G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
annexin A1
G00000691 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000020016 (Vega human gene)
ENSG00000135046 (Ensembl human gene)
301 (Entrez Gene)
1082 (G2Cdb plasticity & disease)
ANXA1 (GeneCards)
151690 (OMIM)
Marker Symbol
Protein Sequence
P04083 (UniProt)

Literature (103)

Pubmed - other

  • Enhanced neutrophil expression of annexin-1 in coronary artery disease.

    Särndahl E, Bergström I, Nijm J, Forslund T, Perretti M and Jonasson L

    Department of Biomedicine, School of Health and Medical Sciences, Orebro University, SE-701 82 Orebro, Sweden. eva.sarndahl@oru.se

    The systemic inflammatory activity in patients with stable coronary artery disease (CAD) is associated with a dysregulated cortisol response. Moreover, an aberrant activation status of neutrophils in CAD has been discussed; and the question of glucocorticoid resistance has been raised. The anti-inflammatory actions of glucocorticoids are mediated by annexin-1 (ANXA1). We investigated the expression of glucocorticoid receptors (GR) and ANXA1, as well as the exogenous effects of ANXA1 on neutrophils in CAD patients and related the data to diurnal salivary cortisol. Salivary cortisol levels were measured in the morning and evening during 3 consecutive days in 30 CAD patients and 30 healthy individuals. The neutrophil expression of GR and ANXA1 was determined by flow cytometry. The effect of exogenous ANXA1 was determined in a neutrophil stimulation assay. The patients showed a flattened diurnal cortisol pattern compared with healthy subjects, involving higher levels in the evening. The neutrophil expression of GR-total and GR-alpha was decreased, whereas the GR-beta expression did not differ compared with controls. The neutrophil expression of ANXA1 was significantly increased in patients. Ex vivo, ANXA1 impaired the leukotriene B(4)-induced neutrophil production of reactive oxygen species in patients but not in controls. Our findings indicate a persistent overactivation of the hypothalamic-pituitary-adrenal axis in CAD patients but do not give any evidence for glucocorticoid resistance, as assessed by the neutrophil expression of GR and ANXA1. The altered neutrophil phenotype in CAD may thus represent a long-term response to disease-related activation.

    Metabolism: clinical and experimental 2010;59;3;433-40

  • Annexin-I overexpression is associated with tumour progression and independently predicts inferior disease-specific and metastasis-free survival in urinary bladder urothelial carcinoma.

    Li CF, Shen KH, Huang LC, Huang HY, Wang YH and Wu TF

    Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan.

    Aims: In our previous studies, comparative proteomics and immunohistochemistry (IHC) demonstrated that annexin-I (ANXA1) is up-regulated in high grade urinary bladder urothelial carcinoma (UBUC) as compared to non-high grade carcinomas. However, the small sample size prohibited further correlation of ANXA1 expression to tumour progression. Therefore, in the present study, 81 primary localised UBUC specimens of various grades and primary tumour (pT) status were examined for ANXA1 expression to further confirm the proteomics data and to clarify the relevance of ANXA1 expression level to the prognosis of UBUC.

    Methods: IHC was implemented to investigate ANXA1 protein expression in 81 primary localised UBUC specimens. The association of ANXA1 expression with tumour progression and prognosis was analysed.

    Results: Our data demonstrated that the ANXA1 expression level was strongly associated with an escalated pT status (p < 0.001) and a higher histological grade (p < 0.001), suggesting that ANXA1 might be related to tumour progression. Moreover, at the univariate level, ANXA1 overexpression, along with higher pT status and histological grade, significantly predicted disease-specific survival (DSS) and metastasis-free survival (MFS). More importantly, multivariate analyses revealed that the association of ANXA1 overexpression and prognosis remained significant for both DSS and MFS.

    Conclusion: The above results reinforced the comparative proteomics results and confirmed the prognostic role of ANXA1 in UBUC.

    Pathology 2010;42;1;43-9

  • Cell surface externalization of annexin A1 as a failsafe mechanism preventing inflammatory responses during secondary necrosis.

    Blume KE, Soeroes S, Waibel M, Keppeler H, Wesselborg S, Herrmann M, Schulze-Osthoff K and Lauber K

    Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany.

    The engulfment of apoptotic cells is of crucial importance for tissue homeostasis in multicellular organisms. A failure of this process results in secondary necrosis triggering proinflammatory cytokine production and autoimmune disease. In the present study, we investigated the role of annexin A1, an intracellular protein that has been implicated in the efficient removal of apoptotic cells. Consistent with its function as bridging protein in the phagocyte synapse, opsonization of apoptotic cells with purified annexin A1 strongly enhanced their phagocytic uptake. A detailed analysis, however, surprisingly revealed that annexin A1 was hardly exposed to the cell surface of primary apoptotic cells, but was strongly externalized only on secondary necrotic cells. Interestingly, while the exposure of annexin A1 failed to promote the uptake of these late secondary necrotic cells, it efficiently prevented induction of cytokine production in macrophages during engulfment of secondary necrotic cells. Our results therefore suggest that annexin A1 exposure during secondary necrosis provides an important failsafe mechanism counteracting inflammatory responses, even when the timely clearance of apoptotic cells has failed.

    Journal of immunology (Baltimore, Md. : 1950) 2009;183;12;8138-47

  • Comparative analysis of annexin-1 in neuroepithelial tumors shows altered expression with the grade of malignancy but is not associated with survival.

    Schittenhelm J, Trautmann K, Tabatabai G, Hermann C, Meyermann R and Beschorner R

    Institute of Brain Research, University of Tuebingen, Tuebingen, Germany. jens.schittenhelm@med.uni-tuebingen.de

    In various types of cancer, the expression of members of the annexin family of calcium- and phospholipid-binding anti-inflammatory proteins is dysregulated. Annexin-1 (ANXA1, lipocortin-1) is involved in proliferation, differentiation and apoptosis. It serves as a substrate for the epidermal growth factor receptor (EGFR), which is frequently amplified in primary gliomas. It is unclear how annexin-1 is expressed in various neuroepithelial tumors, and whether there is any association with tumor malignancy or survival. We studied annexin-1 expression in 394 glial neoplasms of all grades of malignancy and 81 normal brain samples by immunohistochemistry using tissue microarrays. The results were validated using western blot and reverse transcription-PCR (RT-PCR). In the normal human brain, the expression of annexin-1 is limited to ependymal cells and subependymal astrocytes, but is also upregulated in reactive astrocytes. Ependymomas and astrocytomas showed significantly higher mean annexin-1 expression levels in the cytoplasm compared with oligodendrogliomas (both: P<0.0001). In addition, nuclear staining of annexin-1 in oligodendroglial tumor cells was significantly reduced (P=0.0002), which may be used as a diagnostic tool for differentiating between astrocytomas and oligodendrogliomas. Although annexin-1 expression in ependymomas decreased with the grade of malignancy, diffuse astrocytomas showed a significant increase in cytoplasmic annexin-1-positive tumor cells. However, survival analysis showed that the expression of annexin-1 is not associated with patient survival. Similar to the EGFR amplification profile, primary glioblastomas had a higher annexin-1 expression level compared with secondary glioblastomas. Thus, annexin-1 upregulation in astrocytomas may contribute to tumor progression and its expression profile is similar to its substrate, EGFR, suggesting a possible regulation thereof.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2009;22;12;1600-11

  • Identification of annexin 1 as a PU.1 target gene in leukemia cells.

    Iseki Y, Imoto A, Okazaki T, Harigae H and Takahashi S

    Division of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, 1-15-1 Kitasato, Sagamihara City, Kanagawa 228-8555, Japan.

    To identify PU.1 downstream target genes, we first established PU.1-knockdown K562 (K562PU.1KD) cells expressing reduced levels of PU.1 by stably transfected PU.1 siRNAs. From microarray analysis, we found that several genes including annexin 1 were markedly induced in K562PU.1KD cells. Annexin 1 is a calcium- and phospholipid-binding protein and increased expression leads to the constitutive activation of extracellular signal-regulated kinase (ERK). Consistent with this, we observed constitutive activation of ERK in K562PU.1KD cells. Furthermore, we revealed the mRNA expression of annexin 1 was negatively correlated with PU.1 mRNA expression in 43 primary AML specimens (R=-0.31, p<0.042).

    Leukemia research 2009;33;12;1658-63

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.

    Svendsen JM, Smogorzewska A, Sowa ME, O'Connell BC, Gygi SP, Elledge SJ and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Structure-specific endonucleases mediate cleavage of DNA structures formed during repair of collapsed replication forks and double-strand breaks (DSBs). Here, we identify BTBD12 as the human ortholog of the budding yeast DNA repair factor Slx4p and D. melanogaster MUS312. Human SLX4 forms a multiprotein complex with the ERCC4(XPF)-ERCC1, MUS81-EME1, and SLX1 endonucleases and also associates with MSH2/MSH3 mismatch repair complex, telomere binding complex TERF2(TRF2)-TERF2IP(RAP1), the protein kinase PLK1 and the uncharacterized protein C20orf94. Depletion of SLX4 causes sensitivity to mitomycin C and camptothecin and reduces the efficiency of DSB repair in vivo. SLX4 complexes cleave 3' flap, 5' flap, and replication fork structures; yet unlike other endonucleases associated with SLX4, the SLX1-SLX4 module promotes symmetrical cleavage of static and migrating Holliday junctions (HJs), identifying SLX1-SLX4 as a HJ resolvase. Thus, SLX4 assembles a modular toolkit for repair of specific types of DNA lesions and is critical for cellular responses to replication fork failure.

    Funded by: Howard Hughes Medical Institute; NCI NIH HHS: T32 CA009216, T32CA09216; NIA NIH HHS: R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: R01 GM070565, R01 GM070565-04

    Cell 2009;138;1;63-77

  • Down-regulation of plasma membranous Annexin A1 protein expression in premalignant and malignant lesions of the oral cavity: correlation with epithelial differentiation.

    Nomura H, Uzawa K, Yamano Y, Fushimi K, Nakashima D, Kouzu Y, Kasamatsu A, Ogawara K, Shiiba M, Bukawa H, Yokoe H and Tanzawa H

    Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

    Purpose: To determine the potential involvement of ANXA1 in oral squamous-cell carcinoma (OSCC), we evaluated the ANXA1 protein expression in oral premalignant lesions (OPLs) and OSCCs and correlated the results with clinicopathologic variables.

    Methods: Matched normal and tumour specimens of 44 primary OSCCs and 28 OPLs were analyzed for ANXA1 subcellular localization and protein expression level by immunohistochemistry (IHC). Correlations between ANXA1-IHC staining scores of OSCCs and clinicopathologic features were evaluated by Fisher's exact test.

    Results: Markedly down-regulation of ANXA1 protein expression was identified on the plasma membrane of epithelial cells in OSCCs (P < 0.001) and OPLs (P = 0.001) compared with normal counterparts. Moreover, loss of plasma membranous ANXA1 expression was significantly correlated with the poorly differentiated status of OSCC cells (P = 0.012).

    Conclusions: Our findings suggest that loss of ANXA1 is frequent and early event during oral carcinogenesis and that ANXA1 could contribute to maintaining epithelial differentiation in OSCC.

    Journal of cancer research and clinical oncology 2009;135;7;943-9

  • Loss of annexin A1 disrupts normal prostate glandular structure by inducing autocrine IL-6 signaling.

    Inokuchi J, Lau A, Tyson DR and Ornstein DK

    Department of Urology, University of California, Irvine, Orange, CA 92868, USA.

    Annexin A1 (ANXA1) expression is commonly reduced in premalignant lesions and prostate cancer, but a causal relationship of ANAX1 loss with carcinogenesis has not been established. ANXA1 levels have been shown to inversely correlate with interleukin 6 (IL-6) expression in other cell types and IL-6 has been suggested to enhance prostate cancer initiation and promotion. To investigate whether loss of ANXA1 may contribute to prostate carcinogenesis, ANXA1 expression was reduced using RNA interference in non-tumorigenic human prostatic epithelial cells (RWPE-1/rA1). No effect on morphology, apoptosis, migration or anchorage-dependent or -independent growth was detected. However, IL-6 mRNA and secreted protein levels were elevated in RWPE-1/rA1 cells. In addition, re-expression of ANXA1 in these cells suppressed IL-6 secretion, and altering ANXA1 levels in prostate cancer cells had similar effects on IL-6. The effects of ANXA1 loss and increased IL-6 expression on prostate epithelium were examined using an assay of acinar morphogenesis in vitro. Acini formed by RWPE-1/rA1 cells had delayed luminal clearing and larger mean diameters than control cells. The RWPE-1/rA1 phenotype was recapitulated by treating control cells with recombinant IL-6 and was reversed in RWPE-1/rA1 cells by blocking IL-6 bioactivity. Taken together, these data support a direct role for decreased ANXA1 expression in prostate carcinogenesis and enhancing tumor aggressiveness via the upregulation of IL-6 expression and activity.

    Funded by: NIDDK NIH HHS: DK 068137

    Carcinogenesis 2009;30;7;1082-8

  • Identification of annexin A1 as a novel substrate for E6AP-mediated ubiquitylation.

    Shimoji T, Murakami K, Sugiyama Y, Matsuda M, Inubushi S, Nasu J, Shirakura M, Suzuki T, Wakita T, Kishino T, Hotta H, Miyamura T and Shoji I

    Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.

    E6-associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high-risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull-down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP-binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi-mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca(2+)-dependent manner. GST pull-down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP-mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin-proteasome pathway.

    Journal of cellular biochemistry 2009;106;6;1123-35

  • Decreased expression of Annexin A1 correlates with pathologic differentiation grade in oral squamous cell carcinoma.

    Zhang L, Yang X, Zhong LP, Zhou XJ, Pan HY, Wei KJ, Li J, Chen WT and Zhang ZY

    Department of Oral and Maxillofacial Surgery, Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.

    Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB96 cells. In this study, comparative proteomic analysis identified that Annexin A1 was one of the significantly down-regulated genes in the cancerous HB96 cells. To investigate Annexin A1 down-regulation and its potential usefulness as a molecular marker in OSCC, we further screened Annexin A1 expressions with a panel of OSCC lines, and clinical samples of cancerous and the paired adjacent normal tissues from primary OSCC patients. By Western blot analysis and real-time PCR, we showed that both Annexin A1 mRNA and protein expressions decreased in OSCC cell lines except in two cell lines for the mRNA levels. Immunohistochemistry and real-time PCR also showed that both Annexin A1 mRNA and protein expressions decreased in the cancerous tissues from OSCC patients compared with those in the paired adjacent non-malignant epithelia. More importantly, both Annexin A1 mRNA and protein expressions negatively correlated with the pathologic differentiation grades of cancerous tissues. The lower Annexin A1 mRNA or protein expressions correlated with the poorer pathologic differentiation grades. These results suggest that decreased expression of Annexin A1 contributes to the cancerous progression of OSCC, and Annexin A1 may be a potential biomarker for pathologic differentiation grade of OSCC.

    Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 2009;38;4;362-70

  • Immunophenotypic analysis of CD103+ B-lymphoproliferative disorders: hairy cell leukemia and its mimics.

    Dong HY, Weisberger J, Liu Z and Tugulea S

    Genzyme Genetics, 521 W 57th St, New York, NY 10019, USA.

    CD103 is characteristically expressed in hairy cell leukemia (HCL), a B-lymphoproliferative disorder highly responsive to treatment with purine analogs. Other CD103+ diseases are rare and do not respond well to the same therapy, including HCL variant (HCLv) and splenic marginal zone B-cell lymphoma (SMZL) variants. We analyzed 215 cases of CD103+ B-lymphoproliferative disorders to further delineate their immunophenotypic features. Flow cytometric analysis revealed that 78.6% of all cases expressed CD25 and CD103, characteristic of classical HCL. Cases analyzed immunohistochemically were also invariably positive for annexin-A1; a subset coexpressed CD10 (33/169 [19.5%]) or BCL1 (26/65 [36.9%]). In contrast, 21.4% of cases lacked CD25, a subset of which was analyzed and was invariably negative for annexin-A1, CD10, and BCL1. The CD25- cases had variable morphologic features ranging from HCLv and SMZL to prolymphocytic leukemia and diffuse large B-cell lymphoma. Clinically, patients with CD25- disease tended to be older (P= .001), typically had leukocytosis (P= .014), and did not respond well to cladribine or pentostatin. We suggest categorizing CD103+ B-lymphoproliferative disorders into 2 groups. While HCL coexpresses CD25 and annexin-A1, diseases lacking CD25 and annexin-A1 behave clinically differently and can be separated from HCL on diagnosis.

    American journal of clinical pathology 2009;131;4;586-95

  • Annexin-1 regulates growth arrest induced by high levels of estrogen in MCF-7 breast cancer cells.

    Ang EZ, Nguyen HT, Sim HL, Putti TC and Lim LH

    Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    Estrogen, a naturally occurring female steroid growth hormone, has been implicated as a major risk factor for the development of breast cancer. Recent research into this disease has also correlated Annexin-1 (ANXA1), a glucocorticoid-inducible protein, with the development of breast tumorigenesis. ANXA1 is lost in many cancers, including breast cancer, and this may result in a functional promotion of tumor growth. In this study, we investigated the expression of ANXA1 in MCF-7 cells treated with estrogen and the regulation of estrogen functions by ANXA1. Exposure of MCF-7 breast cancer cells to high physiologic levels (up to 100 nmol/L) of estrogen leads to an up-regulation of ANXA1 expression partially through the activation of cyclic AMP-responsive element binding protein and dependency on activation of the estrogen receptor. In addition, treatment of MCF-7 cells with physiologic levels of estrogen (1 nmol/L) induced proliferation, whereas high pregnancy levels of estrogen (100 nmol/L) induced a growth arrest of MCF-7 cells, associated with constitutive activation of extracellular signal-regulated kinase 1/2 and up-regulation of cell cycle arrest proteins such as p21(waf/cip). Silencing of ANXA1 with specific small interfering RNA reverses the estrogen-dependent proliferation as well as growth arrest and concomitantly modulates extracellular signal-regulated kinase 1/2 phosphorylation. We confirm that ANXA1 is lost in clinical breast cancer, indicating that the antiproliferative protective function of ANXA1 against high levels of estrogen may be lost. Finally, we show that ANXA1-deficient mice exhibit faster carcinogen-induced tumor growth. Our data suggest that ANXA1 may act as a tumor suppressor gene and modulate the proliferative functions of estrogens.

    Molecular cancer research : MCR 2009;7;2;266-74

  • Annexin A1 and glucocorticoids as effectors of the resolution of inflammation.

    Perretti M and D'Acquisto F

    William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK. m.perretti@qmul.ac.uk

    Glucocorticoids are widely used for the management of inflammatory diseases. Their clinical application stems from our understanding of the inhibitory effect of the corticosteroid hormone cortisol on several components of the immune system. Endogenous and exogenous glucocorticoids mediate their multiple anti-inflammatory effects through many effector molecules. In this Opinion article, we focus on the role of one such effector molecule, annexin A1, and summarize the recent studies that provide insight into its molecular and pharmacological functions in immune responses. In addition, we propose a model in which glucocorticoids regulate the expression and function of annexin A1 in opposing ways in innate and adaptive immune cells to mediate the resolution of inflammation.

    Funded by: Arthritis Research UK; British Heart Foundation; Medical Research Council: G0400327; Wellcome Trust

    Nature reviews. Immunology 2009;9;1;62-70

  • Loss of annexin A1 expression in breast cancer progression.

    Cao Y, Li Y, Edelweiss M, Arun B, Rosen D, Resetkova E, Wu Y, Liu J, Sahin A and Albarracin CT

    Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

    Background: Annexin A1 (ANXA1) is a potential marker of development of breast cancer. However, previous studies of ANXA1 expression in primary breast carcinoma and lymph node metastasis have yielded conflicting results. Therefore, to accurately characterize the ANXA1 expression pattern, we used microarray analysis and matched patient samples to evaluate progressive alterations in ANXA1 protein expression during malignant transformation and metastasis.

    Design: We constructed a tissue microarray using 82 pairs of primary breast cancers and lymph node metastases from archival materials. We also identified 21 cases of breast carcinoma for which a single slide contained the entire progression from benign breast tissue, carcinoma in situ, to invasive carcinoma. Immunohistochemical staining for ANXA1 and various prognostic markers was performed.

    Result: Microarray analysis revealed that ANXA1 expression was lost in 79% of breast carcinomas, and there was no difference in ANXA1 expression between primary breast carcinoma and lymph node metastasis. Most ANXA1-negative tumors were positive for estrogen and progesterone receptors but negative for HER2/neu and epidermal growth factor receptor. In contrast, most ANXA1-positive tumors were negative for estrogen, progesterone, and HER2/neu. In the whole tissue sections, ANXA1 is heterogeneously expressed in benign epithelium and is lost in both in situ carcinoma and invasive carcinoma.

    Conclusions: The lack of ANXA1 expression in the majority of breast carcinomas and the early loss of ANXA1 expression in in situ carcinoma, which is maintained in both invasive and metastatic tumors, suggests a possible role for ANXA1 in the early events of malignant transformation.

    Funded by: NCI NIH HHS: P50 CA083639, R01 CA131183

    Applied immunohistochemistry & molecular morphology : AIMM 2008;16;6;530-4

  • Annexin A1 subcellular expression in laryngeal squamous cell carcinoma.

    Alves VA, Nonogaki S, Cury PM, Wünsch-Filho V, de Carvalho MB, Michaluart-Júnior P, Moyses RA, Curioni OA, Figueiredo DL, Scapulatempo-Neto C, Parra ER, Polachini GM, Silistino-Souza R, Oliani SM, Silva-Júnior WA, Nobrega FG, Head and Neck Genome Project/GENCAPO, Tajara EH and Zago MA

    Department of Pathology, School of Medicine, USP, São Paulo, Brazil.

    Aims: Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis.

    Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis.

    Conclusions: ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.

    Histopathology 2008;53;6;715-27

  • MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers.

    Luthra R, Singh RR, Luthra MG, Li YX, Hannah C, Romans AM, Barkoh BA, Chen SS, Ensor J, Maru DM, Broaddus RR, Rashid A and Albarracin CT

    Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA. rluthra@mdanderson.org

    Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation -0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation -0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3'-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

    Funded by: NCI NIH HHS: 1P50CA098258-01

    Oncogene 2008;27;52;6667-78

  • Occurrence of autoantibodies to annexin I, 14-3-3 theta and LAMR1 in prediagnostic lung cancer sera.

    Qiu J, Choi G, Li L, Wang H, Pitteri SJ, Pereira-Faca SR, Krasnoselsky AL, Randolph TW, Omenn GS, Edelstein C, Barnett MJ, Thornquist MD, Goodman GE, Brenner DE, Feng Z and Hanash SM

    Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. jiqiu@fhcrc.org

    Purpose: We have implemented a high throughput platform for quantitative analysis of serum autoantibodies, which we have applied to lung cancer for discovery of novel antigens and for validation in prediagnostic sera of autoantibodies to antigens previously defined based on analysis of sera collected at the time of diagnosis.

    Proteins from human lung adenocarcinoma cell line A549 lysates were subjected to extensive fractionation. The resulting 1,824 fractions were spotted in duplicate on nitrocellulose-coated slides. The microarrays produced were used in a blinded validation study to determine whether annexin I, PGP9.5, and 14-3-3 theta antigens previously found to be targets of autoantibodies in newly diagnosed patients with lung cancer are associated with autoantibodies in sera collected at the presymptomatic stage and to determine whether additional antigens may be identified in prediagnostic sera. Individual sera collected from 85 patients within 1 year before a diagnosis of lung cancer and 85 matched controls from the Carotene and Retinol Efficacy Trial (CARET) cohort were hybridized to individual microarrays.

    Results: We present evidence for the occurrence in lung cancer sera of autoantibodies to annexin I, 14-3-3 theta, and a novel lung cancer antigen, LAMR1, which precede onset of symptoms and diagnosis.

    Conclusion: Our findings suggest potential utility of an approach to diagnosis of lung cancer before onset of symptoms that includes screening for autoantibodies to defined antigens.

    Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2008;26;31;5060-6

  • Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles.

    Dalli J, Norling LV, Renshaw D, Cooper D, Leung KY and Perretti M

    The William Harvey Research Institute, Barts and The London Medical School, London, United Kingdom.

    Polymorphonuclear leukocyte (PMN)-derived microparticles display inhibitory properties on target cells as assessed in vitro; since PMNs contain abundant amounts of the endogenous anti-inflammatory protein annexin 1 (AnxA1), we tested here whether biologically active AnxA1 could be present in PMN-derived microparticles. PMN adhesion to human umbilical vein endothelial cell (HUVEC) monolayers led to the generation of microparticles that contained AnxA1, as detected by Western blotting, flow cytometry, and mass spectrometry analyses. Addition of these microparticles to recipient PMNs prior to flow over HUVEC monolayers significantly inhibited cell adhesion, an effect abrogated by a neutralizing anti-AnxA1 antibody, or an antibody raised against the AnxA1 receptor, that is termed lipoxin A(4) receptor or ALX. Intravenous delivery of human PMN-derived microparticles markedly inhibited PMN recruitment to an air pouch inflamed with IL-1beta. This anti-inflammatory effect was also dependent on endogenous AnxA1, since injection of microparticles produced from wild-type PMNs (bone marrow derived), but not from AnxA1-null PMNs, inhibited IL-1beta-induced leukocyte trafficking. In conclusion, PMN-derived microparticles contain functionally active AnxA1 that confers them anti-inflammatory properties; generation of these microparticles in the microcirculation could promote inflammatory resolution by time-dependent dampening of cell recruitment.

    Funded by: Arthritis Research UK: 15775

    Blood 2008;112;6;2512-9

  • Identification of annexin 1 as a novel autoantigen in acute exacerbation of idiopathic pulmonary fibrosis.

    Kurosu K, Takiguchi Y, Okada O, Yumoto N, Sakao S, Tada Y, Kasahara Y, Tanabe N, Tatsumi K, Weiden M, Rom WN and Kuriyama T

    Department of Respirology (B2), Graduate School of Medicine, Chiba University, Chiba, Japan. kurosu@faculty.chiba-u.jp

    Consistent with the hypothesis that pulmonary epithelial apoptosis is the key to the acute exacerbation of idiopathic pulmonary fibrosis (IPF), we conducted serological identification of Ags by recombinant expression cloning (SEREX) analysis using type II alveolar cell carcinoma (A549) cell lines to identify disease-related Abs. In a survey of Abs to the recombinant autoantigens identified by SEREX analysis, five Abs were identified as novel candidates for the acute exacerbation of IPF. Abs to annexin 1 were detected in 47 and 53% of the sera and bronchoalveolar lavage materials from patients with acute exacerbation of IPF. Some identical TCR Vbeta genes were identified in sequential materials obtained at 1-3 mo in all 10 acute exacerbation IPF cases, suggesting that some infiltrating CD4-positive T cells sharing limited epitopes expand by Ag-driven stimulation during disease extension. The CDR3 region of these identical TCR Vbeta genes showed high homology with the N-terminal portion of annexin 1, including in the HLA-DR ligand epitopes predicted by TEPITOPE analysis. By Western blotting analysis and observation of the CD4-positive T cell responses in bronchoalveolar lavage samples, the N-terminal portion of annexin 1 was cleaved and found to induce marked proliferative responses of CD4-positive T cells in three patients. Our study demonstrates that annexin 1 is an autoantigen that raises both Ab production and T cell response in patients with acute exacerbation of IPF, and that the N-terminal portion of annexin 1 plays some role in the pathogenesis of acute exacerbation in IPF patients.

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;1;756-67

  • Decreased expression of annexin A1 during the progression of cervical neoplasia.

    Wang LD, Yang YH, Liu Y, Song HT, Zhang LY and Li PL

    Department of Obstetrics and Gynaecology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China.

    The aim of this study was to evaluate whether the expression of annexin A1 (ANXA1) is associated with the progression of cervical neoplasia. ANXA1 expression was examined by immunohistochemistry in paraffin-embedded cervical tissue samples (n = 234), comprising 52 samples of normal cervical epithelia, 30 of cervical intraepithelial neoplasia (CIN) I, 27 of CIN II, 32 of CIN III, and 93 of invasive squamous cell carcinoma (ISCC). ANXA1 expression was strong in normal cervical squamous epithelium and significantly reduced with increasing progression of cervical neoplasia. Moreover, a close association was observed between ANXA1 expression and tumour cell differentiation in ISCC. These preliminary results indicate that ANXA1 may be an effective candidate for detecting CIN lesions and for evaluating tumour cell differentiation in squamous cell carcinoma of the cervix.

    The Journal of international medical research 2008;36;4;665-72

  • Expression of Annexin-1 in patients with endometriosis.

    Li CY, Lang JH, Liu HY and Zhou HM

    Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China.

    Background: Annexin-1 was identified as an endometriosis-related protein by comparative proteomics in previous study. As an endogenous anti-inflammatory mediator, Annexin-1 has been shown to regulate the immune response, cell proliferation and apoptosis. To investigate whether Annexin-1 is involved in the pathogenesis of endometriosis, we examined the expression of Annexin-1 in eutopic endometrium of women with or without endometriosis, and detected its expression in peritoneal fluids of those with endometriosis.

    Methods: Eutopic endometrium samples from twenty-five women with endometriosis and those from sixteen age-matched women without endometriosis were collected. Peritoneal fluids were obtained from ten patients with endometriosis. The expression of Annexin-1 protein in eutopic endometrium was detected by immunohistochemistry and Western blotting, and mRNA detected by real-time PCR. Annexin-1 protein in the peritoneal fluids was detected by Western blotting.

    Results: Annexin-1 mRNA and protein were overexpressed in eutopic endometrium of endometriosis without significant differences between the proliferative and secretory phase. Immunohistochemistry showed that Annexin-1 protein was expressed mainly in endometrial glandular cells throughout the menstrual cycle. Annexin-1 protein was detected in the peritoneal fluids of all the ten patients with endometriosis.

    Conclusions: Annexin-1 is overexpressed in eutopic endometrium and presents in the peritoneal fluids of patients with endometriosis, and may play a role in the pathogenesis of endometriosis.

    Chinese medical journal 2008;121;10;927-31

  • Nuclear localization of annexin A1 is a prognostic factor in oral squamous cell carcinoma.

    Lin CY, Jeng YM, Chou HY, Hsu HC, Yuan RH, Chiang CP and Kuo MY

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

    To investigate whether annexin A1 (ANXA1) expression is a marker in predicting the prognosis of oral cancer patients.

    Methods: We immunohistochemically examined the expression of ANXA1 in 66 cases of oral epithelial dysplasia (OED) and 115 cases of oral squamous cell carcinoma (OSCC). The results were correlated with the clinicopathological parameters of tumors and overall patient survival.

    Results: In normal oral mucosa, ANXA1 staining was predominantly located on the cell membrane. In OED and OSCC specimens, membranous staining decreased, whereas nuclear staining increased. Positive nuclear staining was observed in 9 of 66 (13.64%) OED cases and 63 of 115 (54.8%) OSCCs. Kaplan-Meier curves showed that OSCC patients with ANXA1 nuclear staining had significantly shorter overall lengths of survival (P = 0.00036 by the log-rank test). Multivariate analysis showed that ANXA1 nuclear staining is a significant predictor of poor overall survival. And oral cancer SAS cells treated with hepatocyte growth factor (HGF) can induce ANXA1 protein translocation from cytoplasm to nucleus. Cells pretreated with LY294002 (PI3K inhibitor) almost completely inhibited (88.3% inhibition) HGF-mediated ANXA1 nuclear translocation.

    Conclusions: The nuclear localization of ANXA1 protein is a frequent event and could be used as a prognostic factor in OSCC.

    Journal of surgical oncology 2008;97;6;544-50

  • Heterogeneity and timing of translocation and membrane-mediated assembly of different annexins.

    Skrahina T, Piljić A and Schultz C

    Gene Expression Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

    Many cell types, including neurons and epithelial cells, express a variety of annexins. Although the overall function has only been partially unravelled, a dominant feature is the formation of two-dimensional assemblies under the plasma membrane in a calcium-dependent manner. Here we show that fluorescently tagged annexins A1, A2, A4, A5, and A6 translocate and assemble at the plasma membrane and the nuclear envelope, except annexin A2, which only attaches to the plasma membrane. All annexins have different response times to elevated calcium levels as was shown by the translocation of co-expressed proteins. Fluorescence recovery after photobleaching revealed the static nature of all annexin assemblies. Analysis of the assemblies by Foerster resonance energy transfer (FRET) using acceptor bleaching demonstrated mostly annexin-specific self-assembly. Heterogeneous assembly formation was shown between annexins A5 and A1, and A5 and A2. The formation of homo- and heterogeneous annexin assemblies may play an important role when high increases in calcium occur, such as after disruption of the plasma membrane.

    Experimental cell research 2008;314;5;1039-47

  • 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

  • Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks beta 2-integrin adhesion in neutrophils.

    Meliton AY, Munoz NM, Zhu X and Leff AR

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

    We examined the effect of glucocorticoid stimulation in blocking beta 2-integrin adhesion of polymorphonuclear leukocytes (PMNs) isolated from human subjects. Surface expression of CD11b and ERK-1/2-mediated gIVaPLA2 phosphorylation, which are required for beta 2-integrin adhesion, were not affected by treatment with < or =10(-6) M fluticasone propionate (FP) for PMNs activated by either 10(-7) M LTB4 or 30 ng/ml TNF-alpha and caused no significant blockade of beta 2-integrin adhesion in vitro. Baseline expression of annexin-1 (ANXA1) synthesis was increased only after 10(-6) M FP for PMNs; by contrast, comparable increase in ANXA1 expression was demonstrated in human eosinophils from the same subjects with 10(-8) M FP. Viability of PMNs was verified by propidium iodide and by the persistence of beta 2-integrin adhesion in treated groups. Exogenous administration of ANXA1 mimetic peptide fragment blocked significantly and comparably the beta 2-integrin adhesion in PMNs activated by LTB4 and TNF-alpha and in eosinophils activated by IL-5. Translocation of gIVaPLA2 from the cytosol to the nucleus also was refractory for activated PMNs treated with > or =10(-7) M FP; by contrast, complete blockade of nuclear translocation of cytosolic gIVaPLA2 was effected by 10(-9) M FP in eosinophils. Our data indicate that the cell surface ANXA1 synthesis is capable of blocking beta 2-integrin adhesion in both PMNs and eosinophils. However, in contrast to eosinophils, FP does not cause either substantial ANXA1 synthesis or nuclear transport of cytosolic gIVaPLA2 in PMNs and thus does not block beta2-integrin adhesion, a necessary step for granulocyte cell migration in vivo.

    Funded by: NHLBI NIH HHS: HL-46368, HL-85779

    Journal of leukocyte biology 2008;83;2;344-51

  • Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic phospholipase A2 in normal and malignant squamous epithelial cells.

    Sakaguchi M, Murata H, Sonegawa H, Sakaguchi Y, Futami J, Kitazoe M, Yamada H and Huh NH

    Department of Cell Biology and Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata-chou, Okayama 700-8558, Japan.

    Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca(2+)-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA(2), resulting in inhibition of cPLA(2) activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.

    The Journal of biological chemistry 2007;282;49;35679-86

  • The confluence-dependent interaction of cytosolic phospholipase A2-alpha with annexin A1 regulates endothelial cell prostaglandin E2 generation.

    Herbert SP, Odell AF, Ponnambalam S and Walker JH

    Faculty of Biological Sciences, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.

    The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A(2)-alpha (cPLA(2)alpha) cleaves phospholipids in a Ca(2+)-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca(2+)-independent association of cPLA(2)alpha with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA(2)alpha and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA(2)alpha is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA(2)alpha and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA(2)alpha activity and endothelial cell prostaglandin generation.

    Funded by: Wellcome Trust

    The Journal of biological chemistry 2007;282;47;34468-78

  • Overexpression of annexin a1 induced by terephthalic acid calculi in rat bladder cancer.

    Cui L, Wang Y, Shi Y, Zhang Z, Xia Y, Sun H, Wang S, Chen J, Zhang W, Lu Q, Song L, Wei Q, Zhang R and Wang X

    Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing, China.

    Prolonged cell proliferation in response to irritation by bladder calculi can evoke malignant transformation of the urothelium. However, the molecular mechanisms responsible for calculi-associated bladder carcinogenesis are unknown. We compared the protein expression pattern of rat bladder transitional cell carcinomas (TCCs) induced by terephthalic acid with that of normal bladder tissues using 2-DE. Comparative analysis of the respective spot patterns on 2-DE showed 146 spots that were markedly changed in TCC samples. Subsequently, 56 of the variant protein spots were identified by MALDI-TOF MS. Among them, overexpression of annexin a1 (ANNA1) in rat TCCs was confirmed by Western blotting and real-time RT-PCR analysis. Immunohistochemical staining revealed that ANNA1, usually a cytoplasmic protein in normal urothelium, was translocated to the nucleus in rat bladder cancer cells. In contrast to the animal studies, examination of human clinical specimens showed that ANNA1 expression was reduced in TCC compared to normal urothelium. The expression of ANNA1 was inversely related to the level of differentiation of TCC. Our data suggest that overexpression of ANNA1 is involved in bladder carcinogenesis induced by bladder calculi and that translocation of the protein may be partly responsible for the effect. ANNA1 may serve as a new marker of differentiation for the histopathological grading of human TCC.

    Proteomics 2007;7;22;4192-202

  • Annexin 1 cleavage in activated neutrophils: a pivotal role for proteinase 3.

    Vong L, D'Acquisto F, Pederzoli-Ribeil M, Lavagno L, Flower RJ, Witko-Sarsat V and Perretti M

    William Harvey Research Institute, Barts and The London, Charterhouse Square, London EC1M 6BQ, United Kingdom.

    Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells, including neutrophils. Here we have developed a cleavage assay using tagged annexin 1 and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells, and proteomic analyses allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N terminus region of the protein, at least in the context of neutrophil activation. Because annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase 3 would augment its homeostatic pro-resolving actions and could represent an opportunity for innovative anti-inflammatory drug discovery.

    Funded by: Arthritis Research UK: 15755; British Heart Foundation: PG/06/153/22042; Medical Research Council: G0400327; Wellcome Trust

    The Journal of biological chemistry 2007;282;41;29998-30004

  • [Expression of annexin I in human pancreatic cancer and the influence of its down-regulation on biology of this cancer].

    Liu Q, Wang CF, Zhou Z, Hu H, Zhao DB, Ni XG, Bai XF, Gao JD, Tian YT and Zhao P

    Department of General Oncology, Cancer Institute (Hospital), Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China.

    Objective: To investigate the expression of annexin in human pancreatic cancer and to elucidate its role in oncogenesis of pancreatic cancer.

    Methods: A pancreatic carcinoma cell line Suit-II with high-expression of annexin I gene was adopted. Three subtypes of annexin I -siRNA sequences and a non-related fragment were combined, and the eukaryotic expression vectors bearing siRNA fragments were constructed. Then they were transfected into pancreatic carcinoma cells to knock down the expression of annexin I by RNAi. After knocking down the expression of annexin I , the growth speed, cell cycling, morphological features and apoptosis of pancreatic carcinoma cells were examined by RT-PCR and MTT test.

    Results: When the expression of annexin I was blocked, the growth speed of pancreatic carcinoma cells was significantly decreased, the morphological features were changed and pronounced apoptosis occurred.

    Conclusion: Annexin I can modulate pancreatic carcinoma cell cycle, promote the cell proliferation, increasingly stimulate the cell growth, and suppress the process of apoptosis in pancreatic carcinoma cells.

    Zhonghua zhong liu za zhi [Chinese journal of oncology] 2007;29;10;738-41

  • [Epstein-Barr virus-encoded latent membrane protein 1 mediates serine-phosphorylation of annexin I by activating protein kinase C].

    Yan GR, Luo W, Luo XJ and Cao Y

    Institutes of Life and Health Engineering, Jinan University, Guangzhou, Guangdong, 510632, P. R. China. jxygr007@126.com

    Twenty-five novel phosphoproteins, including Annexin I, triggered by Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) were identified when we previously combined phosphorylation enrichment with proteomics technology to elucidate signaling pathway activated by LMP1. This study was to map signal transduction pathway between LMP1 and phosphorylation of AnnexinI.

    Methods: The expression of Annexin I in nasopharyngeal carcinoma cell lines (stably expressing LMP1) was analyzed by Western blot. Serine-and tyrosine-phosphorylation levels of AnnexinIwere detected through combining immunoprecipitation with Western blot. The activity of protein kinase C (PKC) was analyzed by non-radioactive protein kinase assay.

    Results: The protein level and tyrosine phosphorylation of Annexin I in CNE1 and CNE1-LMP1 cells were similar; the serine phosphorylation of annexin I was higher in CNE1-LMP1 cells than in CNE1 cells. The relative activity of PKC was significantly lower in CNE1 cells than in CNE1-LMP1 cells (0.97+/-0.05 vs. 1.22+/-0.10, P<0.01). The change of PKC activity was followed by the change of serine phosphorylation level of Annexin I.

    Conclusion: LMP1 mediates serine phosphorylation of Annexin I via PKC pathway.

    Ai zheng = Aizheng = Chinese journal of cancer 2007;26;7;679-82

  • Annexin 1: differential expression in tumor and mast cells in human larynx cancer.

    Silistino-Souza R, Rodrigues-Lisoni FC, Cury PM, Maniglia JV, Raposo LS, Tajara EH, Christian HC and Oliani SM

    Department of Biology, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), São Paulo State University (UNESP), São José do Rio Preto, São Paulo, Brazil.

    Annexin 1 protein (ANXA1) expression was evaluated in tumor and mast cells in human larynx cancer and control epithelium. The effect of the exogenous ANXA1 (peptide Ac 2-26) was also examined during the cellular growth of the Hep-2 human larynx epidermoid carcinoma cell line. This peptide inhibited the proliferation of the Hep-2 cells within 144 hr. In surgical tissue specimens from 20 patients with larynx cancer, ultrastructural immunocytochemistry analysis showed in vivo down-regulation of ANXA1 expression in the tumor and increased in mast cells and Hep-2 cells treated with peptide Ac2-26. Combined in vivo and in vitro analysis demonstrated that ANXA1 plays a regulatory role in laryngeal cancer cell growth. We believe that a better understanding of the regulatory mechanisms of ANXA1 in tumor and mast cells may lead to future biological targets for the therapeutic intervention of human larynx cancer.

    International journal of cancer 2007;120;12;2582-9

  • Activation of the annexin 1 counter-regulatory circuit affords protection in the mouse brain microcirculation.

    Gavins FN, Dalli J, Flower RJ, Granger DN and Perretti M

    The William Harvey Research Institute, Barts and The London, Charterhouse Sq., London, EC1M 6BQ, UK.

    The purpose of this study was to investigate the role of the homeostatic antiinflammatory axis centered on annexin 1 (AnxA1) in cerebral microvascular dysfunction and tissue injury associated with middle cerebral artery (MCA) occlusion and reperfusion. Intravital fluorescence microscopy was used to visualize the mouse cerebral microcirculation: AnxA1 null mice exhibited more white blood cell adhesion in cerebral venules than their wild-type counterparts, and this was accompanied by a larger cerebral infarct vol and worse neurological score. All parameters were rescued by delivery of human recombinant AnxA1. To further explore these findings using pharmacological tools, the effect of a short AnxA1 peptidomimetic was tested. When given during the reperfusion phase, peptide Ac2-26 produced similar cerebroprotection, which was associated with a marked attenuation of cell adhesion and markers of inflammation as measured in tissue homogenates. The pharmacological effects of peptide Ac2-26 occurred via receptors of the formyl-peptide receptor (FPR) family, most likely FPR-rs2, as deduced by displacement assays with transfected cells and in vivo experiments with transgenic mice and receptor antagonists. Our findings indicate that the endogenous antiinflammatory circuit centered on AnxA1 produces significant cerebral protection, and that these properties might have therapeutic potential for stroke treatment.

    Funded by: NHLBI NIH HHS: HL26441; Wellcome Trust

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;8;1751-8

  • Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages.

    Scannell M, Flanagan MB, deStefani A, Wynne KJ, Cagney G, Godson C and Maderna P

    Diabetes Research Centre, School of Medicine and Medical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.

    The resolution of inflammation is a dynamically regulated process that may be subverted in many pathological conditions. Macrophage (Mphi) phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation as this process prevents the exposure of tissues at the inflammatory site to the noxious contents of lytic cells. It is increasingly appreciated that endogenously produced mediators, such as lipoxins, act as potent regulators (nanomolar range) of the phagocytic clearance of apoptotic cells. In this study, we have investigated the intriguing possibility that apoptotic cells release signals that promote their clearance by phagocytes. We report that conditioned medium from apoptotic human polymorphonuclear neutrophils (PMN), Jurkat T lymphocytes, and human mesangial cells promote phagocytosis of apoptotic PMN by Mphi and THP-1 cells differentiated to a Mphi-like phenotype. This prophagocytic activity appears to be dose dependent, sensitive to the caspase inhibitor zVAD-fmk, and is associated with actin rearrangement and release of TGF-beta1, but not IL-8. The prophagocytic effect can be blocked by the formyl peptide receptor antagonist Boc2, suggesting that the prophagocytic factor(s) may interact with the lipoxin A(4) receptor, FPRL-1. Using nanoelectrospray liquid chromatography mass spectrometry and immunodepletion and immunoneutralization studies, we have ascertained that annexin-1 and peptide derivatives are putative prophagocytic factors released by apoptotic cells that promote phagocytosis of apoptotic PMN by M[phi] and differentiated THP-1 cells. These data highlight the role of annexin-1 and peptide derivatives in promoting the resolution of inflammation and expand on the therapeutic anti-inflammatory potential of annexin-1.

    Funded by: Wellcome Trust

    Journal of immunology (Baltimore, Md. : 1950) 2007;178;7;4595-605

  • Annexin 1: the new face of an old molecule.

    Lim LH and Pervaiz S

    Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.

    The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with a significant degree of biological and structural homology (40-60%). First described in the late 1970s and subsequently referred to as macrocortin, renocortin, lipomodulin, lipocortin-1, and more recently Annexin 1, this 37 kDa calcium and phospholipid binding protein is a strong inhibitor of glucocorticoid-induced eicosanoid synthesis and PLA2. Recent interest in the biological activity of this intriguing molecule has unraveled important functional attributes of Annexin 1 in a variety of inflammatory pathways, on cell proliferation machinery, in the regulation of cell death signaling, in phagocytic clearance of apoptosing cells, and most importantly in the process of carcinogenesis. Here we attempt to present a short review on these diverse biological activities of an interesting and important molecule, which could be a potential target for novel therapeutic intervention in a host of disease states.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;4;968-75

  • Annexin-1 modulates T-cell activation and differentiation.

    D'Acquisto F, Merghani A, Lecona E, Rosignoli G, Raza K, Buckley CD, Flower RJ and Perretti M

    William Harvey Research Institute, Bart's and The London, Queen Mary School of Medicine and Dentistry, UK. f.dacquisto@qmul.ac.uk

    Annexin-1 is an anti-inflammatory protein that plays an important homeostatic role in innate immunity; however, its potential actions in the modulation of adaptive immunity have never been explored. Although inactive by itself, addition of annexin-1 to stimulated T cells augmented anti-CD3/CD28-mediated CD25 and CD69 expression and cell proliferation. This effect was paralleled by increased nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NFATs), and activator protein-1 (AP-1) activation and preceded by a rapid T-cell receptor (TCR)-induced externalization of the annexin-1 receptor. Interestingly, differentiation of naive T cells in the presence of annexin-1 increased skewing in Th1 cells; in the collagen-induced arthritis model, treatment of mice with annexin-1 during the immunization phase exacerbated signs and symptoms at disease onset. Consistent with these findings, blood CD4+ cells from patients with rheumatoid arthritis showed a marked up-regulation of annexin-1 expression. Together these results demonstrate that annexin-1 is a molecular "tuner" of TCR signaling and suggest this protein might represent a new target for the development of drugs directed to pathologies where an unbalanced Th1/Th2 response or an aberrant activation of T cells is the major etiologic factor.

    Funded by: Medical Research Council: G0400327, G116/131; Wellcome Trust: 040 269/Z/96/A, 040269, 069 234/Z/02/Z

    Blood 2007;109;3;1095-102

  • Annexin-I as a potential target for green tea extract induced actin remodeling.

    Xiao GS, Jin YS, Lu QY, Zhang ZF, Belldegrun A, Figlin R, Pantuck A, Yen Y, Li F and Rao J

    Department of Clinic Molecular Pharmacology, Comprehensive Cancer Center at City of Hope National Medical Center, Duarte, California, USA.

    Using a multistep human urothelial model, we previously showed that green tea extract (GTE) selectively modulates actin remodeling in transformed cells (MC-T11), which resulted in increased cell adhesion and reduced cell motility (Lu et al., Clin Cancer Res 2005;11:1675-83). This study further analyzed which actin binding proteins (ABPs) might be involved in this process. Proteomic profiles of GTE treated and untreated MC-T11 cells using two-dimensional gel electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC/MS/MS) and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) identified 20 GTE-induced proteins. Among them, 3 were ABPs (tropomodulin, cofilin and annexin-I), and only annexin-I showed a dose- and time-dependent expression. The increased annexin-I correlated with actin remodeling, and was the result of transcription level up-regulation, as determined by RT-PCR, pull-down immunoblot and siRNA analyses. 5-Azacytidine, a DNA methylation inhibitor, exhibited no effect on annexin-I expression when used alone, but had an additive effect for GTE-induced annexin-I expression. Immunohistochemistry of bladder cancer tissue array showed a decrease of annexin-I expression in carcinoma in situ and low grade papillary carcinoma (n = 32, 0% positive) compared to nontumor urothelium (n = 18, 89% positive) (p < 0.001 by Fisher exact test), but increased in some (6 of 15, 40%) high-grade tumors. Together, GTE induced annexin-I expression plays a role in regulating actin remodeling and decreased annexin-I expression is a common event in early stage of bladder cancer development.

    Funded by: NCI NIH HHS: U01CA96116

    International journal of cancer 2007;120;1;111-20

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Decreased expression of annexin A1 is correlated with breast cancer development and progression as determined by a tissue microarray analysis.

    Shen D, Nooraie F, Elshimali Y, Lonsberry V, He J, Bose S, Chia D, Seligson D, Chang HR and Goodglick L

    Gonda/UCLA Breast Cancer Research Laboratory, Revlon/UCLA Breast Center, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.

    Annexin A1 (ANXA1) is a calcium- and phospholipid-binding protein and a known mediator of glucocorticoid-regulated inflammatory responses. Using a combined multiple high-throughput approach, we recently identified a reduced expression of ANXA1 in human breast cancer. The finding was confirmed at the gene level by quantitative reverse transcription-polymerase chain reaction and at the protein level by immunohistochemical staining of normal, benign, and malignant breast tissues. In this study, we constructed and used a high-density human breast cancer tissue microarray to characterize the expressional pattern of ANXA1 according to histopathologies. The tissue microarray contains 1,158 informative breast tissue cores of different histologies including normal tissues, hyperplasia, in situ and invasive tumors, and lymph node metastases. Our results showed that there was a significant decrease in glandular expression of ANXA1 in ductal carcinoma in situ and invasive ductal carcinoma compared with either normal breast tissue or hyperplasia (P < .0001). Moreover, in benign breast tissue, myoepithelial cells showed strong expression of ANXA1. There was a decrease of ANXA1 expression in myoepithelial cells in ductal carcinoma in situ lesions compared with the same cell population in either normal or hyperplastic lesions. These results suggest that suppressed ANXA1 expression in breast tissue is correlated with breast cancer development and progression.

    Funded by: NCI NIH HHS: CA-86366

    Human pathology 2006;37;12;1583-91

  • Requirement for annexin A1 in plasma membrane repair.

    McNeil AK, Rescher U, Gerke V and McNeil PL

    Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.

    Ca2+ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca2+, binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca2+ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca2+ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.

    The Journal of biological chemistry 2006;281;46;35202-7

  • 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

  • Glucocorticoid upregulation of the annexin-A1 receptor in leukocytes.

    Sawmynaden P and Perretti M

    The William Harvey Research Institute, Bart's and the London, Charterhouse Square, London EC1M 6BQ, UK.

    We tested here whether glucocorticoids modulated myeloid cell expression of a specific G-coupled receptor, termed formyl-peptide receptor like-1 (FPRL-1), recently shown to mediate the anti-inflammatory actions of annexin-A1. Real-time PCR and flow cytometry demonstrated rapid up-regulation of mRNA followed by the protein in HL-60 cells incubated with dexamethasone, with peaks at 2 and 24h, respectively. This effect was not restricted to dexamethasone, since reproduced by glucocorticoids. In addition, it was not restricted to the cell line, since replicated with human peripheral blood monocytes. Glucocorticoid ability to upregulate cell surface expression of FPRL-1 was specific, since no effects upon the related receptor FPR or the integrin CD11b could be detected. In view of the wide range of endogenous ligands known to interact with FPRL-1, including the anti-inflammatory protein annexin-A1, we speculate that the novel effect here described may impact on the clinical immunosuppressive and anti-inflammatory properties of glucocorticoids.

    Biochemical and biophysical research communications 2006;349;4;1351-5

  • Proteolytic cleavage of annexin 1 by human leukocyte elastase.

    Rescher U, Goebeler V, Wilbers A and Gerke V

    Center for Molecular Biology of Inflammation, Institute for Medical Biochemistry, von Esmarch-Strasse 56, 48149 Münster, Germany.

    Annexin 1 has been shown to participate through its unique N-terminal domain in the recruitment and activation of leukocytes at sites of inflammation. Peptides derived from this domain are true mimetics of the annexin 1 action in all inflammation models tested and most likely serve as the active entities generated at sites of inflammation. To elucidate mechanisms underlying peptide generation we used isolated blood leukocytes and endothelial cell monolayers. We show that following endothelial adhesion, annexin 1 was externalized from leukocytes and rapidly cleaved. Addition of purified annexin 1 to degranulating leukocytes resulted in the truncation of annexin 1, which seemed to depend on the proteolytic activity of human leukocyte elastase (HLE). The capacity of elastase to proteolytically cleave annexin 1 was confirmed using both purified annexin 1 and HLE. The identification of annexin 1 as a substrate for HLE supports the model in which annexin 1 participates in regulating leukocyte emigration into inflamed tissue through N-terminal peptides generated at inflammatory sites.

    Biochimica et biophysica acta 2006;1763;11;1320-4

  • The impact of altered annexin I protein levels on apoptosis and signal transduction pathways in prostate cancer cells.

    Hsiang CH, Tunoda T, Whang YE, Tyson DR and Ornstein DK

    Department of Urology, University of California, Irvine, California, USA.

    Background: Although reduced expression levels of annexin I (ANX I) protein is a common finding in all stages of prostate cancer a causative relationship between ANX I dysregulation and prostate cancer development has yet to be established.

    Methods: Annexin I expression was restored in LNCaP and MDA PCa 2b that normally express low or undetectable levels of ANX I protein. The impact of restoring ANX I expression on cell viability, colony formation in soft agar, apoptosis, and extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK) activation was examined.

    Results: Restoring ANX I expression reduced cell viability, colony formation, in addition to inducing apoptosis. The proliferative response of epidermal growth factor was blocked by restoring ANX I expression. Furthermore, increasing basal and induced levels of phosphorylated p38 and JNK were observed in prostate cancer cells following restoration of ANX I expression.

    Conclusions: Annexin I may have tumor suppressor functions in prostate cancer. The pro-apoptotic effect of ANX I involves the activation of p38 and JNK, which appears to shift the balance of signal transduction away from proliferation and toward apoptosis.

    Funded by: NIDDK NIH HHS: R21 DK068137-01

    The Prostate 2006;66;13;1413-24

  • Expression of annexin A1 in esophageal and esophagogastric junction adenocarcinomas: association with poor outcome.

    Wang KL, Wu TT, Resetkova E, Wang H, Correa AM, Hofstetter WL, Swisher SG, Ajani JA, Rashid A, Hamilton SR and Albarracin CT

    Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, 77030, USA.

    Purpose: Annexin A1 (ANXA1) is a calcium-binding protein involved in arachidonic acid metabolism and epidermal growth factor receptor tyrosine kinase pathway. ANXA1 has been implicated in early squamous cell carcinogenesis of esophagus and correlates with degree of tumor differentiation. However, the role of ANXA1 in esophageal adenocarcinoma is unclear. Our goal was to evaluate ANXA1 expression and determine its prognostic significance in adenocarcinoma of the esophagus and esophagogastric junction.

    This study included 104 consecutive patients with primary resected esophageal and esophagogastric junction adenocarcinomas (11 stage I, 24 stage II, 53 stage III, and 16 stage IV). ANXA1 protein expression in each tumor was assessed by immunohistochemical staining of tissue microarrays. ANAX1 expression level was classified as high (>/=25% of tumor cells with cytoplasmic staining), low (<25% of tumor cells with cytoplasmic staining), or negative; and was correlated with clinicopathologic features and patients' outcomes.

    Results: High ANXA1 expression was present in 39% (41 of 104) of tumors and was associated with higher pathologic T stage (P = 0.03) and distant metastasis (P = 0.04). High ANXA1 expression correlated with increased recurrence rate (P = 0.004) and decreased overall survival (P = 0.003) in univariate analysis. In multivariate analysis, ANXA1 expression and pN stage significantly correlated with recurrence rate (P = 0.008 and P < 0.001, respectively) and overall survival (P = 0.02 and P < 0.001, respectively) independent of T stage.

    Conclusion: Our results indicate that high ANXA1 expression is frequent in esophageal and esophagogastric junction adenocarcinomas, correlates with more advanced pathologic T stage and the presence of distant metastasis, and is an independent prognostic factor for patient survival.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2006;12;15;4598-604

  • Annexin I regulates SKCO-15 cell invasion by signaling through formyl peptide receptors.

    Babbin BA, Lee WY, Parkos CA, Winfree LM, Akyildiz A, Perretti M and Nusrat A

    Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, USA. bbabbin@emory.edu

    Annexin 1 (AnxA1) is a multifunctional phospholipid-binding protein associated with the development of metastasis in some invasive epithelial malignancies. However, the role of AnxA1 in the migration/invasion of epithelial cells is not known. In this study, experiments were performed to investigate the role of AnxA1 in the invasion of a model epithelial cell line, SKCO-15, derived from colorectal adenocarcinoma. Small interfering RNA-mediated knockdown of AnxA1 expression resulted in a significant reduction in invasion through Matrigel-coated filters. Localization studies revealed a translocation of AnxA1 to the cell surface upon the induction of cell migration, and functional inhibition of cell surface AnxA1 using antiserum (LCO1) significantly reduced cell invasion. Conversely, SKCO-15 cell invasion was increased by approximately 2-fold in the presence of recombinant full-length AnxA1 and the AnxA1 N-terminal-derived peptide mimetic, Ac2-26. Because extracellular AnxA1 has been shown to regulate leukocyte migratory events through interactions with n-formyl peptide receptors (nFPRs), we examined the expression of FPR-1, FPRL-1, and FPRL-2 in SKCO-15 cells by reverse transcriptase-PCR and identified expression of all three receptors in this cell line. Treatment of SKCO-15 cells with AnxA1, Ac2-26, and the classical nFPR agonist, formylmethionylleucylphenylalanine, induced intracellular calcium release consistent with nFPR activation. Furthermore, the nFPR antagonist, Boc2, abrogated the AnxA1 and Ac2-26-induced intracellular calcium release and increase in SKCO-15 cell invasion. Together, these results support an autocrine/paracrine role for membrane AnxA1 in stimulating SKCO-15 cell migration through nFPR activation. The findings in this study suggest that activation of nFPRs stimulates epithelial cell motility important in the development of metastasis as well as wound healing.

    Funded by: NIGMS NIH HHS: T32 GM008169

    The Journal of biological chemistry 2006;281;28;19588-99

  • Annexin-1 downregulation in thyroid cancer correlates to the degree of tumor differentiation.

    Petrella A, Festa M, Ercolino SF, Zerilli M, Stassi G, Solito E and Parente L

    Department of Pharmaceutical Sciences, University of Salerno, Salerno, Italy.

    We investigated the expression of annexin-1 (ANXA1) in thyroid carcinoma cell lines and in thyroid cancers with a different degree of differentiation. The highest level of ANXA1 expression examined by Western blotting was detected in the papillary carcinoma cells (NPA) and in the follicular cells (WRO). On the other hand, the most undifferentiated thyroid carcinoma cells (ARO and FRO) presented the lowest level of ANXA1 expression. In surgical tissue specimens from 32 patients with thyroid cancers, we found high immunoreactivity for ANXA1 in papillary (PTC) and follicular (FTC) thyroid cancers while in undifferentiated thyroid cancers (UTC) the expression of the protein was barely detectable. Control thyroid tissue resulted positive for ANXA1. In summary, 70% of UTC examined weakly expressed ANXA1, whereas 65% of PTC or FTC specimens tested showed high expression of the protein. Thus ANXA1 expression may correlate with the tumorigenesis suggesting that the protein may represent an effective differentiation marker in thyroid cancer.

    Cancer biology & therapy 2006;5;6;643-7

  • Interaction of human neutrophils with endothelial cells regulates the expression of endogenous proteins annexin 1, galectin-1 and galectin-3.

    Gil CD, La M, Perretti M and Oliani SM

    Post-Graduation in Morphology, São Paulo School of Medicine-- UNIFESP, São Paulo, SP, Brazil.

    Annexin 1 (ANXA1), galectin-1 (Gal-1) and galectin-3 (Gal-3) proteins have been identified as important mediators that promote or inhibit leukocyte migration. The expression of these proteins was studied in human neutrophils and endothelial cells (ECs) during a transmigration process induced by IL-8. Upon neutrophil adhesion to EC, a significant increase in the cleaved ANXA1 (LCS3, raised against all ANXA1 isoforms) expression was detected in the plasma membrane of adhered neutrophils and ECs compared to intact ANXA1 isoform (LCPS1, against N-terminus of protein). Adherent neutrophils had elevated Gal-3 levels in the nucleus and cytoplasm, and ECs in their plasma membranes. In contrast, a decrease in the total amounts of Gal-1 was detected in migrated compared to non-migrated neutrophils. Therefore, ANXA1 and Gal-3 changed in their content and localization when neutrophils adhere to endothelia, suggesting a process of sensitive-balance between two endogenous anti- and pro-inflammatory mediators.

    Cell biology international 2006;30;4;338-44

  • In vitro and in vivo studies on CCR10 regulation by Annexin A1.

    Rodrigues-Lisoni FC, Mehet DK, Mehemet DK, Peitl P, John CD, da Silva Júnior WA, Tajara E, Buckingham JC and Solito E

    Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Faculty of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

    The mode of action of annexin A1 (ANXA1) is poorly understood. By using rapid subtraction hybridization we studied the effects of human recombinant ANXA1 and the N-terminal ANXA1 peptide on gene expression in a human larynx cell line. Three genes showed strong downregulation after treatment with ANXA1. In contrast, expression of CCR10, a seven transmembrane G-protein coupled receptor for chemokine CCL27 involved in mucosal immunity, was increased. Moreover the reduction in CCR10 expression induced by ANXA1 gene deletion was rescued by intravenous treatment with low doses of ANXA1. These findings provide new evidence that ANXA1 modulates gene expression.

    Funded by: Wellcome Trust: 069234/B/02/2

    FEBS letters 2006;580;5;1431-8

  • Regulation of annexin I in rheumatoid synovial cells by glucocorticoids and interleukin-1.

    Morand EF, Hall P, Hutchinson P and Yang YH

    Centre or Inflammatory Diseases, Monash Institute for Medical Research , Monash Medical Centre, Locked Bag No 29, Clayton Victoria 3168, Australia.

    The glucocorticoid (GC)-induced antiinflammatory molecule annexin I is expressed in leukocytes and has antiinflammatory effects in animal models of arthritis, but the expression of annexin I in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is unknown. We report the constitutive and dexamethasone (DEX)-inducible expression of annexin I in RA FLS. DEX increased FLS annexin I protein translocation and mRNA expression. Interleukin (IL)-1beta also induced annexin I translocation and mRNA but also increased intracellular protein. DEX and IL-1 had additive effects on annexin I mRNA, but DEX inhibited the inducing effect of IL-1beta on cell surface annexin I. These results indicate that glucocorticoids and IL-1beta upregulate the synthesis and translocation of annexin I in RA FLS, but interdependent signalling pathways are involved.

    Mediators of inflammation 2006;2006;2;73835

  • Decreased annexin I expression in prostatic adenocarcinoma and in high-grade prostatic intraepithelial neoplasia.

    Patton KT, Chen HM, Joseph L and Yang XJ

    Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA.

    Aims: To analyse annexin I expression in prostatic carcinoma. Annexin I belongs to a family of structurally related calcium and phospholipid-binding proteins implicated in signal transduction, DNA replication, cell proliferation and apoptosis. The decreased expression of annexin I, II and VII proteins has been reported in different types of cancer.

    Using immunohistochemistry, we analysed annexin I expression in 77 cases of prostatic adenocarcinoma (Gleason score 6, N = 40; Gleason scores 7-8, N = 27; and Gleason scores 9-10, N = 10) and high-grade prostatic intraepithelial neoplasia (PIN, N = 50). Immunoreactivity of annexin I in tumour cells was evaluated as negative (< 5% of cells), focally positive (5-25% of cells) or positive (> 25% of cells). In contrast to positive staining in adjacent benign prostatic epithelium, annexin I expression was decreased (focally positive) in 76% of cases of high-grade PIN (P < 0.0001) and was decreased or absent in 81% of prostatic adenocarcinomas (P < 0.0001). Annexin I expression in all higher grade tumours (Gleason scores 7-10) was only focally positive or absent.

    Conclusions: Expression of annexin I inversely correlates with the increasing histological grade of prostatic adenocarcinoma. By showing a progressive loss of annexin I expression in high-grade PIN, intermediate-grade and high-grade cancer, our findings suggest that the loss of annexin I expression occurs early in prostatic tumorigenesis and becomes more prominent throughout tumour progression. The loss of expression of annexin I may serve as a useful marker of prostate cancer development and progression.

    Histopathology 2005;47;6;597-601

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis.

    Bruneel A, Labas V, Mailloux A, Sharma S, Royer N, Vinh J, Pernet P, Vaubourdolle M and Baudin B

    Service de Biochimie A, Hôpital Saint-Antoine, AP-HP, Paris, France. arnaud.bruneel@sat.ap-hop-paris.fr

    We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.

    Proteomics 2005;5;15;3876-84

  • Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients.

    Bensalem N, Ventura AP, Vallée B, Lipecka J, Tondelier D, Davezac N, Dos Santos A, Perretti M, Fajac A, Sermet-Gaudelus I, Renouil M, Lesure JF, Halgand F, Laprévote O and Edelman A

    INSERM U467, Faculté de médecine Necker, Université Paris-Descartes, France.

    Cystic fibrosis is a fatal human genetic disease caused by mutations in the CFTR gene encoding a cAMP-activated chloride channel. It is characterized by abnormal fluid transport across secretory epithelia and chronic inflammation in lung, pancreas, and intestine. Because cystic fibrosis (CF) pathophysiology cannot be explained solely by dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), we applied a proteomic approach (bidimensional electrophoresis and mass spectrometry) to search for differentially expressed proteins between mice lacking cftr (cftr(tm1Unc), cftr-/-) and controls using colonic crypts from young animals, i.e. prior to the development of intestinal inflammation. By analyzing total proteins separated in the range of pH 6-11, we detected 24 differentially expressed proteins (>2-fold). In this work, we focused on one of these proteins that was absent in two-dimensional gels from cftr-/- mice. This protein spot (molecular mass, 37 kDa; pI 7) was identified by mass spectrometry as annexin A1, an anti-inflammatory protein. Interestingly, annexin A1 was also undetectable in lungs and pancreas of cftr-/- mice, tissues known to express CFTR. Absence of this inhibitory mediator of the host inflammatory response was associated with colonic up-regulation of the proinflammatory cytosolic phospholipase A2. More importantly, annexin A1 was down-regulated in nasal epithelial cells from CF patients bearing homozygous nonsense mutations in the CFTR gene (Y122X, 489delC) and differentially expressed in F508del patients. These results suggest that annexin A1 may be a key protein involved in CF pathogenesis especially in relation to the not well defined field of inflammation in CF. We suggest that decreased expression of annexin A1 contributes to the worsening of the CF phenotype.

    Molecular & cellular proteomics : MCP 2005;4;10;1591-601

  • Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies.

    Cagliani R, Magri F, Toscano A, Merlini L, Fortunato F, Lamperti C, Rodolico C, Prelle A, Sironi M, Aguennouz M, Ciscato P, Uncini A, Moggio M, Bresolin N and Comi GP

    IRCCS E. Medea, Associazione La Nostra Famiglia, Bosisio Parini, Lecco, Italy.

    Mutations in the DYSF gene underlie two main muscle diseases: Limb Girdle Muscular Dystrophy (LGMD) 2B and Miyoshi myopathy (MM). Dysferlin is involved in muscle membrane-repair and is thought to interact with other dysferlin molecules and annexins A1 and A2 at the sarcolemma. We performed genotype/phenotype correlations in a large cohort of dysferlinopathic patients and explored the possible role of annexins as modifier factors in LGMD-2B and MM. In particular, clinical examination, expression of sarcolemmal proteins and genetic analysis were performed on 27 dysferlinopathic subjects. Expression of A1 and A2 annexins was investigated in LGMD-2B/MM subjects and in patients with other muscle disorders. We identified 24 different DYSF mutations, 10 of them being novel. We observed no clear correlation between mutation type and clinical phenotype, but MM patients were found to display muscle symptoms significantly earlier in life than LGMD subjects. Remarkably, dysferlinopathic patients and subjects suffering from other muscular disorders expressed higher levels of both annexins compared to controls; a significant correlation was observed between annexin expression levels and clinical severity scores. Also, annexin amounts paralleled the degree of muscle histopathologic changes. In conclusion, our data indicate that the pathogenesis of different inherited and acquired muscle disorders involves annexin overexpression, probably because these proteins actively participate in the plasmalemma repair process. The positive correlation between annexin A1 and A2 and clinical severity, as well as muscle histopathology, suggests that their level may be a prognostic indicator of disease.

    Human mutation 2005;26;3;283

  • Localization of annexins I, II, IV and VII in whole prostate sections from radical prostatectomy patients.

    Lehnigk U, Zimmermann U, Woenckhaus C and Giebel J

    Institute of Anatomy, Ernst Moritz Arndt University, Friedrich-Loeffler-Strasse 23c, D-17489 Greifswald, Germany.

    Annexins (ANXs) represent a family of calcium and phospholipid binding proteins that are involved in several physiological processes e.g. signal transduction, cellular differentiation and proliferation. Since they are known to be dysregulated in a variety of cancers we investigated the immunolocalization of ANXs in whole prostate sections containing benign prostatic epithelium (BPE), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) in order to evaluate their possible role during tumorigenesis. Samples were obtained from 28 patients undergoing radical prostatectomy. Gross sections of whole prostates were examined immunohistochemically for the distribution of ANX I, II, IV and VII. In BPE all ANXs were localized to the cell membranes and the cytoplasm of all gland cells. In BPH the immunoreactivity of ANX I and II was restricted to the basal cells of glands and expression pattern of ANX IV and VII was similar to BPE. In PIN only basal cells expressed ANX II. In PCa ANX II immunoreactivity was absent and weak ANX I and ANX IV immunoreactivity was restricted to the cytoplasm of tumor cells. ANX VII immunoreactivity was seen in some but not all tumor cells. Since ANX IV and VII expression did not show significant changes in PCa compared to non-neoplastic tissue and PIN an essential role during prostate tumourigenesis seems unlikely. In contrast, as progression from PIN to PCa is characterized by a reduction of ANX I and II this suggests that downregulation of these proteins could represent an important event in prostate carcinogenesis.

    Histology and histopathology 2005;20;3;673-80

  • Annexin1 regulates the erythroid differentiation through ERK signaling pathway.

    Huo XF and Zhang JW

    National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, The Chinese Academy of Medical Sciences and The Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, People's Republic of China.

    K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.

    Biochemical and biophysical research communications 2005;331;4;1346-52

  • Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5.

    Guzmán-Aránguez A, Olmo N, Turnay J, Lecona E, Pérez-Ramos P, López de Silanes I and Lizarbe MA

    Dpto. Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain.

    Butyrate induces differentiation and alters cell proliferation in intestinal-epithelial cells by modulation of the expression of several genes. Annexins are a superfamily of ubiquitous proteins characterized by their calcium-dependent ability to bind to biological membranes; their involvement in several physiological processes, such as membrane trafficking, calcium signaling, cell motility, proliferation, and differentiation has been proposed. Thus, we have analyzed changes in annexin A1 (AnxA1), annexin A2 (AnxA2), and annexin A5 (AnxA5) levels and localization in human colon adenocarcinoma cells differentiated by butyrate treatment or by culture in glucose-free inosine-containing medium. The acquired differentiated phenotype increased dipeptidyl peptidase-IV (DPP-IV) expression and alkaline phosphatase (ALP) activity, two well known brush border markers. Butyrate induces cell differentiation and growth arrest in BCS-TC2, BCS-TC2.2, HT-29, and Caco-2 cells, increasing the levels of AnxA1 and AnxA5, whereas AnxA2 decreases except in Caco-2 cells. Inosine-differentiated cells present increased amounts of the three studied annexins, as occurs in spontaneously differentiated Caco-2 cells. AnxA2 down-regulation is not due to proteasome activation and seems to be related to the butyrate-induced cell proliferation arrest; AnxA1 and AnxA5 expression is growth-state independent. AnxA1 and AnxA5 are mainly found in the cytoplasm while AnxA2 is localized underneath the plasma membrane in cell-to-cell contacts. Butyrate induces changes in subcellular localization towards a vesicle-associated pattern. Human colon adenocarcinoma cell differentiation is associated with an up-regulation of AnxA1, AnxA2, and AnxA5 and with a subcellular relocation of these proteins. No correlation between annexin levels and tumorigenicity was found. Up-regulation of AnxA1 could contribute to the reported anti-inflammatory effects of butyrate in colon inflammatory diseases.

    Journal of cellular biochemistry 2005;94;1;178-93

  • Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD and Comb MJ

    Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.

    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

    Funded by: NCI NIH HHS: 1R43CA101106

    Nature biotechnology 2005;23;1;94-101

  • Phosphorylation of annexin I by TRPM7 channel-kinase.

    Dorovkov MV and Ryazanov AG

    Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

    TRPM7 is an unusual bifunctional molecule consisting of a TRP ion channel fused to a protein kinase domain. It has been shown that TRPM7 plays a key role in the regulation of intracellular magnesium homeostasis as well as in anoxic neuronal death. TRPM7 channel has been characterized using electrophysiological techniques; however, the function of the kinase domain is not known and endogenous substrates for the kinase have not been reported previously. Here we have identified annexin 1 as a substrate for TRPM7 kinase. Phosphorylation of annexin 1 by TRPM7 kinase is stimulated by Ca2+ and is dramatically increased in extracts from cells overexpressing TRPM7. Phosphorylation of annexin 1 by TRPM7 kinase occurs at a conserved serine residue (Ser5) located within the N-terminal amphipathic alpha-helix of annexin 1. The N-terminal region plays a crucial role in interaction of annexin 1 with other proteins and membranes, and therefore, phosphorylation of annexin 1 at Ser5 by TRPM7 kinase may modulate function of annexin 1.

    Funded by: NCI NIH HHS: R01 CA81102; NIGMS NIH HHS: R01 GM57300

    The Journal of biological chemistry 2004;279;49;50643-6

  • Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain.

    Radke S, Austermann J, Russo-Marie F, Gerke V and Rescher U

    Institute for Medical Biochemistry, Centre for Molecular Biology of Inflammation, ZMBE, IZKF Münster, University of Münster, von-Esmarch-Str. 56, 48149 Münster, Germany.

    Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.

    FEBS letters 2004;578;1-2;95-8

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • [Absence of evidence for the association of single nucleotide polymorphisms in Annexin A1 gene with type 2 diabetes in Chinese].

    Dong Y, Li G, Luo TH, Wu G, Huang W and Luo M

    Institute of Endocrinology, Ruijin Hospital, Shanghai Second Medical University, Shanghai, 200025 P.R.China.

    Objective: To identify single nucleotide polymorphisms (SNPs) in the annexin A1(ANXA1) gene and to analyze the association of these SNPs with type 2 diabetes in Shanghai Han population.

    Methods: SNPs in the promotor and exon regions (including intron sequence near splicing site) in the ANXA1 gene were screened by direct sequencing in 24 type 2 diabetes patients and were further genotyped by direct sequencing in another 171 type 2 diabetes patients and 189 normal control subjects.

    Results: The total sequence of ANXA1 gene is 6798 bp. And 7 SNPs were found; among them, 2 SNPs (-7974 C>T and -7040 G>T) were in promotor region, 3 SNPs in intron regions (+9059 A>G, +9204 C>T, +10486 A>G), 1 SNP in 5'-untranslation region (-6614 A>G) and 1 SNP in coding regions (+1784 A>G). These 7 SNPs were genotyped further and the results revealed that the allele frequencies of these SNPs showed no significant difference between the diabetic and the control groups (P>0.05).

    Conclusion: There is no association of these SNPs in ANXA1 gene with type 2 diabetes in Shanghai Han population.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2004;21;5;508-11

  • Comprehensive characterization of annexin I alterations in esophageal squamous cell carcinoma.

    Hu N, Flaig MJ, Su H, Shou JZ, Roth MJ, Li WJ, Wang C, Goldstein AM, Li G, Emmert-Buck MR and Taylor PR

    Cancer Prevention Studies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-8314, USA.

    Purpose: The purpose is to characterize alterations of the annexin I gene, its mRNA, and protein expression in esophageal squamous cell carcinoma.

    Fifty-six cases of esophageal squamous cell carcinoma were analyzed using four microsatellite markers flanking the annexin I gene (9q11-q21) to identify loss of heterozygosity. In addition, we performed (a) single-strand conformation polymorphism and DNA sequencing along the entire promoter sequence and coding region to identify mutations, (b) real-time quantitative reverse transcription-PCR of RNA from frozen esophageal squamous cell carcinoma tissue (n = 37) and in situ hybridization (n = 5) on selected cases to assess mRNA expression, and (c) immunohistochemistry (n = 44) to evaluate protein expression. The prevalence of the allelic variants identified in the first 56 patients was refined in 80 additional esophageal squamous cell carcinoma patients and 232 healthy individuals.

    Results: Forty-six of 56 (82%) esophageal squamous cell carcinoma patients showed loss of an allele at one or more of the four microsatellite markers; however, only one (silent) mutation was seen. Two intragenic variants were identified with high frequency of allelic loss (A58G, 64%; L109L, 69%). Thirty of 37 (81%) esophageal squamous cell carcinoma patients showed reduced annexin I mRNA expression, which was confirmed by in situ hybridization, whereas annexin I protein expression was reduced in 79% of poorly differentiated tumor cell foci but in only 5% of well-differentiated tumor foci, although allelic loss on chromosome 9 was found in both tumor grades.

    Conclusions: Allelic loss of annexin I occurs frequently, whereas somatic mutations are rare, suggesting that annexin I is not inactivated in esophageal squamous cell carcinoma via a two-hit mechanism. A decrease in annexin I protein expression was confirmed, consistent with a quantitative decrease in mRNA expression, and appeared to be related to tumor cell differentiation. We conclude that annexin I is not the tumor suppressor gene corresponding to the high levels of loss of heterozygosity observed on chromosome 9 in esophageal squamous cell carcinoma; however, dysregulation of mRNA and protein levels is associated with this tumor type.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;18 Pt 1;6013-22

  • An annexin 1 N-terminal peptide activates leukocytes by triggering different members of the formyl peptide receptor family.

    Ernst S, Lange C, Wilbers A, Goebeler V, Gerke V and Rescher U

    Institute for Medical Biochemistry, Center for Molecular Biology of Inflammation, von Esmarch-Strasse 56, D-48149 Münster, Germany.

    The human N-formyl peptide receptor (FPR) is a key modulator of chemotaxis directing granulocytes toward sites of bacterial infections. FPR is the founding member of a subfamily of G protein-coupled receptors thought to function in inflammatory processes. The other two members, FPR-like (FPRL)1 and FPRL2, have a greatly reduced affinity for bacterial peptides or do not bind them at all, with FPRL2 being considered an orphan receptor so far. In this study we show that a peptide derived from the N-terminal domain of the anti-inflammatory protein annexin 1 (lipocortin 1) can activate all three FPR family members at similar concentrations. The annexin 1 peptide initiates chemotactic responses in human monocytes that express all three FPR family members and also desensitizes the cells toward subsequent stimulation with bacterial peptide agonists. Experiments using HEK 293 cells stably expressing a single FPR family member reveal that all three receptors can be activated and desensitized by the N-terminal annexin 1 peptide. These observations identify the annexin 1 peptide as the first endogenous ligand of FPRL2 and indicate that annexin 1 participates in regulating leukocyte emigration into inflamed tissue by activating and desensitizing different receptors of the FPR family.

    Journal of immunology (Baltimore, Md. : 1950) 2004;172;12;7669-76

  • Annexin 1 and neutrophil apoptosis.

    Perretti M and Solito E

    The William Harvey Research Institute, Queen Mary University of London, Bart's and the London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. M.Perretti@qmul.ac.uk

    ANXA1 (annexin 1), a member of the 'annexin' family of calcium- and phospholipid-binding proteins, was originally identified as an endogenous mediator of the anti-inflammatory actions of glucocorticoids. However, this protein exerts multiple inhibitory effects on the host inflammatory response, including a preferential regulation of the adhesion step of blood-borne neutrophil within the microenvironment of an inflamed vasculature. It is now emerging that ANXA1 is endowed with other roles, since the protein is abundant in inflammatory exudates as it is produced and released by the extravasated neutrophil. In the present paper, we review the novel proapoptotic effect of ANXA1 and discuss its potential with respect to the pathophysiology of inflammation and leucocyte recruitment.

    Biochemical Society transactions 2004;32;Pt3;507-10

  • Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes.

    Fan X, Krahling S, Smith D, Williamson P and Schlegel RA

    Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802, USA.

    When cells undergo apoptosis, or programmed cell death, they expose phosphatidylserine (PS) on their surface. Macrophages that efficiently phagocytose apoptotic cells also express PS on their surface, although at a lower level. The PS exposed on both cells is required for phagocytosis, because uptake is inhibited by masking PS on either cell with annexin V, a PS-binding protein. The inhibition is not additive, suggesting that the exposed PS molecules on the two cells participate in a common process. We asked whether this dual requirement reflects bridging of the target cell and macrophage by bivalent, PS-binding annexins. Monoclonal antibodies (mAbs) against annexins I or II stained a variety of live phagocytes. Apoptotic Jurkat T lymphocytes and human peripheral T lymphocytes, but not apoptotic thymocytes, were stained by anti-annexin I but not II. Phagocytosis of apoptotic targets was inhibited by mAbs to annexins I or II, or by pretreatment of macrophages with the same mAbs. Pretreatment of apoptotic thymocytes had no effect, whereas pretreating Jurkat cells with anti-annexin I or removing annexin I with EGTA was inhibitory. Annexin bridging is vectorial, because annexin is bound to PS molecules on targets but not on macrophages, suggesting annexins serve as both ligand and receptor in promoting phagocytosis.

    Funded by: NIAID NIH HHS: R01 AI46261

    Molecular biology of the cell 2004;15;6;2863-72

  • Annexin-I expression modulates drug resistance in tumor cells.

    Wang Y, Serfass L, Roy MO, Wong J, Bonneau AM and Georges E

    Institute of Parasitology, McGill University, Macdonald Campus, Ste-Anne de Bellevue, Que., Canada.

    The use of anti-cancer chemotherapy often leads to the rise of multidrug-resistant (MDR) tumors. We have previously reported the overexpression of a 40kDa protein (P-40) in several MDR tumor cell lines. In this report we describe the cloning of a 1.4kb cDNA with an open reading frame of 344 amino acids that encodes the P-40 protein. Analysis of the P-40 amino acid sequence showed it is identical to the human annexin I (Anx-I) protein. The identity of the isolated P-40 cDNA as Anx-I was confirmed by the specific binding of IPM96 mAb to a 40kDa protein following the in vitro expression of P-40 full-length cDNA. Northern blot analysis of total RNA from drug-sensitive and -resistant cells revealed an increase in P-40 (or Anx-I) mRNA in drug-resistant cells relative to drug-sensitive cells. Transfection of Anx-I cDNA into drug-sensitive MCF-7 cells was carried out without further drug selection and showed 2- to 5-fold increase in resistance of transfected cells to adriamycin, melphalan, and etoposide. Conversely, transfection of reverse Anx-I cDNA into SKOV-3 cells decreased the expression of Anx-I without affecting the expression of other members of the annexin family and showed a 3- to 8-fold increase in sensitivity to these drugs. Of interest was the correlation between the presence of Anx-I and MDR in MDA-MB-231 cells when compared to MCF-7 cells. MDA-MB-231 cells show 3- to 20-fold increase in resistance to adriamycin, melphalan, and etoposide in the absence of detectable levels of P-glycoprotein (P-gp1), the multidrug resistance protein (MRP1) or the breast cancer resistance protein (BCRP). Taken together, these results provide the first direct evidence for the role of Anx-I in MDR of tumor cells.

    Biochemical and biophysical research communications 2004;314;2;565-70

  • Identification of metastasis-associated proteins through protein analysis of metastatic MDA-MB-435 and metastasis-suppressed BRMS1 transfected-MDA-MB-435 cells.

    Cicek M, Samant RS, Kinter M, Welch DR and Casey G

    Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.

    BRMS1 (breast cancer metastasis suppressor 1) was recently identified as a novel breast cancer metastasis suppressor gene. To further characterize BRMS1-mediated metastasis suppression, we applied two-dimensional proteomic and mass spectrometry (LC-tandem MS and MALDI-TOF) analysis to identify proteins differentially expressed between highly metastatic MDA-MB-435 cells and metastasis-suppressed BRMS1-transfected MDA-MB-435 cells. Quadruplicate independent 2D gels were run and analyzed under identical conditions. Following in-gel trypsin digestion of seven differentially expressed proteins, amino acid sequence and mass profiles of the peptides were generated. Proteins were identified from the NCBI non-redundant database using the search program TurboSequest. Differential expression was confirmed for five proteins, including annexin I and alpha B-crystallin, by Northern blot analysis and immunostaining. Furthermore, we showed that both proteins were expressed in vivo in lungs containing metastasized MDA-MB-435 cells but not expressed in normal lung tissue of athymic mice. Our results suggest that annexin I and alpha B-crystallin are important cellular proteins that are down regulated through BRMS1 mediated metastasis suppression.

    Funded by: NCI NIH HHS: CA87728, CA89019

    Clinical & experimental metastasis 2004;21;2;149-57

  • Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.

    Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT and Brown RH

    Day Neuromuscular Research Laboratory, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.

    Mutations in the dysferlin gene cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. We report here the results of expression profile analyses and in vitro investigations that point to an interaction between dysferlin and the Ca2+ and lipid-binding proteins, annexins A1 and A2, and define a role for dysferlin in Ca2+-dependent repair of sarcolemmal injury through a process of vesicle fusion. Expression profiling identified a network of genes that are co-regulated in dysferlinopathic mice. Co-immunofluorescence, co-immunoprecipitation, and fluorescence lifetime imaging microscopy revealed that dysferlin normally associates with both annexins A1 and A2 in a Ca2+ and membrane injury-dependent manner. The distribution of the annexins and the efficiency of sarcolemmal wound-healing are significantly disrupted in dysferlin-deficient muscle. We propose a model of muscle membrane healing mediated by dysferlin that is relevant to both normal and dystrophic muscle and defines the annexins as potential muscular dystrophy genes.

    Funded by: NIA NIH HHS: AG020570, AG08487; NIBIB NIH HHS: EB00768; NINDS NIH HHS: 5PO1NS40828-02, P01 NS040828

    The Journal of biological chemistry 2003;278;50;50466-73

  • Gene expression profiling of alcoholic liver disease in the baboon (Papio hamadryas) and human liver.

    Seth D, Leo MA, McGuinness PH, Lieber CS, Brennan Y, Williams R, Wang XM, McCaughan GW, Gorrell MD and Haber PS

    Drug Health Services, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia. phaber@mail.usyd.edu.au

    The molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD.

    Funded by: NIAAA NIH HHS: AA11115, R01 AA011115; NIDDK NIH HHS: DK56402, R01 DK056402

    The American journal of pathology 2003;163;6;2303-17

  • The surface receptor is involved in annexin I-stimulated insulin secretion in MIN6N8a cells.

    Won JH, Kang NN, Auh CK and Park YM

    Department of Biological Sciences and Institute for Basic Sciences, Sungkyunkwan University, Suwon, South Korea.

    This study investigated the effect of extracellular annexin I on regulating insulin secretion in MIN6N8a (an insulin secreting cell line) cells. The properties of annexin I receptor in MIN6N8a cells were also determined. Annexin I stimulated insulin release in MIN6N8a cells, regardless of the presence or absence of extracellular Ca(2+). Confocal microscopy revealed that annexin I bound to the surface of MIN6N8a cells. In addition, FACs analysis showed that annexin I bound to the surface of MIN6N8a cells in a dose-dependent manner. However, the annexin I-stimulated insulin secretion and the annexin I binding were abolished in MIN6N8a cells treated with proteases. Annexin I receptors were regenerated time-dependently. Furthermore, annexin I-stimulated insulin secretion was inhibited by cycloheximide but not by actinomycin D. These results showed that annexin I binds to the surface receptor in order to regulate the stimulation of insulin release in MIN6N8a cells.

    Biochemical and biophysical research communications 2003;307;2;389-94

  • Subcellular localization of S100A11 (S100C) in LLC-PK1 renal cells: Calcium- and protein kinase c-dependent association of S100A11 with S100B and vimentin intermediate filaments.

    Bianchi R, Giambanco I, Arcuri C and Donato R

    Department of Experimental Medicine and Biochemical Sciences, Section of Anatomy, University of Perugia, 06122 Perugia, Italy.

    The subcellular localization of the Ca(2+)-modulated protein, S100A11, was investigated in the renal cell line LLC-PK1 by immunofluorescence and confocal laser scanning microscopy under varying experimental conditions. In control cells, S100A11 was detected on the plasma membrane, where the protein co-localized with annexin I (ANXA1) at discrete sites, and found diffusely in the cytoplasm. Elevation of the cytosolic Ca(2+) concentration by means of the Ca(2+) ionophore, ionomycin, caused a significant fraction of S100A11 to associate with vimentin intermediate filament (IF)-bound S100B, another member of the S100 protein family. Under these conditions, ANXA1 underwent a quite different kind of relocation. Translocation of S100A11 onto vimentin IF-bound S100B was also observed upon activation of protein kinase C (PKC). Under these conditions, S100A11 appeared to associate directly with vimentin IFs at cell sites displaying low or no abundance of S100B such as cell processes, and, again, S100A11 and ANXA1 underwent a different relocation. Our data suggest the possibility that the intracellular Ca(2+) level might regulate the subcellular localization of S100A11 and its interaction with definite target proteins, and that S100A11 might serve the function of modulating S100B activities. Interestingly, in spite of the known ability of S100A11 to form heterotetramers with ANXA1, the two proteins underwent a different relocation on elevation of the cytosolic Ca(2+) concentration or activation of PKC, pointing to different regulatory activities of individual proteins in renal cells.

    Microscopy research and technique 2003;60;6;639-51

  • Translocation of annexin I from cellular membrane to the nuclear membrane in human esophageal squamous cell carcinoma.

    Liu Y, Wang HX, Lu N, Mao YS, Liu F, Wang Y, Zhang HR, Wang K, Wu M and Zhao XH

    National Lab. of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing P. O. Box 2258, Beijing 100021, China.

    Aim: To investigate the alteration of the annexin I subcellular localization in esophageal squamous cell carcinoma (ESCC) and the correlation between the translocation and the tumorigenesis of ESCC.

    Methods: The protein localization of annexin I was detected in both human ESCC tissues and cell line via the indirect immunofluorescence strategy.

    Results: In the normal esophageal epithelia the annexin I was mainly located on the plasma membrane and formed a consecutive typical trammels net. Annexin I protein also expressed dispersively in cytoplasm and the nuclei without specific localization on the nuclear membrane. In esophageal cancer annexin I decreased very sharply with scattered disappearance on the cellular membrane, however it translocated and highly expressed on the nuclear membrane, which was never found in normal esophageal epithelia. In cultured esophageal cancer cell line annexin I protein was also focused on the nuclear membrane, which was consistent with the result from esophageal cancer tissues.

    Conclusion: This observation suggests that the translocation of annexin I protein in ESCC may correlate with the tumorigenesis of the esophageal cancer.

    World journal of gastroenterology 2003;9;4;645-9

  • Annexin 1 modulates monocyte-endothelial cell interaction in vitro and cell migration in vivo in the human SCID mouse transplantation model.

    Perretti M, Ingegnoli F, Wheller SK, Blades MC, Solito E and Pitzalis C

    William Harvey Research Institute, London, United Kingdom. M.Perretti@qmul.ac.uk

    The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; approximately 50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1alpha (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo.

    Funded by: Arthritis Research UK: 15718; Wellcome Trust

    Journal of immunology (Baltimore, Md. : 1950) 2002;169;4;2085-92

  • Dexamethasone induces the secretion of annexin I in immature lymphoblastic cells by a calcium-dependent mechanism.

    Castro-Caldas M, Duarte CB, Carvalho AP and Lopes MC

    Center for Neuroscience of Coimbra, Department of Zoology, University of Coimbra, Portugal.

    The mechanisms by which glucocorticoids (GC) regulate annexin I (ANXA1) secretion in different cells are still a matter of debate. The aims of this study were to evaluate the ability of dexamethasone (Dex) to induce ANXA1 secretion and to investigate the roles of the intracellular free Ca2+ concentration ([Ca2+]i), and of the GC receptor, on that process. For this purpose, the human immature lymphoblastic CCRF-CEM cell line was used. Treatment of the cells with Dex, for up to 4 h, significantly reduced the intracellular content of ANXA1 and increased the amount of this protein bound to the outer surface of the plasma membrane, whereas exposure of cells to Dex, for 12 h, induced the synthesis of ANXA1. At the same short time periods, Dex also induced a significant increase in the [Ca2+]i. Incubation of the cells with BAPTA-AM (10 microM), a cell-permeant high affinity Ca2+ chelator, completely inhibited Dex-induced ANXA1 secretion. Furthermore, the Ca2+ ionophore, ionomycin, alone induced ANXA1 cleavage, but not its secretion. Additionally, we used brefeldin A to investigate the involvement of the classical endoplasmic reticulum (ER)-Golgi pathway of protein secretion in the release of ANXA1. The GC receptor antagonist, RU486, neither reverted the Dex-dependent ANXA1 secretion nor inhibited the increase of the [Ca2+]i induced by Dex. Together, our results indicate that Dex induces ANXA1 synthesis and secretion in CCRF-CEM cells. ANXA1 secretion in this cell type show the following characteristics: (i) is unlikely to involve the classical ER-Golgi pathway; (ii) requires a Ca(2+)-dependent cleavage of ANXA1; (iii) involves both Ca(2+)-dependent and independent mechanisms; and (iv) is apparently independent of the GC receptor alpha isoform.

    Molecular and cellular biochemistry 2002;237;1-2;31-8

  • 17beta-estradiol promotes the synthesis and the secretion of annexin I in the CCRF-CEM human cell line.

    Castro-Caldas M, Duarte CB, Carvalho AR and Lopes MC

    Center for Neuroscience of Coimbra, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal.

    Aims: Annexin I (ANXA1), a 37kDa member of the annexin family of Ca2+-binding and phospholipid-binding proteins, is particularly abundant in various populations of peripheral blood leukocytes. Since this protein modulates the anti-inflammatory actions of the steroid hormones, the purpose of this study was to investigate the effects of the female sex steroid hormone, 17beta-estradiol (E2beta), on the synthesis and secretion of ANXA1 in the human CCRF-CEM acute lymphoblastic leukemia cell line.

    Methods: Complementary reverse transcription-polymerase chain reaction and Western blot assays were performed to study the effect of E2beta on the expression of mRNA and protein ANXA1, respectively.

    Treatment of CCRF-CEM cells with E2beta, for 30 min, stimulated the synthesis of ANXA1 mRNA molecules, and increased the cellular level of ANXA1 protein. Moreover, when the cells were incubated with E2beta under the same experimental conditions, a significant increase in the amount of ANXA1 secreted from the cells was also detected. ICI 182,780, a selective inhibitor of the intracellular estrogen receptor, had no effect on the E2beta-stimulated expression and externalisation of ANXA1. Taken together, these results indicate that E2beta induces de novo synthesis of ANXA1 and stimulates its secretion in the CCRF-CEM cell line, apparently through a mechanism independent of the intracellular estrogen receptor.

    Mediators of inflammation 2001;10;5;245-51

  • Characterization of the annexin I gene and evaluation of its role in type 2 diabetes.

    Lindgren CM, Nilsson A, Orho-Melander M, Almgren P and Groop LC

    Department of Endocrinology, Wallenberg Laboratory, Malmö University Hospital, Malmö, Sweden. cecilia.lindgren@endo.mas.lu.sc

    In a previous study, we identified suggestive linkage between type 2 diabetes and a locus on chromosome 9p13-q21. This region contains the gene annexin I (ANXA1), encoding a protein suggested to be involved in both insulin secretion and insulin action. In this study, we sequenced the exon/intron boundaries of the human ANXA1 gene and performed mutation screening in 41 individuals from the initial linkage study. We identified five single nucleotide polymorphisms A58G, A401G, intronic variance sequence (IVS)8-28A/G, IVS11 +31A/G, and IVS12-11T/G, which were further tested for association to diabetes in 197 parent/offspring trios using the transmission disequilibrium test. No significant association with type 2 diabetes was observed, although the common A allele of the +58A/G variant gave a 22:12 transmission distortion (P = 0.12). This variant was further genotyped in 481 case and control subjects, but no difference in allele, genotype, or haplotype frequencies were observed between the groups. Further, a novel polymorphic (CA)(15-25) repeat in intron 11 was genotyped in the subjects included in the initial linkage study. No improvement of the original finding was observed. We therefore concluded that the ANXA1 gene is unlikely to harbor variants that contribute to risk of type 2 diabetes.

    Funded by: Wellcome Trust: 090532

    Diabetes 2001;50;10;2402-5

  • An annexin 1 (ANXA1)-derived peptide inhibits prototype antigen-driven human T cell Th1 and Th2 responses in vitro.

    Kamal AM, Smith SF, De Silva Wijayasinghe M, Solito E and Corrigan CJ

    Academic Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Charing Cross Campus, Fulham Palace Road, London W6 8RF, UK.

    Background: Annexin-1 (ANXA1, lipocortin 1) is a pleiotrophic protein produced by many cell types including peripheral blood leucocytes. Although it has been shown to inhibit "macroscopic" inflammatory processes in animal models, its direct effects on antigen-activated human T cells have not been studied.

    Objective: To test the hypothesis that ANXA1-derived peptides inhibit antigen-driven prototype Th1 and Th2-type human T cell responses of clinical relevance and lectin-driven responses in vitro.

    Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 14 atopic subjects sensitized to house dust mite allergen (Dermatophagoides pteronyssinus, Der p) and purified protein derivative (PPD) of Mycobacterium tuberculosis. PBMC (1 x 106/mL) were cultured with phytohaemagglutinin (PHA; 5 microg/mL; 4 days), Der p (25 microg/mL; 6 days), PPD (10 microg/mL, 6 days) or medium control. Two ANXA1-derived peptides, Ac2-26 and AF-2 (5-500 microM), were assessed for possible inhibition of PHA-and antigen-induced T cell proliferation (measured by 3H-thymidine uptake), while Ac2-26 was assessed for inhibition of Der p-induced interleukin (IL)-5 release and PPD-induced interferon-gamma (IFN-gamma) release (measured by ELISA). Comparison was made with dexamethasone as an established inhibitory control. Endogenous production by PBMC of cell surface-associated and intracellular ANXA1 in response to PHA, Der p and PPD in the presence and absence of dexamethasone was measured by specific ELISA.

    Results: Both PHA- and antigen-induced T cellular proliferation were inhibited by dexamethasone. Although neither ANXA1-derived peptide significantly altered PHA-induced proliferation, both effected concentration-dependent reductions in antigen-induced proliferation, Ac2-26 being the more potent. Peptides of identical amino acid composition to Ac2-26 and AF-2, but of random sequence, were ineffective at equivalent concentrations. In addition, Ac2-26 and dexamethasone inhibited Der p-induced IL-5 release and PPD-induced IFN-gamma release in a concentration-dependent fashion. Endogenous ANXA1 was detectable in PBMC, but at concentrations approximately 104-fold lower, in molar terms, than the effective concentrations of the exogenously added, ANXA1-derived inhibitory peptides. Endogenous production was not significantly altered by any of the T cell stimuli employed in this study, in the presence or absence of dexamethasone.

    Conclusion: In prototype Th1 and Th2-type human T cell responses, ANXA1-derived peptides can inhibit antigen-driven cellular proliferation and cytokine production.

    Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 2001;31;7;1116-25

  • Small proline-rich protein 1 is the major component of the cell envelope of normal human oral keratinocytes.

    Lee CH, Marekov LN, Kim S, Brahim JS, Park MH and Steinert PM

    Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD 20892-7252, USA.

    Oral keratinocytes of buccal and gingival tissues undergo a terminal differentiation program to form a protective epithelial barrier as non-keratinized or parakeratinized stratified cells. We have examined the protein composition of cell envelopes (CEs) from normal human buccal and gingival tissues as well as keratinocytes from normal human gingival cells grown in culture. Biochemical and sequencing analyses reveal that the CEs contain 60-70% small proline-rich protein 1a/b (SPR1a/b), together with smaller amounts of involucrin, annexin I and several other known CE proteins. The data imply a specialized role for SPR1 proteins in the unique barrier function requirements of oral epithelia.

    FEBS letters 2000;477;3;268-72

  • A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR.

    Walther A, Riehemann K and Gerke V

    Center for Molecular Biology of Inflammation, Institute for Medical Biochemistry, Münster, Germany.

    The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I-mediated antiinflammatory effects.

    Molecular cell 2000;5;5;831-40

  • Identification of genes (SPON2 and C20orf2) differentially expressed between cancerous and noncancerous lung cells by mRNA differential display.

    Manda R, Kohno T, Matsuno Y, Takenoshita S, Kuwano H and Yokota J

    Biology Division, Pathology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo, 104-0045, Japan.

    mRNA differential display was applied to three small cell lung carcinoma (SCLC) cell lines, six non-small cell lung carcinoma (NSCLC) cell lines, and three normal lung tissues to identify genes differentially expressed between lung carcinoma cells and normal lung tissues and between SCLC cells and NSCLC cells. We isolated five differentially expressed genes, two that were novel and three that were already known. DIL-1 (differentially expressed in cancerous and noncancerous lung cells; HGMW-approved symbol SPON2) and pulmonary surfactant apoprotein A were expressed in normal lung tissues but not in lung carcinoma cell lines, whereas DIL-2 (HGMW-approved symbol C20orf2) and nm23-H1 were expressed in lung carcinoma cell lines but not in normal lung tissues. The remaining gene, Annexin II, was expressed at a lower level in SCLC than in NSCLC and normal lung tissues. These genes were also differentially expressed in primary lung cancers. One of the two novel genes, DIL-1, encodes a secreted protein homologous to the Mindin/F-spondin family. The other, DIL-2, encodes a protein with a putative ATP/GTP binding site motif. These data provide basic information necessary to understand the differences in gene expression profiles between lung carcinoma and normal lung and between SCLC and NSCLC. Further characterization of these genes will help to clarify the molecular mechanisms of human lung carcinogenesis.

    Genomics 1999;61;1;5-14

  • NMR solution structure of domain 1 of human annexin I shows an autonomous folding unit.

    Gao J, Li Y and Yan H

    Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.

    Annexins are excellent models for studying the folding mechanisms of multidomain proteins because they have four-eight homologous helical domains with low identity in sequence but high similarity in folding. The structure of an isolated domain 1 of human annexin I has been determined by NMR spectroscopy. The sequential assignments of the 1H, 13C, and 15N resonances of the isolated domain 1 were established by multinuclear, multidimensional NMR spectroscopy. The solution structure of the isolated domain 1 was derived from 1,099 experimental NMR restraints using a hybrid distance geometry-simulated annealing protocol. The root mean square deviation of the ensemble of 20 refined conformers that represent the structure from the mean coordinate set derived from them was 0. 57 +/- 0.14 A and 1.11 +/- 0.19 A for the backbone atoms and all heavy atoms, respectively. The NMR structure of the isolated domain 1 could be superimposed with a root mean square deviation of 1.36 A for all backbone atoms with the corresponding part of the crystal structure of a truncated human annexin I containing all four domains, indicating that the structure of the isolated domain 1 is highly similar to that when it folded together with the other three domains. The result suggests that in contrast to isolated domain 2, which is largely unfolded in solution, isolated domain 1 constitutes an autonomous folding unit and interdomain interactions may play critical roles in the folding of annexin I.

    Funded by: NCRR NIH HHS: RR08299; NIGMS NIH HHS: GM51901

    The Journal of biological chemistry 1999;274;5;2971-7

  • Lipocortin 1 co-associates with cytokeratins 8 and 18 in A549 cells via the N-terminal domain.

    Croxtall JD, Wu HL, Yang HY, Smith B, Sutton C, Chang BI, Shi GY and Flower R

    Department of Biochemical Pharmacology, The William Harvey Research Institute, The Medical College of St. Bartholomews and the Royal London School of Medicine and Dentistry at Queen Mary and Westfield College, London, UK. j.croxtall@qmw.ac.uk

    An affinity chromatography strategy was used to search for proteins in A549 cells which interact with the N-terminus of lipocortin 1 (annexin 1). Using the biologically active fragment Lc13-25 as the affinity ligand, two proteins of molecular weight (m.w.) 52 and 48kDa were extracted. Affinity blots of these proteins bound iodinated Lc13-25. Partial tryptic digests of these proteins were analysed by matrix assisted laser desorption mass spectrometry and found to display fragmentation patterns with a strong similarity to those of cytokeratin 8 and 18 respectively. Subsequent blotting with a panel of specific cytokeratin antibodies strongly supported the idea that the two proteins were cytokeratin 8 and cytokeratin 18. Cytokeratin 8 was isolated from A549 cells in intermediate filament (IF) preparations which were also found to contain lipocortin 1 as a potential intermediate filament associated protein (IFAP). This association persisted throughout cycles of IF assembly and disassembly. Dual-labelling immuno-histochemistry in A549 cells showed strong co-localization of lipocortin 1 and cytokeratin 8. The implications of this finding are discussed in the light of the biological activity and possible function of lipocortin 1.

    Funded by: Wellcome Trust

    Biochimica et biophysica acta 1998;1401;1;39-51

  • Partial mediation of glucocorticoid antiproliferative effects by lipocortins.

    Almawi WY, Saouda MS, Stevens AC, Lipman ML, Barth CM and Strom TB

    Department of Biochemistry, Faculty of Medicine, American University of Beirut, Lebanon.

    The glucocorticoids (GCs) dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent fashion, profoundly inhibit mitogen-induced proliferation of human peripheral blood mononuclear lymphocytes (PBML). This inhibition was specific for GCs, as non-GC steroids were devoid of any antiproliferative capacity. GCs enhanced the mRNA (Northern blot) and protein (Western blot) expression of the calcium and phospholipid binding proteins lipocortin I, II, and V. As a consequence of mitogenic stimulation, PBML secrete PGE2 and leukotriene B4 (LTB4). Antiproliferative concentrations of both DEX and PRED as well as recombinant lipocortin I abolished PGE2 and LTB4 production, suggesting an involvement of lipocortins in GC-mediated antiproliferative effects, possibly by inhibiting eicosanoid production and, consequently, mitogen-induced cellular proliferation. Whereas 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid mimicked DEX and PRED in inhibiting PGE2 and LTB4 production, neither 5,8,11,14-eicosatetraynoic acid nor nordihydroguaiaretic acid had any effect on mitogen-induced PBML proliferation, indicating that the GC-mediated antiproliferative effect is separate from their effects on eicosanoid release. Furthermore, neutralizing anti-lipocortin I and anti-lipocortin II mAb, while reversing the inhibitory activity of DEX and PRED on PGE2 and LTB4 production, only partially reversed DEX- and PRED-mediated antiproliferative effects. This indicates that the GC-mediated antiproliferative effect is not dependent on inhibition of eicosanoid release by lipocortins and suggests the existence of lipocortin-dependent and lipocortin-independent pathways by which GCs mediate their antiproliferative effects.

    Journal of immunology (Baltimore, Md. : 1950) 1996;157;12;5231-9

  • Calcium-dependent binding of S100C to the N-terminal domain of annexin I.

    Mailliard WS, Haigler HT and Schlaepfer DD

    Department of Physiology and Biophysics, University of California, Irvine 92717, USA.

    The annexin family of proteins is characterized by a conserved core domain that binds to phospholipids in a Ca(2+)-dependent manner. Each annexin also has a structurally distinct N-terminal domain that may impart functional specificity. To search for cellular proteins that interact with the N-terminal domain of annexin I, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of human annexin I (GST-AINT; AINT = annexin I N-terminal). Extracts from metabolically labeled A431 cells contained a single protein (M(r) approximately 10,000) that bound to GST-AINT in a Ca(2+)-dependent manner. A synthetic peptide corresponding to amino acids 2-18 of annexin I inhibited the binding of the 10-kDa protein to GST-AINT with half-maximal inhibition occurring at approximately 15 microM peptide. In cellular extracts, endogenous annexin I and the 10-kDa protein associated in a reversible Ca(2+)-dependent manner. Experiments with other annexins and with N-terminal truncated forms of annexin I indicated that the 10-kDa protein bound specifically to a site within the first 12 amino acids of annexin I. The 10-kDa protein was purified from human placenta by hydrophobic and affinity chromatography. Amino acid sequence analysis indicated that the 10-kDa protein is the human homologue of S100C, a recently identified member of the S100 subfamily of EF-hand Ca(2+)-binding proteins.

    Funded by: NCI NIH HHS: 5T32CA09054

    The Journal of biological chemistry 1996;271;2;719-25

  • A BC200-derived element and Z-DNA as structural markers in annexin I genes: relevance to Alu evolution and annexin tetrad formation.

    Morgan RO and Fernández MP

    Department of Pediatrics, George Washington University, Washington, DC 20010, USA.

    We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.

    Journal of molecular evolution 1995;41;6;979-85

  • Crystal structure of human annexin I at 2.5 A resolution.

    Weng X, Luecke H, Song IS, Kang DS, Kim SH and Huber R

    Department of Molecular and Cell Biology, University of California, Berkeley 94720.

    cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).

    Protein science : a publication of the Protein Society 1993;2;3;448-58

  • Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli.

    Arcone R, Arpaia G, Ruoppolo M, Malorni A, Pucci P, Marino G, Ialenti A, Di Rosa M and Ciliberto G

    CEINGE, Biotecnologie Avanzate, Napoli, Italy.

    Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.

    European journal of biochemistry 1993;211;1-2;347-55

  • Treatment of Haemophilus aphrophilus endocarditis with ciprofloxacin.

    Dawson SJ and White LA

    Department of Microbiology, Southampton General Hospital, U.K.

    A patient with Haemophilus aphrophilus endocarditis was successfully treated with ciprofloxacin. The response to treatment with cefotaxime and netilmicin for 12 days was poor but was satisfactory to a 6 weeks' course of ciprofloxacin.

    The Journal of infection 1992;24;3;317-20

  • Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor.

    Ye RD, Cavanagh SL, Quehenberger O, Prossnitz ER and Cochrane CG

    Department of Immunology, Scripps Research Institute, La Jolla, CA 92037.

    A cDNA of 1650 base pairs was isolated by screening an HL-60 granulocyte library with an N-formyl peptide receptor (NFPR) cDNA probe under low stringency conditions. The cDNA encodes a protein of 351 amino acids tentatively named FPR2, with a calculated molecular weight of 39 kDa. Sequence analysis revealed that FPR2 is 69% identical in sequence to the human NFPR and shares extensive homology to several other chemoattractant receptors. FPR2 expressed in transfected cells mediated formyl peptide-stimulated calcium mobilization at micromolar concentrations of ligand. FPR2 messenger is detected in granulocytic HL-60 cells, but not in undifferentiated HL-60 cells. These findings suggest that FPR2 is a novel receptor for formyl peptide ligand and a new member of the chemoattractant receptor gene family.

    Funded by: NIGMS NIH HHS: GM46572

    Biochemical and biophysical research communications 1992;184;2;582-9

  • Correlation of gene and protein structure of rat and human lipocortin I.

    Kovacic RT, Tizard R, Cate RL, Frey AZ and Wallner BP

    Biogen Inc., Cambridge, Massachusetts 02142.

    Lipocortins (annexins) are a family of calcium-dependent phospholipid-binding proteins with phospholipase A2 inhibitory activity. The characteristic primary structure of members of this family consists of a core structure of four or eight repeated domains, which have been implicated in calcium-dependent phospholipid binding. In two lipocortins (I and II) a short amino-terminal sequence distinct from the core structure has potential regulatory functions which are dependent on its phosphorylation state. We have isolated the rat and the human lipocortin I genes and found that they both consist of 13 exons with a striking conservation of their exon-intron structure and their promoter and amino acid sequences. Both lipocortin I genes are at least 19 kbp in length with exons ranging from 57 to 123 bp interrupted by introns as large as 5 kbp. Each of the four repeat units of lipocortin I are encoded by two consecutive exons while individual exons code for the highly conserved putative calcium-binding domains. The promoter sequences in the rat and in human genes are highly conserved and contain nucleotide sequences characterized as enhancer sequences in other genes. The structure of the lipocortin I gene lends support to the hypothesis that the lipocortin genes arose by a duplication of a single domain.

    Biochemistry 1991;30;37;9015-21

  • Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids.

    Ando Y, Imamura S, Owada MK and Kannagi R

    Department of Dermatology, Faculty of Medicine, Kyoto University, Japan.

    Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.

    The Journal of biological chemistry 1991;266;2;1101-8

  • Characterization of Ca2(+)-dependent phospholipid binding, vesicle aggregation and membrane fusion by annexins.

    Blackwood RA and Ernst JD

    Department of Pediatrics, San Francisco General Hospital, University of California 94143-0868.

    The annexins are a family of structurally similar, Ca2(+)-dependent, phospholipid-binding proteins. We compared six members of this family (calpactin I heavy chain, lipocortins I and III, endonexin II, p68 and protein II) to determine their phospholipid-binding specificities, as well as their ability to promote aggregation and fusion of phospholipid vesicles. The Ca2+ requirement for all of the proteins was lowest for binding to vesicles composed of phosphatidic acid, followed by phosphatidylserine and then phosphatidylinositol. Only protein II, p68, lipocortin III and endonexin II bound to vesicles composed of phosphatidylethanolamine, and none bound to phosphatidylcholine. Both calpactin I heavy chain and lipocortin I promoted aggregation of phosphatidylserine- or phosphatidylinositol-containing vesicles in the presence of less than 10 microM-Ca2+. Lipocortin I promoted fusion of liposome membranes by lowering threshold Ca2+ concentrations. Although calpactin I heavy chain did not affect threshold Ca2+ concentrations, it did increase the rate and extent of spontaneous fusion. In contrast, p68 inhibited fusion at threshold Ca2+ concentrations. Whereas previous reports have emphasized properties that the annexins have in common, these findings reveal quantitative and qualitative differences among the annexins which may relate to distinct intracellular functions.

    Funded by: NIAID NIH HHS: AI23697; NIAMS NIH HHS: AR20684; NIGMS NIH HHS: GM11909

    The Biochemical journal 1990;266;1;195-200

  • A dimeric form of lipocortin-1 in human placenta.

    Pepinsky RB, Sinclair LK, Chow EP and O'Brine-Greco B

    Biogen Inc., Cambridge, MA 02142.

    We have characterized a 68 kDa lipocortin from human placenta that was identified as a covalently linked homodimer of lipocortin-1 by peptide mapping and sequence analysis. The site of cross-linking was localized within the 3 kDa N-terminal tail region, an exposed domain that contains the phosphorylation sites for protein tyrosine kinase and protein kinase C and is sensitive to proteolysis. Sequence analysis of the corresponding peptide revealed that glutamine-18 was modified, suggesting that the cross-link may be generated by a transglutaminase. By incubating lipocortin-1 with placental membranes and with labelled glycine ethyl ester we observed a Ca2+-dependent labelling of lipocortin-1 within the tail region, supporting this notion. Like lipocortin-1, the dimer inhibits phospholipase Ad2 activity, is a substrate for the epidermal-growth-factor (EGF) receptor/kinase, and display Ca2+-dependent binding to phosphatidylserine-containing vesicles. In preparations from human placenta the dimer is particularly abundant, accounting for approx. 20% of the lipocortin-1.

    The Biochemical journal 1989;263;1;97-103

  • Diversity in the lipocortin/calpactin family.

    Crompton MR, Moss SE and Crumpton MJ

    Imperial Cancer Research Fund Laboratories, London, England.

    Cell 1988;55;1;1-3

  • Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein.

    Kaplan R, Jaye M, Burgess WH, Schlaepfer DD and Haigler HT

    Division of Molecular Biology, Rorer Biotechhology Inc., Springfield, Virginia 22151.

    Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.

    Funded by: NIGMS NIH HHS: GM357844

    The Journal of biological chemistry 1988;263;17;8037-43

  • Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C.

    Varticovski L, Chahwala SB, Whitman M, Cantley L, Schindler D, Chow EP, Sinclair LK and Pepinsky RB

    Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.

    Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.

    Funded by: NIDDK NIH HHS: DK 07542; NIGMS NIH HHS: GM 36624

    Biochemistry 1988;27;10;3682-90

  • Chromosomal localization of the human genes for lipocortin I and lipocortin II.

    Huebner K, Cannizzaro LA, Frey AZ, Hecht BK, Hecht F, Croce CM and Wallner BP

    Wistar Institute, Philadelphia, PA 19104.

    The human genes which code for Lipocortin I and Lipocortin II, proteins that inhibit phospholipase A2 (PLA2) activity, have been regionally localized in the human genome by chromosomal in situ hybridization and segregation analysis in somatic cell hybrids using cDNA clones for Lipocortin I and II. Lipocortin I, the 35 kd substrate for the epidermal growth factor (EGF) receptor/kinase, maps to chromosome region 9q11- greater than q22. The Lipocortin II cDNA probe detects at least four independently segregating loci which map to human chromosome regions 4q21-q31.1, 9pter-q34 proximal to c-abl, 10q proximal to 10q24 and 15q21-q22 proximal to the 15q22 translocation breakpoint characteristic of acute promyelocytic leukemia (APL). Thus, Lipocortin I and one locus detected by Lipocortin II cDNA are syntenic on chromosome 9; one Lipocortin II locus is perhaps not far from the genes for EGF and IL-2 on 4q; and another of the Lipocortin II loci is on 15q, perhaps not far from the APL breakpoint.

    Funded by: NCI NIH HHS: CA-10805, CA-21124, CA-39860; ...

    Oncogene research 1988;2;4;299-310

  • Characterization by tandem mass spectrometry of structural modifications in proteins.

    Biemann K and Scoble HA

    Tandem mass spectrometry can be used to solve a number of protein structural problems that are not amenable to conventional methods for amino acid sequencing. Typical problems that use this approach involve characterization of peptides with blocked amino termini or peptides that have been otherwise posttranslationally processed, such as, by phosphorylation or sulfation. The structure and homogeneity of synthetic peptides can also be evaluated. Since peptides can be selectively characterized in the presence of other peptides or contaminants, the need for extensive purification is reduced or eliminated.

    Funded by: NCRR NIH HHS: RR00317; NIGMS NIH HHS: GM05472

    Science (New York, N.Y.) 1987;237;4818;992-8

  • Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity.

    Wallner BP, Mattaliano RJ, Hession C, Cate RL, Tizard R, Sinclair LK, Foeller C, Chow EP, Browing JL, Ramachandran KL et al.

    The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2 inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2 hydrolysis of phospholipids. Lipocortin-like proteins have been isolated from various cell types, including monocytes, neutrophils and renal medullary cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2 activity.

    Nature 1986;320;6057;77-81

Gene lists (3)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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