1 pathway, respectively, and regulate diverse cellular processes including embryonic development, cell differentiation and apoptosis. Here, we show that inhibition of GSK-3 using GSK-3 inhibitors or RNA interference (RNAi) significantly induced the phosphorylation of ERK1/2 in human colon cancer cell lines HT29 and Caco-2. Pretreatment with the PKCdelta-selective inhibitor rottlerin or transfection with PKCdelta siRNA attenuated the phosphorylation of ERK1/2 induced by the GSK-3 inhibitor SB-216763 and, furthermore, treatment with SB-216763 or transfection with GSK-3alpha and GSK-3beta siRNA increased PKCdelta activity, thus identifying a role for PKCdelta in the induction of ERK1/2 phosphorylation by GSK-3 inhibition. Treatment with SB-216763 increased expression of cyclooxygenase-2 (COX-2) and IL-8, which are downstream targets of ERK1/2 activation; this induction was abolished by MEK/ERK inhibition, suggesting GSK-3 inhibition induced COX-2 and IL-8 through ERK1/2 activation. The transcriptional induction of COX-2 and IL-8 by GSK-3 inhibition was further demonstrated by the increased COX-2 and IL-8 promoter activity after SB-216763 treatment or transfection with GSK-3alpha or GSK-3beta siRNA. Importantly, our findings identify GSK-3, acting through PKCdelta, as a negative regulator of ERK1/2, thus revealing a novel crosstalk mechanism between these critical signaling pathways.

    Funded by: NIA NIH HHS: R37 AG010885, R37 AG10885; NIDDK NIH HHS: P01 DK035608, P01 DK35608, R01 DK048498, R01 DK48498

    Oncogene 2006;25;1;43-50

  • Crystal structure of human ERK2 complexed with a pyrazolo[3,4-c]pyridazine derivative.

    Kinoshita T, Warizaya M, Ohori M, Sato K, Neya M and Fujii T

    Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan. kinotk@b.s.osakafu-u.ac.jp

    A series of pyrazolopyridazine compounds were briefly investigated as ERK2 inhibitors. The crystal structure of ERK2 complexed with an allyl derivative was determined. The compound induces structural change including movement of the glycine-rich loop and peptide flip between Met108-Glu109. As a result, the newly formed subsite can recognize small hydrophobic substituents but not hydrophilic ones.

    Bioorganic & medicinal chemistry letters 2006;16;1;55-8

  • A role of p44/42 mitogen-activated protein kinases in formyl-peptide receptor-mediated phospholipase D activity and oxidant production.

    Paruch S, El-Benna J, Djerdjouri B, Marullo S and Périanin A

    Département de Biologie Cellulaire de l'Institut Cochin, The Institut National de la Recherche Médicale (INSERM U567), The Centre National de la Recherche Scientifique (UMR 8104), and the Université René Descartes, Paris France.

    Phosphatidylcholine-specific phospholipase D (PLD) is a major cellular source of phosphatidic acid and choline, which regulate various physiopathological processes. PLD activation mediated by chemoattractants involves protein phosphorylation. This study provides pharmacological and biochemical evidence of a major role of p44/42 MAP kinases (ERK1/2) in PLD activation induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). ERK1/2 inhibition by the MEK1/2 antagonist U0126 in neutrophilic HL-60 cells or HEK 293T cells stably expressing fMLP receptors abolished fMLP-mediated PLD activity. Conversely, a constitutively activated MEK1 mutant expressed in HEK 293T cells potentiated fMLP-induced PLD activity. Expression of inactive PLD mutants showed that PLD2, but not PLD1, contributed to fMLP-mediated PLD activity. PLD2 co-immunoprecipitated with ERK1/2 and became phosphorylated on MAP kinase consensus sites in fMLP-stimulated cells. In cell-free systems, ERK2 gave rise to strong ATP-dependent PLD activity and directly phosphorylated PLD2 that generated two phosphopeptides only after tryptic digestion. Finally, pharmacological inhibition of ERK activation and the inhibition of PLD expression by antisense oligonucleotides in HL-60 cells suggest that the ERK/PLD2 pathway contributes to fMLP-mediated oxidant production. In conclusion, the fMLP-mediated PLD activity is regulated by ERK1/2, involving a predominant contribution of PLD2. The ERK/PLD2 coupling may provide potential pharmacological targets to control PLD-associated cellular dysfunctions.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;1;142-4

  • Dihydrosphingosine 1-phosphate stimulates MMP1 gene expression via activation of ERK1/2-Ets1 pathway in human fibroblasts.

    Bu S, Yamanaka M, Pei H, Bielawska A, Bielawski J, Hannun YA, Obeid L and Trojanowska M

    Division of Rheumatology & Immunology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes formation of important regulators of inter- and intracellular signaling, sphingosine-1 phosphate (S1P), and dihydrosphingosine 1-phosphate (dhS1P). In this study, we investigated the role of SphK1 in the regulation of expression of matrix metalloproteinase 1 (MMP1) in dermal fibroblasts, a key event in regulation of extra cellular matrix. We show that overexpression of SphK1 up-regulated MMP1 protein, MMP1 mRNA, and MMP1 promoter activity, and this action of SphK1 required activation of the ERK1/2-Ets1 and NF-kappaB pathways. Furthermore, experiments using SphK1 specific siRNA demonstrated that SphK1 is required for the TNF-alpha stimulation of MMP1. Additional data revealed a specific role of dhS1P, and not S1P, as a mediator of SphK1-dependent activation of ERK1/2 and up-regulation of MMP1. The stimulatory effect of dhS1P was sensitive to pertussis toxin, suggesting a possible involvement of a G-protein-coupled receptor. In contrast, S1P, but not dhS1P, stimulated the induction of COX-2, which demonstrated selective actions of these two closely related bioactive lipids. In conclusion, this study describes a novel mode of SphK1 signaling through generation of dhS1P with a key role in mediating transcriptional responses to TNF-alpha. This is the first report of selective function of dhS1P as compared with the better studied S1P.

    Funded by: NIA NIH HHS: AG 16583; NIAMS NIH HHS: P60 AR049459

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;1;184-6

  • Opposite effect of ERK1/2 and JNK on p53-independent p21WAF1/CIP1 activation involved in the arsenic trioxide-induced human epidermoid carcinoma A431 cellular cytotoxicity.

    Huang HS, Liu ZM, Ding L, Chang WC, Hsu PY, Wang SH, Chi CC and Chuang CH

    Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. huanghs@mail.ncku.edu.tw

    While arsenic trioxide (As2O3) is an infamous carcinogen, it is also an effective chemotherapeutic agent for acute promyelocytic leukemia and some solid tumors. In human epidermoid carcinoma A431 cells, we found that As2O3 induced cell death in time- and dose-dependent manners. Similarly, dependent regulation of the p21WAF1/CIP1 (p21) promoter, mRNA synthesis, and resultant protein expression was also observed. Additionally, transfection of a small interfering RNA of p21 could block the As2O3-induced cell growth arrest. The As2O3-induced p21 activation was attenuated by inhibitors of EGFR and MEK in a dose-dependent manner. Using a reporter assay, we demonstrated the involvement of the EGFR-Ras-Raf-ERK1/2 pathway in the promoter activation. In contrast, JNK inhibitor enhanced the As2O3-induced p21 activation, also in a dose-dependent fashion. Over-expression of a dominant negative JNK plasmid likewise also enhanced this activation. Furthermore, MEK inhibitor attenuated the anti-tumor effect of As2O3. In contrast, in combination with JNK inhibitor and As2O3 enhanced cellular cytotoxicity. Therefore, we conclude that in A431 cells the ERK1/2 and JNK pathways might differentially contribute to As2O3-induced p21 expression and then due to cellular cytotoxicity.

    Journal of biomedical science 2006;13;1;113-25

  • Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.

    Adams DG, Coffee RL, Zhang H, Pelech S, Strack S and Wadzinski BE

    Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

    Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Balpha/delta regulatory subunits by approximately 3-fold or knock-down of Balpha by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Balpha/delta subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Balpha knock-down cells but elevated in Balpha- and Bdelta-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Balpha or Bdelta. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABalphaC and ABdeltaC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABalphaC and ABdeltaC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.

    Funded by: NCI NIH HHS: CA68485; NIDDK NIH HHS: 5T32DK07563, DK20593; NIGMS NIH HHS: GM51366, GM62265; NIMH NIH HHS: MH19732

    The Journal of biological chemistry 2005;280;52;42644-54

  • New insights into the catalytic activation of the MAPK phosphatase PAC-1 induced by its substrate MAPK ERK2 binding.

    Zhang Q, Muller M, Chen CH, Zeng L, Farooq A and Zhou MM

    Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, One Gustave L. Levy Place, New York, NY 10029, USA.

    PAC-1 is an inducible, nuclear-specific, dual-specificity mitogen-activated protein (MAP) kinase phosphatase that has been shown recently to be a transcription target of the human tumor-suppressor protein p53 in signaling apoptosis and growth suppression. However, its substrate specificity and regulation of catalytic activity thus far remain elusive. Here, we report in vitro characterization of PAC-1 phosphatase activity with three distinct MAP kinase subfamilies. We show that the recombinant PAC-1 exists in a virtually inactive state when alone in vitro, and dephosphorylates extracellular signal-regulated kinase 2 (ERK2) but not p38alpha or c-Jun NH(2)-terminal kinase 2 (JNK2). ERK2 dephosphorylation by PAC-1 requires association of its amino-terminal domain with ERK2 that results in catalytic activation of the phosphatase. p38alpha also interacts with but does not activate PAC-1, whereas JNK2 does not bind to or cause catalytic activation by PAC-1. Moreover, our structure-based analysis reveals that individual mutation of the conserved Arg294 and Arg295 that likely comprise the phosphothreonine-binding pocket in PAC-1 to either alanine or lysine results in a nearly complete loss of its phosphatase activity even in the presence of ERK2. These results suggest that Arg294 and Arg295 play an important role in PAC-1 catalytic activation induced by ERK2 binding.

    Funded by: NCI NIH HHS: CA80938

    Journal of molecular biology 2005;354;4;777-88

  • Genistein-induced neuronal differentiation is associated with activation of extracellular signal-regulated kinases and upregulation of p21 and N-cadherin.

    Hung SP, Hsu JR, Lo CP, Huang HJ, Wang JP and Chen ST

    Department of Life Sciences, National Cheng Kung University, Tainan City 701, Taiwan.

    Neuronal differentiation in the mammalian CNS is driven by multiple events. When treated with retinoic acid (RA), hNTera-2 (NT-2) cells undergo postmitotic neuronal differentiation. Here, we show that a prolonged exposure of NT-2 cells with non-cytotoxic doses of genistein, a protein tyrosine kinase (PTK) inhibitor, induced differentiation of NT-2 cells. Additionally, genistein enhanced RA-induced neuronal differentiation by increasing the activation of extracellular signal-related kinase 1/2 (ERK1/2) via phosphorylation at Thr183 and Tyr185 in 3-7 days. Meanwhile, genistein also upregulated N-cadherin and p21 (a Cdk inhibitor), but downregulated proliferating cell nuclear antigen protein (PCNA). MEK1/2 inhibitors, such as PD98059 and U0126, reduced RA-induced ERK1/2 activity, but could not block the genistein effects. Our observations indicate that genistein-induced neuronal differentiation is not dependent of the MEK-ERK signaling cascade. Instead, genistein-upregulated ERK activation is likely due to this chemical's direct effect on chromosome and gene transcription, rather than its inhibition on tyrosine kinases. Failure of inhibition of ERK1/2 activation by the MEK1/2 inhibitors PD98059 and U0126 suggests presence of an unknown activator for ERK1/2 in neuronal cells.

    Journal of cellular biochemistry 2005;96;5;1061-70

  • Phosphorylation of the homeotic tumor suppressor Cdx2 mediates its ubiquitin-dependent proteasome degradation.

    Gross I, Lhermitte B, Domon-Dell C, Duluc I, Martin E, Gaiddon C, Kedinger M and Freund JN

    Development and Physiopathology of the Intestine and Pancreas, 3, avenue Molière, 67200 Strasbourg, France.

    The Caudal-related homeodomain transcription factor Cdx2 plays a key role in intestinal cell fate determination. Reduction of Cdx2 expression is a feature of many human colon carcinomas and inactivation of one cdx2 allele facilitates the development of invasive adenocarcinoma in the murine colon. Here, we investigated the post-translational regulation of Cdx2. We showed that various forms of Cdx2 coexist in the intestine and colon cancer cell lines, some of them being phosphorylated forms. We found that cyclin-dependent kinase 2 phosphorylated Cdx2 in vitro and in vivo. Using site-specific mutagenesis, we identified serine 281 as a new key residue for Cdx2 phosphorylation. Intriguingly, serine 281 belongs to a conserved motif of four evenly spaced serines (the 4S motif) similar to the one controlling beta-catenin degradation by the proteasome pathway. A nonphosphorylated mutant Cdx2 lacking the 4S motif (4S>A) exhibited reduced polyubiquitination upon proteasome inhibition and increased stability compared to wild-type Cdx2. In addition, we found that this mutant was less efficient to suppress colony formation than wild-type Cdx2. Thus, our data highlight a novel post-translational mechanism controlling Cdx2 degradation via phosphorylation and polyubiquitination, which may be of importance for intestinal development and cancer.

    Oncogene 2005;24;54;7955-63

  • A mutation in the common docking domain of ERK2 in a human cancer cell line, which was associated with its constitutive phosphorylation.

    Arvind R, Shimamoto H, Momose F, Amagasa T, Omura K and Tsuchida N

    Department of Molecular Cellular Oncology and Microbiology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 113-8549 Tokyo, Japan.

    The EGFR/Ras/Raf/MEK/ERK pathway is a major pathway involved in the control of growth signals, cell survival and differentiation. Mutations of signaling components, such as EGFR (c-erbB1), Ras, and B-Raf, have been shown to play roles in the genesis of human cancer, while point mutation of ERK has not been reported. In this study, we present evidence for a mutation in an oral squamous cell carcinoma cell line, HSC6. PCR-amplification of cDNA, cloning and sequencing resulted in the identification of glutamic acid to lysine substitution at codon 322 (E322K) that occurred in the common docking (CD) domain of ERK2. The mutant protein contributed towards faster-migration in SDS-PAGE, and constitutive phosphorylation in a MEK-dependent manner. The transient transfection of the mutant ERK2 in 293T cells resulted in the expression of the same faster-migrating band in SDS-PAGE as was detected in HSC6 cells, which was preferentially phosphorylated relative to endogenous wild-type ERK2. The present study is the first to report ERK2 substitution mutation in a human cancer cell line which resulted in constitutive phosphorylation.

    International journal of oncology 2005;27;6;1499-504

  • ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1.

    Tárrega C, Ríos P, Cejudo-Marín R, Blanco-Aparicio C, van den Berk L, Schepens J, Hendriks W, Tabernero L and Pulido R

    Centro de Investigación Príncipe Felipe, Valencia, Spain.

    The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.

    The Journal of biological chemistry 2005;280;45;37885-94

  • Platelet-derived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts.

    Svegliati S,