G2Cdb::Gene report

Gene id
G00001875
Gene symbol
GJA1 (HGNC)
Species
Homo sapiens
Description
gap junction protein, alpha 1, 43kDa
Orthologue
G00000626 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000015479 (Vega human gene)
Gene
ENSG00000152661 (Ensembl human gene)
2697 (Entrez Gene)
1052 (G2Cdb plasticity & disease)
GJA1 (GeneCards)
Literature
121014 (OMIM)
Marker Symbol
HGNC:4274 (HGNC)
Protein Sequence
P17302 (UniProt)

Synonyms (4)

  • CX43
  • ODD
  • ODOD
  • SDTY3

Literature (201)

Pubmed - other

  • The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein.

    Giepmans BN and Moolenaar WH

    Division of Cellular Biochemistry The Netherlands Cancer Institute Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

    Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated by growth factors and oncogenes [3-5], although the underlying mechanisms are poorly understood as cellular proteins that interact with connexins have yet to be identified. The carboxy-terminal cytosolic domain of Cx43 contains several phosphorylation sites and potential signalling motifs. We have used a yeast two-hybrid protein interaction screen to identify proteins that bind to the carboxy-terminal tail of Cx43 and thereby isolated the zona occludens-1 (ZO-1) protein. ZO-1 is a 220 kDa peripheral membrane protein containing multiple protein interaction domains including three PDZ domains and a Src homology 3 (SH3) domain [6-9]. The interaction of Cx43 with ZO-1 occurred through the extreme carboxyl terminus of Cx43 and the second PDZ domain of ZO-1. Cx43 associated with ZO-1 in Cx43-transfected COS7 cells, as well as endogenously in normal Rat-1 fibroblasts and mink lung epithelial cells. Confocal microscopy revealed that endogenous Cx43 and ZO-1 colocalised at gap junctions. We suggest that ZO-1 serves to recruit signalling proteins into Cx43-based gap junctions.

    Current biology : CB 

  • Mutation analysis of Connexon43 gene in Chinese patients with congenital heart defects.

    Wang B, Wen Q, Xie X, Liu S, Liu M, Tao Y, Li Z, Suo P, Shen A, Wang J and Ma X

    International journal of cardiology 2010;145;3;487-9

  • Rac1-induced connective tissue growth factor regulates connexin 43 and N-cadherin expression in atrial fibrillation.

    Adam O, Lavall D, Theobald K, Hohl M, Grube M, Ameling S, Sussman MA, Rosenkranz S, Kroemer HK, Schäfers HJ, Böhm M and Laufs U

    Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarland, Homburg/Saar, Germany. oliver.adam@uks.eu

    Objectives: We studied the signal transduction of atrial structural remodeling that contributes to the pathogenesis of atrial fibrillation (AF).

    Background: Fibrosis is a hallmark of arrhythmogenic structural remodeling, but the underlying molecular mechanisms are incompletely understood.

    Methods: We performed transcriptional profiling of left atrial myocardium from patients with AF and sinus rhythm and applied cultured primary cardiac cells and transgenic mice with overexpression of constitutively active V12Rac1 (RacET) in which AF develops at old age to characterize mediators of the signal transduction of atrial remodeling.

    Results: Left atrial myocardium from patients with AF showed a marked up-regulation of connective tissue growth factor (CTGF) expression compared with sinus rhythm patients. This was associated with increased fibrosis, nicotinamide adenine dinucleotide phosphate oxidase, Rac1 and RhoA activity, up-regulation of N-cadherin and connexin 43 (Cx43) expression, and increased angiotensin II tissue concentration. In neonatal rat cardiomyocytes and fibroblasts, a specific small molecule inhibitor of Rac1 or simvastatin completely prevented the angiotensin II-induced up-regulation of CTGF, Cx43, and N-cadherin expression. Transfection with small-inhibiting CTGF ribonucleic acid blocked Cx43 and N-cadherin expression. RacET mice showed up-regulation of CTGF, Cx43, and N-cadherin protein expression. Inhibition of Rac1 by oral statin treatment prevented these effects, identifying Rac1 as a key regulator of CTGF in vivo.

    Conclusions: The data identify CTGF as an important mediator of atrial structural remodeling during AF. Angiotensin II activates CTGF via activation of Rac1 and nicotinamide adenine dinucleotide phosphate oxidase, leading to up-regulation of Cx43, N-cadherin, and interstitial fibrosis and therefore contributing to the signal transduction of atrial structural remodeling.

    Journal of the American College of Cardiology 2010;55;5;469-80

  • Genetic susceptibility to febrile seizures: case-control association studies.

    Kira R, Ishizaki Y, Torisu H, Sanefuji M, Takemoto M, Sakamoto K, Matsumoto S, Yamaguchi Y, Yukaya N, Sakai Y, Gondo K and Hara T

    Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. kirari@pediatr.med.kyushu-u.ac.jp

    Objective: A genetic predisposition to febrile seizures (FS) has long been recognized. The inheritance appears to be polygenic in small families or sporadic cases of FS encountered in daily clinical practice. To determine whether candidate genes are responsible for the susceptibility to FS, we have performed genetic association studies in FS patients and controls.

    Methods: The single-nucleotide polymorphisms (SNPs) of genes involved in immune response (interleukin (IL) 1B), endocannabinoid signaling (CNR1), acid-base balance (SLC4A3, SLC9A1, SLC9A3), gap junction channel (CX43), and GABA(A) receptor trafficking (PRIP1) were examined in 249 FS patients (186 simple and 63 complex FS) and 225 controls.

    Results: There were no significant differences in the allele frequencies of the SNPs between controls and all FS, simple FS, and complex FS patients. When the simple FS patients were divided into two groups according to either having (familial) or not having a family history of FS in close relatives (sporadic), there was a significant association between IL1B -511 SNP and sporadic simple FS (p=0.003).

    Conclusions: These data suggest that cytokine genes may act as enhancers or attenuators of FS susceptibility. Genetic association study may be an effective approach to understanding the molecular basis of FS at least in a subgroup of patients.

    Brain & development 2010;32;1;57-63

  • Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium.

    Smyth JW, Hong TT, Gao D, Vogan JM, Jensen BC, Fong TS, Simpson PC, Stainier DY, Chi NC and Shaw RM

    Cardiovascular Research Institute, Department of Medicine, University of California-San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143, USA.

    Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with ad 1e0f herens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias.

    Funded by: NHLBI NIH HHS: HL075449, HL094414, HL31113, HL54737, K08 HL075449, K08 HL075449-04, K08 HL075449-05, K08 HL096836, R01 HL031113, R01 HL054737, R01 HL094414, R01 HL094414-01A1

    The Journal of clinical investigation 2010;120;1;266-79

  • Bouvier D, Spagnol G, Chenavas S, Kieken F, Vitrac H, Brownell S, Kellezi A, Forge V and Sorgen PL

    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.

    Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the "particle-receptor" model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.

    Funded by: NCI NIH HHS: P30 CA036727, P30CA036727; NHLBI NIH HHS: HL039707, P01 HL039707; NIGMS NIH HHS: GM072631, R01 GM072631

    The Journal of biological chemistry 2009;284;49;34257-71

  • Asymmetric dimethylarginine damages connexin43-mediated endothelial gap junction intercellular communication.

    Jia SJ, Zhou Z, Zhang BK, Hu ZW, Deng HW and Li YJ

    Department of Pharmaceutics, The Third XiangYa Hospital of Central South University, Changsha, China.

    Asymmetric dimethylarginine (ADMA), a major endogenous inhibitor of nitric oxide synthase, is recently defined as a novel atherogenic factor. Communication via gap junction (GJIC) is involved in the regulation of a variety of endothelial activities, such as cell differentiation and senescence. The aim of this study is to explore the effects of ADMA on connexin43 (Cx43) mediated endothelial GJIC. Lysophosphatidylcholine (LPC) caused the downregulation of Cx43 expression and GJIC dysfunction in cultured human umbilical vein endothelial cells (HUVECs), which were significantly ameliorated by decreasing ADMA accumulation. Furthermore, we found that ADMA (10 micromol x L(-1), 24 h) markedly downregulated Cx43 expression and damaged GJIC function in HUVECs. ADMA also increased production of intracellular reactive oxygen species (ROS) and induced phosphorylation of p38 MAPK. Furthermore, the inhibitory effect of ADMA on Cx43-mediated GJIC could be attenuated by NADPH oxidase inhibitor diphenyleneiodonium and apocynin as well as p38 MAPK inhibitor SB203580, respectively. In conclusion, our present results suggest that ADMA inhibits endothelial GJIC function via downregulating Cx43 expression, which suggesting a novel mechanism linking between elevated ADMA level and progression of atherosclerosis.

    Biochemistry and cell biology = Biochimie et biologie cellulaire 2009;87;6;867-74

  • Tyrosine-dependent basolateral targeting of human connexin43-eYFP in Madin-Darby canine kidney cells can be disrupted by the oculodentodigital dysplasia mutation L90V.

    Chtchetinin J, Gifford WD, Li S, Paznekas WA, Jabs EW and Lai A

    Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.

    Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286-289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin-Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43-eYFP, and analyzed the membrane localization of Cx43-eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43-eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43-eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284-289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43-eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V oculodentodigital dysplasia-associated mutations could affect polarized sorting of Cx43-eYFP, we analyzed the expression of these Cx43-eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.

    Funded by: NIDCR NIH HHS: R01 DE013849, R01 DE013849-09, R01 DE13849

    The FEBS journal 2009;276;23;6992-7005

  • Matricellular protein CCN3 (NOV) regulates actin cytoskeleton reorganization.

    Sin WC, Tse M, Planque N, Perbal B, Lampe PD and Naus CC

    Department of Cellular and Physiological Sciences and The Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada. wcsin@interchange.ubc.ca

    CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.

    Funded by: Canadian Institutes of Health Research: MOP 81202; NIGMS NIH HHS: GM55632, R01 GM055632

    The Journal of biological chemistry 2009;284;43;29935-44

  • Comparative evaluation of estrogen and progesterone receptor expression with connexins 26 and 43 in endometrial cancer.

    Lesniewicz T, Kanczuga-Koda L, Baltaziak M, Jarzabek K, Rutkowski R, Koda M, Wincewicz A, Sulkowska M and Sulkowski S

    Department of General Pathomorphology, Medical University of Bialystok, Waszyngtona, Bialystok, Poland.

    Progression of numerous neoplasms could involve alterations of gap junction channels composed of connexins (Cxs). Disorders of expression and cellular displacement of Cxs were also found in endometrial cancer. Gap junctional intercellular communication can be regulated by wide array of agents, for instance, growth factors, oncogenes, and steroid hormones. Nevertheless, expressions of Cxs and progesterone receptor (PR) were not compared in human tissues. This study focused on assessment of expression of estrogen receptor alpha (ERalpha) and PRs in relation to the expression of Cx26 and Cx43 in 88 cases of endometrial cancer and analysis of these proteins' expression in comparison with anatomoclinical features. Positive ERalpha and PR nuclear staining was present in 66 (75%) and 60 (68.2%) of all studied tumors, respectively. Positive correlation was found between expression of PR and histopathologic type of tumor (P = 0.026), and negative correlation was drawn with grading (G) (P = 0.002). There were positive reactions to Cx26 and Cx43 of mainly cytoplasmic location in 60 (68.2%) and 66 (75%) of studied cancers, respectively. Progesterone receptor expression correlated negatively with Cx26 in endometrial cancers (P = 0.016, r = -0.256). Moreover, ERalpha expression positively correlated with PR expression (P < 0.001, r = 0.678). On the ground of our findings, disorders of Cx expression and altered distribution pattern occur during endometrial carcinogenesis, and it seems that PR could participate in this fact. Loss of functional gap junctions may occur because of the aberrant expression and localization of Cx26 and Cx43 in endometrial cancer.

    International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 2009;19;7;1253-7

  • Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding.

    Myllykoski M, Kuczera K and Kursula P

    Department of Biochemistry, FIN-90014, University of Oulu, Oulu, Finland.

    Gap junctions are formed by a family of transmembrane proteins, connexins. Connexin43 is a widely studied member of the family, being ubiquitously expressed in a variety of tissues and a target of a large number of disease mutations. The intracellular loop of connexin43 has been shown to include a calmodulin binding domain, but detailed 3-dimensional data on the structure of the complex are not available. In this study, we used a synthetic peptide from this domain to reveal the conformation of the calmodulin-peptide complex by small angle X-ray scattering. Upon peptide binding, calmodulin lost its dumbbell shape, adopting a more globular conformation. We also studied the energetics of the interaction using calorimetry and computational methods. All our data indicate that calmodulin binds to the peptide from cx43 in the classical 'collapsed' conformation.

    Biophysical chemistry 2009;144;3;130-5

  • A case of oculodentodigital dysplasia syndrome with novel GJA1 gene mutation.

    Himi M, Fujimaki, Yokoyama T, Fujiki K, Takizawa T and Murakami A

    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.

    Purpose: To report a case of oculodentodigital dysplasia syndrome (ODDD) with a heterozygous mutation in GJA1 (connexin 43) gene.

    Methods: A 9-year-old girl visited our hospital complaining of visual disturbances. The patient had microphthalmia, a small nose with hypoplastic alae nasi, and syndactyly. Visual acuity with prescribed glasses improved to 0.5 (1.2) OU 2 months after the first visit. She was satisfied with the new glasses and the improvement in visual acuity. Genomic DNA was extracted from leukocytes of the patient's peripheral blood in accordance with standard procedures, after obtaining parental informed consent. We amplified GJA1 exon 2 from her genomic DNA by the PCR method, and sequenced the product using the dye terminator method.

    Results: S5C (c. 13A > T), a novel mutation in exon 2 of GJA1, was found in the patient. The parents had no mutation of GJAI, nor was there any sign of abnormality in other family members. No similar mutation could be found in the 50 genotyped normal subjects in the control group.

    Conclusions: A novel GJA1 mutation was detected in a Japanese ODDD patient. Glaucoma complications associated with ODDD have already been reported. Careful long-term monitoring and treatment are also necessary.

    Japanese journal of ophthalmology 2009;53;5;541-5

  • Increased connexin 43 expression as a potential mediator of the neuroprotective activity of the corticotropin-releasing hormone.

    Hanstein R, Trotter J, Behl C and Clement AB

    Institute of Pathobiochemistry, University Medical Center, Johannes Gutenberg-University, 55099 Mainz, Germany.

    CRH is a major central stress mediator, but also a potent neuroprotective effector. The mechanisms by which CRH mediates its neuroprotective actions are largely unknown. Here, we describe that the gap junction molecule connexin43 (Cx43) mediates neuroprotective effects of CRH toward experimentally induced oxidative stress. An enhanced gap junction communication has been reported to contribute to neuroprotection after neurotoxic insults. We show that CRH treatment up-regulates Cx43 expression and gap junctional communication in a CRH receptor-dependent manner in IMR32 neuroblastoma cells, primary astrocytes, and organotypic hippocampal slice cultures. MAPKs and protein kinase A-cAMP response element binding protein -coupled pathways are involved in the signaling cascade from CRH to enhanced Cx43 function. Inhibition of CRH-promoted gap junction communication by the gap junction inhibitor carbenoxolone could prevent neuroprotective actions of CRH in cell and tissue culture models suggesting that gap junction molecules are involved in the neuroprotective effects of CRH. The extent of oxidative stress-induced protein carbonylation and cell death inversely correlated with Cx43 protein levels as shown by Cx43 small interfering RNA knockdown experiments. Coculture studies of primary neurons and astrocytes revealed that astrocytic Cx43 likely contributes to the neuroprotective effects of CRH. To our knowledge this is the first description of Cx43 as a potential mediator of the neuroprotective actions of CRH.

    Molecular endocrinology (Baltimore, Md.) 2009;23;9;1479-93

  • Overexpression of protease-activated receptor-1 contributes to melanoma metastasis via regulation of connexin 43.

    Villares GJ, Dobroff AS, Wang H, Zigler M, Melnikova VO, Huang L and Bar-Eli M

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

    Protease-activated receptor-1 (PAR-1) is a key player in melanoma metastasis with higher expression seen in metastatic melanoma cell lines and tissue specimens. cDNA microarray and Western blot analyses reveal that the gap junctional intracellular communication molecule connexin 43 (Cx-43), known to be involved in tumor cell diapedesis and attachment to endothelial cells, is signific 802 antly decreased after PAR-1 silencing in metastatic melanoma cell lines. Furthermore, Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells, suggesting that PAR-1 regulates Cx-43 at the transcriptional level. Chromatin immunoprecipitation studies showed a reduction in the binding of SP-1 and AP-1 transcription factors to the promoter of Cx-43. Both transcription factors have been shown previously to be required for maximal Cx-43 promoter activity. These results were corroborated by mutating the AP-1 and SP-1 binding sites resulting in decreased Cx-43 promoter activity in PAR-1-positive cells. Moreover, as Cx-43 has been shown to facilitate arrest of circulating tumor cells at the vascular endothelium, melanoma cell attachment to endothelial cells was significantly decreased in PAR-1-silenced cells, with this effect being abrogated after PAR-1 rescue. Herein, we report that up-regulation of PAR-1 expression, seen in melanoma progression, mediates high levels of Cx-43 expression. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Indeed, Cx-43 expression was restored following PAR-1 rescue in PAR-1-silenced cells. Taken together, our data support the tumor promoting function of Cx-43 in melanoma.

    Funded by: NCI NIH HHS: R01 CA076098, R01 CA076098-06, R01 CA076098-07, R01 CA076098-08, R01 CA076098-09A1, R01 CA076098-10, R01 CA76098

    Cancer research 2009;69;16;6730-7

  • [Expression of nNOS, Pax3 and Cx43 proteins in early developing posterior horn of embryonic and fetal human spinal cord].

    Zhang Y and Liu XH

    School of Medicine, Shaoxing University, Shaoxing 312000, China. hhren6588@sina.com

    Objective: To investigate the distribution pattern of the expressions neuronal nitric oxide synthase (nNOS), Pax3 and connexin 43 (Cx43) proteins in the early developing posterior horn of embryonic and fetal human spinal cord.

    Methods: Immuno 18d9 histochemistry was used to detect the expressions of nNOS, Pax3 and Cx43 proteins in the posterior horn of the spinal cord during the second, third and fourth month of human embryonic and fetal development.

    Results: In the second to fourth month of gestation, the expressions of nNOS and Pax3 proteins increased gradually from weak expression to strong expression in the posterior horn of the spinal cord. In the second to third month of development, Cx43 protein expression was negative in the posterior horn of the spinal cord, but positive in the myelin sheath. In the fourth month, positive Cx43 expression was detected in some of the cells in the posterior horn of the spinal cord.

    Conclusion: nNOS, Pax3 and Cx43 proteins are closely related to the growth and development of the spinal cord in human embryos and fetuses.

    Nan fang yi ke da xue xue bao = Journal of Southern Medical University 2009;29;8;1651-3

  • p54nrb is a transcriptional corepressor of the progesterone receptor that modulates transcription of the labor-associated gene, connexin 43 (Gja1).

    Dong X, Yu C, Shynlova O, Challis JR, Rennie PS and Lye SJ

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1X5. xdong@prostatecentre.com

    The progesterone receptor (PR) plays important roles in the establishment and maintenance of pregnancy. By dynamic interactions with coregulators, PR represses the expression of genes that increase the contractile activity of myometrium and contribute to the initiation of labor. We have previously shown that PTB-associated RNA splicing factor (PSF) can function as a PR corepressor. In this report, we demonstrated that the PSF heterodimer partner, p54nrb (non-POU-domain-containing, octamer binding protein), can also function as a transcription corepressor, independent of PSF. p54nrb Interacts directly with PR independent of progesterone. In contrast to PSF, p54nrb neither enhances PR protein degradation nor blocks PR binding to DNA. Rather, p54nrb recruits mSin3A through its N terminus to the PR-DNA complex, resulting in an inhibition of PR-mediated transactivation of the progesterone-response element-luciferase reporter gene. PR also repressed transcription of the connexin 43 gene (Gja1), an effect dependent on the presence of an activator protein 1 site within the proximal Gja1 promoter. Mutation of this site abolished PR-mediated repression and decreased the recruitment of PR and p54nrb onto the Gja1 promoter. Furthermore, knockdown p54nrb expression by small interfering RNA alleviated PR-mediated repression on Gja1 transcription, whereas overexpression of p54nrb enhanced it. In the physiological context of pregnancy, p54nrb protein levels decrease with the approach of labor in the rat myometrium. We conclude that p54nrb is a transcriptional corepressor of PR. Decreased expression of p54nrb at the time of labor may act to derepress PR-mediated inhibition on connexin 43 expression and contribute to the initiation of labor.

    Molecular endocrinology (Baltimore, Md.) 2009;23;8;1147-60

  • Novel pharmacophores of connexin43 based on the "RXP" series of Cx43-binding peptides.

    Verma V, Larsen BD, Coombs W, Lin X, Spagnol G, Sorgen PL, Taffet SM and Delmar M

    Center for Arrhythmia Research, University of Michigan Medical School, 5025 Venture Dr, Ann Arbor MI 48104, USA.

    Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function.

    Funded by: NHLBI NIH HHS: HL087226, HL39707, P01 HL039707, P01 HL039707-180011, P01 HL087226, P01 HL087226-010001; NIGMS NIH HHS: GM072631, GM57691, R01 GM057691, R01 GM057691-12, R01 GM072631

    Circulation research 2009;105;2;176-84

  • Effect of high-frequency electromagnetic fields on trophoblastic connexins.

    Cervellati F, Franceschetti G, Lunghi L, Franzellitti S, Valbonesi P, Fabbri E, Biondi C and Vesce F

    Department of Biology and Evolution, Section of General Physiology, University of Ferrara, via L. Borsari 46, 44100 Ferrara, Italy.

    Connexins (Cx) are membrane proteins able to influence trophoblast functions. Here we investigated the effect of high-frequency electromagnetic fields (HF-EMF) on Cx expression and localization in extravillous trophoblast cell line HTR-8/SVneo. We also analysed cell ultrastructural changes induced by HF-EMF exposure. Samples were exposed to pulse-modulated 1817 MHz sinusoidal waves (GSM-217 Hz; 1h: SAR of 2 W/kg). Cx mRNA expression was assessed through semi-quantitative RT-PCR, protein expression by Western blotting, protein localization by indirect immunofluorescence, cell ultrastructure using electron microscopy. HF-EMF exposure significantly and selectively increased Cx40 and Cx43, without altering protein expression. Nevertheless, Cx40 and Cx43 lost their punctuate fluorescence within the cell membrane, becoming diffuse after HF-EMF exposure. Electron microscopy evidenced a sharp decrease in intercellular gap junction-like structures. This study is the first to indicate that exposure of extravillous trophoblast to GSM-217 Hz signals can modify Cx gene expression, Cx protein localization and cellular ultrastructure.

    Reproductive toxicology (Elmsford, N.Y.) 2009;28;1;59-65

  • A large-scale genome-wide association study of Asian populations uncovers genetic factors influencing eight quantitative traits.

    Cho YS, Go MJ, Kim YJ, Heo JY, Oh JH, Ban HJ, Yoon D, Lee MH, Kim DJ, Park M, Cha SH, Kim JW, Han BG, Min H, Ahn Y, Park MS, Han HR, Jang HY, Cho EY, Lee JE, Cho NH, Shin C, Park T, Park JW, Lee JK, Cardon L, Clarke G, McCarthy MI, Lee JY, Lee JK, Oh B and Kim HL

    Center for Genome Science, National Institute of Health, Seoul, Korea.

    To identify genetic factors influencing quantitative traits of biomedical importance, we conducted a genome-wide association study in 8,842 samples from population-based cohorts recruited in Korea. For height and body mass index, most variants detected overlapped those reported in European samples. For the other traits examined, replication of promising GWAS signals in 7,861 independent Korean samples identified six previously unknown loci. For pulse rate, signals reaching genome-wide significance mapped to chromosomes 1q32 (rs12731740, P = 2.9 x 10(-9)) and 6q22 (rs12110693, P = 1.6 x 10(-9)), with the latter approximately 400 kb from the coding sequence of GJA1. For systolic blood pressure, the most compelling association involved chromosome 12q21 and variants near the ATP2B1 gene (rs17249754, P = 1.3 x 10(-7)). For waist-hip ratio, variants on chromosome 12q24 (rs2074356, P = 7.8 x 10(-12)) showed convincing associations, although no regional transcript has strong biological candidacy. Finally, we identified two loci influencing bone mineral density at multiple sites. On chromosome 7q31, rs7776725 (within the FAM3C gene) was associated with bone density at the radius (P = 1.0 x 10(-11)), tibia (P = 1.6 x 10(-6)) and heel (P = 1.9 x 10(-10)). On chromosome 7p14, rs1721400 (mapping close to SFRP4, a frizzled protein gene) showed consistent associations at the same three sites (P = 2.2 x 10(-3), P = 1.4 x 10(-7) and P = 6.0 x 10(-4), respectively). This large-scale GWA analysis of well-characterized Korean population-based samples highlights previously unknown biological pathways.

    Nature genetics 2009;41;5;527-34

  • Conformational maturation and post-ER multisubunit assembly of gap junction proteins.

    Vanslyke JK, Naus CC and Musil LS

    Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA.

    For all previously well-characterized oligomeric integral membrane proteins, folding, multisubunit assembly, and recognition of conformationally immature molecules for degradation occurs at their organelle of synthesis. This cannot, however, be the case for the gap junction-forming protein connexin43 (Cx43), which when endogenously expressed undergoes multisubunit assembly into connexons only after its transport to the trans-Golgi network. We have developed two novel assays to assess Cx43 folding and assembly: acquisition of resistance of disulfide bonds to reduction by extracellularly added DTT and Triton X-114 detergent phase partitioning. We show that Cx43 synthesized at physiologically relevant levels undergoes a multistep conformational maturation process in which folding of connexin monomers within the ER is a prerequisite for multisubunit assembly in the TGN. Similar results were obtained with Cx32, disproving the widely reported contention that the site of endogenous beta connexin assembly is the ER. Exogenous overexpression of Cx43, Cx32, or Cx26 allows these events to take place within the ER, the first example of the TGN and ER as alternative sites for oligomeric assembly. Our findings also constitute the first biochemical evidence that defective connexin folding is a cause of the human disorder X-linked Charcot-Marie-Tooth disease.

    Funded by: NEI NIH HHS: R01 EY014622; NINDS NIH HHS: R01 NS040740, R01NS40740-01

    Molecular biology of the cell 2009;20;9;2451-63

  • ERp29 restricts Connexin43 oligomerization in the endoplasmic reticulum.

    Das S, Smith TD, Sarma JD, Ritzenthaler JD, Maza J, Kaplan BE, Cunningham LA, Suaud L, Hubbard MJ, Rubenstein RC and Koval M

    Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus.

    Funded by: NHLBI NIH HHS: HL-083120, R01 HL083120, R01 HL083120-03; NIAAA NIH HHS: AA-013528, AA-013757, P50 AA013757, T32 AA013528; NIDDK NIH HHS: DK-58046, R01 DK058046; NIGMS NIH HHS: GM-61012, R01 GM061012

    Molecular biology of the cell 2009;20;10;2593-604

  • Ocular manifestations in oculodentodigital dysplasia resulting from a heterozygous missense mutation (L113P) in GJA1 (connexin 43).

    Musa FU, Ratajczak P, Sahu J, Pentlicky S, Fryer A, Richard G and Willoughby CE

    Birmingham and Midland Eye Centre, City Hospital, Birmingham, UK. fayyaz@doctors.net.uk

    Purpose: To characterize the ophthalmic findings, intrafamilial variability, and molecular genetic basis of oculodentodigital dysplasia (ODDD; MIM no. 164200).

    Methods: Ophthalmic examination included best-corrected visual acuity, slit-lamp biomicroscopy, direct and indirect ophthalmoscopy, Goldmann applanation tonometry and A-scan ultrasonography. Blood samples were taken for DNA extraction and mutation screening of GJA1 (connexin 43).

    Results: All three affected individuals had characteristic features of ODDD. The ophthalmic features were epicanthus, microcornea, and the presence of glaucoma. The ocular phenotype resulted from a heterozygous T>C transition at nucleotide 338 in GJA1 (L113P) that was not detected in 120 chromosomes of unaffected individuals. The L113P mutation results in a nonconservative substitution in the cytoplasmic loop of Cx43 (GJA1) and is predicted to disrupt the high-order structure of Cx43.

    Conclusions: This report describes the ocular phenotype in a molecularly characterized ODDD syndrome family. The ocular features in this family highlight the key role Cx43 plays in eye development and in the development of glaucoma. L113P represents a pathogenic mutation in GJA1 (Cx43) and results in ODDD with marked intrafamilial variation in glaucoma type and severity.

    Eye (London, England) 2009;23;3;549-55

  • Cx43 has distinct mobility within plasma-membrane domains, indicative of progressive formation of gap-junction plaques.

    Simek J, Churko J, Shao Q and Laird DW

    Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada.

    Connexin 43 (Cx43) is a dynamic molecule, having a short half-life of only a few hours. In this study, we use fluorescent-protein-tagged Cx43 variants to examine Cx43 delivery to the cell surface, its residency status in various cell-surface membrane domains and its mobility characteristics. Rapid time-lapse imaging led to the identification of Cx43 being delivered to cell-surface domains that lacked a contacting cell, and also to its localization within membrane protrusions. Fluorescence recovery after photobleaching (FRAP) was used to investigate the mobility state of cell-surface-localized Cx43. Cx43 mobility within clustered cell-surface profiles of Cx43 could be categorized into those with generally a high degree of lateral mobility and those with generally a low degree of lateral mobility. Cx43 mobility was independent of cluster size, yet the C-terminal domain of Cx43 regulated the proportion of gap-junction-like clusters that acquired a low Cx43 mobility state. Collectively, these studies show that Cx43 establishes residency at all cell-surface membrane domains, and progressively acquires assembly states that probably reflect differences in either channel packing and/or its interactions with Cx43-binding proteins.

    Journal of cell science 2009;122;Pt 4;554-62

  • Melatonin synergizes with oxytocin to enhance contractility of human myometrial smooth muscle cells.

    Sharkey JT, Puttaramu R, Word RA and Olcese J

    Florida State University College of Medicine, Tallahassee, Florida 32306-4300, USA.

    Context: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night.

    Objective: The purpose of this study was to determine the effects of MEL on contractility and the contractile machinery in telomerase-immortalized human myometrial cells.

    Design: To ascertain the effect of MEL on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodomelatonin +/- oxytocin (OT). The effects of iodomelatonin on gap junctions were also investigated. Additionally, expression levels of the type 2 MEL receptor (MT2R) were assessed in myometrial biopsies from term pregnant women with or without labor.

    Results: MEL was found to synergistically enhance OT-induced contractility via the MT2R, which is coupled to a protein kinase C-dependent increase in phosphorylation of the myosin light chain protein. MT2R expression was markedly elevated in samples from pregnant women who had entered labor, as compared to matched nonlaboring pregnant women. MEL increased expression of the gap junction protein, connexin 43. In vitro dye spread assays showed that MEL-treated cells displayed substantially increased intercellular coupling. Increases in connexin 43 mRNA and cell to cell coupling were also found to be mediated via the MT2R in a protein kinase C-dependent manner.

    Conclusions: MEL synergizes with OT to promote myometrial cell contractions and to facilitate gap junction activity in vitro. Such a synergy in vivo would promote coordinated and forceful contractions of the late term pregnant uterus necessary for parturition.

    Funded by: PHS HHS: PPG 11149

    The Journal of clinical endocrinology and metabolism 2009;94;2;421-7

  • Perturbing plasma membrane hemichannels attenuates calcium signalling in cardiac cells and HeLa cells expressing connexins.

    Verma V, Hallett MB, Leybaert L, Martin PE and Evans WH

    Department of Medical Biochemistry and Immunology, Cardiff University Medical School, Heath Park, Cardiff, Wales, UK.

    Many cell signalling pathways are driven by changes in cytosolic calcium. We studied the effects of a range of inhibitors of connexin channels on calcium signalling in cardiac cells and HeLa cells expressing connexins. Gap 26 and 27, peptides that mimic short sequences in each of the extracellular loops of connexin 43, and anti-peptide antibodies generated to extracellular loop sequences of connexins, inhibited calcium oscillations in neonatal cardiac myocytes, as well as calcium transients induced by ATP in HL-1 cells originating from cardiac atrium and HeLa cells expressing connexin 43 or 26. Comparison of single with confluent cells showed that intracellular calcium responses were suppressed by interaction of connexin mimetic peptides and antibodies with hemichannels present on unapposed regions of the plasma membrane. To investigate how inhibition of hemichannels in the plasma membrane by the applied reagents was communicated to calcium store operation in the endoplasmic reticulum, we studied the effect of Gap 26 on calcium entry into cells and on intracellular IP3 release; both were inhibited by Gap 26. Calcium transients in both connexin 43- and connexin 26-expressing HeLa cells were inhibited by the peptides suggesting that the extended cytoplasmic carboxyl tail domain of larger connexins and their interactions with intracellular scaffolding/auxiliary proteins were unlikely to feature in transmitting peptide-induced perturbations at hemichannels in the plasma membrane to IP3 receptor channel central to calcium signalling. The results suggest that calcium levels in a microenvironment functionally connecting plasma membrane connexin hemichannels to downstream IP3-dependent calcium release channels in the endoplasmic reticulum were disrupted by the connexin mimetic peptide, although implication of other candidate hemichannels cannot be entirely discounted. Since calcium signalling is fundamental to the maintenance of cellular homeostasis, connexin hemichannels emerge as therapeutic targets open to manipulation by reagents interacting with external regions of these channels.

    Funded by: British Heart Foundation: PG/01/1298

    European journal of cell biology 2009;88;2;79-90

  • Splice variant IVS2-2A>G in the SLC26A5 (Prestin) gene in five Estonian families with hearing loss.

    Teek R, Oitmaa E, Kruustük K, Zordania R, Joost K, Raukas E, Tõnisson N, Gardner P, Schrijver I, Kull M and Ounap K

    Department of Oto-Rhino-Laryngology, University of Tartu, Tartu, Estonia.

    Objective: The aim of our study was to identify the IVS2-2A>G sequence change in the SLC26A5 (Prestin) gene in Estonian individuals with hearing loss and in their family members.

    Methods: In the years 2005-2007 we have screened 194 probands with early onset hearing loss and 68 family members with an arrayed primer extension (APEX) microarray, which covers 201 mutations in six nuclear genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5) and two mitochondrial genes encoding 12S rRNA and tRNA-Ser (UCN).

    Results: In four probands with early onset hearing loss and in five unaffected family members from five families we identified the IVS2-2A>G change in one allele of the SLC26A5 gene. We did not find any homozygosity for this splice variant. IVS2-2A>G was identified in 2.1% of probands. One of these probands, however, is also homozygous for the 35delG mutation in the GJB2 gene and a second patient has Down syndrome, which is also associated with hearing impairment. Therefore, in those two cases the etiology of the hearing loss is probably not associated with the IVS2-2A>G sequence change in the SLC26A5 gene.

    Conclusion: Our data support the hypothesis that heterozygosity for the mutation IVS2-2A>G in SLC26A5 gene may not, by itself, be sufficient to cause hearing loss.

    International journal of pediatric otorhinolaryngology 2009;73;1;103-7

  • TGF-beta1 mediates glucose-evoked up-regulation of connexin-43 cell-to-cell communication in HCD-cells.

    Hills CE, Bland R, Bennett J, Ronco PM and Squires PE

    Department of Infection, Immunity and Inflammation, MSB building, University of Leicester.

    In the current study we examined if the multifunctional cytokine TGF-beta1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD).

    Methods: RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-beta1 and Cx43 in HCD-cells. The effect of TGF-beta1 and high glucose (25 mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-beta1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry.

    Results: At 24 hrs, high glucose (25 mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-beta1 (2 ng/ml) at low glucose (5 mM). Both high glucose and TGF-beta1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-beta. Furthermore, high glucose-evoked changes were inhibited by a TGF-beta1-specific monoclonal antibody. Mannitol (25 mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-beta1 evoked changes in Cx43 expression were biphasic. An early (4-8 hr) transient decrease in expression was followed by an increase in protein expression (12-24 hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24 hrs coincided with a TGF-beta1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells.

    Conclusions: High glucose evoked a TGF-beta1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2009;24;3-4;177-86

  • Functional consequences of overexpressing the gap junction Cx43 in the cardiogenic potential of pluripotent human embryonic stem cells.

    Moore JC, Tsang SY, Rushing SN, Lin D, Tse HF, Chan CW and Li RA

    Department of Cell Biology & Human Anatomy, University of California, Davis Shriners Hospital, Sacramento, CA 95817, USA.

    Gap junctions, encoded by the connexin (Cx) multi-gene family, couple adjacent cells and underlie cell-cell communications. Previous mouse studies suggest that Cxs play an important role in development but their role in human cardiogenesis is undefined. Human embryonic stem cells (hESC) provide a unique model for studying human differentiation. Lentivirus-mediated stable overexpression of Cx43 in hESC (Cx43-hESC) did not affect colony morphology, karyotype and expression of pluripotency genes such as Oct4 but completely suppressed the formation of spontaneously beating, cardiomyocyte-containing clusters in embryoid bodies (EBs). Unlike control hEBs, the transcripts of several mesodermal markers (kallikrein, delta-globin, and CMP), ventricular myosin light chain and cardiac troponin I were absent or delayed. Transcriptomic and pathway analyses showed that 194 genes crucial for movement, growth, differentiation and maintenance were differentially expressed in Cx43-hESC. We conclude that Cx43 mediates the expression of an array of genes involved in human cardiogenesis, in addition to intercellular communication.

    Funded by: NHLBI NIH HHS: F32 HL078330, R01 HL-72857

    Biochemical and biophysical research communications 2008;377;1;46-51

  • Bystander effect in glioblastoma cells with a predominant cytoplasmic localization of connexin43.

    Cottin S, Ghani K and Caruso M

    Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Centre Hospitalier Universitaire de Québec, Québec, Canada.

    Herpes simplex virus thymidine kinase (TK) gene transfer followed by ganciclovir (GCV) administration is an approach investigated for glioblastoma treatment. The bystander effect (BE) enhances the cytotoxic effect of this strategy by allowing the diffusion of phosphorylated GCV from TK-expressing cells toward neighboring TK negative cells. This transfer of toxic metabolites is mainly mediated via gap junctions that are composed of connexins. Downregulation and/or cytoplasmic localization of connexins are common in tumors, and should be detrimental to the success of the TK/GCV strategy. In this study, we investigated the level of expression, the localization and the functionality of connexin43 (Cx43) in three glioblastoma cell lines. We showed that Cx43 was predominantly located in lysosomes and late endosomes, with only few gap junctions present at the cell surface. Surprisingly, the gap-junctional intercellular communication (GJIC) and the BE capacity were preserved, and in two of the cell lines analyzed, it was at least twice as high as compared to a control HeLa transfectant that expresses high levels of Cx43 at the cell membrane. Experiments performed in the presence of alpha-glycyrrhetinic acid or small interfering RNA confirmed that Cx43 was responsible for the GJIC and the BE. Our results indicate for the first time that the very limited numbers of gap junctions present in glioblastoma cells are highly functional. We thus conclude that the TK/GCV strategy is still a valuable therapeutic option to be developed for the treatment of glioblastoma patients.

    Cancer gene therapy 2008;15;12;823-31

  • Characterization of the molecular phenotype of two arrhythmogenic right ventricular cardiomyopathy (ARVC)-related plakophilin-2 (PKP2) mutations.

    Joshi-Mukherjee R, Coombs W, Musa H, Oxford E, Taffet S and Delmar M

    Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York, USA.

    Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to mutations in desmosomal proteins, including plakophilin-2 (PKP2). Little is known about the changes in cellular function and structure that follow expression of ARVC-relevant PKP2 mutations.

    Objective: The purpose of this study was to investigate the function and distribution of an ARVC-relevant PKP2 mutant where arginine at position 79 was replaced by a stop codon (R79x).

    Methods: Results were compared with those obtained with mutation 179fs (frameshift at position 179). Mutant constructs were introduced by adenoviral infection into neonatal rat ventricular myocytes in culture.

    Results: Both mutant proteins failed to preferentially localize to sites of cell-cell apposition. Their expression did not disrupt localization of endogenous PKP2, connexin-43 (Cx43), or desmoplakin (DP). However, we observed reduced abundance of Cx43 after R79x expression. Early truncation of PKP2 at position 79 also prevented its physical interaction with both DP and Cx43. Finally, R79x expression correlated with loss of expression of HSP90, a protein relevant to cardiomyocyte apoptosis.

    Conclusion: These results provide the first observations of the cellular/molecular phenotype consequent to these PKP2 mutations and give insight into the possible cellular substrates that lead to ARVC.

    Funded by: NHLBI NIH HHS: HL087226, HL39707, P01 HL039707, P01 HL039707-180011, P01 HL087226, P01 HL087226-020001, R01 HL106632; NIGMS NIH HHS: GM57691, R01 GM057691, R01 GM057691-11

    Heart rhythm : the official journal of the Heart Rhythm Society 2008;5;12;1715-23

  • A pilot study of Connexin 43 (Cx43) in human bladder tissue in patients with idiopathic detrusor overactivity.

    Kuhn A, Stadlmayr W, Monga A, Cameron I and Anthony F

    Frauenklinik, Inselspital Bern und Universität Bern, Switzerland. annette.kuhn@insel.ch

    Objective: Aim of the study was to compare Connexin 43 (Cx43) in human bladder tissue of urodynamically proven idiopathic detrusor overactivity to those of uro 103f dynamically stable bladders.

    We compared bladder biopsies of patients with detrusor overactivity and those with stable bladder analysing Cx43 message by RNA extraction and PCR amplification. All patients had multichannel urodynamics prior to the biopsies.

    Results: We investigated the bladder biopsies of 15 female patients with and 15 patients without detrusor overactivity. Cx43 could be detected in nine patients of the detrusor overactivity group and in eight patients of the control group which was not statistically significant. 42 cycles of PCR were necessary to demonstrate Cx43 presence in the positive specimen. The presence of Cx43 was not consistent in the samples from the bladder dome and the side walls meaning there were Cx43 positive results in the dome and negative ones in the side walls of the same patient and vice versa.

    Conclusion: In conclusion, Cx43 is present in human bladder tissue both of overactive bladders and those of controls. However, it is expressed in very small amounts and is not always detectable. The role of Cx43 for the origin of detrusor overactivity remains unclear.

    European journal of obstetrics, gynecology, and reproductive biology 2008;141;1;83-6

  • Novel mutations in GJA1 cause oculodentodigital syndrome.

    Fenwick A, Richardson RJ, Butterworth J, Barron MJ and Dixon MJ

    Faculty of Life Sciences and Dental School, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.

    Oculodentodigital syndrome (ODD) is a rare, usually autosomal-dominant disorder that is characterized by developmental abnormalities of the face, eyes, teeth, and limbs. The most common clinical findings include a long, narrow nose, short palpebral fissures, type III syndactyly, and dental abnormalities including generalized microdontia and enamel hypoplasia. Recently, it has been shown that mutations in the gene GJA1, which encodes the gap junction protein connexin 43, underlie oculodentodigital syndrome. Gap junction communication between adjacent cells is known to be vital during embryogenesis and subsequently for normal tissue homeostasis. Here, we report 8 missense mutations in the coding region of GJA1, 6 of which have not been described previously, in ten unrelated families diagnosed with ODD. In addition, immunofluorescence analyses of a developmental series of mouse embryos and adult tissue demonstrates a strong correlation between the sites of connexin 43 expression and the clinical phenotype displayed by individuals affected by ODD.

    Funded by: Wellcome Trust: 075945

    Journal of dental research 2008;87;11;1021-6

  • Clathrin and Cx43 gap junction plaque endoexocytosis.

    Nickel BM, DeFranco BH, Gay VL and Murray SA

    Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, 324 South Biomedical Science Tower, Pittsburgh, PA 15261, USA.

    In earlier transmission electron microscopic studies, we have described pentilaminar gap junctional membrane invaginations and annular gap junction vesicles coated with short, electron-dense bristles. The similarity between these electron-dense bristles and the material surrounding clathrin-coated pits led us to suggest that the dense bristles associated with gap junction structures might be clathrin. To confirm that clathrin is indeed associated with annular gap junction vesicles and gap junction plaques, quantum dot immuno-electron microscopic techniques were used. We report here that clathrin associates with both connexin 43 (Cx43) gap junction plaques and pentilaminar gap junction vesicles. An important finding was the preferential localization of clathrin to the cytoplasmic surface of the annular or of the gap junction plaque membrane of one of the two contacting cells. This is consistent with the possibility that the direction of gap junction plaque internalization into one of two contacting cells is regulated by clathrin.

    Biochemical and biophysical research communications 2008;374;4;679-82

  • Domain-swapped dimerization of ZO-1 PDZ2 generates specific and regulatory connexin43-binding sites.

    Chen J, Pan L, Wei Z, Zhao Y and Zhang M

    Department of Biochemistry, Molecular Neuroscience Center, Hong Kong University of Science and Technology, Kowloon, Hong Kong.

    PDZ domain scaffold proteins are capable of assembling macromolecular protein complexes in diverse cellular processes through PDZ-mediated binding to a short peptide fragment at the carboxyl tail of target proteins. How each PDZ domain specifically recognizes its target protein(s) remains a major conceptual question, as at least a few out of the several hundred PDZ domains in each eukaryotic genome share overlapping binding properties with any given target protein. Here, we show that the domain-swapped dimerization of zonula occludens-1 PDZ2 generates a distinct interface that functions together with the well-separated canonical carboxyl tail-binding pocket in each PDZ unit in binding to connexin43 (Cx43). We further demonstrate that the charge-charge interaction network formed by residues in the PDZ dimer interface and upstream residues of the Cx43 peptide not only provides the unprecedented interaction specificity for the complex but may also function as a phosphorylation-mediated regulatory switch for the dynamics of the Cx43 gap junctions. Finally, we provide evidence that such domain-swapped dimer assembly also occurs in other PDZ domain scaffold proteins. Therefore, our findings present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.

    The EMBO journal 2008;27;15;2113-23

  • Activation of endothelial cells to pathological status by down-regulation of connexin43.

    Wang HH, Kung CI, Tseng YY, Lin YC, Chen CH, Tsai CH and Yeh HI

    Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan.

    Aims: We investigated the effects of connexin43 (Cx43) down-regulation on endothelial function.

    We used two different sequences of Cx43-specific small interference RNA (siRNA) to reduce de novo synthesis of Cx43 in human aortic endothelial cells and then examined the expression profiles, proliferation activity and viability, and angiogenic potential. The involvement of mitogen-activated protein kinase signalling pathways was analysed. In parallel, the effect of inhibition of gap-junctional communication by connexin-mimetic peptides was evaluated. During the down-regulation of Cx43 by the siRNA, the cells exhibited impaired gap-junctional communication, proliferation, viability, and angiogenic potential. In addition, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor were up-regulated. Furthermore, c-jun N-terminal kinase (JNK) and its downstream target c-jun were activated, while caspase-3, p38, and extracellular signal-regulated kinase remained unchanged. Inhibition of JNK by SP600125 blocked the siRNA-induced increased expression of PAI-1 and partially recovered the impaired angiogenic potential. Short-term inhibition of Cx43 channels by connexin-mimetic peptides did not activate JNK.

    Conclusion: Down-regulation of Cx43 inhibits gap-junctional communication and activates endothelial cells to pathological status, as characterized by up-regulation of coagulatory molecules and impairment of proliferation, viability, and angiogenesis. The processes are associated with activation of JNK signalling pathways and rectified by inhibition of the activation. These results suggest that inadequate expression of Cx43 per se impairs endothelial function by the activation of stress-activated protein kinase.

    Cardiovascular research 2008;79;3;509-18

  • Expanding the neurologic phenotype of oculodentodigital dysplasia in a 4-generation Hispanic family.

    Amador C, Mathews AM, Del Carmen Montoya M, Laughridge ME, Everman DB and Holden KR

    Department of Neurology, Hospital Escuela, Tegucigalpa, Honduras.

    We report a 4-generation Hispanic family with oculodentodigital dysplasia whose members were found to have typical phenotypic characteristics of this disorder, as well as a variable expression of neurologic manifestations in multiple generations ranging from a mild spastic gait to moderate to severe spastic tetraparesis/quadriplegia with epilepsy and an abnormal brain and spinal cord magnetic resonance imaging result. Gene testing documented a previously reported missense mutation in GJA1 (connexin 43) exon 2 (c.389T>C;p.I130T). Our evaluation not only expands the phenotypes associated with GJA1 gene mutations but also demonstrates that a great degree of variability in neurological defects can exist within a single family without evidence of genetic anticipation. A gen 1009 otype-phenotype correlation between the p.I130T mutation and neurologic dysfunction appears more likely with the addition of this report's neurologic and GJA1 gene mutation findings. These findings expand the neurologic phenotype and prognosis and underscore the importance of counseling families with oculodentodigital dysplasia about the possibility of neurologic involvement.

    Journal of child neurology 2008;23;8;901-5

  • TNF-alpha represses connexin43 expression in HaCat keratinocytes via activation of JNK signaling.

    Tacheau C, Laboureau J, Mauviel A and Verrecchia F

    INSERM U697, Paris, France.

    We used both pharmacological and gene knockout approaches to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NFkappaB pathways downstream of TNF-alpha in the context of connexin43 (Cx43) gene expression. We demonstrate that TNF-alpha reduces the expression of Cx43 in HaCat cell lines at the protein and mRNA levels, and transcriptionally. We also demonstrate that TNF-alpha decreases gap junctional intercellular communication (GJIC) between HaCat cells. Using pharmacological inhibitors of the NFkappaB signaling pathway, we determined that the NFkappaB signaling cascade is not implicated in TNF-alpha effect on Cx43 expression and in the subsequent decrease of GJIC. Conversely, in NFkappaB essential modulator (NEMO(-)) fibroblasts, lack of NFkappaB activation did not influence both the effect of TNF-alpha on Cx43 expression and on GJIC. In contrast, pharmacologic inhibition of JNK abolishes TNF-alpha-driven repression of Cx43 gene expression and GJIC between HaCat cells. Using JNK(1) (-/-)-JNK(2) (-/-) (JNK(-/-)) fibroblasts, we demonstrate that similar regulatory mechanisms take place in fibroblasts. Together, these results identify JNK and not NFkappaB, as a critical mediator of TNF-alpha repressory effect on Cx43 gene expression.

    Journal of cellular physiology 2008;216;2;438-44

  • Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium.

    Elzarrad MK, Haroon A, Willecke K, Dobrowolski R, Gillespie MN and Al-Mehdi AB

    Department of Pharmacology and Center for Lung Biology, University of South Alabama, North University Boulevard, Mobile, AL 36688, USA. mke303@jaguar1.usouthal.edu

    Background: The modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium.

    Methods: Tumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 1f40 or dominant-negatively mutated connexin-43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function.

    Results: Adhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin-43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci.

    Conclusion: Connexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis.

    Funded by: NCCIH NIH HHS: P50 AT000428, P50 AT00428-01

    BMC medicine 2008;6;20

  • Expression of astrocytic markers aquaporin 4 and connexin 43 is altered in brains of subjects with autism.

    Fatemi SH, Folsom TD, Reutiman TJ and Lee S

    Department of Psychiatry, University of Minnesota, Minneapolis, Minnesota 55455, USA. fatem002@umn.edu

    Neuroanatomical studies have revealed extensive structural brain abnormalities in subjects with autism. Recently, studies have provided evidence of neuroglial responses and neuroinflammation in autism. The current study investigated whether two astrocytic markers, aquaporin 4 and connexin 43, are altered in brains from subjects with autism. Postmortem brain tissues from Brodmann's Area 40 (BA40, parietal cortex), Brodmann's Area 9 (BA9, superior frontal cortex), and cerebella of subjects with autism and matched controls were subject to SDS-PAGE and western blotting. Connexin 43 expression was increased significantly in BA9. Aquaporin 4 expression was decreased significantly in cerebellum. These data suggest that changes are apparent in markers for abnormal glial-neuronal communication (connexin 43 and aquaporin 4) in brains of subjects with autism.

    Funded by: NICHD NIH HHS: 1R01HD052074-01A2, R01 HD052074, R01 HD052074-02

    Synapse (New York, N.Y.) 2008;62;7;501-7

  • ODDD-linked Cx43 mutants reduce endogenous Cx43 expression and function in osteoblasts and inhibit late stage differentiation.

    McLachlan E, Plante I, Shao Q, Tong D, Kidder GM, Bernier SM and Laird DW

    Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada.

    Introduction: Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD.

    We expressed human and mouse ODDD-linked Cx43 mutants in MC3T3-E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin.

    Results: We showed that ODDD-linked Cx43 mutants are loss-of-function and dominant-negative to co-expressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation.

    Conclusions: Expression of human and mouse ODDD-linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43-based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2008;23;6;928-38

  • Report of a black South African child with oculodentodigital dysplasia and a novel GJA1 gene mutation.

    Feller L, Wood NH, Sluiter MD, Noffke C, Raubenheimer EJ, Lemmer J and van Rensburg EJ

    Department of Periodontology and Oral Medicine, School of Dentistry, University of Limpopo (Medunsa Campus), Pretoria, South Africa. lfeller@medunsa.ac.za

    American journal of medical genetics. Part A 2008;146A;10;1350-3

  • A genome-wide association study identifies protein quantitative trait loci (pQTLs).

    Melzer D, Perry JR, Hernandez D, Corsi AM, Stevens K, Rafferty I, Lauretani F, Murray A, Gibbs JR, Paolisso G, Rafiq S, Simon-Sanchez J, Lango H, Scholz S, Weedon MN, Arepalli S, Rice N, Washecka N, Hurst A, Britton A, Henley W, van de Leemput J, Li R, Newman AB, Tranah G, Harris T, Panicker V, Dayan C, Bennett A, McCarthy MI, Ruokonen A, Jarvelin MR, Guralnik J, Bandinelli S, Frayling TM, Singleton A and Ferrucci L

    Department of Epidemiology and Public Health, Institute of Biomedical and Clinical Sciences, Peninsula College of Medicine and Dentistry, University of Exeter, Devon, United Kingdom.

    There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts - cis effects, and elsewhere in the genome - trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8x10(-57)), CCL4L1 (p = 3.9x10(-21)), IL18 (p = 6.8x10(-13)), LPA (p = 4.4x10(-10)), GGT1 (p = 1.5x10(-7)), SHBG (p = 3.1x10(-7)), CRP (p = 6.4x10(-6)) and IL1RN (p = 7.3x10(-6)) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8x10(-40)), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These include the presence of strong genetic effects in cis locations. The identification of protein quantitative trait loci (pQTLs) may be a powerful complementary method of improving our understanding of disease pathways.

    Funded by: Intramural NIH HHS; NIA NIH HHS: N01-AG-6-2101, N01-AG-6-2103, N01-AG-6-2106, R01 AG024233, R01 AG24233-01

    PLoS genetics 2008;4;5;e1000072

  • Involvement of connexin 43 in angiotensin II-induced migration and proliferation of saphenous vein smooth muscle cells via the MAPK-AP-1 signaling pathway.

    Jia G, Cheng G, Gangahar DM and Agrawal DK

    Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA.

    Proliferation and migration of vascular smooth muscle cells (VSMCs) lead to intimal thickening and influence the long-term patency of venous graft post coronary arterial bypass graft. There is increasing evidence that connexins are involved in the development of intimal hyperplasia and restenosis. We assessed connexin 43 (Cx43) expression and its role in angiotensin II-induced proliferation and migration of smooth muscle cells and the signal pathways involved in human saphenous vein bypass conduits. Angiotensin II significantly increased gap junctional intercellular communication and induced the expression of Cx43 in human saphenous vein SMCs in a dose- and time-dependent manner through angiotensin II type 1 receptor. The effect of angiotensin II was blocked by siRNA of ERK 1/2, p38 MAPK and JNK, respectively. Overexpression of Cx43 markedly increased the proliferation of saphenous vein SMCs. However, siRNA for Cx43 inhibited angiotensin II-induced proliferation, cyclin E expression and migration of human saphenous vein SMCs. In dual-luciferase reporter assay, angiotensin II markedly activated AP-1 transcription factor, which was significantly attenuated by a dominant-negative AP-1 (A-Fos) with subsequent inhibition of angiotensin II-induced transcriptional expression of Cx43. These data demonstrate the role of Cx43 in the proliferation and migration of human saphenous vein SMCs and angiotensin II-induced Cx43 expression via mitogen-activated protein kinases (MAPK)-AP-1 signaling pathway.

    Funded by: NHLBI NIH HHS: R01 HL070885, R01 HL073349, R01 HL090580, R01 HL090580-01A1, R01HL070885, R01HL073349

    Journal of molecular and cellular cardiology 2008;44;5;882-90

  • Extracellular-loop peptide antibodies reveal a predominant hemichannel organization of connexins in polarized intestinal cells.

    Clair C, Combettes L, Pierre F, Sansonetti P and Tran Van Nhieu G

    Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris Cedex 15, France.

    Shigella, the causative agent of bacillary dysentery, invades colonic epithelial cells to elicit an intense inflammatory reaction leading to destruction of the mucosa. ATP-dependent paracrine signalling induced by connexin (Cx) hemichannel opening was previously shown to favor Shigella flexneri invasion and dissemination in transfectants of HeLa cells [G. Tran Van Nhieu, C. Clair, R. Bruzzone, M. Mesnil, P. Sansonetti and L. Combettes. (2003). Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat. Cell Biol. 5, 720-726.]. However, although Cxs have been described in polarized epithelial cells, little is known about their structural organization and the role of hemichannels during S. flexneri invasion. We show here that polarized Caco-2/TC7 cells express significant amounts of Cx26, Cx32 and Cx43, but that unexpectedly, cell-cell coupling assessed by dye-transfer experiments is inefficient. Consistent with a predominant Cx organization in hemichannels, dye loading induced by low calcium was readily observed, with preferential loading at the basolateral side. Antibodies (Abs) against connexin extracellular loop peptides (CELAbs) demonstrated the importance of hemichannel signalling since they inhibited dye uptake at low calcium and at physiological calcium concentrations during S. flexneri invasion. Importantly, CELAbs allowed the visualization of hemichannels at the surface of epithelial cells, as structures distinct from gap intercellular junctions.

    Funded by: Howard 1a47 Hughes Medical Institute

    Experimental cell research 2008;314;6;1250-65

  • Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1.

    Bruce AF, Rothery S, Dupont E and Severs NJ

    National Heart and Lung Institute, Imperial College London, Guy Scadding Building, London, UK.

    Aims: Remodelling of gap junctions, involving reduction of total gap junction quantity and down-regulation of connexin43 (Cx43), contributes to the arrhythmic substrate in congestive heart failure. However, little is known of the underlying mechanisms. Recent studies from in vitro systems suggest that the connexin-interacting protein zonula occludens-1 (ZO-1) is a potential mediator of gap junction remodelling. We therefore examined the hypothesis that ZO-1 contributes to reduced expression of Cx43 gap junctions in congestive heart failure.

    Left ventricular myocardium from healthy control human hearts (n = 5) was compared with that of explanted hearts from transplant patients with end-stage congestive heart failure due to idiopathic dilated cardiomyopathy (DCM; n = 5) or ischaemic cardiomyopathy (ICM; n = 5). Immunoconfocal and immunoelectron microscopy showed that ZO-1 is specifically localized to the intercalated disc of cardiomyocytes in control and failing ventricles. ZO-1 protein levels were significantly increased in both DCM and ICM (P = 0.0025), showing a significant, negative correlation to Cx43 levels (P = 0.0029). There was, however, no significant alteration of ZO-1 mRNA (P = 0.537). Double immunolabelling demonstrated that a proportion of ZO-1 label is co-localized with Cx43, and that co-localization of Cx43 with ZO-1 is significantly increased in the failing ventricle (P = 0.003). Interaction between the two proteins was confirmed by co-immunoprecipitation. The proportion of Cx43 that co-immunoprecipitates with ZO-1 was significantly increased in the failing heart.

    Conclusion: Our findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.

    Funded by: British Heart Foundation: PG/05/003

    Cardiovascular research 2008;77;4;757-65

  • Caveolin-1 and -2 interact with connexin43 and regulate gap junctional intercellular communication in keratinocytes.

    Langlois S, Cowan KN, Shao Q, Cowan BJ and Laird DW

    Department of Anatomy and Cell Biology, University of Western Ontario, London, ON N6A 5C1, Canada.

    Connexin43 (Cx43) has been reported to interact with caveolin (Cav)-1, but the role of this association and whether other members of the caveolin family bind Cx43 had yet to be established. In this study, we show that Cx43 coimmunoprecipitates and colocalizes with Cav-1 and Cav-2 in rat epidermal keratinocytes. The colocalization of Cx43 with Cav-1 was confirmed in keratinocytes from human epidermis in vivo. Our mutation and Far Western analyses revealed that the C-terminal tail of Cx43 is required for its association with Cavs and that the Cx43/Cav-1 interaction is direct. Our results indicate that newly synthesized Cx43 interacts with Cavs in the Golgi apparatus and that the Cx43/Cavs complex also exists at the plasma membrane in lipid rafts. Using overexpression and small interfering RNA approaches, we demonstrated that caveolins regulate gap junctional intercellular communication (GJIC) and that the presence of Cx43 in lipid raft domains may contribute to the mechanism modulating GJIC. Our results suggest that the Cx43/Cavs association occurs during exocytic transport, and they clearly indicate that caveolin regulates GJIC.

    Molecular biology of the cell 2008;19;3;912-28

  • A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43.

    Li X, Su V, Kurata WE, Jin C and Lau AF

    Natural Products and Cancer Biology Program, Cancer Research Center of Hawaii, Honolulu, Hawaii 96813, USA.

    The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys 264 and Asn 302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.

    Funded by: NCI NIH HHS: CA052098, R01 CA052098, R01 CA052098-18

    The Journal of biological chemistry 2008;283;9;5748-59

  • Closing the gap on autosomal dominant connexin-26 and connexin-43 mutants linked to human disease.

    Laird DW

    Departments of Anatomy & Cell Biology and Physiology & Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada. dale.laird@schulich.uwo.ca

    Cells within the vast majority of human tissues communicate directly through clustered arrays of intercellular channels called gap junctions. Gene ablation studies in mouse models have revealed that these intercellular channels are necessary for a variety of organ functions and that some of these genes are essential for survival. Molecular genetics has uncovered that germ line mutations in nearly half of the genes that encode the 21-member connexin family of gap junction proteins are linked to one or more human diseases. Frequently, these mutations are autosomal recessive, whereas in other cases, autosomal dominant mutations manifest as disease. Given the broad and overlapping distribution of connexins in a wide arrangement of tissues, it is hard to predict where connexin-linked diseases will clinically manifest. For instance, the most prevalent connexin in the human body is connexin-43 (Cx43), yet autosomal dominant mutations in the GJA1 gene, which encodes Cx43, exhibit modest developmental disorders resulting in a disease termed oculodentodigital dysplasia. Autosomal recessive mutations in the gene encoding Cx26 result in moderate to severe sensorineural hearing loss, whereas autosomal dominant mutations produce hearing loss and a wide range of skin diseases, including palmoplantar keratoderma. Here, we will focus on autosomal dominant mutations of the genes encoding Cx26 and Cx43 in relation to models that link genotypes to phenotypic outcomes with particular reference to how these approaches provide insight into human disease.

    The Journal of biological chemistry 2008;283;6;2997-3001

  • Connexin 43 contributes to differentiation of retinal pigment epithelial cells via cyclic AMP signaling.

    Kojima A, Nakahama K, Ohno-Matsui K, Shimada N, Mori K, Iseki S, Sato T, Mochizuki M and Morita I

    Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.

    Retinal pigment epithelium (RPE) cells play important roles in the visual system that supports neurosensory retina homeostasis. Connexin (Cx) 43-mediated gap-junctional intercellular communication (GJIC) participates in the regulation of retinal organogenesis, but much of the function of Cx43 on the differentiation of RPE cells is unclear. Here, we report the involvement of Cx43 in RPE differentiation. Knockdown of Cx43 in RPE cells dramatically inhibited the differentiation, whereas Cx43-overexpression successfully induced RPE cell differentiation under de-differentiation conditions. From the experiments using GJIC inhibitors and C-terminus-truncated mutant of Cx43, it was clearly demonstrated that the regulation of RPE cell differentiation by Cx43 did not result from Cx43-mediated GJIC. The RPE cell differentiation induced by Cx43-overexpression was abolished by a cAMP antagonist. In contrast, the treatment with forskolin and phosphodiesterase inhibitor rolipram induced RPE cell differentiation under de-differentiation conditions. These findings indicate that Cx43 contributes to RPE differentiation via cAMP signaling.

    Biochemical and biophysical research communications 2008;366;2;532-8

  • Connexin 43 expression delineates two discrete pathways in the human atrioventricular junction.

    Hucker WJ, McCain ML, Laughner JI, Iaizzo PA and Efimov IR

    Washington University, Saint Louis, Missouri 63130, USA.

    Gap junction expression has been studied in the atrioventricular junction (AVJ) of many species, however, their distribution in the human AVJ is unknown. The AVJ expression of the gap junction protein connexin 43 (Cx43) is species dependent; therefore we investigated its distribution in the human AVJ. Using Masson trichrome histology, we reconstructed the AVJ of three normal human hearts and one with dilated cardiomyopathy in three dimensions. Cx43 was immunolabeled with vimentin and alpha-actinin to determine the cellular origin of Cx43 and was quantified in the following structures: interatrial septum (IAS), His bundle, compact node (CN), lower nodal bundle (LNB), leftward and rightward nodal extensions (LE and RE), and inferior, endocardial, and left-sided transitional cells. Histology revealed two nodal extensions in three of four hearts. Cx43 was found in the myocytes, but not fibroblasts, of the AVJ. LE and CN Cx43 was lower than the IAS (P < 0.05) and the RE, LNB, and His all expressed Cx43 similarly, with approximately half of IAS expression (RE: 44 +/- 36%; LNB: 50 +/- 26%; His: 48 +/- 12%, P = NS compared with IAS). Cx43 levels in transitional cells were similar to the IAS (P = not significant). Cx43 was found in myocytes of the human AVJ, and its expression pattern delineates two separate continuous structures: one consists of the LE and CN with little Cx43, and the other consists of the His, LNB, and RE expressing approximately half the Cx43 of the IAS. The differential Cx43 expression may provide each structure with unique conduction properties, contributing to arrhythmias arising from the AVJ.

    Funded by: NHLBI NIH HHS: R01 HL085369, R01 HL085369-01A2, R01 HL114395

    Anatomical record (Hoboken, N.J. : 2007) 2008;291;2;204-15

  • Ultraviolet A induced modulation of gap junctional intercellular communication by P38 MAPK activation in human keratinocytes.

    Bellei B, Mastrofrancesco A, Briganti S, Aspite N, Ale-Agha N, Sies H and Picardo M

    San Gallicano Dermatological Institute IRCCS, Rome, Italy. citolab@ifo.it

    Aberrant gap junctional intercellular communication (GJIC) has been implicated in tumor development and progression. UltravioletA (UVA)-induced oxidative stress has been associated with skin carcinogenesis. We report a potential link between GJIC and the cellular stress response induced by UVA in normal human keratinocytes (NHK). In this study, UVA irradiation (10 J/cm(2)) compromised GJIC integrity in absence of cytotoxic effects as demonstrated by the absence of cell death and by the reversibility of GJIC down-regulation. Inhibition of communication by UVA was associated with hyperphosphorylation and decreased expression of connexin43 (Cx43), the most abundant gap junction protein expressed by keratinocytes. Cx43 hyperphosphorylation induced by UVA is, at least in part, mediated through mitogen-activated protein kinase (MAPK) activation as Ser279 and Ser282 sites, two downstream direct targets of p38 MAPK were found to be phosphorylated after UVA treatment. However, inhibition of p38 MAPK activity did not significantly protect from cell-cell communication inhibition because 267 of a strong cellular cytotoxicity observed with SB202190 and SB203580, two selective inhibitors of p38 MAPK, in combination with UVA that compromises the outcome of dye transfer assay. By contrast, in Hacat cell line, inhibition of p38 activity reduced both phosphorylation and degradation of Cx43, demonstrating that these events are correlated.

    Experimental dermatology 2008;17;2;115-24

  • Endothelin-1 i 1f40 nhibits human osteoblastic cell differentiation: influence of connexin-43 expression level.

    Niger C, Geneau G, Fiorini C, Defamie N, Pointis G, Mesnil M and Cronier L

    Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA.

    Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during developmental and differentiation processes. In bone, the involvement of the gap junctional protein, connexin-43 (Cx43), and of GJIC in osteoblastic differentiation and mineralization of the extracellular matrix has been previously demonstrated. Former studies have shown that endothelin-1 (ET-1) was also implicated in the control of osteoblastic proliferation and differentiation. However, depending on the cellular models, ET-1 has been shown to decrease or increase osteoblastic differentiation markers. As no data were available on the ET-1 effect on GJIC and Cx43 expression in osteoblastic cells, we analyzed here the possible crosstalk between Cx43 and ET-1 in a human cell line (hFOB 1.19) which displays different Cx43 expression levels and phenotypes when cultured at 33.5 or 39 degrees C. The presence of ET-1 (10(-8) M) for 2-12 days of culture did not significantly alter the proliferation rate of hFOB cells whatever their phenotype. In contrast, ET-1 induced a differential inhibitory effect on the biochemical differentiation markers (alkaline phosphatase activity and osteocalcin expression) with a significant reduction in the differentiated phenotype at 39 degrees C, whereas no effects were measured at 33.5 degrees C. The inhibitory effect was linked to a decrease of GJIC and of Cx43 both at transcriptional and protein levels. Altogether, our results suggest that Cx43 expression level could influence the action of ET-1 on human osteoblastic cell differentiation. Our data also indicate that the gap junctional protein could play a pivotal role in the response of osteoblasts to mitogenic factors implicated in bone pathologies.

    Journal of cellular biochemistry 2008;103;1;110-22

  • Alterations in Cx43 and OB-cadherin affect breast cancer cell metastatic potential.

    Li Z, Zhou Z and Donahue HJ

    Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.

    Emerging evidence suggests that gap junctional intercellular communication (GJIC) and expression of connexins (Cx) contribute to the metastatic potential of breast cancer cells. To more directly address this, an aggressive bone metastasis breast cancer cell line, MDA-MET (MET), was stably transfected with human Cx43 cDNA (MET/Cx43(+)). Focusing on clone 28 of MET/Cx43(+), we demonstrated that GJIC, Cx43 protein and Cx43 mRNA were significantly increased in MET/Cx43(+) cells relative to MET, the plasmid control for the Cx43 transfectants (MET/HY) and a metastatic breast cancer cell that is less metastatic to bone than MET, MDA-MB-231. Cx26 mRNA was also increased in MET/Cx43(+ )clone 28 cells while mRNA for Cx32, Cx37, Cx40 and Cx45 were not detected in any of the breast cancer cell lines examined. MET/Cx43(+ )clone 28 invasiveness was decreased by 33% relative to MET/HY, while their ability to migrate was unchanged. The ability of MET/Cx43(+ )clone 28 cells to adhere to hFOB and HUV-EC-C cells was decreased approximately 30% and 70%, respectively, relative to MET and MET/HY. E-cadherin and N-cadherin proteins were not detected in MET, MDA-MB-231, MET/Cx43(+ )clone 28 and MET/HY cells. However, OB-cadherin protein levels were decreased approximately 43% in MET/Cx43(+ )clone 28 relative to MET/HY cells. These findings suggest that GJIC and Cx43 expression contribute to breast cancer cell adhesion and migration, possibly through a mechanism involving OB-cadherin, and these changes in turn regulate the metastatic potential of breast cancer cells, especially to bone.

    Funded by: NCI NIH HHS: R01-CA 90991

    Clinical & experimental metastasis 2008;25;3;265-72

  • Connexin 43 recruits E-cadherin expression and inhibits the malignant behaviour of lung cancer cells.

    Xu HT, Li QC, Zhang YX, Zhao Y, Liu Y, Yang ZQ and Wang EH

    Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang 110001, China.

    The interaction of connexin 43 and E-cadherin may play an important role in carcinogenesis and malignant behaviour of tumours. In this study, we examined the relationship between connexin 43 and E-cadherin in human non-small cell lung cancers (NSCLC). Expression levels of connexin 43 and E-cadherin were examined in 107 NSCLC specimens by immunohistochemistry. The connexin 43 gene was transfected into lung cancer LH7 cells. The protein localizations and levels of connexin 43 and E-cadherin were detected using immunofluorescence staining and western blot. Cell cycle and proliferation of lung cancer cells were examined using flow cytometry and MTT. We found that reduced expression of both connexin 43 and E-cadherin significantly correlated to poor differentiation, advanced TNM stage, and lymph note metastasis of NSCLCs. Connexin 43 and E-cadherin expression significantly correlated with each other. Over-expression of connexin 43 significantly induced E-cadherin expression. Moreover, connexin 43-transfected LH7 cells showed significantly decreased cell proliferation. The percentage of cells in G1 phase increased, while the number of cells in S and G2 phases significantly decreased. We concluded that concurrent reduction of connexin 43 and E-cadherin may contribute to the development of lung cancer. Connexin 43 may induce E-cadherin expression and inhibit cell proliferation and progression of lung cancer.

    Folia histochemica et cytobi 1f40 ologica 2008;46;3;315-21

  • Expressing connexin 43 in breast cancer cells reduces their metastasis to lungs.

    Li Z, Zhou Z, Welch DR and Donahue HJ

    Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.

    Recently the concept that gap junctions play a role in cancer cell metastasis has emerged. However, the mechanism by which this might occur is unknown. To examine this issue a metastatic breast cancer cell line, MDA-MB-435, was stably transfected with human Cx43 cDNA. Four clones of 435 transfectants (435/Cx43(+) c1, c6, c8, c14) and two clones of plasmid control (435/hy) were isolated and examined in this study. We found that expressing Cx43 in MDA-MB-435 cells decreased their expression of Cx32 but did not affect gap junctional intercellular communication, migration or invasion through Matrigel((R)). However, forced expression of Cx43 decreased the growth of MDA-MB-435 cells, decreased expression of N-cadherin, which is frequently associated with an aggressive phenotype, and increased MDA-MB-435 sensitivity to apoptosis. More importantly, there were fewer lung metastases in mice injected with 435/Cx43(+) cells relative to mice injected with 435/hy. These results suggest that expressing Cx43 in breast cancer cells decreases their metastatic potential through a mechanism independent of gap junctional communication but, rather, related to N-cadherin expression and apoptosis.

    Funded by: NCI NIH HHS: CA87228, CA90991, R01 CA087728, R01 CA087728-04, R01 CA087728-05A1, R01 CA087728-06, R01 CA087728-07, R01 CA087728-08, R01 CA090991; NIA NIH HHS: AG13087, R01 AG013087

    Clinical & experimental metastasis 2008;25;8;893-901

  • Genetic analysis of presbycusis by arrayed primer extension.

    Rodriguez-Paris J, Ballay C, Inserra M, Stidham K, Colen T, Roberson J, Gardner P and Schrijver I

    Department of Pathology, L235, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.

    Using the Hereditary Hearing Loss arrayed primer extension (APEX) array, which contains 198 mutations across 8 hearing loss-associated genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5, 12S-rRNA, and tRNA Ser), we compared the frequency of sequence variants in 94 individuals with early presbycusis to 50 unaffected controls and aimed to identify possible genetic contributors. This cross-sectional study was performed at Stanford University with presbycusis samples from the California Ear Institute. The patients were between ages 20 and 65 yr, with adult-onset sensorineural hearing loss of unknown etiology, and carried a clinical diagnosis of early presbycusis. Exclusion criteria comprised known causes of hearing loss such as significant noise exposure, trauma, ototoxic medication, neoplasm, and congenital infection or syndrome, as well as congenital or pediatric onset. Sequence changes were identified in 11.7% and 10% of presbycusis and control alleles, respectively. Among the presbycusis group, these solely occurred within the GJB2 and SLC26A4 genes. Homozygous and compound heterozygous pathogenic mutations were exclusively seen in affected individuals. We were unable to detect a statistically significant difference between our control and affected populations regarding the frequency of sequence variants detected with the APEX array. Individuals who carry two mild mutations in the GJB2 gene possibly have an increased risk of developing early presbycusis.

    Annals of clinical and laboratory science 2008;38;4;352-60

  • A new GJA1 (connexin 43) mutation causing oculodentodigital dysplasia associated to uncommon features.

    de la Parra DR and Zenteno JC

    Research Unit, Institute of Ophthalmology Conde de Valenciana, Mexico City, Mexico.

    Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder that includes a clinical spectrum of craniofacial, neurologic, limb, and ocular malformations. The disease is caused by heterozygous mutations in the 6q22-q23 located GJA1 gene, that encodes connexin 43 (Cx43). In this paper we describe a novel Cx43 mutation (G2V) found in a Mexican eight-year-old boy. This de novo mutation predicts a missense substitution at the second amino acid of Cx43, in the first intracellular domain, and is the most amino-terminal located mutation reported so far. Umbilical hernia and congenital optociliary veins, two uncommon ODDD-associated features, were recognized in our patient. The phenotype of three previously described patients with Cx43 first intracellular domain mutation is discussed and compared with that observed in our patient. This case expands the phenotypic and genotypic spectrum of ODDD.

    Ophthalmic genetics 2007;28;4;198-202

  • Corticotropin-releasing hormone activates connexin 43 via activator protein-1 transcription factor in human myometrial smooth muscle cells.

    Wu X, Shen H, Yu L, Peng M, Lai WS and Ding YL

    Dept. of Gynecology and Obstetrics, The Second Xiangya Hospital, Central South Univ., Changsha, Hunan 410011, China.

    Corticotropin-releasing hormone (CRH) and connexin 43 (Cx43) play crucial roles in uterine contraction and the onset of labor. The aim of the present study was to investigate the regulatory effects of CRH on Cx43 expression in human myometrial smooth muscle cells (SMCs) and, potentially, its activation of the c-Fos/activator protein (AP)-1 signaling pathway. Human myometrial SMCs collected from nonpregnant women were treated with different concentrations of CRH. Transient transfection of AP-1 decoy oligodeoxynucleotide (ODN) was used to block AP-1 sites of Cx43. The transcriptional activity of AP-1 was detected by luciferase assay. Cx43 protein expression was visualized by immunofluorescence staining. mRNA and protein expression of c-Fos and Cx43 were demonstrated by real-time quantitative RT-PCR and Western blot, respectively. CRH facilitated Cx43 expression and enhanced AP-1 promoter activity in human uterine SMCs. After CRH treatment, Cx43 expression in the 1f40 cytoplasm increased significantly. CRH significantly increased mRNA and protein expression of c-Fos and Cx43 in a dose-dependent manner (P < 0.01). A transient transfection of AP-1 decoy ODN did not affect CRH regulation of c-Fos (P > 0.05) but almost completely abolished CRH-induced enhancement of Cx43 expression (P < 0.01). In human primary myometrial SMCs, CRH enhances Cx43 mRNA and protein expression through upregulation of c-Fos expression. Blockade of AP-1 sites to the Cx43 promoter can neutralize the CRH-induced upregulation of Cx43.

    American journal of physiology. Endocrinology and metabolism 2007;293;6;E1789-94

  • Intracellular calcium regulation of connexin43.

    Lurtz MM and Louis CF

    Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA. monica.lurtz@ucr.edu

    The mechanism by which intracellular Ca(2+) concentration ([Ca(2+)](i)) regulates the permeability of gap junctions composed of connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca(2+) in the presence of the Ca(2+) ionophore ionomycin produced a sustained elevation in [Ca(2+)](i) that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC(50) of 360 nM Ca(2+)). The Ca(2+) dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins (Cx43, Cx44, and Cx49). The intracellular Ca(2+)-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action but not by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II or protein kinase C. In experiments that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43(Delta257)), cell-to-cell coupling was similarly decreased by an elevation of [Ca(2+)](i) (IC(50) of 310 nM Ca(2+)) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca(2+)](i) regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.

    Funded by: NEI NIH HHS: EY05684

    American journal of physiology. Cell physiology 2007;293;6;C1806-13

  • Identification of the calmodulin binding domain of connexin 43.

    Zhou Y, Yang W, Lurtz MM, Ye Y, Huang Y, Lee HW, Chen Y, Louis CF and Yang JJ

    Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, USA.

    Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-depende 1f40 nt manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.

    Funded by: NEI NIH HHS: EY-05684; NIGMS NIH HHS: GM62999, R01 GM062999

    The Journal of biological chemistry 2007;282;48;35005-17

  • Enhanced transmural fiber rotation and connexin 43 heterogeneity are associated with an increased upper limit of vulnerability in a transgenic rabbit model of human hypertrophic cardiomyopathy.

    Ripplinger CM, Li W, Hadley J, Chen J, Rothenberg F, Lombardi R, Wickline SA, Marian AJ and Efimov IR

    Department of Biomedical Engineering, Washington University, St Louis, MO 63130, USA.

    Human hypertrophic cardiomyopathy, characterized by cardiac hypertrophy and myocyte disarray, is the most common cause of sudden cardiac death in the young. Hypertrophic cardiomyopathy is often caused by mutations in sarcomeric genes. We sought to determine arrhythmia propensity and underlying mechanisms contributing to arrhythmia in a transgenic (TG) rabbit model (beta-myosin heavy chain-Q403) of human hypertrophic cardiomyopathy. Langendorff-perfused hearts from TG (n=6) and wild-type (WT) rabbits (n=6) were optically mapped. The upper and lower limits of vulnerability, action potential duration (APD) restitution, and conduction velocity were measured. The transmural fiber angle shift was determined using diffusion tensor MRI. The transmural distribution of connexin 43 was quantified with immunohistochemistry. The upper limit of vulnerability was significantly increased in TG versus WT hearts (13.3+/-2.1 versus 7.4+/-2.3 V/cm; P=3.2e(-5)), whereas the lower limits of vulnerability were similar. APD restitution, conduction velocities, and anisotropy were also similar. Left ventricular transmural fiber rotation was significantly higher in TG versus WT hearts (95.6+/-10.9 degrees versus 79.2+/-7.8 degrees; P=0.039). The connexin 43 density was significantly increased in the mid-myocardium of TG hearts compared with WT (5.46+/-2.44% versus 2.68+/-0.77%; P=0.024), and similar densities were observed in the endo- and epicardium. Because a nearly 2-fold increase in upper limit of vulnerability was observed in the TG hearts without significant changes in APD restitution, conduction velocity, or the anisotropy ratio, we conclude that structural remodeling may underlie the elevated upper limit of vulnerability in human hypertrophic cardiomyopathy.

    Funded by: NHLBI NIH HHS: P50 HL054313, P50 HL054313-060012, P50 HL054313-070012, P50 HL054313-080012, P50 HL054313-08S10012, P50 HL054313-090012, P50 HL054313-100012, R01 HL067322, R01 HL067322-05A2, R01 HL068884, R01 HL074283, R01-HL074283, R01-HL67322, R01-HL68884

    Circulation research 2007;101;10;1049-57

  • The proteasome regulates the interaction between Cx43 and ZO-1.

    Girao H and Pereira P

    Centre of Ophthalmology, Biomedical Institute for Research in Light and Image (IBILI), Faculty of Medicine, University of Coimbra, 3000-354 Coimbra, Portugal.

    Gap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO-1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO-1. Overexpression of the PDZ2 domain of ZO-1 and the expression of Cx-43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO-1 and the C-terminus of Cx43. When interaction between Cx43 and ZO-1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non-junctional communication with extracellular environment, contributing to cell and tissue dysfunction.

    Journal of cellular biochemistry 2007;102;3;719-28

  • Cutting edge: Lack of evidence for connexin-43 expression in human epidermal Langerhans cells.

    Zimmerli SC, Masson F, Cancela J, Meda P and Hauser C

    Division of Immunology and Allergy, Department of Internal Medicine, Geneva University Hospitals and Medical School, Switzerland. Simone.Zimmerli@hcuge.ch

    A provocative study has shown that viral peptides may be transferred in vitro from epithelial cells to APC through connexin-43 gap junction channels. In support of this cross-presentation pathway, the study also reported that human dendritic cells, including Langerhans cells of skin, express connexin-43. In this report we show that if this was the case, the levels of connexin-43 are below those detectable by immunofluorescence, flow cytometry, quantitative PCR of purified CD1a+ cells, and electron microscopy, raising questions about the relevance of the connexin-43-dependent mechanism for Langerhans cells of noninflamed human skin.

    Journal of immunology (Baltimore, Md. : 1950) 2007;179;7;4318-21

  • Connexin 43 expression in human and mouse testes with impaired spermatogenesis.

    Kotula-Balak M, Hejmej A, Sadowska J and Bilinska B

    Department of Endocrinology and Tissue Culture, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Kraków, Poland.

    Connexin 43 (Cx43) belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelter's syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelter's patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelter's patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought.

    European journal of histochemistry : EJH 2007;51;4;261-8

  • [Decreased expression of Cx32 and Cx43 and their function of gap junction intercellular communication in gastric cancer].

    Wu J, Zhou HF, Wang CH, Zhang B, Liu D, Wang W and Sui GJ

    The Seventh Medical Department, Tumor Hospital of Harbin Medical University, Harbin 150040, China.

    Objective: The aim of this study was to investigate the expression of connexin (Cx) and the function of gap junction intercellular communication (GJIC) in the carcinogenesis, progression and metastasis of gastric cancers.

    Methods: Immunohistochemistry was used to detect the expression of Cx32 and Cx43 proteins in tissue samples. Indirect immunofluorescence assay was used to investigate the expression of Cx32 and Cx43 proteins in several gastric cancer lines of various differentiation grades. The expression of Cx43 in samples of gastric cancer tissue, adjacent normal tissue and in the gastric cancer cell lines of various differentiation grades was detected by Western blot. Scrape-loading dye transfer (SLDT) technique was used to detect the function of gap junction intercellular communication (GJIC) in the various cell lines.

    Results: In the normal gastric mucosa the expression rates of both Cx32 and Cx43 were 100%. In gastric cancers, the expression rates of Cx32 andCx43 were 49.5% (55/111) and 39.6% (44/111), respectively. There was a significant difference between their expression in normal and cancer tissues (P < 0.05). Age of the patients was not significantly correlated with the expression level of Cx32 and Cx43 (P > 0.05). Cx43 expression was significantly associated with the TMN stage, histological type, depth of infiltration and distant metastasis (P < 0.05), but Cx32 expression was not significantly correlated with depth of infiltration ( P > 0.05). In the cancer cell line 1696 s, a positive expression of Cx32 and Cx43 was detected in transfected human stomach mucosal cell line (CES-1) and human well differentiated stomach cancer cell line (N87), but negative in the poorly differentiated stomach cancer cell line (BGC-823) at all. Both Cx32 and Cx43 expression rates were 100% in the cell line GES-1. Cx32 expression rate was 49.0% and Cx43 expression rate was 55.0% in the cell line N87. But in the poorly differentiated cancer cell line BGC-823 both Cx32 and Cx43 expression was negative. GJIC function detection showed: GES-1 showed well GIJC function but no GIJC function in the cell lines N87 and BGC-823. The intensity of fluorescence was gradually decreasing from GES-1 cells to N87 cells and almost no fluorescence in BGC-823 cells. Western blotting showed that Cx43 expression in normal tissue was higher than that in gastric cancer tissue, and in the cell lines GES-1, N87 and BGC-823, the bands seemed decreasing progressively. There was very low expression in BGC-823 cells.

    Conclusion: The decreasing expression of connexin Cx32 and Cx43 is obviously correlated with the occurrence, development and metastatic potential of stomach cancers.

    Zhonghua zhong liu za zhi [Chinese journal of oncology] 2007;29;10;742-7

  • Some oculodentodigital dysplasia-associated Cx43 mutations cause increased hemichannel activity in addition to deficient gap junction channels.

    Dobrowolski R, Sommershof A and Willecke K

    Institute of Genetics, University of Bonn, Roemerstrasse 164, 53117, Bonn, Germany.

    Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels.

    The Journal of membrane biology 2007;219;1-3;9-17

  • Angiotensin II and IGF-1 regulate connexin43 expression via ERK and p38 signaling pathways in vascular smooth muscle cells of coronary artery bypass conduits.

    Jia G, Mitra AK, Cheng G, Gangahar DM and Agrawal DK

    Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska 68178, USA.

    Background: Changes in connexin expression have been found in vascular smooth muscle cells (VSMCs) during the progression of atherosclerotic lesion and intimal hyperplasia. It is our hypothesis that increased connexin43 expression following stimulation of VSMCs with Ang II and IGF-1 contributes to more proliferation in saphenous vein (SV) than in the internal mammary artery (IMA).

    Using immunohistochemistry, Western blot, and reverse-transcription polymerase chain reaction, we assessed the effect of Ang II and IGF-1 stimulation on connexin43 expression and the signaling pathways involved in VSMCs of SV and IMA.

    Results: Immunostaining demonstrated strong expression of connexin43 in SV compared with IMA after stimulation with Ang II and IGF-1. Ang II up-regulated the expression of connexin43 in VSMCs of SV in a dose- and time-dependent manner. This was inhibited by p38 and ERK MAP kinase inhibitors, SB203580 and PD98059, respectively. In the VSMCs of IMA, the connexin43 expression was markedly low and maintained at a reduced level even after 3 h stimulation. IGF-1 dose-dependently induced mRNA expression of connexin43 in the VSMCs of SV, which was blocked by PD98059. However, in VSMCs of IMA there was no significant effect of IGF-1 on the connexin43 mRNA expression.

    Conclusion: These data suggest that connexin43 expression can be influenced by Ang II and IGF-1 through ERK and p38 pathways and may contribute to the pathogenesis of vein graft disease following coronary artery bypass grafting.

    Funded by: NHLBI NIH HHS: R01HL070885, R01HL073349

    The Journal of surgical research 2007;142;1;137-42

  • Pressure induces loss of gap junction communication and redistribution of connexin 43 in astrocytes.

    Malone P, Miao H, Parker A, Juarez S and Hernandez MR

    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, USA.

    Astrocytes, the major glia in the nonmyelinated optic nerve head (ONH), connect via gap junctions built of connexin-43 (Cx43) to form a functional syncytium allowing communication and control of ionic and metabolic homeostasis of retinal ganglion cells (RGCs) axon. We examined gap junction intercellular communication (GJIC) by scrape loading assays in human ONH astrocytes exposed to hydrostatic (HP) or ambient pressure (CP) in vitro. Immunostaining, immunoprecipitation, and immunoblots were used to detect Cx43 distribution and phosphorylation in astrocytes exposed to HP with/without EGF receptor (EGFR) tyrosine kinase inhibitors AG1478 and AG82 and MAPK inhibitors U0126, PD98059, and SB203580. The data indicates that upon exposure to HP, astrocytes decrease GJIC and exhibit altered cellular localization and phosphorylation of Cx43. Inhibition of EGFR blocked the effects of HP on GJIC and HP-induced Cx43 tyrosine phosphorylation. Inhibitors of MAPK- ERK1/2 and -p38 caused partial closure of GJIC under CP and HP, which was maintained for 6 h. Inhibition of Big Mitogen-Activated Kinase 1/ERK5 (BMK1/ERK5) caused partial closure under CP and HP followed by full recovery after 6 h. Inhibition of MAPK did not affect the HP-induced increase in Cx43 serine 279/282 phosphorylation. We conclude that activation of the EGFR pathway in response to HP leads to decrease of GJIC via tyrosine phosphorylation of Cx43 in ONH astrocytes. In glaucoma under conditions of elevated intraocular pressure (IOP), astrocytes may lose GJIC altering the homeostasis of RGC axons, adopting the reactive phenotype, contributing to glaucomatous neuropathy.

    Funded by: NEI NIH HHS: EY-06416

    Glia 2007;55;10;1085-98

  • [Connexin 43 expression and interacellular communicating function in acute leukemia bone marrow stroma cells].

    Liu Y, Zhang X, Si YJ, Gao L, Gao L and Chen XH

    Department of Hematology, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037, China.

    This study was purposed to investigate the connexin 43 (Cx43) expression level in acute leukemia bone marrow stromal cells (ABMSCs) and normal bone marrow stromal cells (NBMSCs), and to explore the difference in communicating functions between these cells. The Cx43 expression levels of ABMSCs and NBMSCs were detected by using immunohistochemistry and computer gray scale assay, and the difference of gap junction intercellular communication (GJIC) was examined through dry transfer technique. The results showed that expression level of Cx43 in ABMSCs was lower than that in NBMSCs and its function of GJIC in ABMSCs was also weaker than that in NBMSCs. It is concluded that cell-cell communication function is lowered in ABMSCs.

    Zhongguo shi yan xue ye xue za zhi 2007;15;4;679-82

  • Increased communication among nasal epithelial cells in air-liquid interface culture.

    Yeh TH, Tsai CH, Chen YS, Hsu WC, Cheng CH, Hsu CJ and Lee SY

    Department of Otolaryngology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan. yth@ha.mc.ntu.edu.tw

    The retinoid acid (RA) sufficient air-liquid interface (ALI) cell culture model, but not the classical submerged single layer (SSL) cell culture model, can achieve ciliary differentiation of nasal epithelial cells. Because gap junction mediated intercellular communication (GJIC) may contribute to differentiation in numerous cell types, this study compared the extent of GJIC and the expression of Connexin 43 (Cx43) in nasal epithelial cells in both SSL and ALI cultures.

    Methods: Cell morphology was examined via optical and scanning electron microscope, and the number of cells with ciliary beating were counted. Lucifer Yellow dye transfer test using the scrape loading method was performed to assess the GJIC. Cx43 expression was measured with reverse-transcription polymerase chain reaction (RT-PCR) and quantitative (Q)-PCR.

    Results: Nasal epithelial cells in ALI culture exhibited increased numbers of ciliated cells compared with SSL culture during the 3-week culture period. On day 20, GJIC was increased in ALI culture (ALI % - SSL % = 9.6 +/- 1.2%, n = 5). Accordingly, Cx43 expression was increased via RT-PCR (4.22-fold) and Q-PCR (5.3 +/- 1.1-fold, n = 5) examination.

    Conclusions: RA sufficient ALI culture manifested more differentiated nasal epithelial cell status with ciliogenesis. Cx43, being the responsible molecule for GJIC, increased in parallel. Consequently, as in primary cultured limbal epithelial cells, Cx43 expression and extent of GJIC may serve as markers for the differentiation status of nasal epithelial cells.

    The Laryngoscope 2007;117;8;1439-44

  • Connexin 43 expression promotes malignancy of HuH7 hepatocellular carcinoma cells via the inhibition of cell-cell communication.

    Zhang D, Kaneda M, Nakahama K, Arii S and Morita I

    Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan.

    In normal liver, Connexin (Cx) 43 is not detected, but up-regulated in some liver cancers. We herein investigated the role of Cx43 in hepatoma cell carcinogenesis. Cx43-silenced HuH7 cells using shRNA showed lower growth and higher differentiation, and Cx43-overexpressing cells exhibited rapid growth and low differentiation. Unexpectedly, gap junctional intercellular communication (GJIC) was inversely correlated with Cx43 levels. Furthermore, the expression level and promoter activity of Cx32 was negatively regulated by the expression of Cx43. From these data, Cx43 expression may be in part responsible for the malignancy of hepatoma cells through a decrease in GJIC composed of Cx32.

    Cancer letters 2007;252;2;208-15

  • Endothelial connexin 37, connexin 40, and connexin 43 respond uniquely to substrate and shear stress.

    Johnson TL and Nerem RM

    Georgia Tech/Emory Center for the Engineering of Living Tissues, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA.

    Endothelial connexins have been linked to atherosclerosis and hypertension; however, little is know about their sensitivity to stimuli and individual functions. This study investigates the responses of endothelial connexin 37, connexin 40, and connexin 43 (Cx37, Cx40, and Cx43) to shear stress and substrate. Human endothelial cells were seeded on adsorbed collagen or a collagen gel containing smooth muscle cells and exposed to static or laminar shear stress. Connexin mRNA, protein, and gap junction communication were examined. Endothelial monolayers were treated with connexin-specific short interfering RNA (siRNA) and evaluated for communication, proliferation, and morphology under static and shear stress. Results show differential responses of Cx37, Cx40, and Cx43 to substrate and shear stress with reduced communication after shear exposure. RNA interference of individual connexins resulted in expression change of nontarget connexins, which suggests linked expression. Gap junction communication under static conditions is reduced following Cx43 siRNA treatment. Endothelial cells are more elongated with RNA interference (RNAi) targeting Cx40. In conclusion, endothelial connexins demonstrated novel sensitivity to mechanical environment and substrate. Individual isotypes show differential responses and RNAi knockdown provides new insight into connexin function and potential roles in the vasculature.

    Endothelium : journal of endothelial cell research 2007;14;4-5;215-26

  • Zonula occludens-1 and connexin 43 expression in the failing human heart.

    Kostin S

    Core Lab for Molecular and Structural Biology, W. G. Kerckhoff Institute, Max-Planck Institute for Heart and Lung Research, Parkstrasse 1, 61231 Bad Nauheim, Germany. sawa.kostin@mpi-bn.mpg.de

    Focal disorganization of gap junctional distribution and down-regulation of the major gap junctional protein connexin 43 are typical features of myocardial remodelling in the failing human heart. Increasing evidence indicates that connexin 43 interacts with zonula-occludens-1 (ZO-1), and it has recently been shown that ZO-1 promotes the formation and growth of gap junctional plaques. In the present study, distribution patterns of ZO-1 and connexin 43 were studied in normal and in heart failure patients using double-label immunohistochemistry and confocal microscopy. ZO-1 was found to be co-localized with connexin 43 at intercalated disks. Importantly, in patients with heart failure due to dilated or ischaemic cardiomyopathy, areas of diminished connexin 43 expression were characterized by a markedly reduced ZO-1 staining. Based on these data it is concluded that in patients with heart failure, down-regulation of ZO-1 matches the diminished expression levels of connexin 43, suggesting that ZO-1 plays an important role in gap junction formation and gap junction plaque stability.

    Journal of cellular and molecular medicine 2007;11;4;892-5

  • Differential potency of dominant negative connexin43 mutants in oculodentodigital dysplasia.

    Gong XQ, Shao Q, Langlois S, Bai D and Laird DW

    Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada.

    Oculodentodigital dysplasia (ODDD) is a congenital autosomal dominant disorder with phenotypic variability, which has been associated with mutations in the GJA1 gene encoding connexin43 (Cx43). Given that Cx43 mutants are thought to be equally co-expressed with wild-type Cx43 in ODDD patients, it is imperative to examine the consequence of these mutants in model systems that reflect this molar ratio. To that end, we used differential fluorescent protein tagging of mutant and wild-type Cx43 to quantitatively monitor the ratio of mutant/wild-type within the same putative gap junction plaques and co-immunoprecipitation to determine if the mutants interact with wild-type Cx43. Together the fluorescence-based assay was combined with patch clamp analysis to assess the dominant negative potency of Cx43 mutants. Our results revealed that the ODDD-linked Cx43 mutants, G21R and G138R, as well as amino terminus green fluorescent protein-tagged Cx43, were able to co-localize with wild-type Cx43 at the gap junction plaque-like structures and to co-immunoprecipitate with wild-type Cx43. All Cx43 mutants demonstrated dominant negative action on gap junctional conductance of wild-type Cx43 but not that of Cx32. More interestingly, these Cx43 mutants demonstrated different potencies in inhibiting the function of wild-type Cx43 with the G21R mutant being two times more potent than the G138R mutant. The potency difference in the dominant negative properties of ODDD-linked Cx43 mutants may have clinical implications for the various symptoms and disease severity observed in ODDD patients.

    The Journal of biological chemistry 2007;282;26;19190-202

  • Gene expres 103a sion response to cisplatin treatment in drug-sensitive and drug-resistant ovarian cancer cells.

    Li J, Wood WH, Becker KG, Weeraratna AT and Morin PJ

    Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, Baltimore, MD 21224, USA.

    The molecular pathways activated in response to acute cisplatin exposure, as well as the mechanisms involved in the long-term development of cisplatin-resistant cancer cells remain unclear. Using whole genome oligonucleotide microarrays, we have examined the kinetics of gene expression changes in a cisplatin-sensitive cell line, A2780, and its cisplatin-resistant derivative, ACRP. Both sensitive and resistant cell lines exhibited a very similar response of p53-inducible genes as early as 16 h after treatment. This p53 response was further increased at the 24-h time point. These experiments identify p53 as the main pathway producing a large-scale transcriptional response after cisplatin treatment in these cells containing wild-type p53. Consistent with a role for the p53 response in cisplatin sensitivity, knockdown of the p53 protein with small interfering RNA led to a twofold decrease in cell survival in the resistant cells. In addition, our analysis also allowed the identification of several genes that are differentially expressed between sensitive and resistant cells. These genes include GJA1 (encoding connexin 43 (Cx43)) and TWIST1, which are highly upregulated in cisplatin-resistant cells. The importance of Cx43 in drug resistance was demonstrated through functional analyses, although paradoxically, inhibition of Cx43 function in high expressing cells led to an increase in drug resistance. The pathways important in cisplatin response, as well as the genes found differentially expressed between cisplatin-resistant and -sensitive cells, may represent targets for therapy aimed at reversing drug resistance.

    Funded by: Intramural NIH HHS

    Oncogene 2007;26;20;2860-72

  • The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2.

    Yang B, Lin H, Xiao J, Lu Y, Luo X, Li B, Zhang Y, Xu C, Bai Y, Wang H, Chen G and Wang Z

    Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, Heilongjiang 150086, China. yangbf@ems.hrbmu.edu.cn

    MicroRNAs (miRNAs) are endogenous noncoding RNAs, about 22 nucleotides in length, that mediate post-transcriptional gene silencing by annealing to inexactly complementary sequences in the 3'-untranslated regions of target mRNAs. Our current understanding of the functions of miRNAs relies mainly on their tissue-specific or developmental stage-dependent expression and their evolutionary conservation, and therefore is primarily limited to their involvement in developmental regulation and oncogenesis. Of more than 300 miRNAs that have been identified, miR-1 and miR-133 are considered to be muscle specific. Here we show that miR-1 is overexpressed in individuals with coronary artery disease, and that when overexpressed in normal or infarcted rat hearts, it exacerbates arrhythmogenesis. Elimination of miR-1 by an antisense inhibitor in infarcted rat hearts relieved arrhythmogenesis. miR-1 overexpression slowed conduction and depolarized the cytoplasmic membrane by post-transcriptionally repressing KCNJ2 (which encodes the K(+) channel subunit Kir2.1) and GJA1 (which encodes connexin 43), and this likely accounts at least in part for its arrhythmogenic potential. Thus, miR-1 may have important pathophysiological functions in the heart, and is a potential antiarrhythmic target.

    Nature medicine 2007;13;4;486-91

  • Gating properties of heterotypic gap junction channels formed of connexins 40, 43, and 45.

    Rackauskas M, Kreuzberg MM, Pranevicius M, Willecke K, Verselis VK and Bukauskas FF

    Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

    Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, V(j). Cx40/Cx45 heterotypic GJ channels preferentially exhibit V(j)-dependent gating transitions between open and residual states with a conductance of approximately 42 pS; transitions between fully open and closed states with conductance of approximately 52 pS in magnitude occur at substantially lower ( approximately 10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.

    Funded by: NIMH NIH HHS: MH66199, R01 MH066199; NINDS NIH HHS: R01 NS036706, R01 NS036706-09

    Biophysical journal 2007;92;6;1952-65

  • Connexin expression in mouse lung tumor.

    Udaka N, Miyagi Y and Ito T

    Department of Pathology and Experimental Medicine, Kumamoto University Graduate School of Medical Sciences, 1-1-1 Honjyo, Kumamoto, Japan. naoudaka@kaiju.medic.kumamoto-u.ac.jp

    Gap junction intercellular communication (GJIC) is considered to play roles in regulation of homeostasis, development and differentiation of many tissues. In the present study, using reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR, we examined expression of Connexins (Cx26, 32, 37, 40, 43 and 45) in the normal lung and lung tumors of mice to determine whether their expressions change during lung tumorigenesis. Cx26, 32 and 40 were expressed similarly in the normal lung tissue and tumors with smaller size (0.5-1.5mm) though expression of Cx32 and 40 decreased in tumors with larger size (>2.5mm). Cx26 was undetectable in larger size tumors. Cx37 and 45 were expressed in both normal lung and larger size tumors but no expression was seen in smaller size tumors. Cx43 was similarly detectable in normal lung, smaller size tumor and larger size tumor, but western blotting showed that Cx43 was phosphorylated during lung tumorigenesis. Thus, it is likely that alteration of expression of these Cx may be involved in expression of the neoplastic phenotype.

    Cancer letters 2007;246;1-2;224-9

  • Connexin 43 is localized with actin in tenocytes.

    Wall ME, Otey C, Qi J and Banes AJ

    Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    Varieties of cell-matrix or cell-cell adhesions are associated with the actin cytoskeleton. However, for gap junctions, which are both channels and adhesions, there has been little evidence for such an association. The purpose of this study was to determine if connexin 43 (Cx43) associates with actin and to determine if this association is altered under mechanical load in tenocytes, a mechanically sensitive cell. Avian tenocytes were subjected to multiple cyclic strain regimens and then fixed and examined immunohistochemically at various times poststrain to determine where Cx43 protein was localized within the cells in relation to actin filaments. Four percent of tenocytes had colocalization of actin filaments and Cx43, which was significantly increased with 5% cyclic strain. To confirm this phenomenon, human tenocytes and COS-7 cells were also subjected to cyclic strain and then fixed at the same times after strain. As with avian tenocytes, Cx43 was colocalized with actin in human tenocytes and COS-7 cells. The colocalization increased significantly after cyclic strain in human tenocytes but not in COS-7 cells. The lack of detectable changes in COS-7 cells may indicate that they are less mechanosensitive than tenocytes perhaps due to the less robust actin cytoskeleton seen in the COS-7 cells when compared to the tenocytes. Furthermore, inhibiting myosin II activity greatly reduced the immunohistochemically-detectable Cx43 on actin filaments. Connexins may associate with actin to stabilize gap junctions at the plasma membrane, ensuring that tenocytes remain coupled during periods of prolonged or intense mechanical loading.

    Funded by: NIAMS NIH HHS: AR38121

    Cell motility and the cytoskeleton 2007;64;2;121-30

  • Combination of non-viral connexin 43 gene therapy and docetaxel inhibits the growth of human prostate cancer in mice.

    Fukushima M, Hattori Y, Yoshizawa T and Maitani Y

    Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan.

    Docetaxel (DTX) is used for the treatment of advanced hormone refractory prostate cancer. Connexin 43 (Cx43) is a tumor suppressor gene, and transfection of the Cx43 gene increases sensitivity to several chemotherapeutic agents. The objective of this study was to evaluate the effectiveness of combination therapy of Cx43-expressing plasmid DNA (pCMV-Cx43) and DTX both in vitro and in vivo using a non-viral vector in human prostate cancer PC-3 cells. Transfection of pCMV-Cx43 into the cells neither inhibited tumor growth nor increased gap junctional intercellular communication; however, combination therapy of pCMV-Cx43 and DTX significantly inhibited cell growth. Forced expression of Cx43 in the cells induced apoptotic cells by down-regulation of Bcl-2 expression and significantly more up-regulation of caspase-3 activity than either treatment alone. The combination of repeated intratumoral injection of pCMV-Cx43 (10 microg/tumor) with non-viral vector and a single intravenous injection of DTX (15 mg/kg) was compared with a repeated injection of Cx43 alone and a single injection of DTX alone on PC-3 tumor xenografts. Significant antitumoral effects were observed in mice receiving combined treatment, compared with DTX alone. The data presented here provide a rational strategy for treating patients with advanced hormone refractory prostate cancer.

    International journal of oncology 2007;30;1;225-31

  • Identification of mutations in members of the connexin gene family as a cause of nonsyndromic deafness in Taiwan.

    Yang JJ, Huang SH, Chou KH, Liao PJ, Su CC and Li SY

    Genetics Laboratory and Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan, ROC.

    Connexins (Cx), a large family of membrane proteins, are key components of gap junction channels. These channels are critical intercellular pathways through which ions or small molecules are passed, regulating a variety of physiological and developmental processes. One of these processes is hearing. In the current study, a genetic survey was made on 380 Taiwanese individuals, 260 with nonsyndromic deafness and 120 with normal hearing. All the 380 Taiwanese were screened for the presence of mutations in 8 genes of the Cx gene family. These genes included Cx26 (GJB2), Cx29 (GJE1), Cx30 (GJB6), Cx30.3 (GJB4), Cx31 (GJB3), Cx32 (GJB1), Cx43 (GJA1) and pseudogene [rho] of Cx43 (rho GJA1). Mutations were identified in 7 out of the 8 screened genes of the Cx family from 62 of the 260 deaf subjects (23.85%). Of the 17 mutations observed in the Cx gene family, 11 were novel mutations. Fourteen polymorphisms that were not associated with hearing loss were identified in the Cx gene family. The first 2 most frequently occurring mutations were found in the Cx26 (28/62; 45.16%) and the rho Cx43 (17/62; 27.42%), respectively. Nine cases of mutations were found in the Cx30.3 (9/62; 14.52%). In the Cx30, 1 novel mutation was identifie 16e9 d in 1 case (1/62; 1.61%). Two patients with mutations of each of Cx29 and Cx43 were found (2/62; 3.23%). One novel mutation of Cx31 was identified in 3 patients with nonsyndromic deafness (3/62; 4.84%). The Cx32 was the only gene without detecting any mutation or polymorphism.Our study provides information for understanding the importance of genetic factors in nonsyndromic deafness of the Taiwanese and may be of use in the improvement of genetic diagnosis of hearing loss in Taiwan.

    Audiology & neuro-otology 2007;12;3;198-208

  • The TLR3 ligand polyI: C downregulates connexin 43 expression and function in astrocytes by a mechanism involving the NF-kappaB and PI3 kinase pathways.

    Zhao Y, Rivieccio MA, Lutz S, Scemes E and Brosnan CF

    Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10464, USA.

    Toll-like receptor 3 (TLR3) is a component of the innate immune response that responds to dsRNA viruses and virus replication intermediates. In this study we show that activation of astrocytes with the dsRNA mimetic polyinosinic-cytidylic acid (pI:C) results in loss of expression of connexin43 (Cx43) mRNA and protein while upregulating the expression of the ionotropic P2 receptor P2X(4)R. Analysis of the signaling pathways involved failed to demonstrate a role for the p38 MAP kinase, ERK, or JNK signaling pathways whereas an inhibitor of the PI3 kinase/Akt pathway effectively blocked the action of pI:C. Using adenoviral vectors containing a super-repressor of NF-kappaB (NF-kappaB SR) construct or a dominant negative interferon regulatory factor 3 (dnIRF3) construct showed that inhibition of both transcription factors also blocked the effects of pI:C. To explore the functional consequences of pI:C activation we used a pore-forming assay for P2X(4)R activity and a scrape loading assay for gap junction intercellular communication (GJIC). No pore-forming activity consistent with functional P2X(4)R expression was detected in either control or activated astrocytes. In contrast, robust Lucifer yellow transfer indicative of GJIC was detected in resting cells that was lost following pI:C activation. The dnIRF3 construct failed to restore GJIC whereas the NF-kappaB SR or the NF-kappaB inhibitor BAY11-7082 and the PI3K inhibitor LY294002 all significantly reversed the effect of pI:C on GJ connectivity. We conclude that activation of the innate immune response in astrocytes is associated with functional loss of GJIC through a pathway involving NF-kappaB and PI3 kinase.

    Funded by: NINDS NIH HHS: R01 NS040137, R01 NS041023, R01 NS041023-05, T32 NS007098

    Glia 2006;54;8;775-85

  • Glucose-evoked alterations in connexin43-mediated cell-to-cell communication in human collecting duct: a possible role in diabetic nephropathy.

    Hills CE, Bland R, Wheelans DC, Bennett J, Ronco PM and Squires PE

    Molecular Physiology, Biomedical Research Institute, Dept. of Biological Sciences, Univ. of Warwick, Coventry, CV4 7AL, UK. psquires@bio.warwick.ac.uk

    Aberrant sodium absorption has been linked to the development of hypertension in both renal disease and diabetes. Efficient absorption depends on coordination of cellular activity across the entire epithelium via cell-to-cell coupling. In the current study we have utilized a model human collecting duct cell line (HCD) to assess the role of connexin43 (Cx43)-mediated gap junctions in the transfer of intracellular Ca(2+) transients within coupled cell clusters. HCD cells express Cx43 mRNA and protein, as well as that for the mechanosensitive transient receptor potential receptor (TRPV4). Mechanical stimulation of individual cells within a cluster evoked a transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) that propagated between cells via a heptanol-sensitive mechanism. The rise in [Ca(2+)](i) was dependent on both store release and Ca(2+)-influx pathways. Lucifer yellow dye transfer and Cx43 knockdown experiments confirmed direct cell-to-cell communication. Application of the Ca(2+) ionophore ionomycin, or an increase in glucose (5 to 25 mM), produced a time-dependent (48 h) increase in Cx43 protein expression. The transmission rate of touch-evoked Ca(2+) transients between coupled cells was accelerated after exposure to high glucose, providing a functional correlate to increased Cx43 expression. These data suggest a pivotal role for Cx43-mediated gap junctions in the synchronization of activity between HCD cells in response to stimuli that mimic osmotic and physical changes. Cx43 expression and cell-to-cell communication increased in response to high glucose and may protect the collecting duct from renal damage associated with more established diabetic nephropathy.

    American journal of physiology. Renal physiology 2006;291;5;F1045-51

  • Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma.

    Gong XQ, Shao Q, Lounsbury CS, Bai D and Laird DW

    Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada.

    A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.

    The Journal of biological chemistry 2006;281;42;31801-11

  • A potential novel mechanism involving connexin 43 gap junction for control of sertoli cell proliferation by thyroid hormones.

    Gilleron J, Nebout M, Scarabelli L, Senegas-Balas F, Palmero S, Segretain D and Pointis G

    INSERM U 670, Faculté de Médecine, Université de Paris V René Descartes, Paris, France.

    There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation.

    Journal of cellular physiology 2006;209;1;153-61

  • Enhanced connexin 43 immunoreactivity in penumbral areas in the human brain following ischemia.

    Nakase T, Yoshida Y and Nagata K

    Department of Neurology, Research Institute for Brain and Blood Vessels, 6-10 Sensyu Kubota Machi, Akita 010-0874, Japan. nakase@akita-noken.go.jp

    Astrocytes support neurons not only physically but also chemically by secreting neurotrophic factors and energy substrates. Moreover, astrocytes establish a glial network and communicate through gap junctions in the brain. Connexin 43 (Cx43) is one of major component proteins in astrocytic gap junctions. Heterozygote Cx43 KO mice and astrocyte specific Cx43 KO mice exhibited amplified brain damage after ischemic insults, suggesting a neuroprotective role for astrocytic gap junctions. However, some reports mentioned unfavorable effects of gap junctions in neuronal support. Therefore, the role of astrocytic gap junctions under ischemic condition remains controversial. Since these studies have been performed using animal models, we investigated the Cx43 expression in human brain after stroke. Brain slice sections were prepared from pathological samples in our hospital. Embolic stroke brains sectioned because of the stroke were considered as acute ischemic models. Multiple infarction brains sectioned because of pneumonia or cancer were considered as chronic models. We observed the levels of Cx43 in both lesioned and intact areas, and compared them with acute and chronic models. As the results, astrocytes were strongly activated in penumbral lesions both of acute and chronic ischemic models. The Cx43 immunoreactivity was significantly amplified in the penumbra of chronic model compared to that of the acute model. Neurons were well preserved in chronic model compared to acute model. These findings suggested that the brain may generate neuronal protection by increasing the levels of Cx43 and amplifying the astrocytic gap junctional intercellular communication under hypoxic condition.

    Glia 2006;54;5;369-75

  • Interaction of zonula occludens-1 (ZO-1) with alpha-actinin-4: application of functional proteomics for identification of PDZ domain-associated proteins.

    Chen VC, Li X, Perreault H and Nagy JI

    Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.

    The use of recombinant "bait" proteins to capture protein-binding partners, followed by identification of protein interaction networks by mass spectrometry (MS), has gained popularity and widespread acceptance. We have developed an approach using recombinant PDZ protein interaction modules of the membrane-associated guanylate kinase (MAGUK) protein zonula occludens-1 (ZO-1) to pull-down and screen for proteins that interact with these modules via their PDZ domain binding motifs. Identification of proteins by MS of pull-down material was achieved using a vacuum-based chromatography sample preparation device designed for matrix-assisted laser desorption/ionization (MALDI) MS. MS analysis of tryptic fragments in pull-down material revealed a number of potential ZO-1 interacting candidates, including the presence of peptides corresponding to the cortical membrane scaffolding protein alpha-actinin-4. Interaction of alpha-actinin-4 with ZO-1 was confirmed by coimmunoprecipitation of these two proteins from cultured cells, as well as from brain, liver, and heart, and by immunoblot detection of alpha-actinin-4 after pull-down with the first PDZ domain of ZO-1. In contrast, the highly homologous alpha-actinin family member, alpha-actinin-1, displayed no association with ZO-1. Immunofluorescence showed colocalization of alpha-actinin-4 with ZO-1 in cultured HeLa and C6 glioma cells, as well as in a variety of tissues in vivo, including brain, heart, liver, and lung. This study demonstrates the utility of MS-based functional proteomics for identifying cellular components of the ZO-1 scaffolding network. Our finding of the interaction of ZO-1 with alpha-actinin-4 provides a mechanism for linking the known protein recruitment and signaling activities of ZO-1 with alpha-actinin-4-associated plasma membrane proteins that have regulatory activities at cell-cell and cell-extracellular matrix contacts.

    Journal of proteome research 2006;5;9;2123-34

  • Leukemogenic AML1-ETO fusion protein upregulates expression of connexin 43: the role in AML 1-ETO-induced growth arrest in leukemic cells.

    Li X, Xu YB, Wang Q, Lu Y, Zheng Y, Wang YC, Lübbert M, Zhao KW and Chen GQ

    Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, China.

    AML1-ETO, a fusion protein generated by the chromosomal translocation t(8;21), is frequently associated with acute myeloid leukemia (AML). In addition to blocking differentiation, AML1-ETO is also shown to induce growth arrest in AML cells, which is unfavorable for leukemogenesis harboring the t(8;21) translocation. However, its precise mechanism is still unclear. Here we provide the first demonstration that the conditional expression of AML1-ETO by the ecdysone-inducible system dramatically increases the expression of connexin 43 (CX43), together with growth arrest at G1 phase in leukemic U937 cells. We also show that the CX43 induction inhibits the proliferation of U937 cells at G1 phase, while the suppression of CX43 expression by small interfering RNA (siRNA) effectively overcomes the growth-inhibitory effect of AML1 -ETO in leukemic cells. Furthermore, either AML1-ETO or CX43 induction elevates cell-cycle negative regulator P27(kip1) protein by inhibiting its degradation, which is antagonized by siRNA against CX43. Taken together, our data indicate that CX43 plays a role in AML1-ETO-induced growth arrest possibly through the accumulation of P27(kip1) protein. The potential mutation or/and epigenetic alterations of CX43 and its related gene(s) deserve to be explored in AML1-ETO-positive AML patients.

    Journal of cellular physiology 2006;208;3;594-601

  • Connexin 43 transfection in basaloid squamous cell carcinoma cells.

    Shima K, Muramatsu T, Abiko Y, Yamaoka Y, Sasaki H and Shimono M

    Department of Pathology, Tokyo Dental College, Chiba 261-8502, Japan.

    To investigate the relationship between the expression of connexin in basaloid squamous cell carcinomas (BSCC) and their rapid proliferation and invasive potential, we examined the effect of overexpression of connexin 43 (Cx43) in a BSCC-derived cell line (BSC-OF). BSC-OF was transfected with Cx43 to obtain 15 clones with a stable expression of Cx43. In these cells, although Cx43 was distributed throughout the cytoplasm, it did not form connexon plaque. In almost all of the clones, cell proliferation was clearly suppressed. Furthermore, we investigated cell migration and invasion in three clones that showed a remarkable down-regulation in cell growth, and found that Cx43 transfection showed no significant effect on either. These results suggest that Cx43 plays a role as a tumor suppressor in the cytoplasm of Cx43-transfected BSC-OF cells. However, no definite correlation was found between Cx43 and cell migration and invasion.

    Oncology reports 2006;16;2;285-8

  • A nonsense mutation in the first transmembrane domain of connexin 43 underlies autosomal recessive oculodentodigital syndrome.

    Richardson RJ, Joss S, Tomkin S, Ahmed M, Sheridan E and Dixon MJ

    Faculty of Life Sciences and Dental School, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK. mike.dixon@manchester.ac.uk.

    Background: Oculodentodigital syndrome (ODD) is a pleiotropic congenital disorder characterised by abnormalities of the face, eyes, dentition, and limbs. ODD, which is inherited as an autosomal dominant trait, results from missense mutations in the gap junction protein connexin 43.

    Objective: To analyse a family with a history of ODD which is inherited in an autosomal recessive manner

    Results: ODD in this family resulted from the homozygous mutation R33X in the first transmembrane domain of connexin 43.

    Conclusions: The findings provide clear genetic evidence that ODD can be inherited in an autosomal recessive manner and that a dominant negative mechanism underlies autosomal dominant ODD.

    Funded by: Wellcome Trust: 058423, 069243

    Journal of medical genetics 2006;43;7;e37

  • Downregulation of connexin 43 expression by high glucose induces senescence in glomerular mesangial cells.

    Zhang X, Chen X, Wu D, Liu W, Wang J, Feng Z, Cai G, Fu B, Hong Q and Du J

    Department of Nephrology, Kidney Center and Key Lab of PLA, General Hospital of PLA, 28 Fuxing Road, Beijing 100853, People's Republic of China.

    Connexin 43 (Cx43) plays an important role in cell differentiation and growth control, but whether it can be regulated by high glucose and whether it can mediate in glomerular mesangial cells (GMC) the phenotype alterations that are induced by high glucose still remain to be explored. In this study, RNA interference and gene transfer techniques were used to knock down and overexpress Cx43 gene in rat GMC to determine the contribution of Cx43 to GMC senescence that was induced by high glucose. The results show that high glucose (30 mM) not only downregulated Cx43 mRNA and protein expression (P<0.05) but also increased the percentage of senescence-associated beta-galactosidase (SA-beta-gal) stained cells and expression of p21cip1 and p27kip1 (P<0.05), indicating that high glucose promoted rat GMC senescence. Knocking down Cx43 gene expression significantly increased the percentage of SA-beta-gal stained cells and p27kip1 and p21cip1 expression in GMC (P<0.05), whereas overexpression of Cx43 significantly decreased the percentage of SA-beta-gal stained cells (P<0.05). These results demonstrate for the first time that downregulation of Cx43 expression by high glucose promotes the senescence of GMC, which may be involved in the pathogenesis of diabetic nephropathy.

    Journal of the American Society of Nephrology : JASN 2006;17;6;1532-42

  • Transition from preinvasive carcinoma in situ to seminoma is accompanied by a reduction of connexin 43 expression in Sertoli cells and germ cells.

    Brehm R, Rüttinger C, Fischer P, Gashaw I, Winterhager E, Kliesch S, Bohle RM, Steger K and Bergmann M

    Institute of Veterinary Anatomy, Histology and Embryology, University of Giessen, Giessen, Germany. Ralph.H.Brehm@vetmed.uni-giessen.de

    Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap junctional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and ge 481 rm cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated, that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

    Neoplasia (New York, N.Y.) 2006;8;6;499-509

  • Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue.

    Cluff AH, Byström B, Klimaviciute A, Dahlqvist C, Cebers G, Malmström A and Ekman-Ordeberg G

    Department of Woman and Child Health, Division for Obstetrics and Gynaecology, Karolinska University Hospital Solna, S-171 76 Stockholm, Sweden. ann.hjelm@home.se

    Background: Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour.

    Methods: Uterine isthmic biopsies were collected from non-pregnant (n = 7), term pregnant women not in labour (n = 14), in normal labour (n = 7) and in prolonged labour (n = 7). mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy.

    Results: In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p < 0.05). In term-pregnant tissue, the expression of syndecan 3 and connexin 43 did not differ significantly compared to non-pregnant and normal labour. The immunoreactivity of syndecan 3 was strong at normal labour, in contrast to prolonged labour, where both a weaker expression and an irregular distribution were detected. The immunoreactivity of connexin 43 increased until term and further stronger staining occurred at normal labour. At prolonged labour, the immunoreactivity was weaker and more unevenly distributed. At labour, a co-localization of syndecan 3 and connexin 43 could be demonstrated in the smooth muscle by confocal microscopy.

    Conclusion: The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility.

    Reproductive biology and endocrinology : RB&E 2006;4;24

  • Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels.

    Ebong EE, Kim S and DePaola N

    Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.

    Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and 7ee differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.

    Funded by: NHLBI NIH HHS: HL-64728

    American journal of physiology. Heart and circulatory physiology 2006;290;5;H2015-23

  • Gap junction protein connexin 43 serves as a negative marker for a stem cell-containing population of human limbal epithelial cells.

    Chen Z, Evans WH, Pflugfelder SC and Li DQ

    Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas 77030, USA.

    This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151-187), a sequence that is highly homologous in Cx43, the Cx43(dim) and Cx43(bright) cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43(dim) and Cx43(bright) cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43(dim) cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin beta1 and negative for differentiation markers K3 and involucrin. The Cx43(dim) cells possessed a greater proliferative potential than Cx43(bright) cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells.

    Funded by: NEI NIH HHS: EY014553, EY11915, R01 EY011915, R03 EY014553, R03 EY014553-02

    Stem cells (Dayton, Ohio) 2006;24;5;1265-73

  • Loss of communication in ovarian cancer.

    Toler CR, Taylor DD and Gercel-Taylor C

    Division of Gynecologic Oncology, Department of Obstetrics, Gynecology and Women's Health, University of Louisville School of Medicine, Louisville, KY, USA.

    Objective: This study was undertaken to investigate the expression of connexins in ovarian cancer cell lines and assess correlation with chemosensitivity.

    Eight cell lines were used in this study. Cytotoxicity assays with carboplatin and paclitaxel were performed. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the expression of gap junction proteins. Western blotting and immunohistochemical staining of selective gap junction proteins (connexin 26 and 43) were performed.

    Results: All 11 connexins were detected with RT-PCR profiling in ovarian cancer cells. Further experiments were performed to analyze protein expression with connexins 26 and 43. Levels of connexin proteins did not correlate with gene expression obtained with RT-PCR. Results obtained with immunoblotting were confirmed by immunohistochemistry. Carboplatin and paclitaxel sensitivities were determined and connexin 43 expression was associated with sensitivity to paclitaxel.

    Conclusion: Expression of connexins is regulated at the translational level in ovarian cancer. Sensitivity to paclitaxel maybe associated with connexin 43.

    American journal of obstetrics and gynecology 2006;194;5;e27-31

  • Structural assessment of PITX2, FOXC1, CYP1B1, and GJA1 genes in patients with Axenfeld-Rieger syndrome with developmental glaucoma.

    Cella W, de Vasconcellos JP, de Melo MB, Kneipp B, Costa FF, Longui CA and Costa VP

    Department of Ophthalmology, Faculty of Medical Sciences, State University of Campinas, Campinas, SP, Brazil. wenercella@ig.com.br

    Purpose: Axenfeld-Rieger (AR) is an autosomal dominant disorder with phenotypic heterogeneity characterized by anterior segment dysgenesis, facial bone defects, and redundant periumbilical skin. The PITX2 gene, on chromosome 4q25, and the FOXC1 gene, on chromosome 6p25, have been implicated in the different phenotypes of the syndrome through mutational events. Recently, the CYP1B1 gene was found to be associated with Peters' anomaly, and the gene associated with oculodentodigital dysplasia syndrome, which presents some similarities with AR, was identified (connexin 43--GJA1 gene). The purpose of this study was to evaluate PITX2, FOXC1, CYP1B1, and GJA1 gene mutations in Brazilian families with AR.

    Methods: Eight unrelated patients affected by AR (all eight with glaucoma and three with systemic manifestations) and their families were ophthalmologically evaluated and their blood was collected for DNA extraction purposes. The coding regions of PITX2, FOXC1, CYP1B1, and GJA1 genes were completely evaluated through direct sequencing.

    Results: The frequency of mutations in the FOXC1, GJA1, PITX2, and CYP1B1 genes in this study were 25%, 12.5%, 0% and 0%, respectively. In the FOXC1 gene, two GGC triplet insertions (GGC375ins and GGC447ins) defined as a polymorphism, and two new mutations--a deletion (718 to 719delCT) and a nonsense mutation (Trp152STOP)--were identified. One polymorphism (Ala253Val) was identified in the GJA1 gene in the same family presenting the Trp152STOP mutation in the FOXC1 gene. In this family harboring both structural alterations, two patients who carried the GJA1 (Ala253Val) and FOXC1 (Trp152STOP) mutations developed less severe glaucoma compared with family members presenting the FOXC1 (Trp152STOP) mutation alone.

    Conclusions: Two new structural alterations in the FOXC1 gene and a polymorphism in the GJA1 gene were first described in Brazilian patients with AR and developmental glaucoma. A polymorphism in the GJA1 gene (Ala253Val), for the first time identified in association with AR, raises the possibility of its participation as a modifier gene.

    Investigative ophthalmology & visual science 2006;47;5;1803-9

  • Up-regulation of connexin43 correlates with increased synthetic activity and enhanced contractile differentiation in TGF-beta-treated human aortic smooth muscle cells.

    Rama A, Matsushita T, Charolidi N, Rothery S and Dupont E

    Cardiac Medicine, National Heart and Lung Institute (Imperial College), Guy Scadding Building, Dovehouse Street, London SW3 6LY, UK.

    Up-regulation of the gap-junctional protein connexin43 (Cx43) in arterial smooth muscle cells (SMCs) features in response to injury and in atherosclerosis, in parallel with phenotypic transition to the synthetic state. TGF-beta1 is known to have a role in SMC differentiation and extracellular matrix (ECM) synthesis, key characteristics of phenotypic state. Here, we set out to examine the effects of TGF-beta1 on Cx43-gap junction expression in relation to SMC differentiation, ECM synthesis and growth. Cx43 expression was analysed by immunoconfocal microscopy and Western blotting in primary human aortic SMCs treated with TGF-beta1 over a 48-h period, with assessment of gap-junctional communication by cell-to-cell transfer of microinjected ethidium bromide. In parallel, synthetic activity was analysed by Northern blotting for ECM components alpha-1(I) and alpha1(III) procollagen transcripts, contractile differentiation was assessed by immunoconfocal microscopy and Western blotting of the markers smooth muscle alpha-actin, calponin and smooth muscle heavy chain isoform 1 (SM1), and growth was measured by BrdU incorporation. Our results demonstrate that TGF-beta1 significantly up-regulates Cx43 expression and intercellular communication, in concert with increased expression of alpha-actin, calponin and SM1. Concomitant with contractile protein expression, ECM synthesis was increased rather than decreased, TGF-beta1 inducing a significant up-regulation of both procollagen transcripts. These effects were independent of growth. We conclude that in human aortic SMCs, TGF-beta1 treatment leads to up-regulation of Cx43-mediated gap-junctional communication and increased synthetic activity yet, somewhat paradoxically, also enhanced contractile differentiation.

    European journal of cell biology 2006;85;5;375-86

  • Modulation of connexin43 alters expression of osteoblastic differentiation markers.

    Li Z, Zhou Z, Saunders MM and Donahue HJ

    Division of Musculoskeletal Sciences, Department of Orthopaedics & Rehabilitation, Pennsylvania State University College of Medicine 500 University Dr., Hershey, 17033, USA. zhongyongli@psu.edu

    Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43(-)). hFOB/Cx43(-) cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication re 829 lative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43(-) cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43(-) cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor alpha1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43(-). Osteopontin mRNA levels were increased in hFOB/Cx43(-) relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43(-) and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells.

    Funded by: NIA NIH HHS: AG-13087

    American journal of physiology. Cell physiology 2006;290;4;C1248-55

  • Reduced atrial connexin43 expression after pediatric heart surgery.

    Pavlovic M, Schaller A, Ammann RA, Sanz J, Pfammatter JP, Carrel T, Berdat P and Gallati S

    Division of Pediatric Cardiology, University Children's Hospital, Berne, Switzerland. mladen.pavlovic@insel.ch

    Myocardial dysfunction and arrhythmias may be induced by congenital heart defects, but also be the result of heart surgery with cardiopulmonary bypass (CPB), potentially caused by differential expression of connexin40 (Cx40) and connexin43 (Cx43). In 16 pediatric patients undergoing corrective heart surgery, connexin mRNA expression was studied in volume overloaded (VO group, n=8) and not overloaded (NO group, n=8) right atrial myocardium, excised before and after CPB. Additionally, in eight of these patients ventricular specimens were investigated. The atrial Cx43 expression decreased during CPB, which was restricted to the VO group (p=0.008). In contrast, atrial Cx40 mRNA did not change during CPB. In ventricular myocardium compared to atrial mRNA levels, Cx40 was lower (p=0.006) and Cx43 higher (p=0.017) expressed, without significant change during CPB. This study revealed a significant influence of CPB and the underlying heart defect on Cx43 expression.

    Biochemical and biophysical research communications 2006;342;1;310-5

  • Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel.

    Beahm DL, Oshima A, Gaietta GM, Hand GM, Smock AE, Zucker SN, Toloue MM, Chandrasekhar A, Nicholson BJ and Sosinsky GE

    Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA.

    Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the alpha- and beta-connexin subgroups but not the gamma-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.

    Funded by: NCI NIH HHS: CA048049; NCRR NIH HHS: P41RR02250, RR04050; NIGMS NIH HHS: GM048773, GM065937, GM072881, R01 GM065937

    The Journal of biological chemistry 2006;281;12;7994-8009

  • Connexin43: emerging role in erectile function.

    Pointis G

    INSERM U 670, Faculté de Médecine, IFR 50, 28 Avenue de Valombrose, 06107 Nice Cedex 02, France. pointis@unice.fr

    Connexins, that have their main function as part of gap junction channels, are proteins expressed in a large number of tissues such as endocrine, nervous, vascular, and muscular tissues. Gap junctions are implicated in tissue homeostasis and control of cell proliferation and differentiation. Interestingly, mutations of connexin genes have been reported in several human diseases (peripheral neuropathies, cardiovascular and dermatological diseases, hereditary cataract, and deafness) and altered expression of connexins have been associated with tumoral progression. Today, several lines of study argue for a critical role of gap junctions in corporal smooth muscle relaxation and erectile response. The present review highlights the emerging role of connexin43, one of these membranous proteins, in the physiology and physiopathology of human erectile function and its possible medical application.

    The international journal of biochemistry & cell biology 2006;38;10;1642-6

  • 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

  • Molecular cloning and functional analysis of a novel Cx43 partner protein CIP150.

    Akiyama M, Ishida N, Ogawa T, Yogo K and Takeya T

    Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0129, Japan.

    We identified and cloned a novel gene encoding a partner protein, CIP150, of connexin 43 (Cx43). CIP150 associates with Cx43 through its carboxyl terminal domain. Conversely, a region consisting of 16 amino acids in the juxtamembrane region (amino acids 227-242) in the carboxyl terminal tail of Cx43 was identified to be responsible for the association. A variant of Cx43 lacking this region was expressed only in a nonphosphorylated form and appeared to lose the capacity to localize to the region of cell-cell contact and dye transfer activity. When the expression of CIP150 was suppressed using small interfering RNA, the localization to the plasma membrane as well as dye transfer activity of Cx43 was significantly reduced. These results suggest that the newly identified domain is essential for the proper phosphorylation and localization of Cx43, and CIP150 is a novel partner protein targeting this domain.

    Biochemical and biophysical research communications 2005;335;4;1264-71

  • A novel mutation in the GJA1 gene in a family with oculodentodigital dysplasia.

    Vasconcellos JP, Melo MB, Schimiti RB, Bressanim NC, Costa FF and Costa VP

    Department of OphthalmologyState University of Campinas, Campinas, Brazil. cabraljp@uol.com.br

    Objectives: To describe a Brazilian family with oculodentodigital dysplasia (ODDD) and to screen for mutations in the gap junction protein alpha 1 (GJA1) gene in this family.

    Methods: Twelve members of a 3-generation family with ODDD underwent screening for mutations of the GJA1 gene and a comprehensive ophthalmic examination. We defined ODDD on the basis of clinical characteristics described in this syndrome (microdontia, caries, enamel hypoplasia, thin nose, and syndactyly) and eye abnormalities such as microphthalmos, iris atrophy, and glaucoma. Direct sequencing of the GJA1 gene was performed using DNA collected from peripheral blood. A control group of 60 healthy individuals underwent evaluation by means of enzyme digestion.

    Results: Among the 8 members of this family who were characterized as having ODDD, 2 showed chronic angle-closure glaucoma, and 1 had open-angle glaucoma. A new mutation in the GJA1 gene was identified, consisting of a change from proline to histidine at codon 59. This mutation segregated through members with the ODDD phenotype. Analysis of the control group by means of restriction fragment length polymorphism (MvaI enzyme) did not disclose this mutation.

    Conclusion: Our results demonstrate a new mutation (P59H) in the GJ1A gene, identified in a family with ODDD syndrome. Clinical Relevance The presence of different forms of glaucoma in families with ODDD may indicate a new mutation in the GJA1 gene.

    Archives of ophthalmology (Chicago, Ill. : 1960) 2005;123;10;1422-6

  • New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43.

    Nicchia GP, Srinivas M, Li W, Brosnan CF, Frigeri A and Spray DC

    Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, Bari, Italy. p.nicchia@biologia.uniba.it

    Aquaporin-4 (AQP4), the main water channel in the brain, is expressed in the perivascular membranes of mouse, rat, and human astrocytes. In a previous study, we used small interfering RNA (siRNA) to specifically knock down AQP4 in rat astrocyte primary cultures and found that together with reduced osmotic permeability, AQP4 knockdown (KD) led to altered cell morphology. However, a recent report on primary cultured astrocytes from AQP4 null mice (KO) showed no morphological differences compared with wild types. In this study, we compared the effect of AQP4 KD in mouse, rat, and human astrocyte primary cultures and found that AQP4 KD in human astrocytes resulted in a morphological phenotype similar to that found in rat. In contrast, AQP4 KD in mouse astrocytes caused only very mild morphological changes. The actin cytoskeleton of untreated astrocytes exhibited strong species-specific differences, with F-actin being organized in cortical bands in mouse and in stress fibers in rat and human astrocytes. Surprisingly, as a consequence of AQP4 KD, F-actin cytoskeleton was depolymerized in rat and human whereas it was completely rearranged in mouse astrocytes. Although AQP4 KD induced alterations of the cell cytoskeleton, we found that the expression of dystrophin (DP71), beta-dystroglycan, and alpha-syntrophin was not altered. AQP4 KD in cultured mouse astrocytes produced strong down-regulation of connexin43 (Cx43) with a concomitant reduction in cell coupling while no major alterations in Cx43 expression were found in rat and human cells. Taken together, these results demonstrate that with regard to these properties, human astrocytes in culture are more similar to rat than to mouse astrocytes. Moreover, even though AQP4 KD in mouse astrocytes did not result in a dramatic morphological phenotype, it induced a remarkable rearrangement of F-actin, not related to disruption of the dystrophin complex, indicating a primary role of this water channel in the cytoskeleton changes observed. Finally, the strong down-regulation of Cx43 and cell coupling in AQP4 KD mouse astrocytes indicate that a functional relationship likely exists between water channels and gap junctions in brain astrocytes.

    Funded by: NINDS NIH HHS: NS41282

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;12;1674-6

  • Novel GJA1 mutations in patients with oculo-dento-digital dysplasia (ODDD).

    P, Van Esch H, Huysmans C, Pijkels E, De Smet L, Van de Ven W, Devriendt K and Fryns JP

    Department of Orthopaedics, University Hospital Pellenberg, Weligerveld 1, B-3212 Pellenberg, Belgium. philippe.debeer@uz.kuleuven.ac.be

    European journal of medical genetics 2005;48;4;377-87

  • Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins.

    Wang M, Martínez AD, Berthoud VM, Seul KH, Gemel J, Valiunas V, Kumari S, Brink PR and Beyer EC

    Department of Pediatrics, Section of Hematology/Oncology and Stem Cell Transplantation, University of Chicago, Chicago, IL, USA.

    Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.

    Funded by: NEI NIH HHS: EY08368, R01 EY008368, R01 EY008368-15A2; NHLBI NIH HHS: HL45466, HL59199, R01 HL045466, R01 HL059199, R01 HL059199-06; NICHD NIH HHS: P01 HD009402, P01 HD009402-290012

    Biochemical and biophysical research communications 2005;333;4;1185-93

  • Mutations of connexin43 in fetuses with congenital heart malformations.

    Chen P, Xie LJ, Huang GY, Zhao XQ and Chang C

    Pediatric Heart Center, Children's Hospital of Fudan University, Shanghai 200032, China.

    Background: Gap junction channels formed by connexin43 (Cx43) protein are important in cardiac morphogenesis, and Cx43 gene is thought to be associated with congenital heart malformation (CHM). This study was undertaken to detect the mutations of Cx43 in fetuses with CHM.

    Methods: Cx43 extron DNA was amplified by PCR from 16 fetuses with a variety of CHM. The PCR products were analyzed by SSCP and DNA sequencing. Thirty children who had no CHM were selected as controls.

    Results: Eight homozygous mutations of Cx43 were observed in a fetus with double outlet right ventricule (DORV), five of the 8 mutations were missense mutations including Arg239Trp, Ser251Thr, Ala253Pro, Pro283Leu and Thr290Asn, and the remaining 3 were silent polymorphisms including Gly252Gly, Pro256Pro and Thr275Thr. No mutations were found in other fetuses and the control group.

    Conclusions: Mutations of Cx43 may be associated with congenital conotruncal anomalies. PCR-SSCP is an effective method for screening the mutations of Cx43.

    Chinese medical journal 2005;118;12;971-6

  • Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum.

    Maza J, Das Sarma J and Koval M

    Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.

    Funded by: NHLBI NIH HHS: HL019737-300018, P01 HL019737, P01 HL019737-300018, P01-HL019737-26; NIGMS NIH HHS: GM061012-06, GM61012, R01 GM061012, R01 GM061012-06

    The Journal of biological chemistry 2005;280;22;21115-21

  • Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.

    Déglise S, Martin D, Probst H, Saucy F, Hayoz D, Waeber G, Nicod P, Ris HB, Corpataux JM and Haefliger JA

    Department of Thoracic and Vascular Surgery, University Hospital, Lausanne, Switzerland.

    Objective: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity.

    Methods: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis.

    Results: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression.

    Conclusions: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia.

    Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.

    Journal of vascular surgery 2005;41;6;1043-52

  • The phosphorylated form of connexin43 is up-regulated in breast hyperplasias and carcinomas and in their neoformed capillaries.

    Gould VE, Mosquera JM, Leykauf K, Gattuso P, Dürst M and Alonso A

    Department of Patholgy, Rush Medical College, Chicago, IL 60611, USA.

    We applied an antiserum (SA226P) specifically recognizing the phosphorylated form of connexin43 (P-Cx43) to human breast samples including normal breast samples, with fibrocystic disease (FCD), fibroadenomas (FA), in situ and infiltrating carcinomas of all major types, and miscellaneous extramammary tumors. The findings were compared with those obtained with commercial antisera recognizing all Cx43 forms (pan-Cx43). A subset of samples was stained for Her2-neu and p44/42 to mitogen-activated protein kinase. Paraffin step sections were used. Immunoblots were performed on frozen samples of a representative subset of cases. In the normal breast, FCD, and FA, SA226P stained strongly and extensively most myoepithelial cells (MECs); luminal cells remained unstained. In proliferative FCD and some cellular FA, SA226P stained MEC and the capillary endothelium (CE). In ductal and lobular in situ carcinomas, SA226P reacted strongly and diffusely with the remaining MEC, the CE, and the transformed luminal cells. SA226P stained all infiltrating carcinomas except the tubular variant. In all breast carcinomas, the CE within and adjacent to tumors and some myofibroblasts stained with SA226P. By contrast, pan-Cx43 stained weakly and sporadically the MEC and rare samples of invasive carcinomas. Notably, Mab p44/42 reacted in parallel with the samples stained with SA226P, whereas reactions with Her2 were negative. Immunoblot findings paralleled those obtained immunohistochemically. We conclude that P-Cx43, restricted to MEC in the normal breast, is up-regulated in the same cells in hyperplasias and dysplasias and FA and is strongly up-regulated in invasive carcinomas. Notably, in some proliferative FCD and in most in situ and infiltrating carcinomas, P-Cx43 is strongly expressed in CE within and adjacent to the lesions but not away from them. These findings were paralleled by the strong nuclear reactions noted with Mab p44/42. These phenomena, although not exclusive to malignancy, are particularly conspicuous in breast carcinomas and seemingly reflect active proliferation associated with abnormal gap junctional intercellular communication.

    Human pathology 2005;36;5;536-45

  • Restoration of functional gap junctions through internal ribosome entry site-dependent synthesis of endogenous connexins in density-inhibited cancer cells.

    Lahlou H, Fanjul M, Pradayrol L, Susini C and Pyronnet S

    INSERM U531, Institut Louis Bugnard, CHU Rangueil, TSA 50032, 31059 Toulouse cedex 9, France.

    Gap junctions are composed of connexins and are critical for the maintenance of the differentiated state. Consistently, connexin expression is impaired in most cancer cells, and forced expression of connexins following cDNA transfection reverses the tumor phenotype. We have found that the restoration of density inhibition of human pancreatic cancer cells by the antiproliferative somatostatin receptor 2 (sst2) is due to overexpression of endogenous connexins Cx26 and Cx43 and consequent formation of functional gap junctions. Immunoblotting along with protein metabolic labeling and mRNA monitoring revealed that connexin expression i a11 s enhanced at the level of translation but is not sensitive to the inhibition of cap-dependent translation initiation. Furthermore, we identified a new internal ribosome entry site (IRES) in the Cx26 mRNA. The activity of Cx26 IRES and that of the previously described Cx43 IRES are enhanced in density-inhibited cells. These data indicate that the restoration of functional gap junctions is likely a critical event in the antiproliferative action of the sst2 receptor. We further suggest that the existence of IRESes in connexin mRNAs permits connexin expression in density-inhibited or differentiated cells, where cap-dependent translation is generally reduced.

    Molecular and cellular biology 2005;25;10;4034-45

  • Novel rab GAP-like protein, CIP85, interacts with connexin43 and induces its degradation.

    Lan Z, Kurata WE, Martyn KD, Jin C and Lau AF

    Natural Products and Cancer Biology Program, Cancer Research Center of Hawaii, Honolulu, Hawaii 96813, USA.

    Gap junctions play critical roles in tissue function and homeostasis. Connexin43 (Cx43) is a major gap junction protein expressed in the mammalian heart and other tissues and may be regulated by its interaction with other cellular proteins. Using the yeast two-hybrid screen, we identified a novel Cx43-interacting protein of 85-kDa, CIP85, which contains a single TBC, SH3, and RUN domain, in addition to a short coiled coil region. Homologues containing this unique combination of domains were found in human, D. melanogaster, and C. elegans. CIP85 mRNA is expressed ubiquitously in mouse and human tissues. In vitro interaction assays and in vivo co-immunoprecipitation experiments confirmed the interaction of endogenous CIP85 with Cx43. In vitro interaction experiments using CIP85 mutants with in-frame deletions of the TBC, SH3, and RUN domains indicated that the SH3 domain of CIP85 is involved in its interaction with Cx43. Conversely, analysis of Cx43 mutants with proline to alanine substitutions in the two proline-rich regions of Cx43 revealed that the P(253)LSP(256) motif is an important determinant of the ability of Cx43 to interact with CIP85. Laser-scanning confocal microscopy showed that CIP85 colocalized with Cx43 at the cell periphery, particularly in areas reminiscent of gap junction plaques. The functional importance of the interaction between CIP85 and Cx43 was suggested by the observation that CIP85 appears to induce the turnover of Cx43 through the lysosomal pathway.

    Funded by: NCI NIH HHS: CA52098, R01 CA052098, R01 CA052098-16

    Biochemistry 2005;44;7;2385-96

  • A 2-bp deletion in the GJA1 gene is associated with oculo-dento-digital dysplasia with palmoplantar keratoderma.

    van Steensel MA, Spruijt L, van der Burgt I, Bladergroen RS, Vermeer M, Steijlen PM and van Geel M

    Department of Dermatology, University Hospital Maastricht, Maastricht, The Netherlands. mvst@sder.azm.nl

    Oculo-dento-digital dysplasia (ODDD, OMIM no. 164210) is a pleiotropic disorder characterized mainly by ocular anomalies, varying degrees of finger and toe syndactyly, and enamel defects. It is caused by missense mutations in the gene coding for the gap junction protein connexin 43 or GJA1. Other types of mutations have so far not been reported. Here we describe a Dutch kindred with ODDD showing a new symptom, palmoplantar keratoderma, and associated with a novel 2-bp deletion mutation of GJA1. The dinucleotide deletion 780_781delTG is located in the cytoplasmic C-terminal loop and leads to a frameshift. This is predicted to lead to the production of a slightly truncated protein with 46 incorrect amino acids in the C-terminal cytoplasmic loop (C260fsX307). This novel mutation may explain the presence of skin symptoms.

    American journal of medical genetics. Part A 2005;132A;2;171-4

  • Connexin 43 downregulation and dephosphorylation in nonischemic heart failure is associated with enhanced colocalized protein phosphatase type 2A.

    Ai X and Pogwizd SM

    Department of Medicine, University of Illinois at Chicago, IL 60612, USA.

    In nonischemic heart failure (HF), ventricular tachycardia initiates by a nonreentrant mechanism, but there is altered conduction (that could lead to re-entry) that could arise from changes in gap junctional proteins, especially connexin43 (Cx43). We studied Cx43 expression and phosphorylation state in the left ventricle (LV) from an arrhythmogenic rabbit model of nonischemic HF and from patients with HF attributable to idiopathic dilated cardiomyopathy. We also investigated the role of protein phosphatases that dephosphorylate Cx43--PP1 and PP2A. In HF rabbit LV, Cx43 mRNA and total protein were decreased by 29% and 34%, respectively (P<0.05 and P<0.001). In controls, Cx43 was primarily in the phosphorylated state, but with HF there was a 64% increase in nonphosphorylated Cx43 (Cx43-NP, normalized to total Cx43; P<0.05). Similar results were noted in HF rabbit myocytes (P<0.05) and in human idiopathic dilated cardiomyopathy LV (P<0.05). We found that PP1 and PP2A colocalized with Cx43 in rabbit LV. With HF, the level of colocalized PP2A increased >2.5-fold (P<0.002), whereas colocalized PP1 was unchanged. We also found intercellular coupling (assessed by Lucifer Yellow dye transfer) was markedly reduced in HF. However, okadaic acid (10 nmol/L) reduced the amount of Cx43-NP and significantly improved cell coupling in HF. Thus, in nonischemic HF in rabbits and humans, there is a decrease in both Cx43 expression and phosphorylation that contributes to uncoupling. Increased levels of PP2A that colocalize with Cx43 can underlie enhanced levels of Cx43-NP in HF. Modulation of Cx43 phosphorylation may be a potential therapeutic target to improve conduction in HF.

    Circulation research 2005;96;1;54-63

  • The expression of connexin 43 in children with Tetralogy of Fallot.

    Kołcz J, Drukała J, Bzowska M, Rajwa B, Korohoda W and Malec E

    Department of Pediatric Cardiac Surgery, Polish-American Children's Hospital, Collegium Medicum, Jagiellonian University, Wielicka 265, 30-663 Kraków, Poland.

    Abnormalities in the expression and distribution of Connexin 43 (Cx43) in cardiomyocytes may lead to anomalous conotruncal embryogenesis and disturbances in the maturation and function of the heart. Tetralogy of Fallot (TOF) is the most frequent, cyanotic congenital heart defect in which conotruncal anomalies, right ventricle dysfunction and life-threatening arrhythmias occur. In this study, age-related changes in the expression and spatial distribution of Cx43 in cardiomyocytes from TOF children compared to patients without right ventricular outflow tract pathology were determined Confocal microscopy and flow cytometry were used to assess the changes. Disturbances in both the expression and distribution of Cx43 were found. In the group of infants with TOF, a lower level of expression of the protein was determined. Cardiomyocytes from TOF hearts were found to have Cx43 distributed over their entire surface, which is the pattern seen in immature tissue. In the controls, Cx43 was located within the intercalated disks. Expression of Cx43 in TOF hearts increases with the age of the subject, whereas its spatial distribution remains the same in both infants and older children. Disturbances in Cx43 expression and localization may influence heart embryogenesis and maturation, contribute to hypertrophy and dysfunction of the right ventricle and induce arrhythmias in children with TOF. Early redistribution of Cx43 and functional maturation of the heart muscle support a strategy of early total correction of the defect.

    Cellular & molecular biology letters 2005;10;2;287-303

  • Connexin 43 mediated gap junctional communication enhances breast tumor cell diapedesis in culture.

    Pollmann MA, Shao Q, Laird DW and Sandig M

    Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada. mpollman@uwo.ca

    Introduction: Metastasis involves the emigration of tumor cells through the vascular endothelium, a process also known as diapedesis. The molecular mechanisms regulating tumor cell diapedesis are poorly understood, but may involve heterocellular gap junctional intercellular communication (GJIC) between tumor cells and endothelial cells.

    Method: To test this hypothesis we expressed connexin 43 (Cx43) in GJIC-deficient mammary epithelial tumor cells (HBL100) and examined their ability to form gap junctions, establish heterocellular GJIC and migrate through monolayers of human microvascular endothelial cells (HMVEC) grown on matrigel-coated coverslips.

    Results: HBL100 cells expressing Cx43 formed functional heterocellular gap junctions with HMVEC monolayers within 30 minutes. In addition, immunocytochemistry revealed Cx43 localized to contact sites between Cx43 expressing tumor cells and endothelial cells. Quantitative analysis of diapedesis revealed a two-fold increase in diapedesis of Cx43 expressing cells compared to empty vector control cells. The expression of a functionally inactive Cx43 chimeric protein in HBL100 cells failed to increase migration efficiency, suggesting that the observed up-regulation of diapedesis in Cx43 expressing cells required heterocellular GJIC. This finding is further supported by the observation that blocking homocellular and heterocellular GJIC with carbenoxolone in co-cultures also reduced diapedesis of Cx43 expressing HBL100 tumor cells.

    Conclusion: Collectively, our results suggest that heterocellular GJIC between breast tumor cells and endothelial cells may be an important regulatory step during metastasis.

    Breast cancer research : BCR 2005;7;4;R522-34

  • Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368.

    Richards TS, Dunn CA, Carter WG, Usui ML, Olerud JE and Lampe PD

    Division of Basic and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

    Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site-specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

    Funded by: NIAID NIH HHS: T32 AI007509, T32-AI07509; NIAMS NIH HHS: AR47963, R01 AR047963; NIDDK NIH HHS: DK59221, R01 DK059221; NIGMS NIH HHS: GM55632, R01 GM055632

    The Journal of cell biology 2004;167;3;555-62

  • Are gap junction gene connexins 26, 32 and 43 of prognostic values in hepatocellular carcinoma? A prospective study.

    Sheen IS, Jeng KS, Wang PC, Shih SC, Chang WH, Wang HY, Chen CC and Shyung LR

    Department of Surgery, Mackay Memorial Hospital, No.92, Sec 2,Chung-San North Road, Taipei, Taiwan, China. issheen.jks@msa.hinet.net

    Aim: To investigate the prognostic value of the expression of connexin (Cx) 26, 32 and 43 messenger RNA (mRNA) in hepatocellular carcinoma (HCC) tissues.

    Methods: Using a reverse-transcriptase polymerase chain reaction (RT-PCR), Cx 26, Cx 32 and Cx 43 mRNAs were determined in the liver tissues of 15 controls and in HCC tissues of 25 patients undergoing curative hepatic resection. The patients were followed up clinically.

    Results: Cx 26 and Cx 32 mRNAs were significantly lower in HCC tissues compared with controls (both P<0.01). By multivariate analysis, a lower level of Cx 26 and Cx 32 mRNA correlated significantly with a risk of HCC recurrence (P = 0.033) and recurrence-related mortality (P = 0.031, P = 0.031). Cx 43 mRNA was higher in HCC tissues compared with controls but did not correlate with postoperative recurrence or recurrence-related mortality. Other significant predictors of HCC recurrence included cellular dedifferentiation (P = 0.033), less encapsulation (P = 0.050), vascular permeation (P = 0.046), and daughter nodules (P = 0.046). Significant variables related to recurrence-related mortality consisted of cell dedifferentiation (P = 0.031), vascular permeation (P = 0.048), and daughter nodules (P = 0.048). The levels of Cx 26 and Cx 32 mRNAs correlated significantly with cell differentiation (P = 0.031).

    Conclusion: A low expression of Cx 26 and Cx 32 mRNAs in HCC tissues is predictive of postoperative recurrence of HCCs.

    World journal of gastroenterology 2004;10;19;2785-90

  • 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

  • Connexin43 interacts with NOV: a possible mechanism for negative regulation of cell growth in choriocarcinoma cells.

    Gellhaus A, Dong X, Propson S, Maass K, Klein-Hitpass L, Kibschull M, Traub O, Willecke K, Perbal B, Lye SJ and Winterhager E

    Institute of Anatomy and Cell Biology, University Hospital Essen, 55 Hufelandstrasse, 45122 Essen, Germany.

    The gap junction protein connexin43 (Cx43) is thought to be involved in growth control in several tissues. Using the doxycycline inducible tet-on system, we generated human malignant trophoblast Jeg3 cells transfected with either Cx40, Cx43, or C-terminal truncated Cx43 (trCx43). Cx43, but not Cx40 or trCx43, displayed a reduced cell growth of Jeg3 cells in vitro and tumor growth in nude mice, suggesting a role of the C terminus of Cx43 in growth regulation. Using gene array analysis, the growth regulator NOV (CCN3), a member of the CCN gene family, was found to be up-regulated only in the Cx43-transfected cells. Validation by reverse transcriptase-PCR confirmed an up-regulation of the NOV transcript exclusively upon Cx43 induction. In contrast to Cx40 or trCx43, induction of Cx43 led to a switch in localization of NOV from the nucleus to the cell membrane, where it is colocalized with Cx43. Coimmunoprecipitation showed a binding of NOV to the C terminus of Cx43 in vitro as well as in transfected cells. Jeg3 cells transfected only with NOV revealed that NOV itself acts as a growth regulator. We suggest that Cx43 is able to regulate cell growth via an up-regulation of NOV transcription, a change in localization of the NOV protein and a binding of NOV to the C terminus of Cx43.

    Funded by: NICHD NIH HHS: 1R01 HD 42558-01

    The Journal of biological chemistry 2004;279;35;36931-42

  • Novel Connexin 43 (GJA1) mutation causes oculo-dento-digital dysplasia with curly hair.

    Kjaer KW, Hansen L, Eiberg H, Leicht P, Opitz JM and Tommerup N

    Wilhelm Johannsen Centre for Functional Genome Research, The Panum Institute Building 24.4, Department of Medical Genetics, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. klaus@medgen.ku.dk

    Oculo-dento-digital dysplasia (ODDD) [OMIM 164200] is a rare autosomal dominant pleiotropic disorder comprising ocular, craniofacial, and digital anomalies, caused by mutations in the gap junction alpha-1 gene (GJA1 or Connexin 43 (CX43)) [Paznekas et al., 2003]. In a Danish family affected over five generations, we found a novel mutation, 286G --> A, resulting in Val96Met. We provide an easy method for mutation detection by use of the restriction enzyme Nde1 and discuss possible pathogenetic mechanisms, arguing that loss of function cannot be excluded. This is the second article reporting ODDD mutations.

    American journal of medical genetics. Part A 2004;127A;2;152-7

  • Connexins 26, 30, and 43: differences among spontaneous, chronic, and accelerated human wound healing.

    Brandner JM, Houdek P, Hüsing B, Kaiser C and Moll I

    Department of Dermatology and Venerology, University Hospital Hamburg-Eppendorf, Hamburg, Germany. brandner@uke.uni-hamburg.de

    Gap junctions (GJ) are known to be involved in spontaneous wound healing in rodent skin. We analyzed the staining patterns of the GJ proteins Cx26, Cx30, and Cx43 in human cutaneous wound healing and compared ex vivo spontaneous wound healing to non-healing wounds (chronic leg ulcers) and to ex vivo accelerated wound healing after transplantation of cultured keratinocytes. We demonstrate a loss of Cx43 staining at the wound margins during initial wound healing and after transplantation of keratinocytes. In contrast, Cx43 remains present at the margins of most non-healing wounds. We show a subsequent induction of Cx26 and Cx30 near the wound margins in spontaneous wound healing and-even earlier-after the transplantation of keratinocytes. The cells at the wound margins remain negative until the commencement of epidermal regeneration. Cx26/30 are present at the wound margins of most non-healing wounds. Cx stainings are absent in the transplanted keratinocytes during early wound healing, but there is a subsequent induction. Our results suggest that the downregulation of Cx43 is an important event in human wound healing. We discuss the assumption that direct cell-cell communication via GJ contribute to the acceleration of wound healing after the transplantation of keratinocytes.

    The Journal of investigative dermatology 2004;122;5;1310-20

  • Human mesenchymal stem cells make cardiac connexins and form functional gap junctions.

    Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR and Cohen IS

    Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.

    Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated 1f40 canine ventricular myocytes. The junctional currents (I(j)) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (V(j)) dependence. I(j) records from hMSC-HeLaCx43 and hMSC-HeLaCx40 cell pairs also showed symmetrical and asymmetrical V(j) dependence, while hMSC-HeLaCx45 pairs always produced asymmetrical I(j) with pronounced V(j) gating when the Cx45 side was negative. Symmetrical I(j) suggests that the dominant functional channel is homotypic, while the asymmetrical I(j) suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (gamma(j)) of 30-80 pS. The macroscopic I(j) obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical V(j) dependence, while single channel events revealed gamma(j) of the size range 40-100 pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.

    Funded by: NEI NIH HHS: R01 EY014604; NHLBI NIH HHS: P01 HL028958, R01 HL020558, R01 HL067101, R37 HL020558; NIDDK NIH HHS: P01 DK060037; NIGMS NIH HHS: R01 GM055263

    The Journal of physiology 2004;555;Pt 3;617-26

  • A homozygous GJA1 gene mutation causes a Hallermann-Streiff/ODDD spectrum phenotype.

    Pizzuti A, Flex E, Mingarelli R, Salpietro C, Zelante L and Dallapiccola B

    Ospedale Casa Sollievo della Sofferenza and Istituto CSS-Mendel, Roma, Italy. a.pizzuti@css-mendel.it

    Oculodentodigital dysplasia (ODDD) and Hallermann-Streiff syndrome (HSS) share several clinical characteristics. However, while ODDD is a dominantly inherited disorder due to mutations in the connexin 43 gene GJA1, the inheritance pattern of the HSS syndrome is still debated. Overlapping phenotypes have been described. In one of such cases we found a homozygous change at the very conserved R76 codon (c.227G>A, p.R76H), the clinically normal parents being heterozigous carriers of the same mutation. A different base change at the same codon (p.R76S) leads to a complete dominant ODDD phenotype. A case of full-blown HSS phenotype was also analysed but GJA1 mutations were not found. GJA1 homozygous hypomorphic mutations can result in a phenotype in the HSS/ODDD spectrum.

    Human mutation 2004;23;3;286

  • Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes.

    Doble BW, Dang X, Pi 10f ng P, Fandrich RR, Nickel BE, Jin Y, Cattini PA and Kardami E

    Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg MB R3E 3J7, Canada.

    Mitogenic stimulation of cardiomyocytes is associated with decreased gap junction coupling and protein kinase C (PKC)-mediated phosphorylation of the gap junction protein connexin43 (Cx43). Identification of and interference with the amino acid(s) that becomes phosphorylated in response to stimulation are important steps towards defining the relationship between Cx43 phosphorylation and cell cycle. Using immunoblotting and phosphospecific antibodies we were able to show that serine-262 (S262) on Cx43 becomes phosphorylated in response to growth factor or PKC stimulation of cardiomyocytes. To examine the effect of Cx43, S262 phosphorylation and cell-cell contact (and/or coupling) on DNA synthesis, we overexpressed wild-type (wt) or mutant Cx43, carrying a S262-to-alanine (S262A, simulating the unphosphorylated state) or a S262-to-aspartate (S262D, simulating constitutive phosphorylation) substitutions in cultures of cell-cell contact forming or isolated cardiomyocytes. Overexpression of wt-Cx43 caused a significant decrease in DNA synthesis irrespective of the presence of cell-cell contact. In cell-cell contact forming cultures, the S262D mutation reversed while the S262A mutation increased the inhibitory effect of Cx43. In the absence of cell-cell contact, the S262-Cx43 mutations had no significant effect on Cx43 inhibition of DNA synthesis. Dye-coupling, evaluated by scrape-loading, indicated increased gap junction permeability in S262A (compared to wt or S262D) overexpressing myocytes. We conclude that Cx43 inhibits cardiomyocyte DNA synthesis irrespectively of cell-cell contact or coupling. Cell-cell contact, and possibly gap junction-mediated communication is required, however, in order to reverse Cx43 inhibition of DNA synthesis by S262 phosphorylation.

    Journal of cell science 2004;117;Pt 3;507-14

  • Expression of Gja1 correlates with the phenotype observed in oculodentodigital syndrome/type III syndactyly.

    Richardson R, Donnai D, Meire F and Dixon MJ

    Journal of medical genetics 2004;41;1;60-7

  • Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study.

    Kanczuga-Koda L, Sulkowski S, Koda M, Sobaniec-Lotowska M and Sulkowska M

    Department of Pathology, Medical University, Bialystok, Poland.

    Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs), which consist of proteins called connexins (Cxs). GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of colorectal tumours. Tissue samples (50 cases) were collected from normal colon, at the maximum distance from the tumor. Using antibodies for Cx26, Cx32 and Cx43, immunohistochemical detection was made. In epithelial cells, strong Cx26 immunoreactivity was found, whereas Cx32 and Cx43 were sparsely distributed. Strong Cx43 immunostaining in muscularis mucosae was observed. In the circular layer of muscularis externa, expression of Cx43 and Cx26 was seen, but only in the portion closest to the submucosa. No immunoreactivity was found in the longitudinal muscle layer. Small vessels stained positively only for Cx43. Furthermore, there was no difference in staining between samples derived from various sections of the colon. This study showed immunohistochemically for the first time the expression of Cx26 in human colon mucosa.

    Folia histochemica et cytobiologica 2004;42;4;203-7

  • Correlation of connexin43 expression and late ventricular potentials in nonischemic dilated cardiomyopathy.

    Kitamura H, Yoshida A, Ohnishi Y, Okajima K, Ishida A, Galeano EJ, Kubo S, Fukuzawa K, Takano T and Yokoyama M

    Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.

    Nonischemic dilated cardiomyopathy (DCM) is associated with a high risk of sudden cardiac death. Signal-averaged electrocardiography (SAECG) is a useful clinical tool for detecting late ventricular potentials (LP). Gap junction alterations have recently been shown to be involved in the pathogenesis of ventricular arrhythmias in DCM; however, the possible relationship between gap junctional connexin43 (C x 43) expression and SAECG has not yet been evaluated. In the present study 16 patients (47+/-13 years) with DCM who had undergone SAECG testing were evaluated. In each patient, the expression of C x 43 proteins was qualitatively and quantitatively determined using immunoconfocal microscopy and right ventricular biopsy specimens. The level of expression of C x 43 protein was defined as the proportion of tissue area occupied by C x 43 (percent tissue area) in each test area. The abundance and distribution of the C x 43 signal was assessed in relation to LP. Late ventricular potentials were positive in 5 patients (LP (+) group) and negative in 11 patients (LP (-) group). The inci 1f40 dence of sustained ventricular tachycardia in the LP (+) group was higher than that in the LP (-) group (80% vs 18%, p=0.04). The percent tissue area of C x 43 in the LP (+) group was significantly lower than that in the LP (-) group (p=0.02). Furthermore, C x 43 protein in the LP (+) group was distributed more heterogeneously than that in the LP (-) group (p=0.001). The heterogeneous expression of C x 43 protein may contribute to impaired ventricular conduction, which may be related to the LP detected on SAECG.

    Circulation journal : official journal of the Japanese Circulation Society 2003;67;12;1017-21

  • Low dose ionizing radiation-induced activation of connexin 43 expression.

    Glover D, Little JB, Lavin MF and Gueven N

    Queensland Institute of Medical Research, Herston, Australia.

    Purpose: Connexin 43 has been implicated in the cellular response to ionizing radiation by enabling cell-to-cell communication. It is established here that the expression of connexin 43 is affected by ionizing radiation and the mechanism involved is investigated.

    The human connexin 43 promoter was cloned into a Luciferase reporter plasmid and activation by ionizing radiation was measured in normal human fibroblasts as well as HeLa cells. The regions responsible for the radiation inducibility were defined using deletion and point mutations of the construct. The results were confirmed by Northern and Western blotting.

    Results: Ionizing radiation activates the human connexin 43 promoter in a time- and dose-dependent manner with a maximal induction (4.2-fold +/-0.58) after 6 h and a dose of 0.5 Gy. Higher doses up to 5 Gy led to a less marked increase (2-fold) over the same period. This promoter activation was associated with comparable increases in both connexin 43 mRNA and protein levels. The low dose radiation response of the promoter is mainly dependent on consensus binding sites for nuclear factor of activated T-cells (NFAT) and activator protein (AP1) in a region -2537 and -2110 bp from the transcriptional start site as determined by mutation analysis.

    Conclusions: Low doses of ionizing radiation induce the transcriptional upregulation of connexin 43 expression employing NFAT and AP1 sites.

    International journal of radiation biology 2003;79;12;955-64

  • Significance of mRNA levels of connexin37, connexin43, and connexin45 in luteinized granulosa cells of controlled hyperstimulated follicles.

    Tsai MY, Lan KC, Huang KE, Huang FJ, Kung FT and Chang SY

    Department of Obstetrics and Gynecology, Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung County, Taiwan.

    Objective: To evaluate the mRNA levels of connexins in different sizes of luteinized follicles.

    Design: Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the connexin mRNA levels in the granulosa cells of 91 luteinized follicles.

    Setting: Academic tertiary care medical center and research unit of university.

    Ninety-one female patients on controlled ovarian hyperstimulation (COH).

    Sonoguided aspiration to collect the oocytes and the granulosa cells simultaneously.

    The RT-PCR data are normalized by comparing the intensity of the connexins to the intensity of internal controls (beta-actin). The follicles are grouped according to the size and the mRNA levels of the connexins. The correlations among the size of the follicles, the outcome of oocytes, and the mRNA levels of the connexins are compared by Fisher's exact test and Mann-Whitney test.

    The mRNA levels of the connexins are low in the follicles equal or larger than 5.5 mL. High cx43 levels are linked to good prognosis of oocytes.

    The luteinized granulosa cells from the large follicles are relatively quiescent in the connexin mRNA expression. In addition to the volume, the mRNA levels of cx43 may serve as a marker to predict the outcome of oocytes.

    Fertility and sterility 2003;80;6;1437-43

  • Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells.

    Spinella F, Rosanò L, Di Castro V, Nicotra MR, Natali PG and Bagnato A

    Laboratories of Molecular Pathology and Ultrastructure and Immunology, Regina Elena Cancer Institute, Rome 00158, Italy.

    Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

    The Journal of biological chemistry 2003;278;42;41294-301

  • Gating and regulation of connexin 43 (Cx43) hemichannels.

    Contreras JE, Sáez JC, Bukauskas FF and Bennett MV

    Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

    Connexin 43 (Cx43) nonjunctional or "unapposed" hemichannels can open under physiological or pathological conditions. We characterize hemichannels comprised of Cx43 or Cx43-EGFP (Cx43 with enhanced GFP fused to the C terminus) expressed in HeLa cells. Channel opening was induced at potentials greater than +60 mV. Open probability appeared to be very low. No comparable opening was detected in the parental, nontransfected HeLa cells. Conductance of fully open single hemichannels was approximately 220 pS, which is approximately double that of Cx43 cell-cell channels. Cx43 hemichannels exhibited two types of gating: fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS and slow transitions (>5 ms) between either open state and the fully closed state. Cx43-EGFP hemichannels exhibited only slow transitions (>5 ms) between closed and fully open states. These properties resemble those of the corresponding Cx43 and Cx43-EGFP cell-cell channels. Cx43 with EGFP on the N terminus (EGFP-Cx43) inserted into the surface and formed plaques but did not form hemichannels or cell-cell channels. Hemichannel blockers, 18beta-glycyrrhetinic acid or La3+, blocked depolarization-induced currents. Uptake of ethidium bromide (i) was faster in Cx43 and Cx43-EGFP than parental and EGFP-Cx43 cells, (ii) was directly correlated with Cx43-EGFP expression, (iii) was reduced by hemichannel blockers, and (iv) occurred at the same low rate in EGFP-Cx43 and parental cells. Although hemichannel opening was not detected electrophysiologically at the resting potential, infrequent or brief opening could account for ethidium bromide uptake. Opening of Cx43 hemichannels may mediate normal signaling or be deleterious.

    Funded by: NINDS NIH HHS: NS36706, NS45837, R01 NS036706, R21 NS045837

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;20;11388-93

  • The correlation between aberrant connexin 43 mRNA expression induced by promoter methylation and nodal micrometastasis in non-small cell lung cancer.

    Chen JT, Cheng YW, Chou MC, Sen-Lin T, Lai WW, Ho WL and Lee H

    Department of Pathology, Veteran General Hospital-Taichung, Taichung 402, Taiwan, ROC.

    Reduced connexin (Cx) 43 gene expression has been shown in most of lung tumors and cancer cell lines. Although aberrant Cx43 gene expression was linked with lung tumorigenesis, our understanding to the mechanism was still limited. We hypothesized that the evidence of aberrant Cx43 gene expression was gradually intensified from adjacent normal lung tissues surrounding tumors toward tumor tissues. In this study, 90 lung tumors and adjacent normal tissues were collected to examine Cx43 mRNA expression by reverse transcription-PCR (RT-PCR). Our data showed that Cx43 mRNA expression in adjacent normal lung tissue was significantly correlated with nodal involvement (P = 0.03), but the similar trend was not observed in tumor tissues. To verify whether lack of Cx43 mRNA expression resulted from promoter methylation, PCR-based methylation assay was performed for Cx43 promoter methylation analysis. A higher frequency of promoter methylation was observed in Cx43 mRNA-negative patients (21 of 33, 63.7%) compared with Cx43 mRNA-positive patients (3 of 57, 5.3%, P < 0.0001). To elucidate whether aberrant Cx43 gene expression originated from adjacent normal lung tissues, 25 lung tumors and each of five adjacent normal tissues at various distances from tumor tissues were collected to examine Cx43 mRNA and protein expression by RT-PCR and Western blot, respectively. The results show that Cx43 mRNA and protein expressions gradually decreased from adjacent normal lung tissues to tumor tissues with a positive correlation to the distance from the tumor tissues. Gel-shift assay data also revealed that shifted band binding with AP1 was only observed in adjacent normal tissues, which were far from the tumor tissues. These results indicate that promoter methylation may interfere with AP1 binding to the promoter to cause aberrant Cx43 gene expression. Thus, Cx43 mRNA in adjacent normal tissue surrounding lung tumor simply detected by RT-PCR may act as a molecular marker of nodal micrometastasis in non-small cell lung cancer.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2003;9;11;4200-4

  • Involvement of connexin 43 in human trophoblast cell fusion and differentiation.

    Frendo JL, Cronier L, Bertin G, Guibourdenche J, Vidaud M, Evain-Brion D and Malassine A

    Institut National de la Santé et de la Recherche Médicale, U427, Paris, France.

    The syncytiotrophoblast is the principal component of the human placenta involved in feto-maternal exchanges and hormone secretion. The syncytiotrophoblast arises from the fusion of villous cytotrophoblasts. We recently showed that functional gap junctional intercellular communication (GJIC) is an important prerequisite for syncytiotrophoblast formation and that connexin 43 (Cx43) is present in cytotrophoblasts and in the syncytiotrophoblast. To determine whether Cx43 is directly involved in trophoblast fusion, we used an antisense strategy in primary cultures of human villous cytotrophoblasts that spontaneously differentiate into the syncytiotrophoblast by cell fusion. We assessed the morphological and functional differentiation of trophoblasts by desmoplakin immunostaining, by quantifying hCG (human chorionic gonadotropin) production and by measuring the expression of specific trophoblast genes (hCG and HERV-W). Furthermore, we used the gap-FRAP (fluorescence recovery after photobleaching) method to investigate functional GJIC. Cytotrophoblasts treated with Cx43 antisense aggregated and fused poorly. Furthermore, less HERV-W env mRNA, hCGbeta mRNA and hCG secretion were detected in Cx43 antisense-treated cytotrophoblasts than in cells treated with scrambled antisense. Treatment with Cx43 antisense dramatically reduced the percentage of coupled trophoblast cells. Taken together, these results suggest that Cx43 is directly involved in human trophoblast cell-cell communication, fusion and differentiation.

    Journal of cell science 2003;116;Pt 16;3413-21

  • Lysosomal and proteasomal degradation play distinct roles in the life cycle of Cx43 in gap junctional intercellular communication-deficient and -competent breast tumor cells.

    Qin H, Shao Q, Igdoura SA, Alaoui-Jamali MA and Laird DW

    Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada.

    The present study was designed to determine the specific roles played by lysosomes and proteasomes in the degradation of Cx43 in both gap junctional intercellular communication-deficient MDA-MB-231 and -competent BICR-M1Rk cells. In MDA-MB-231 cells, immunolocalization and brefeldin A protein transport blocking studies revealed that there was a propensity for newly synthesized Cx43 to be transported to lysosomes. On the other hand, light and electron microscopic analysis of BICR-M1Rk cells showed that Cx43 gap junctions were prevalent with a subpopulation of intracellular Cx43 localized to lysosomes. In both cell types, Western blots revealed a notable increase in total cellular Cx43 in response to lysosome inhibitors. Interestingly, lactacystin inhibition of proteosomal degradation in MDA-MB-231 cells resulted in a marked increase in phosphorylated Cx43 at the expense of non-phosphorylated Cx43, and this change corresponded with an increase in "oversized" gap junction plaques. In BICR-M1Rk cells, lactacystin treatment partially prevented the BFA-induced loss of gap junctions. Together, our data suggests that lysosomes play a key role in not only degrading internalized gap junction in BICR-M1Rk cells but also in degrading Cx43 delivered from early secretory compartments to lysosomes in MDA-MB-231 cells. Overall proteasomal degradation regulates the stability of phosphorylated Cx43 and appears to promote the internalization of Cx43 from the cell surface.

    The Journal of biological chemistry 2003;278;32;30005-14

  • Functioning of cx43 hemichannels demonstrated by single channel properties.

    Contreras JE, Sáez JC, Bukauskas FF and Bennett MV

    Department of Neuroscience, Albert Einstein College of Medicine, NY 10461, USA.

    It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels.

    Funded by: NINDS NIH HHS: NS36706, NS45837

    Cell communication & adhesion 2003;10;4-6;245-9

  • Mutations in the second extracellular region of connexin 43 prevent localization to the plasma membrane, but do not affect its ability to suppress cell growth.

    Olbina G and Eckhart W

    Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, San Diego, CA 92186-5800, USA.

    Connexin 43 (Cx43), the most widely expressed gap junction protein, has a role in regulation of cell growth. In this study, we demonstrate that the point mutations F199L, R202E, and E205R in the second extracellular region of Cx43 prevent localization of the mutant proteins to the plasma membrane. The mutants were aberrantly localized in the cytoplasm if expressed in HeLa cells, which lack Cx43. Coexpression with wild-type Cx43 promoted localization of the F199L and R202E mutant proteins to the plasma membrane. By dye transfer assay, we showed that gap junctional intercellular communication (GJC) is decreased in cells expressing the mutants, compared to Cx43 wild-type-expressing cells. However, the F199L mutant does not appear to have a dominant-negative effect on GJC. Despite the loss of GJC, the ability of the F199L Cx43 mutant to inhibit growth of either Cx43-/- cells or two cancer cell lines, HeLa and C6 glioma cells, was similar to that of the wild-type Cx43. In addition, we showed that both R202E and E205R Cx43 mutant expressions cause growth retardation of HeLa cells. Therefore, the point mutations in the second extracellular region of Cx43 do not affect the ability of the mutant proteins in vitro to suppress cell growth, although they prevent localization to the plasma membrane. The results support the concept that regulation of cell growth by Cx43 does not necessarily require GJC and suggest that the growth-suppressive properties of Cx43 may be independent of the second extracellular loop.

    Funded by: NCI NIH HHS: CA 14195, CA13886

    Molecular cancer research : MCR 2003;1;9;690-700

  • Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart.

    Kiewitz R, Acklin C, Schäfer BW, Maco B, Uhrík B, Wuytack F, Erne P and Heizmann CW

    Departm 6fb ent of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zürich, Steinwiesstr. 75, CH-8032, Zürich, Switzerland.

    The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.

    Biochemical and biophysical research communications 2003;306;2;550-7

  • A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover.

    Thomas MA, Zosso N, Scerri I, Demaurex N, Chanson M and Staub O

    Institute of Pharmacology and Toxicology, University of Lausanne, 1005 Lausanne, Switzerland.

    The gap junction protein connexin43 is known to have a rapid turnover, involving degradation by both the proteasomal and lysosomal systems, but the structural features of connexin43 that govern these actions are not known. The connexin43 C-terminal sequence contains a proline-rich region corresponding to the consensus of a protein-protein interaction PY-motif (xPPxY), and an overlapping putative tyrosine-based sorting signal (Yxxphi; =hydrophobic), known to play a role in the intracellular trafficking of many membrane proteins. As both motifs may control turnover of connexin43, we used a combination of metabolic radiolabelling, immuno-precipitation and functional assays to determine the possible role of these motifs in controlling degradation of human connexin43 expressed in SKHep1 cells. Mutation V289D in the tyrosine-based sorting motif increased the steady-state pool of connexin43 by approximately 3.5-fold, while mutation P283L in the PY-motif produced a comparatively modest augmentation (1.7-fold). No additive effect was observed when the overlapping tyrosine was mutated. In pulse-chase experiments, the Y286A substitution increased the half-life of connexin43 from 2 to 6 hours, indicating that the increased steady-state levels reflected reduced protein degradation. Moreover, expression at the junctional membrane, as well as gap junction-mediated intercellular communication (GJC), were nearly abolished by lysosomal inhibitors and Brefeldin A in cells expressing wild-type connexin43, but were unaffected in the tyrosine mutant. These results provide strong evidence that the tyrosine-based motif of human connexin43 is a prime determinant controlling connexin43 stability, and consequently GJC, by targeting connexin43 for degradation in the endocytic/lysosomal compartment.

    Journal of cell science 2003;116;Pt 11;2213-22

  • Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways.

    Matsuda A, Suzuki Y, Honda G, Muramatsu S, Matsuzaki O, Nagano Y, Doi T, Shimotohno K, Harada T, Nishida E, Hayashi H and Sugano S

    Laboratory for Biology, Institute for Life Science Research, Health Care Company, ASAH1 KASEI Corporation, 2-1 Samejima, Fuji-shi, Shizuoka 416-8501, Japan. matsuda.ab@om.asahi-kasei.co.jp

    We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.

    Oncogene 2003;22;21;3307-18

  • Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5 but not ERK1/2 kinase activation.

    Cameron SJ, Malik S, Akaike M, Lerner-Marmarosh N, Yan C, Lee JD, Abe J and Yang J

    Department of Pharmacology/Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.

    The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.

    Funded by: NHLBI NIH HHS: R01 HL66919-02

    The Journal of biological chemistry 2003;278;20;18682-8

  • Signal transduction of gap junctional genes, connexin32, connexin43 in human hepatocarcinogenesis.

    Ma XD, Ma X, Sui YF, Wang WL and Wang CM

    Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, 17 Changle Xilu, Xi'an 710033, Shaanxi Province, China. maping@fmmu.edu.cn

    Aim: To investigate gap junctional intercellular communication (GJIC) in hepatocellular carcinoma cell lines, and signal transduction mechanism of gap junction genes connexin32(cx32),connexin43(cx43) in human hepatocarcinogenesis.

    Methods: Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium (Ca(2+))i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot.

    Results: SLDT showed that ability of GJIC function was higher in QZG cell than that in HHCC and SMMC-7721 cell lines. By laser scanning confocal microscopy, concentrations of intracellular free calcium (Ca(2+))i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13 nmol/L) and SMMC-7721 (47.08 nmol/L) cells. Western blot suggested that only QZG cells had unphosphorylated tyrosine in Cx32 protein of 32 ku and Cx43 protein of 43 ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein.

    Conclusion: The results indicated that carcinogenesis and development of human hepatocellular carcinoma related with the abnormal expression of cx genes and disorder of its signal transduction pathway, such as decrease of (Ca(2+))i, post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.

    World journal of gastroenterology 2003;9;5;946-50

  • Unexpected induction of the human connexin 43 promoter by the ras signaling pathway is mediated by a novel putative promoter sequence.

    Carystinos GD, Kandouz M, Alaoui-Jamali MA and Batist G

    Department of Pharmacology & Therapeutics and the Montreal Centre for Experimental Therapeutics in Cancer, Lady Davis Institute of the Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada.

    Connexin 43 (Cx43) is essential for survival and is tightly regulated at the transcriptional and post-transcriptional levels. A number of previous studies have demonstrated altered expression in malignant tissues, and in the presence of carcinogenic factors. We examined the effect of protooncogenes of Cx43 expression, and found no effect on Cx43 promoter activity in cells transformed with Src or erbB2. On the other hand, we identified and characterized a novel sequence that mediates Cx43 promoter regulation in cell lines engineered to overexpress H-Ras. Compared with wild-type NIH3T3 cells, both Cx43 mRNA and protein levels are increased in NIH3T3-Ras cells. The H-Ras+ cells also have enhanced Cx43 promoter activation, which is inhibited by the MEK1 inhibitor 2'-amino-3'-methoxyflavone (PD98059), suggesting that Ras-mediated Cx43 overexpression is via the mitogen activated protein kinase kinase/extracellular signal-regulated pathway. Deletion analysis of the Cx43 promoter revealed a 200-bp region downstream of the Cx43 transcription start site as the minimal sequence essential for the Ras-mediated Cx43 up-regulation. Using this 200-base pair fragment in electrophoretic mobility shift assays, we identified one main protein complex that binds efficiently and is more abundant in nuclear extracts from NIH3T3-Ras and MCF7-Ras cells compared with their matched controls. This complex selectively recognizes a consensus sequence, AGTTCAATCA, located at positions +149 to +158 of the Cx43 promoter. Supershift assays identified the 90-kDa heat shock protein (HSP90) and c-Myc as constituents of this DNA-binding complex. Treatment of cells with the HSP90 inhibitor geldanamycin resulted in repression of the Cx43 promoter activity, and inhibits binding of the complex to the Cx43 promoter. Coimmunoprecipitation studies confirmed the interaction between endogenous HSP90 and c-Myc. This study provides evidence that the transcriptional up-regulation of Cx43 by Ras-Raf-MAPK is mediated via the interaction of a novel Cx43 promoter element with a protein complex that contains both HSP90 and c-Myc.

    Molecular pharmacology 2003;63;4;821-31

  • Defective activation of c-Src in cystic fibrosis airway epithelial cells results in loss of tumor necrosis factor-alpha-induced gap junction regulation.

    Huang S, Dudez T, Scerri I, Thomas MA, Giepmans BN, Suter S and Chanson M

    Laboratory of Clinical Investigation III, Department of Pediatrics, Geneva University Hospitals, Switzerland.

    Tumor necrosis factor-alpha (TNF-alpha) signaling is central to the transmission of the innate immune response and subsequent activation of the adaptive immune system. The functioning of both systems is required for optimal clearance of pathogens from the airways. In cystic fibrosis (CF), dysfunction of the CF transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections despite an intense inflammatory and immune response. We reported recently that TNF-alpha decreased gap junction connectivity in non-CF airway cells, a mechanism that was absent in CF cells expressing the DeltaPhe-508 mutant of CFTR. We have now identified the tyrosine kinase c-Src as a possible pathway between the mediators of inflammation and the gap junction protein connexin43 (Cx43). Indeed, TNF-alpha increased the proportion of activated c-Src in non-CF airway cells. Moreover, pharmacological antagonists and expression in non-CF cells of a dominant negative construct of c-Src prevented Cx43 channel closure by TNF-alpha. Finally, gap junction channel closure was prevented by expression of a Cx43 mutant lacking tyrosine phosphorylation sites for c-Src. Additional experiments showed that activation of c-Src was defective in CF airway cells but rescued in CFTR-corrected CF cells. These data suggest that CFTR dysfunction is associated with altered TNF-alpha signaling, resulting in the persistence of gap junction connectivity in CF airway cells. We propose that altered regulation of c-Src may contribute to the dysregulated inflammatory response that is characteristic of the CF phenotype.

    The Journal of biological chemistry 2003;278;10;8326-32

  • Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia.

    Paznekas WA, Boyadjiev SA, Shapiro RE, Daniels O, Wollnik B, Keegan CE, Innis JW, Dinulos MB, Christian C, Hannibal MC and Jabs EW

    Department of Pediatrics, Center for Craniofacial Development and Disorders, McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

    Gap junctions are assemblies of intercellular channels that regulate a variety of physiologic and developmental processes through the exchange of small ions and signaling molecules. These channels consist of connexin family proteins that allow for diversity of channel composition and conductance properties. The human connexin 43 gene, or GJA1, is located at human chromosome 6q22-q23 within the candidate region for the oculodentodigital dysplasia locus. This autosomal dominant syndrome presents with craniofacial (ocular, nasal, and dental) and limb dysmorphisms, spastic paraplegia, and neurodegeneration. Syndactyly type III and conductive deafness can occur in some cases, and cardiac abnormalities are observed in rare instances. We found mutations in the GJA1 gene in all 17 families with oculodentodigital dysplasia that we screened. Sixteen different missense mutations and one codon duplication were detected. These mutations may cause misassembly of channels or alter channel conduction properties. Expression patterns and phenotypic features of gja1 animal mutants, reported elsewhere, are compatible with the pleiotropic clinical presentation of oculodentodigital dysplasia.

    Funded by: NCRR NIH HHS: M01 RR000052, M01 RR0052; NICHD NIH HHS: P30 HD024061, R01 HD24061; NIDCR NIH HHS: R01 DE013849, R01 DE13849

    American journal of human genetics 2003;72;2;408-18

  • The carboxy-tail of connexin-43 localizes to the nucleus and inhibits cell growth.

    Dang X, Doble BW and Kardami E

    Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.

    Gap junctions are plasma membrane intercellular communication channels that in addition to ensuring electrical coupling and coordinated mechanical activity, can act as growth suppressors. To define the role of a non-channel forming domain of connexin-43 (Cx43), the main constituent of cardiomyocyte gap junctions, on growth regulation, we expressed its C-terminal portion (CT-Cx43) in cardiomyocytes and HeLa cells. In addition to broad cytoplasmic localization, CT-Cx43 was also localized to the nucleus of both cell types, detected by immunofluorescence as well as immunoblotting of subcellular fractions. Furthermore, stable expression of CT-Cx43 in HeLa cells induced a significant decrease in proliferation. It is therefore suggested that plasma membrane localization and formation of channels are not required for growth inhibition by Cx43, and that nuclear localization of CT-Cx43 may exert effects on gene expression and growth.

    Molecular and cellular biochemistry 2003;242;1-2;35-8

  • Gap junction remodeling and altered connexin43 expression in the failing human heart.

    Kostin S, Rieger M, Dammer S, Hein S, Richter M, Klövekorn WP, Bauer EP and Schaper J

    Department of Experimental Cardiology, Max-Planck-Institute, Bad Nauheim, Germany. skostin@kerckhoff.mpg.de

    Gap junctions (GJ) are important determinants of cardiac conduction and the evidence has recently emerged that altered distribution of these junctions and changes in the expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes and likely contribute to arrhythmogenesis. However, it is largely unknown whether changes in the expression and distribution of the major cardiac GJ protein, Cx43, is a general feature of diverse chronic myocardial diseases or is confined to some particular pathophysiological settings. In the present study, we therefore set out to investigate qualitatively and quantitatively the distribution and expression of Cx43 in normal human myocardium and in patients with dilated (DCM), ischemic (ICM), and inflammatory cardiomyopathies (MYO). Left ventricular tissue samples were obtained at the time of cardiac transplantation and investigated with immunoconfocal and electron microscopy. As compared with the control group, Cx43 labeling in myocytes bordering regions of healed myocardial 1f40 infarction (ICM), small areas of replacement fibrosis (DCM) and myocardial inflammation (MYO) was found to be highly disrupted instead of being confined to the intercalated discs. In all groups, myocardium distant from these regions showed an apparently normal Cx43 distribution at the intercalated discs. Quantitative immunoconfocal analysis of Cx43 in the latter myocytes revealed that the Cx43 area per myocyte area or per myocyte volume is significantly decreased by respectively 30 and 55% in DCM, 23 and 48% in ICM, and by 21 and 40% in MYO as compared with normal human myocardium. In conclusion, focal disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling that may play an important role in the development of an arrhythmogenic substrate in human cardiomyopathies.

    Molecular and cellular biochemistry 2003;242;1-2;135-44

  • BMP regulation of the mouse connexin43 promoter in osteoblastic cells and embryos.

    Chatterjee B, Meyer RA, Loredo GA, Coleman CM, Tuan R and Lo CW

    Laboratory of Developmental Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8019, USA.

    We examined BMP regulation of the gap junction gene Gjal (Cx43alpha1) using a series of lacZ reporter constructs containing up to 6.7 kbs of mouse Cx43alpha1 promoter sequence. Using transient transfection assays, we showed that BMP2, BMP4, and GDF5, but not BMP6 or BMP7, can modulate Cx43alpha1 promoter activity in the osteosarcoma cell line ROS17/2.8. Positive regulatory elements were found at the proximal and distal ends of the 6.7 kb promoter fragment, while negative regulatory elements were found in the intervening region. Comparison of Cx43alpha1 promoter sequences from the human vs. mouse showed five regions with significant sequence conservation, two of which contained Smad binding elements in conjunction with a BMP response element. Analysis of a transgenic mouse line containing a Cx43alpha1 promoter driven lacZ reporter construct revealed lacZ expression in the developing joints, an expression pattern similar to that previously reported for Gdf5. LacZ expression was also observed in axial regions of the skeletal anlage, which in situ hybridization analysis confirmed as sites of Gdf5 transcript expression. When the Cx43alpha1 promoter driven lacZ transgene was bred into the brachypodism mouse Gdf5(bpJ)(bp) harboring a Gdf5 loss of function mutation, lacZ expression was extinguished. This was observed in homozygous and heterozygous bp animals, suggesting that Cx43alpha1 promoter regulation by GDF5 is subject to haploinsufficiency. Overall, these observations are consistent with recent studies by others indicating a role for Cx43alpha1 in osteogenesis and osteoblastic function during mouse development.

    Cell communication & adhesion 2003;10;1;37-50

  • Impaired trafficking of connexins in androgen-independent human prostate cancer cell lines and its mitigation by alpha-catenin.

    Govindarajan R, Zhao S, Song XH, Guo RJ, Wheelock M, Johnson KR and Mehta PP

    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

    Gap junctions, composed of connexins, provide a pathway of direct intercellular communication for the diffusion of small molecules between cells. Evidence suggests that connexins act as tumor suppressors. We showed previously that expression of connexin-43 and connexin-32 in an indolent prostate cancer cell line, LNCaP, resulted in gap junction formation and growth inhibition. To elucidate the role of connexins in the progression of prostate cancer from a hormone-dependent to -independent state, we introduced connexin-43 and connexin-32 into an invasive, androgen-independent cell line, PC-3. Expression of these proteins in PC-3 cells resulted in intracellular accumulation. Western blot analysis revealed a lack of Triton-insoluble, plaque-assembled connexins. In contrast to LNCaP cells, connexins could not be cell surface-biotinylated and did not reside in the cell surface derived endocytic vesicles, in PC-3 cells, suggesting impaired trafficking to the cell surface. Intracellular accumulation of connexins was observed in several androgen-independent prostate cancer cell lines. Transient expression of alpha-catenin facilitated the trafficking of both connexins to the cell surface and induced gap junction assembly. Our results suggest that impaired trafficking, and not the inability to form gap junctions, is the major cause of communication deficiency in human prostate cancer cell lines.

    Funded by: NCI NIH HHS: CA 73769

    The Journal of biological chemistry 2002;277;51;50087-97

  • Immunohistochemical and ultrastructural study of gap junction proteins connexin26 and 43 in human arachnoid villi and meningeal tumors.

    Arishima H, Sato K and Kubota T

    Department of Neurosurgery, Fukui Medical University, Fukui, Japan.

    Human arachnoid villi and meningiomas are known to have gap junctions formed by connexin (Cx) proteins. We examined the expression and localization of Cxs in normal human arachnoid villi and meningeal tumors (meningiomas and hemangiopericytomas) by immunohistochemistry and Western blots. In arachnoid villi, strong immunopositivity for connexin26 (Cx26) and connexin43 (Cx43) was detected in the cap cell layer, cap cell cluster, and central core. They were weakly expressed in the fibrous capsule. In meningiomas they were strongly expressed in the meningotheliomatous area and weakly positive in the fibrous area. None of them were expressed in hemangiopericytomas. By immunoelectron microscopy, Cx26 and Cx43 were distributed on the cell membranes in arachnoid villi and meningiomas. In the Western blots in arachnoid villi and meningiomas, Cx26 and Cx43 were shown at bands with molecular weights of 26 kD and 42-47 kD, respectively. The degree of positivity for Cxs was different between subtypes of meningiomas. These findings suggest that expression of Cx26 and Cx43 might be related to the differentiation of the arachnoid villi and meningiomas, and exhibit the different origin of various subtypes of meningiomas. We proposed that Cx expression is one of the useful markers for the differentiation of meningioma and hemangiopericytoma.

    Journal of neuropathology and experimental neurology 2002;61;12;1048-55

  • Casein kinase 1 regulates connexin-43 gap junction assembly.

    Cooper CD and Lampe PD

    Fred Hutchinson Cancer Research Center and Department of Pathobiology, University of Washington, Seattle, Washington 98109-1024, USA.

    Phosphorylation of members of the connexin family of gap junction proteins has been correlated with gap junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) gap junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330. (32)P(i)-Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas gap junctional (Triton-insoluble) Cx43 decreased, and non-junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased gap junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for delta or epsilon isoforms. These data suggest CK1de 1676 lta could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.

    Funded by: NIGMS NIH HHS: GM55632

    The Journal of biological chemistry 2002;277;47;44962-8

  • Identification and functional analysis of novel phosphorylation sites in Cx43 in rat primary granulosa cells.

    Yogo K, Ogawa T, Akiyama M, Ishida N and Takeya T

    Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.

    The gap junctional intercellular communication mediated by Cx43 plays indispensable roles in both germ line development and postnatal folliculogenesis. In this study, we focused on the effect of follicle-stimulating hormone (FSH) on the Cx43 protein in rat primary granulosa cells and found that FSH stimulation elevated the phosphorylation in addition to the protein level of Cx43. Serine residues in the carboxyl-terminal region were exclusively phosphorylated in this system and we identified Ser365, Ser368, Ser369 and Ser373 as major phosphorylation sites by FSH stimulation. A Cx43 variant containing mutations at all these serine residues was found to severely reduce dye transfer activity when assayed in HeLa cells. The present study revealed a novel regulatory mechanism of Cx43-mediated gap junctional intercellular communication through phosphorylation in the carboxyl-terminus.

    FEBS letters 2002;531;2;132-6

  • Connexin expression and gap junctional intercellular communication in human first trimester trophoblast.

    Cronier L, Defamie N, Dupays L, Théveniau-Ruissy M, Goffin F, Pointis G and Malassiné A

    LBSC, CNRS UMR 6558, Université de Poitiers, 86022 Poitiers cedex.

    Connexin (Cx) expression and gap junctional intercellular communication (GJIC) are involved in development and differentiation processes, and recently mutation of connexin genes has been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cells coexist and lead to a fusion phenotype (villous trophoblast) and a proliferative/invasive phenotype (extravillous trophoblast). Here we characterized in situ and in vitro the expression of Cx transcripts and proteins in the villous and extravillous trophoblast of first trimester placenta. In addition, the GJIC functionality was investigated using the gap-fluorescence recovery after photobleaching (gap-FRAP) method. We demonstrated in the villous trophoblast the presence of Cx43 mRNA and of Cx43 protein localized between cytotrophoblastic cells and between cytotrophoblastic cells and syncytiotrophoblast. In vitro, a transient functional gap junctional intertr 1f40 ophoblastic communication was demonstrated during the trophoblast fusion leading to the multinucleated syncytiotrophoblast. During the proliferative process of the extravillous trophoblast, Cx40 is expressed in the proximal part of the cell columns. When cytotrophoblastic cells were cultured on Matrigel for 2 days, alpha5beta1 integrin expression was observed concomitant with the presence of Cx40 mRNA and of Cx40 protein between the cells. No evidence for a GJIC was detected in this induced extravillous phenotype. In addition, Cx32 was detected between some aggregated cells after 72 h of culture. Our data show that the presence of Cx43 allows an inter-trophoblastic GJIC and is associated with the fusion process leading to the villous syncytiotrophoblast and that the presence of Cx40 does not allow GJIC and is associated with the extravillous phenotype.

    Molecular human reproduction 2002;8;11;1005-13

  • Inducible expression of the gap junction protein connexin43 decreases the neoplastic potential of HT-1080 human fibrosarcoma cells in vitro and in vivo.

    King TJ, Fukushima LH, Yasui Y, Lampe PD and Bertram JS

    Cancer Prevention Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

    Numerous studies have demonstrated a correlation between dysregulation/loss of connexin expression or gap junction intercellular communication (GJIC) function and decreased growth control both in human tumors and tumor cell lines. Likewise, restoration of constitutive connexin expression/function is correlated with increased growth control/decreased tumorigenicity. Here, we show for the first time that inducible restoration of connexin43 (Cx43) expression and GJIC function in a human tumor line of mesenchymal origin (HT-1080, fibrosarcoma) resulted in a lowered neoplastic potential. Specifically, HT-1080 cells induced to express Cx43 demonstrated diminished foci formation when in co-culture with normal fibroblasts, decreased colony formation under anchorage-independent conditions, and reduced tumor growth when injected into immunodeficient mice. These results, obtained utilizing an inducible system that helps address issues of clonal heterogeneity, strongly implicate Cx43 as a tumor suppressor in human tissue of mesenchymal origin and GJIC as a regulatory mechanism for cellular growth control both in vitro and in vivo. This study also further supports the hypothesis that loss of Cx43/GJIC in human tumors may play an important role in the dysregulation of normal growth control.

    Funded by: NCI NIH HHS: CA74669; NIAID NIH HHS: T32-AI07509; NIAMS NIH HHS: AR47963; NIGMS NIH HHS: GM55632

    Molecular carcinogenesis 2002;35;1;29-41

  • Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication.

    Qin H, Shao Q, Curtis H, Galipeau J, Belliveau DJ, Wang T, Alaoui-Jamali MA and Laird DW

    Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada.

    The mechanism by which gap junction proteins, connexins, act as potent tumor suppressors remains poorly understood. In this study human breast tumor cells were found to exhibit diverse gap junction phenotypes including (a) undetectable Cx43 and no intercellular communication (HBL100); (b) low levels of Cx43 and sparse intercellular communication (MDA-MB-231); and (c) significant levels of Cx43 and moderate intercellular communication (Hs578T). Although retroviral delivery of Cx43 and Cx26 cDNAs to MDA-MB-231 cells did not achieve an expected substantial rescue of intercellular communication, overexpression of connexin genes did result in a dramatic suppression of tumor growth when connexin-expressing MDA-MB-231 cells were implanted into the mammary fat pad of nude mice. Subsequent immunolocalization studies on xenograph sections revealed only cytoplasmic stores of Cx43 and no detectable gap junctions. Moreover, DNA array and Western blot analysis demonstrated that overexpression of Cx43 or Cx26 in MDA-MB-231 cells down-regulated fibroblast growth factor receptor-3. Surprisingly, these results suggest that Cx43 and Cx26 induce their tumor-suppressing properties by a mechanism that is independent of significant gap junctional intercellular communication and possibly through the down-regulation of key genes involved in tumor growth. Moreover, our studies show that retroviruses are effective vehicles for delivering connexins to human breast tumor cells, facilitating potential gene therapy applications.

    The Journal of biological chemistry 2002;277;32;29132-8

  • Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells.

    Lauf U, Giepmans BN, Lopez P, Braconnot S, Chen SC and Falk MM

    Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    Certain membrane channels including acetylcholine receptors, gap junction (GJ) channels, and aquaporins arrange into large clusters in the plasma membrane (PM). However, how these channels are recruited to the clusters is unknown. To address this question, we have investigated delivery of GJ channel subunits (connexons) assembled from green fluorescent protein (GFP)-tagged connexin 43 (Cx43) to the PM and GJs in living cells. Fluorescence-photobleaching of distinct areas of Cx43-GFP GJs demonstrated that newly synthesized channels were accrued to the outer margins of channel clusters. Time-lapse microscopy further revealed that connexons were delivered in vesicular carriers traveling along microtubules from the Golgi to the PM. Routing and insertion of connexons occurred predominantly into the nonjunctional PM. These PM connexons can move laterally as shown by photo-bleaching and thus, can reach the margins of channel clusters. There, the apposing PMs are close enough to allow connexons to dock into complete GJ channels. When connexon delivery to the PM was inhibited by brefeldin A, or nocodazole pretreatment, the PM pool initially enabled connexon accrual to the clusters but further accrual was inhibited upon depletion. Taken together, our results indicate that GJ channel clusters grow by accretion at their outer margins from connexon subunits that were delivered to the nonjunctional PM, and explain how connexons in the PM can function in intra-/extracellular signaling before GJ channel formation and direct cell-cell communication.

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;16;10446-51

  • [Expression of connexin43 and connexin45 in nasopharyngeal carcinoma].

    Xiang Q, Fan SQ, Li J, Tan C, Xiang JJ, Zhang QH, Wang R and Li GY

    Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha 410078, P. R. China.

    The gap junction plays an important role in the exchange of nutrients, ions, and regulatory molecules between cells. It will result in an uncontrolled cell proliferation if it is abnormal. It was reported that some cancer tissues and cancer cells had abnormal gap junction and restoration of the gap junction function could revert the cancer cells to a normal phenotype. Therefore, to investigate the expression of connexin(cx) in human nasopharyngeal carcinoma, may provide a new way for clinical diagnosis and the pathogenesis of nasopharyngeal carcinoma.

    Method: The expressions of Cx43 and Cx45 in the biopsies of nasopharyngeal carcinoma(NPC) and chronic nasopharyngitis tissues were determined by using the immunohistochemical staining.

    Results: 1. Cx43 and Cx45 expressed differentially in NPC and chronic nasopharyngitis tissues(P < 0.01). In NPC, expression of Cx43 and Cx45 were 44.8% and 46.6%, respectively, while in columnar cells of chronic nasopharyngitis tissues, their expressions were 86.5% and 100% respectively, which were higher than those in NPC, 2. The percentage in squamous epithelial cells of chronic nasopharyngitis tissues was 29.7%, which was lower than that in NPC, (56.9%). 3. The expressions of Cx43 and Cx45 in paracancer columnar epithelial cells were lower than that in paracancer squamous epithelial cells (P < 0.001), but higher than that in NPC cells (P < 0.01).

    Conclusions: The abnormal expression of Cx43 and Cx45 in nasopharynx tissues may be associated with cancerization and squamatization of human nasopharynx tissue.

    Ai zheng = Aizheng = Chinese journal of cancer 2002;21;6;593-6

  • Paradoxical overexpression and translocation of connexin43 in homocysteine-treated endothelial cells.

    Li H, Brodsky S, Kumari S, Valiunas V, Brink P, Kaide J, Nasjletti A and Goligorsky MS

    Department of Medicine, State University of New York, Stony Brook, 11794, USA.

    Hyperhomocysteinemia is an established cause of defective vasorelaxation. Gene expression screening of human umbilical vein endothelial cells (HUVEC) treated with homocysteine (Hcy) revealed that connexin43 (Cx43) was upregulated. Expression of Cx43 was increased more than twofold in Hcy-treated HUVEC. Gap junctional communication (Lucifer yellow and whole cell patch clamp) was not enhanced in Hcy-treated HUVEC. HUVEC expressing chimeric Cx43-green fluorescent protein exhibited it at cell-cell contacts in control but showed redistribution to the intracellular compartment(s) in Hcy-treated cells. Confocal microscopy of HUVEC stained with anti-Cx43, mitochondrial, and endoplasmic reticulum fluorescent markers showed the localization of Cx43 to the plasma membrane of control cells and its colocalization with the mitochondrial marker in Hcy-treated HUVEC. Studies of isolated mitochondria confirmed overexpression of Cx43 in the mitochondria of Hcy-treated HUVEC. Microdissected renal interlobar arteries, which normally exhibit endothelium-derived hyperpolarizing factor-induced vasorelaxation, showed reduced nitric oxide synthase- and cyclooxygenase-independent vasorelaxation to acetylcholine after pretreatment with Hcy. In summary, Hcy-induced upregulation of Cx43 transcript and protein expression are associated with unaltered intercellular communication, redistribution of Cx43 in HUVEC, and reduced nitric oxide- and prostanoid-independent vascular responses to acetylcholine in Hcy-treated arteries.

    Funded by: NIDDK NIH HHS: DK-45462, DK-52783, DK-54602, T32-DK-07521-14

    American journal of physiology. Heart and circulatory physiology 2002;282;6;H2124-33

  • Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology.

    Huang R, Lin Y, Wang CC, Gano J, Lin B, Shi Q, Boynton A, Burke J and Huang RP

    Department of Gynecology and Obstetrics, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, GA 30322, USA.

    Previously, we demonstrated that connexin 43 (cx43) suppressed the growth of human glioblastoma cells. To investigate the molecular mechanisms involved in tumor suppression by cx43, we developed a human cytokine array system, which simultaneously detects the expression of 43 cytokines. By using this new technology, we analyzed the cx43-regulated genes in cx43-transfected cells. The cytokine arrays showed that expression of monocyte chemotactic protein-1 (MCP-1) was profoundly reduced in cx43-transfected cells. RT-PCR, immuno-Western blot, and cDNA microarrays further confirmed this observation. Addition of conditioned medium from control-transfected cells and recombinant MCP-1 to cx43-transfected cells significantly enhanced cx43-transfected cell proliferation and colony formation in soft agar. In contrast, addition of neutralization antibody against MCP-1 significantly inhibited cell proliferation in control-transfected cells. Our results suggested that MCP-1 is involved in the suppression of human glioblastoma cell growth by cx43.

    Funded by: NCI NIH HHS: CA 39745, CA 89273

    Cancer research 2002;62;10;2806-12

  • Connexin family members target to lipid raft domains and interact with caveolin-1.

    Schubert AL, Schubert W, Spray DC and Lisanti MP

    Department of Molecular Pharmacology and Albert Einstein Cancer Center and Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

    Lipid rafts are cholesterol-sphingolipid-rich microdomains that function as platforms for membrane trafficking and signal transduction. Caveolae are specialized lipid raft domains that contain the structural proteins known as the caveolins. Connexins are a family of transmembrane proteins that self-associate to form cell-cell connections known as gap junctions and that are linked to cytosolic proteins, forming a protein complex or Nexus. To determine the extent to which these intracellular compartments intersect, we have systematically evaluated whether connexins are associated with lipid rafts and caveolin-1. We show that connexin 43 (Cx43) colocalizes, cofractionates, and coimmunoprecipitates with caveolin-1. A mutational analysis of Cx43 reveals that the hypothesized PDZ- and presumptive SH2/SH3-binding domains within the Cx43 carboxyl terminus are not required for this targeting event or for its stable interaction with caveolin-1. Furthermore, Cx43 appears to interact with two distinct caveolin-1 domains, i.e., the caveolin-scaffolding domain (residues 82-101) and the C-terminal domain (135-178). We also show that other connexins (Cx32, Cx36, and Cx46) are targeted to lipid rafts, while Cx26 and Cx50 are specifically excluded from these membrane microdomains. Interestingly, recombinant coexpression of Cx26 with caveolin-1 recruits Cx26 to lipid rafts, where it colocalizes with caveolin-1. This trafficking event appears to be unique to Cx26, since the other connexins investigated in this study do not require caveolin-1 for targeting to lipid rafts. Our results provide the first evidence that connexins interact with caveolins and partition into lipid raft domains and indicate that these interactions are connexin specific.

    Funded by: NIGMS NIH HHS: T32-GM 07491

    Biochemistry 2002;41;18;5754-64

  • Transduction of cell survival signals by connexin-43 hemichannels.

    Plotkin LI, Manolagas SC and Bellido T

    Division of Endocrinology and Metabolism, the Center for Osteoporosis and Metabolic Bone Diseases, and the Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

    Bisphosphonates, drugs used widely in the treatment of bone diseases, prevent osteoblast and osteocyte apoptosis by a mechanism involving extracellular signal-regulated kinase (ERK) activation. We report herein that hexameric connexin (Cx)-43 hemichannels, but not gap junctions, are the essential transducers of the ERK-activating/anti-apoptotic effects of bisphosphonates. Transfection of Cx-43, but not other Cxs, into Cx-43 naive cells confers de novo responsiveness to the drugs. The signal-transducing property of Cx-43 requires the pore forming as well as the C-terminal domains of the protein, the activation of both Src and ERK kinases, and the SH2 and SH3 domains of Src. This evidence adds Cx-43 to the list of transmembrane proteins capable of transducing survival signals in response to extracellular cues and raises the possibility that it may serve in this capacity for endogenously produced molecules or even other drugs.

    Funded by: NIA NIH HHS: P01-AG13918; NIAMS NIH HHS: KO2-AR02127, R29-AR43453

    The Journal of biological chemistry 2002;277;10;8648-57

  • Conductance and permeability of the residual state of connexin43 gap junction channels.

    Bukauskas FF, Bukauskiene A and Verselis VK

    Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA. fbukausk@aecom.yu.edu

    We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.

    Funded by: NIGMS NIH HHS: GM54179, R01 GM054179; NINDS NIH HHS: NS36706, R01 NS036706

    The Journal of general physiology 2002;119;2;171-85

  • In vivo and in vitro expression of connexin 43 in human teeth.

    About I, Proust JP, Raffo S, Mitsiadis TA and Franquin JC

    Laboratoire IMEB, Faculté d'Odontologie, 27 Boulevard Jean Moulin, 13385 Marseille, France. imad.about@odontologie.univ-mrs.fr

    Gap junctions are composed of transmembrane proteins belonging to the connexin family. These proteins permit the exchange of mall regulatory molecules directly between cells for the control of growth, development and differentiation. Although the presence of gap junctions in teeth has been already evidenced, the involved connexins have not yet been identified in human species. Here, we examined the distribution of connexin 43 (Cx43) in embryonic and permanent intact and carious human teeth. During tooth development, Cx43 localized both in epithelial and mesenchymal dental cells, correlated with cytodifferentiation gradients. In adult intact teeth, Cx43 was distributed in odontoblast processes. While Cx43 expression was downregulated in mature intact teeth, Cx43 appeared to be upregulated in odontoblasts facing carious lesions. In cultured pulp cells, Cx43 expression was related to the formation of mineralized nodules. These results indicate that Cx43 expression is developmentally regulated in human dental tissues, and suggest that Cx43 may participate in the processes of dentin formation and pathology.

    Connective tissue research 2002;43;2-3;232-7

  • Mutations in GJA1 (connexin 43) are associated with non-syndromic autosomal recessive deafness.

    Liu, Xia XJ, Adams J, Chen ZY, Welch KO, Tekin M, Ouyang XM, Kristiansen A, Pandya A, Balkany T, Arnos KS and Nance WE

    Department of Human Genetics, Medical College of Virginia of Virginia Commonwealth University, Richmond, VA 23298-0033, USA. xliu@med.miami.edu

    Mutations in four members of the connexin gene family have been shown to underlie distinct genetic forms of deafness, including GJB2 [connexin 26 (Cx26)], GJB3 (Cx31), GJB6 (Cx30) and GJB1 (Cx32). We have found that alterations in a fifth member of this family, GJA1 (Cx43), appear to cause a common form of deafness in African Americans. We identified two different GJA1 mutations in four of 26 African American probands. Three were homozygous for a Leu-->Phe substitution in the absolutely conserved codon 11, whereas the other was homozygous for a Val-->Ala transversion at the highly conserved codon 24. Neither mutation was detected in DNA from 100 control subjects without deafness. Cx43 is expressed in the cochlea, as is demonstrated by PCR amplification from human fetal cochlear cDNA and by RT-PCR of mouse cochlear tissues. Immunohistochemical staining of mouse cochlear preparations showed immunostaining for Cx43 in non-sensory epithelial cells and in fibrocytes of the spiral ligament and the spiral limbus. To our knowledge this is the first alpha connexin gene to be associated with non-syndromic deafness. Cx43 must also play a critical role in the physiology of hearing, presumably by participating in the recycling of potassium to the cochlear endolymph.

    Funded by: NIDCD NIH HHS: DC 02530, DC 03929, DC 04293, DC 04530, DC 04546, DC 05575

    Human molecular genetics 2001;10;25;2945-51

  • Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43).

    Huang R, Liu YG, Lin Y, Fan Y, Boynton A, Yang D and Huang RP

    Division of Research, Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Connexin 43 (Cx43), a structural component of gap junctions, is believed to function as a tumor suppressor gene. Previously, we showed that expression of Cx43 suppresses cell proliferation and tumorigenicity of human glioblastoma cells [Huang et al., Cancer Res 58:5089-5096, 1998] and enhances apoptosis in response to chemotherapeutic agents [Huang et al., Int J Cancer 92:130-138, 2001]. In the present study, we demonstrated that expression of Cx43 in human glioblastoma cells potentiated an apoptotic program under low-serum conditions. The Cx43-mediated effect was coupled with a decreased expression of the specific apoptosis-inhibitor bcl-2. Overexpression of bcl-2 in Cx43-transfected cells conferred resistance to apoptosis induced under low-serum conditions, suggesting that the Cx43-mediated apoptosis under low-serum conditions is regulated, in part, through the downregulation of bcl-2 expression. Furthermore, application of the phosphatidylinositol-3'-OH kinase inhibitor LY294002 specifically induced apoptosis in Cx43-transfected cells. Our results demonstrate a new role of Cx43 in the mediation of apoptosis under low serum conditions.

    Funded by: NCI NIH HHS: CA 39745, CA 89273

    Molecular carcinogenesis 2001;32;3;128-38

  • Multimeric connexin interactions prior to the trans-Golgi network.

    Das Sarma J, Meyer RA, Wang F, Abraham V, Lo CW and Koval M

    Institute for Environme 6e3 ntal Medicine, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial beta-galactosidase fused to the C terminus of connexin43 (Cx43/beta-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/beta-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a subhexameric complex. Cx43/beta-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/beta-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/beta-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

    Funded by: NICHD NIH HHS: HD 36457; NIGMS NIH HHS: GM 61012, R01 GM061012, R01 GM061012-02

    Journal of cell science 2001;114;Pt 22;4013-24

  • Gap junction channels formed by coexpressed connexin40 and connexin43.

    Valiunas V, Gemel J, Brink PR and Beyer EC

    Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794, USA.

    Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.

    Funded by: N 13f9 HLBI NIH HHS: HL-45466; NHLBI NIH HHS: HL-59199; NIGMS NIH HHS: GM-55263

    American journal of physiology. Heart and circulatory physiology 2001;281;4;H1675-89

  • v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication.

    Lin R, Warn-Cramer BJ, Kurata WE and Lau AF

    Molecular Carcinogenesis Section, Cancer Research Center of Hawaii, Honolulu, HI 96813, USA.

    The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 ae4 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

    Funded by: NCI NIH HHS: CA52098, R01 CA052098

    The Journal of cell biology 2001;154;4;815-27

  • Identification of connexin43 (alpha1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE).

    Dasgupta C, Martinez AM, Zuppan CW, Shah MM, Bailey LL and Fletcher WH

    Department of Biochemistry, School of Medicine, Loma Linda University, Loma Linda, CA 92354, USA.

    Gap junction channels formed by the connexin43 protein are considered to play crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to severe heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that represent a minor population of connexin43 alleles, which could be reliably detected by using denaturing gradient gel electrophoresis (DGGE) to visualize normal and mutant DNAs that were separately sequenced. In contrast, sequencing of total PCR products without DGGE-pre-selection failed to consistently identify these mutations. Forty-six controls and 20 heart transplant recipients were examined in this study. In the latter group, 14 children had hypoplastic left heart syndrome (HLHS) in which connexin43 gene defects were detected in eight. The remaining six transplant patients with HLHS and all controls showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that were missense, replacing arginine codons at positions 362 and 376 with codons for glutamines. All four of these substitutions are identical to the nucleotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5' to the mutation site in a 63bp domain that is 100% identical in the gene and pseudogene. Results from in vitro phosphorylation indicate that the absence of arginines 362 and 376 completely abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the pathologies associated with HLHS.

    Funded by: NHLBI NIH HHS: HL53374

    Mutation research 2001;479;1-2;173-86

  • Protein kinase C-alpha and -epsilon modulate connexin-43 phosphorylation in human heart.

    Bowling N, Huang X, Sandusky GE, Fouts RL, Mintze K, Esterman M, Allen PD, Maddi R, McCall E and Vlahos CJ

    Cardiovascular Research, Discovery Research, Eli Lilly and Company, Indianapolis, IN 46285, USA.

    We have previously demonstrated that protein kinase C (PKC)- alpha expression is significantly elevated in failing human left ventricle, with immunostaining showing increased PKC- alpha localization at the intercalated disks of cardiomyocytes. In the present study we sought to determine, in the failing heart, if PKC- alpha interacted with connexin-43 (Cx-43) both spatially and functionally, and to compare the association of PKC- alpha/Cx-43 with that of PKC- epsilon, a PKC isozyme that does not significantly increase in failing hearts. The possibility of a PKC- alpha or PKC- epsilon/Cx-43 association in non-failing hearts was also investigated. Co-immunoprecipitation of PKC- alpha or PKC- epsilon and Cx-43 in non-failing and failing left ventricle was achieved using antibodies to PKC- alpha or Cx-43. Confocal microscopy confirmed that PKC- alpha distribution within the cardiomyocyte included co-localization with connexin-43 in both failing and non-failing myocardium. In a similar manner, confocal imaging of PKC- epsilon showed cardiomyocyte distribution in both cytosol and membrane, and colocalization of PKC- epsilon with Cx-43. Recombinant PKC- alpha or - epsilon increased PKC activity significantly above endogenous levels in the co-immunoprecipitated Cx-43 complexes (P<0.05). However, phosphorylation of purified human Cx-43 (isolated from failing human left ventricle) by recombinant PKC- alpha or PKC- epsilon resulted in only PKC- epsilon mediated Cx-43 phosphorylation. Thus, in the human heart PKC- alpha, PKC- epsilon, and Cx-43 appear to form a closely associated complex. Whereas only PKC- epsilon directly phosphorylates Cx-43, both PKC isoforms result in increased phosphorylation within the Cx-43 co-immunoprecipitated complex.

    Journal of molecular and cellular cardiology 2001;33;4;789-98

  • Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell-cell communication.

    Giepmans BN, Hengeveld T, Postma FR and Moolenaar WH

    Division of Cellular Biochemistry, The Netherlands Cancer Institute and Centre for Biomedical Genetics, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

    Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK(+)) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK(+) and, to a lesser extent, with wild-type c-Src, but not with kinase-dead c-Src. Mutation of residue Cx43 Tyr(265) (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK(+) in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43Delta263) that lacks residue Tyr(265). Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr(265), resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.

    The Journal of biological chemistry 2001;276;11;8544-9

  • c-Src regulates the interaction between connexin-43 and ZO-1 in cardiac myocytes.

    Toyofuku T, Akamatsu Y, Zhang H, Kuzuya T, Tada M and Hori M

    Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. toyofuku@mr-path.med.osaka-u.ac.jp

    Connexin-43 is known to interact directly with ZO-1 in cardiac myocytes, but little is known about the role of ZO-1 in connexin-43 function. In cardiac myocytes, constitutively active c-Src inhibited endogenous interaction between connexin-43 and ZO-1 by binding to connexin-43. In HEK293 cells, by contrast, a connexin-43 mutant lacking the Src phosphorylation site (Tyr265) interacted with ZO-1 despite cotransfection of a constitutively active c-Src. Moreover, in vitro binding assays using recombinant proteins synthesized from regions of connexin-43 and ZO-1 showed that the tyrosine-phosphorylated C terminus of connexin-43 interacts with the c-Src SH2 domain in parallel with the loss of its interaction with ZO-1. Cell surface biotinylation revealed that, by phosphorylating Tyr265, constitutively active c-Src reduces total and cell surface connexin-43 down to the levels seen in cells expressing a mutant connexin-43 lacking the ZO-1 binding domain. Finally, electrophysiological analysis showed that both the tyrosine phosphorylation site and the ZO-1-binding domain of connexin-43 were involved in the regulation of gap junctional function. We therefore conclude that c-Src regulates the interaction between connexin-43 and ZO-1 through tyrosine phosphorylation and through the binding of its SH2 domain to connexin-43.

    The Journal of biological chemistry 2001;276;3;1780-8

  • Aggregated DsRed-tagged Cx43 and over-expressed Cx43 are targeted to lysosomes in human breast cancer cells.

    Qin H, Shao Q, DJ and Laird DW

    Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada.

    To investigate if either wild-type or aggregated Cx43 is abnormally targeted to lysosomes in human breast tumor cells, we examined the fate of DsRed-tagged Cx43 and over-expressed Cx43 in communication-deficient HBL-100 and MDA-MB-231 cells. DsRed-tagged Cx43 was assembled into gap junctions in control normal rat kidney cells that express endogenous Cx43 but not in Cx43-negative HBL-100 cells. However, when HBL-100 cells were engineered to coexpress wild-type Cx43 a population of DsRed-tagged Cx43 was rescued and assembled into gap junctions. Co-expression of wild-type Cx26 failed to rescue the assembly of DsRed-tagged Cx43 into gap junctions. Immunolocalization studies revealed that DsRed-tagged Cx43 was aggregated and partially localized to lysosomes. Interestingly, when human MDA-MB-231 breast tumor cells over-expressed wild-type Cx43, Cx43 protein primarily localized to lysosomes. Together, these studies provide evidence for Cx43 being targeted to lysosomes as a result of misfolding and aggregation, while in other cases, the delivery of wild-type Cx43 to lysosomes appears to be due to defects innate to the breast tumor cell type.

    Cell communication & adhesion 2001;8;4-6;433-9

  • Bisphosphonate-induced, hemichannel-mediated, anti-apoptosis through the Src/ERK pathway: a gap junction-independent action of connexin43.

    Plotkin LI and Bellido T

    Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences, Little Rock 72205, USA.

    Preservation of the mechanosensory function of osteocytes by inhibiting their apoptosis might contribute to the beneficial effects of bisphosphonates in bone. We report herein a mechanism by which connexin43 hemichannel opening by bisphosphonates triggers the activation of the kinases Src and ERKs and promotes cell survival. Bisphosphonate-induced anti-apoptosis requires connexin channel integrity, but not gap junctions. Osteocytic cells express functional hemichannels that are opened by bisphosphonates, as demonstrated by dye uptake, regulation by established agonists and antagonists, and cell surface biotinylation. The anti-apoptotic effect of bisphosphonates depends on connexin43 expression in mouse embryonic fibroblasts and osteoblastic cells. Transfection of connexin43, but not other connexins, into connexin43 naïve cells confers de novo responsiveness to the drugs. The signal transducing property of connexin43 requires the pore-forming, as well as the C-terminal domains of the protein, the interaction of connexin43 with Src. and the activation of both Src and ERK kinases. These studies establish a role for connexin43 hemichannels in bisphosphonate action, and a novel function of connexin43--b 14e1 eyond gap junction communication--in the regulation of survival signaling pathways.

    Funded by: NIA NIH HHS: P01-AG13918; NIAMS NIH HHS: K02-AR02127, R29-AR43453

    Cell communication & adhesion 2001;8;4-6;377-82

  • Comparison of connexin 43, 40 and 45 expression patterns in the developing human and mouse hearts.

    Kaba RA, Coppen SR, Dupont E, Skepper JN, Elneil S, Haw MP, Pepper JR, Yacoub MH, Rothery S and Severs NJ

    Cardiac Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom. r.kaba@ic.ac.uk

    The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.

    Cell communication & adhesion 2001;8;4-6;339-43

  • Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.

    Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG and Lau AF

    Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. plampe@fhcrc.org

    Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a conc 457 omitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

    Funded by: NCI NIH HHS: R01 CA052098; NHLBI NIH HHS: HL58732, R01 HL058732; NIGMS NIH HHS: GM46277, GM55632, R01 GM046277, R01 GM055632

    The Journal of cell biology 2000;149;7;1503-12

  • Connexin expression and turnover : implications for cardiac excitability.

    Saffitz JE, Laing JG and Yamada KA

    Departments of Pathology and Medicine and the Center for Cardiovascular Research, Washington University, St. Louis, MO 63110, USA. saffitz@pathbox.wustl.edu

    Electrical activation of the heart requires current transfer from one cell to another via gap junctions, arrays of densely packed intercellular channels. The extent to which cardiac myocytes are coupled is determined by multiple mechanisms, including tissue-specific patterns of expression of diverse gap junction channel proteins (connexins), and regulatory pathways that control connexin synthesis, intracellular trafficking, assembly into channels, and degradation. Many connexins, including those expressed in the heart, have been found to turn over rapidly. Recent studies in the intact adult heart suggest that connexin43, the principal cardiac connexin, is surprisingly short-lived (half-life approximately 1.3 hours). Both the proteasome and the lysosome participate in connexin43 degradation. Other ion channel proteins, such as those forming selected voltage-gated K(+) channels, may also exhibit rapid turnover kinetics. Regulation of connexin degradation may be an important mechanism for adjusting intercellular coupling in the heart under normal and pathophysiological conditions.

    Circulation research 2000;86;7;723-8

  • Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations.

    Oviedo-Orta E, Hoy T and Evans WH

    Department of Medical Biochemistry and Wales Heart Research Institute, University of Wales College of Medicine, Cardiff, UK.

    The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil-derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil-derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequenc 1f40 es of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and alpha-glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co-cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T- and B-lymphocyte co-cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes.

    Immunology 2000;99;4;578-90

  • The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation.

    Doble BW, Ping P and Kardami E

    Institute of Cardiovascular Sciences, University of Manitoba, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.

    Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.

    Circulation research 2000;86;3;293-301

  • Sporadic cases of dilated cardiomyopathies associated with atrioventricular conduction defects are not linked to mutation within the connexins 40 and 43 genes.

    Haefliger JA, Goy JJ and Waeber G

    European heart journal 1999;20;24;1843

  • Linkage analysis narrows the critical region for oculodentodigital dysplasia to chromosome 6q22-q23.

    Boyadjiev SA, Jabs EW, LaBuda M, Jamal JE, Torbergsen T, Ptácek LJ, Rogers RC, Nyberg-Hansen R, Opjordsmoen S, Zeller CB, Stine OC, Stalker HJ, Zori RT and Shapiro RE

    Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland 21287-3914, USA.

    Oculodentodigital dysplasia (ODDD) is an autosomal dominant condition with high penetrance and variable expressivity. The anomalies of the craniofacial region, eyes, teeth, and limbs indicate abnormal morphogenesis during early fetal development. Neurologic abnormalities occur later in life and appear to be secondary to white matter degeneration and basal ganglia changes. In familial cases, the dysmorphic and/or neurodegenerative components of the phenotype can be more severe and/or present at a younger age in subsequent generations, suggesting genetic anticipation. These clinical features suggest that the ODDD gene is pleiotropic with important functions throughout pre- and postnatal development. We have performed two-point linkage analysis with seven ODDD families and 19 microsatellite markers on chromosome 6q spanning a genetic distance of approximately 11 cM in males and 20 cM in females. We have refined the location of the ODDD gene between DNA markers D6S266/D6S261 (centromeric) and D6S1639 (telomeric), an interval of 1.01 (male) to 2.87 (female) cM. The strongest linkage was to DNA marker D6S433 (Zmax = 8.96, thetamax = 0.001). Families show significant linkage to chromosome 6q22-q23 and no evidence for genetic heterogeneity.

    Funded by: NCRR NIH HHS: M01 RR00052, RR00064; NIDCR NIH HHS: P50 DE 11131; ...

    Genomics 1999;58;1;34-40

  • Identification of a domain on the beta-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin.

    Grunwald ME, Yu WP, Yu HH and Yau KW

    Department of Neuroscience and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.

    The cGMP-gated cation channel mediating phototransduction in retinal rods has recently been shown to be inhibited by calcium-calmodulin, through direct binding of the latter to the beta-subunit of the heterotetrameric channel complex. Here, we report the characterization of this inhibition and the identification of a domain crucial for this modulation. Heterologous expression of the alpha- and beta-subunits of the human rod channel in HEK 293 cells produced a cGMP-gated current that was highly sensitive to calcium-calmodulin, with half-maximal inhibition at approximately 4 nM. In biochemical and electrophysiological experiments on deletion mutants of the beta-subunit, we have identified a region on its cytoplasmic N terminus that binds calmodulin and is necessary for the calmodulin-mediated inhibition of the channel. However, in gel shift assays and fluorescence emission experiments, peptides derived from this region indicated a low calmodulin affinity, with dissociation constants of approximately 3-10 microM. On the C terminus, a region was also found to bind calmodulin, but it was likewise of low affinity, and its deletion did not abolish the calmodulin-mediated inhibition. We suggest that although the identified region on the N terminus of the beta-subunit is crucial for the calmodulin effect, other regions are likely to be involved as well. In this respect, the rod channel appears to differ from the olfactory cyclic nucleotide-gated channel, which is also modulated by calcium-calmodulin.

    The Journal of biological chemistry 1998;273;15;9148-57

  • Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase.

    Warn-Cramer BJ, Cottrell GT, Burt JM and Lau AF

    Molecular Carcinogenesis Section, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, Hawaii 96813, USA. bwcramer@crch.hawaii.edu

    Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.

    Funded by: NCI NIH HHS: CA 52098; NHLBI NIH HHS: HL 303470

    The Journal of biological chemistry 1998;273;15;9188-96

  • Intercellular calcium signaling via gap junction in connexin-43-transfected cells.

    Toyofuku T, Yabuki M, Otsu K, Kuzuya T, Hori M and Tada M

    Department of Medicine and Pathophysiology, Osaka University Medical School, Japan. toyofuku@mr-path.med.osaka.u.ac.jp

    In excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+ propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187 and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.

    The Journal of biological chemistry 1998;273;3;1519-28

  • connexin43 gene mutations and heterotaxy.

    Tóth T, Hajdú J, Marton T, Nagy B and Papp Z

    Circulation 1998;97;1;117-8

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Tyrosine phosphorylation of connexin 43 by v-Src is mediated by SH2 and SH3 domain interactions.

    Kanemitsu MY, Loo LW, Simon S, Lau AF and Eckhart W

    Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

    Reduction of gap junctional communication in v-src transformed cells is accompanied by tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). Cx43 is phosphorylated on tyrosine by v-Src. The Src homology 3 (SH3) and Src homology 2 (SH2) domains of v-Src mediate interactions with substrate proteins. SH3 domains interact with proline-rich peptide motifs. SH2 domains associate with short amino acid sequences containing phosphotyrosine. We present evidence that the SH3 and SH2 domains of v-Src bind to proline-rich motifs and a phosphorylated tyrosine residue in the C-terminal tail of Cx43. Cx43 bound to the SH3 domain of v-Src, but not c-Src, in vitro. Tyrosine-phosphorylated Cx43 bound to the SH2 domain of v-Src in vitro. v-Src coprecipitated with Cx43 from v-src-transformed Rat-1 fibroblasts. Mutations in the SH3 and SH2 domains of v-Src, and in the proline-rich region or tyrosine 265 of Cx43, reduced interactions between v-Src and Cx43 in vivo. Tyrosine phosphorylation of Cx43 was dependent on the association of v-Src and Cx43. T 1f40 hese results provide further evidence for the direct involvement of v-Src in tyrosine phosphorylation of Cx43 and inhibition of gap junctional communication in v-src-transformed cells.

    Funded by: NCI NIH HHS: CA09370, CA13884, CA14195; ...

    The Journal of biological chemistry 1997;272;36;22824-31

  • Absence of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visceroatrial heterotaxy.

    Penman Splitt M, Tsai MY, Burn J and Goodship JA

    Department of Human Genetics, University of Newcastle upon Tyne, United Kingdom.

    Objective: To determine the frequency of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visceroatrial heterotaxy.

    Design: Mutation screening of the terminal 200 base pairs of connexin43 gene coding sequence in a series of patients from tertiary care centres.

    Patients: 48 patients with visceroatrial heterotaxy attending UK Regional Paediatric Cardiology Centres.

    Results: No changes from the published connexin43 consensus sequence were found in any of the 48 patients studied.

    Conclusions: Germline mutations of the phosphorylation sites in teh regulatory domain of the connexin43 gene are rare in patients with visceroatrial heterotaxy.

    Heart (British Cardiac Society) 1997;77;4;369-70

  • Failure to detect connexin43 mutations in 38 cases of sporadic and familial heterotaxy.

    Gebbia M, Towbin JA and Casey B

    Department of Pathology, Baylor College of Medicine, Houston, Tex 77030, USA.

    Background: Heterotaxy results from failure to establish normal left/right asymmetry during embryonic development. Typical manifestations include complex heart defects and malpositioning of abdominal organs. Missense base substitutions clustered in a 150-base pair region of the gap-junction gene connexin43 (cx43) have been implicated in the pathogenesis of heterotaxy.

    cx43 was studied in 38 cases of sporadic and familial heterotaxy. A 400-base pair region containing the previously reported mutation sites was amplified and directly sequenced in 19 patients. Nineteen additional patients were tested for restriction fragments predicted by two of the previously reported missense substitutions. No difference from normal control subjects was detected in any of the patients.

    Conclusions: Randomly selected cases of heterotaxy are unlikely to be the result of mutations in cx43.

    Funded by: NICHD NIH HHS: P30 HD24064

    Circulation 1996;94;8;1909-12

  • Activation of protein kinase C in human uterine smooth muscle induces connexin-43 gene transcription through an AP-1 site in the promoter sequence.

    Geimonen E, Jiang W, Ali M, Fishman GI, Garfield RE and Andersen J

    Department of Obstetrics, Gynecology and Reproductive Medicine, School of Medicine, State University of New York, Stony Brook, New York 11794-8091, USA.

    Myometrial connexin-43 gap junctions are scarce throughout gestation but appear in large numbers at term to facilitate contractions during labor. The mechanisms that regulate this process are incompletely characterized. This report investigates the effects of protein kinase C activation on the regulation of connexin-43 gene transcription in human uterine smooth muscle cells. In primary myometrial cells treated with phorbol ester, transient increases in c-Fos and c-Jun protein levels were observed at 2-4 h, followed by significant increases in connexin-43 protein levels at 6-8 h. Nuclear run-on transcription analysis showed an increase in connexin-43 transcription 3 h after phorbol ester treatment. AP-1 sites were identified in the sequence of the 5'-flanking promoter region of the human connexin-43 gene at 44 and 1000 base pairs upstream of transcription start. Transcription from a reporter plasmid containing the proximal human connexin-43 promoter was increased in transfected primary cultures treated with phorbol ester. Mutation of the proximal AP-1 site in the promoter abolished the phorbol ester-dependent transactivation. This work provides evidence that transcription of the human connexin-43 gene is induced through protein kinase C activation in uterine smooth muscle cells, and that the induction involves up-regulation and activation of c-Jun and c-Fos.

    Funded by: NHLBI NIH HHS: HL02391; NICHD NIH HHS: HD 30482

    The Journal of biological chemistry 1996;271;39;23667-74

  • Characterization of the mitogen-activated protein kinase phosphorylation sites on the connexin-43 gap junction protein.

    Warn-Cramer BJ, Lampe PD, Kurata WE, Kanemitsu MY, Loo LW, Eckhart W and Lau AF

    Department of Molecular Carcinogenesis, Cancer Research Center of Hawaii, School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813, USA.

    We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increase in serine phosphorylation on the connexin-43 gap junction protein in T51B rat liver epithelial cells. The kinase(s) responsible for phosphorylation and specific serine targets in connexin-43 have not been identified. There are three consensus mitogen-activated protein (MAP) kinase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of peptides from connexin-43 phosphorylated in vivo in cells treated with epidermal growth factor. These data suggested that MAP kinase may phosphorylate connexin-43 directly in vivo. We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to characterize MAP kinase phosphorylation. Site-directed mutagenesis, phosphotryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser255, Ser279, and Ser282, which correspond to the consensus sites recognized earlier. Characterization of MAP kinase-mediated phosphorylation of connexin-43 has defined potential targets for phosphorylation in vivo following activation of the epidermal growth factor receptor and has provided the basis for studies of the effects of phosphorylation, at specific molecular sites, on the regulation of gap junctional communication.

    Funded by: NCI NIH HHS: CA 09370, CA 13884, CA 52098; ...

    The Journal of biological chemistry 1996;271;7;3779-86

  • Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality.

    Britz-Cunningham SH, Shah MM, Zuppan CW and Fletcher WH

    Department of Physiology, Loma Linda University, CA 92354, USA.

    Background: Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Connexin43, the major protein of gap junctions in the heart, is targeted by several protein kinases that regulate myocardial cell-cell coupling. We hypothesized that mutations altering sites critical to this regulation would lead to functional or developmental abnormalities of the heart.

    Methods: Connexin43 DNA from 25 normal subjects and 30 children with a variety of congenital heart diseases was amplified by the polymerase chain reaction and sequenced. Mutant DNA was expressed in cell culture and examined for its effect on the regulation of cell-cell communication.

    Results: The 25 normal subjects and 23 of the 30 children with heart disease had no amino acid substitutions in connexin43. All six children with syndromes that included complex heart malformations had substitutions of one or more phosphorylatable serine or threonine residues. Four of these children had two independent mutations, suggesting an autosomal recessive disorder. Five of these children had substitutions of proline for serine at position 364. A seventh child, with a different heart condition, also had a point mutation in connexin43. Transfected cells expressing the Ser364Pro mutant connexin43 sequence showed abnormalities in the regulation of cell-cell communication, as compared with cells expressing normal connexin43.

    Conclusions: Mutations in the connexin43 gap-junction gene, which lead to abnormally regulated cell-cell communication, are associated with visceroatrial heterotaxia.

    The New England journal of medicine 1995;332;20;1323-9

  • Functional analysis of the connexin43 gene promoter in vivo and in vitro.

    De Leon JR, Buttrick PM and Fishman GI

    Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.

    Connexin43 is the principal gap junction protein found in the mammalian heart. The regulation of connexin43 gene expression may be an important determinant of normal and altered cardiac development and electrophysiology. We constructed a series of chimeric luciferase reporter genes containing nested deletions from the human connexin43 gene (-2400 to -50 basepairs, relative to transcription initiation). The transcriptional activity of the chimeric genes was assayed in several cell types and systems, including rat cardiocytes in vitro and adult rat heart in vivo. In both systems, high levels of luciferase activity required at least 175 base-pairs of 5'-flanking sequence. Constructs including 2400 base-pairs of upstream sequence increased activity two-fold in vivo, but not in vitro. Chimeric genes were also assayed in cultured cardiac non-myocytes. This population of cells accumulates significant levels of connexin43 mRNA when propagated in vitro and promoter constructs were correspondingly active. Elements within the proximal promoter may also confer tissue-specificity. Transfectional analysis of the SKHep1 cell line, which does not express connexin43, demonstrated reduced reporter gene activity, especially for the longest constructs. These studies begin to define the cis-acting elements of the connexin43 gene which regulate its strength and specificity of transcription.

    Funded by: NHLBI NIH HHS: 5K11HL02391, HL37412, HL46034; ...

    Journal of molecular and cellular cardiology 1994;26;3;379-89

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • Human connexin43 gene locus, GJA1, sublocalized to band 6q21-->q23.2.

    Corcos IA, Meese EU and Loch-Caruso R

    Department of Biology, University of Michigan, Ann Arbor.

    Gap junctions permit transfer of small molecules directly between cells. Several laboratories have mapped the human gap junction protein subunit, connexin43 (locus designation, GJA1), to overlapping regions of chromosome 6, localizing GJA1 to 6q14-->q24. We have sublocalized human GJA1 to a very small region. 6q21-->q23.2, using a human x rodent somatic cell hybrid mapping panel and a rat connexin43 cDNA probe.

    Funded by: NICHD NIH HHS: HD18258; NIEHS NIH HHS: ES04424

    Cytogenetics and cell genetics 1993;64;1;31-2

  • Functional analysis of human cardiac gap junction channel mutants.

    Fishman GI, Moreno AP and Spray DC

    Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.

    The connexins form a family of membrane spanning proteins that assemble into gap junction channels. The biophysical properties of these channels are dependent upon the constituent connexin isoform. To begin identifying the molecular basis for gap junction channel behavior in the human heart, a tissue that expresses connexin43, we used site-directed mutagenesis to generate mutant cDNAs of human connexin43 with shortened cytoplasmic tail domains. Premature stop codons were inserted, resulting in proteins corresponding in length to the mammalian isoforms connexin32 and connexin26, which are expressed primarily in liver. All constructs restore intercellular coupling when they are transfected into SKHep1 cells, a human hepatoma line that is communication deficient. Whereas wild-type connexin43 transfectants display two distinct unitary conductance values of about 60 and 100 pS, transfectants expressing the mutant proteins, from which 80 and 138 amino acids have been deleted, exhibit markedly different single-channel properties, with u 647 nitary conductance values of about 160 and 50 pS, respectively. Junctional conductance of channels composed of wild-type connexin43 is less voltage-sensitive compared with transfectants expressing wild-type connexin32. However, neither of the connexin43 truncation mutants alters this relative voltage insensitivity. These results suggest that the cytoplasmic tail domain is an important determinant of the unitary conductance event of gap junction channels but not their voltage dependence. Furthermore, since the mutant connexins are missing several consensus phosphorylation sites, modification of these particular sites may not be required for membrane insertion or assembly of human connexin43 into functional channels.

    Funded by: NHLBI NIH HHS: 1K11HL02391, HL38449; NINDS NIH HHS: NS16542

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;9;3525-9

  • The human connexin gene family of gap junction proteins: distinct chromosomal locations but similar structures.

    Fishman GI, Eddy RL, Shows TB, Rosenthal L and Leinwand LA

    Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.

    Connexins are protein subunits that constitute gap junction channels. Two members of this gene family, connexin43 (Cx43) and connexin32 (Cx32), are abundantly expressed in the heart and liver, respectively. Human genomic DNA analysis revealed the presence of two loci for Cx43: an expressed gene and a processed pseudogene. The expressed gene (GJA1) was mapped to human chromosome 6 and the pseudogene (GJA1P) to chromosome 5. To determine whether Cx32 was linked to Cx43, somatic cell hybrids were analyzed by polymerase chain reaction and hybridization, resulting in the assignment of the gene for Cx32 (GJB1) to the X chromosome at Xp11----q22. Comparison of the structures of connexin genes suggests that members of this multigene family arose from a single precursor, but evolved to distinct chromosomal locations.

    Funded by: NHGRI NIH HHS: HG00333; NHLBI NIH HHS: 1K11HL02391, HL37412

    Genomics 1991;10;1;250-6

  • Molecular characterization and functional expression of the human cardiac gap junction channel.

    Fishman GI, Spray DC and Leinwand LA

    Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.

    Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.

    Funded by: NHLBI NIH HHS: HL07071, P01HL137412; NIGMS NIH HHS: GM29090

    The Journal of cell biology 1990;111;2;589-98

Gene lists (6)

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
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© 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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