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At least 19 recordsLinked to original sources
PubMed1998

Tumour necrosis factor alpha alters the expression of connexin43, connexin40, and connexin37 in human umbilical vein endothelial cells.

Tumour necrosis factor alpha (TNF-alpha) plays an important role in orchestrating inflammatory responses with the vascular endothelium as main target cell type, and was found to promote migration of endothelial cells, as occurs in wound healing processes. Substantial evidence exists that endothelial cell migration in wound healing is related to changes in cell coupling by means of gap junctions. Gap junctions are agglomerates of cell-to-cell channels that allow direct electrical and metabolic communication between cells. The authors have investigated whether TNF-alpha alters the expression of gap junction proteins (connexins, Cx) between human umbilical vein endothelial cells (HUVEC), thereby changing the extent of intercellular communication, as measured by dye coupling. Under control conditions, Cx43, Cx40, and Cx37 protein and mRNA were present in HUVEC. After exposure to 0.5 nM TNF-alpha for 48 h, however, the authors were no longer able to detect Cx37 and Cx40 protein, whereas Cx43 levels seemed unaltered but showed more perinuclear staining. After 24 and 48 h exposure to TNF-alpha, levels of Cx37 and Cx40 mRNA, were reduced, while the level of Cx43 mRNA remained unaltered, suggesting transcriptional regulation. If TNF-alpha was removed from the medium, Cx37 and Cx40 expression was restored within 24 h. The modulation of connexin expression by TNF-alpha resulted in a decrease in dye coupling of 40%.

Cells, Cultured↗
PubMed1997

Gap junctions in human umbilical cord endothelial cells contain multiple connexins.

We investigated the expression pattern of gap junctional proteins (connexins, Cx) in situ and in vitro and their functional characteristics in cultured human umbilical vein endothelial cells (HUVEC) and cultured human umbilical artery endothelial cells (HUAEC). In both arteries and veins, Cx37, Cx40, and Cx43 could be detected in situ and in vitro (passages 2-4). Distribution patterns of Cx40 and Cx43 were homogeneous in situ but more heterogeneous in vitro. Cx37 is heterogeneously expressed both in situ and in vitro. Among most cells, no Cx37 staining could be detected; when present, it was found as bright spots between some clusters of cells. Cx40 was more abundant in cultured arterial endothelium than in cultured venous endothelium. Dye-coupling experiments with Lucifer yellow CH revealed extensive dye spread in HUVEC (15.2 +/- 0.4, mean +/- SE, n = 110) but was significantly restricted in HUAEC (9.8 +/- 0.3, n = 110). Electrophysiological gap junctional characteristics were determined in cultured HUVEC and HUAEC pairs by use of the dual voltage-clamp technique. In contrast to the dye-coupling experiments, mean macroscopic electrical conductance was significantly larger for HUAEC pairs (31.4 +/- 6.0 nS, n = 12) than for HUVEC pairs (16.6 +/- 2.8, n = 18). In HUVEC, we measured multiple single gap junctional channel conductances in the range of 19-75 pS. Interestingly, additional conductances of 80-200 pS were measured in HUAEC, possibly partially reflecting activity of channels formed of Cx40, which are more abundant in the cultured arterial endothelial cells.

Cells, Cultured↗
PubMed1997

Characterization of gap junction channels in adult rabbit atrial and ventricular myocardium.

For effective cardiac output, it is essential that electrical excitation spread rapidly throughout the atria and ventricles. This is effected by electrical coupling through gap junction channels at contact sites between myocytes. These channels form a low-resistance pathway between adjacent myocytes and consist of connexin proteins. The connexin family is a large multigene family, and the channels formed by different members of this family have distinct electrical and regulatory properties. We have studied gap junction channels between adult rabbit atrial and ventricular myocytes using immunocytochemical and electrophysiological methods. Gap junctions of ventricular myocytes were immunoreactive to antibodies directed against connexin43 (Cx43) and Cx45, but not to antibodies against Cx37 or Cx40. Gap junctions between atrial myocytes showed immunostaining with anti-Cx40, -Cx43, and -Cx45 antibodies, but not with anti-Cx37 antibody. Endocardial and endothelial tissue were labeled with both Cx37 and Cx40 antibodies. The conductance of rabbit myocardial gap junctions was measured using the double whole-cell voltage-clamp method. The average macroscopic junctional conductance, corrected for series resistance, of atrial and ventricular cell pairs did not differ significantly (169+/-146 and 175+/-147 nS, respectively), and both were at most only slightly sensitive to the applied transjunctional potential difference. The difference in connexin expression between atrial and ventricular myocytes was reflected in the distribution of single gap junction channel conductances. A single population of unitary channel conductances with an average of 100 pS was observed between ventricular myocyte pairs. In addition to this population, a population with an average conductance of 185 pS was present between atrial myocyte pairs. The observed difference in connexin expression between atrial and ventricular myocardium may enable differential regulation of conduction in these tissues.

Action Potentials↗
PubMed1997

Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system.

The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ-hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.

Amino Acid Sequence↗
PubMed1998

Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed.

Vascular endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vessel wall. The aim of this study was to determine the distribution and identity of the gap junction structural proteins (connexins) expressed by endothelial cells in situ. Connexin expression in different regions of the rat aortic endothelium was analyzed with the use of indirect immunofluorescence microscopy and Western blotting. Connexin40 and connexin37 were present in most, if not all, of the thoracic and abdominal aortic endothelia in the form of maculae at cell-cell appositions. In contrast, connexin43 was undetectable in most endothelia but extremely abundant in small numbers of cells localized at the downstream edge of the ostia of branching vessels and at flow dividers, regions that experience turbulent shear stress from disturbed blood flow. To examine the relationship of shear stress and connexin43 expression, localized stress was induced by surgical coarctation of the aorta, which was sufficient to cause striking local upregulation of connexin43 within 8 days. Thus, increases in connexin43 levels are an endothelial response to mechanical stress.

Animals↗
PubMed1997

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37.

Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage (Gj/Vj) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The Gj/Vj data imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.

Animals↗
PubMed1993

Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins.

Alcohols↗
PubMed1994

CTL induction by a tumour-associated antigen octapeptide derived from a murine lung carcinoma.

Many mouse and human tumours express major histocompatibility complex (MHC) class I-associated antigens that constitute targets for syngeneic cytotoxic T lymphocytes (CTL). Genes encoding such antigens were isolated from a mouse mastocytoma and from human melanomas by genetic methods. Isolation and characterization of MHC class I-associated peptides has enabled specific anchor residues to be identified that are typical of peptides that bind to distinct class I molecules. Moreover, CTL specific to particular MHC-peptide combinations have been used to identify naturally occurring antigenic peptides in cell extracts and enabled them to be sequenced directly. Most known MHC ligands are of viral origin or are self peptides derived from normal proteins. Here we use total acid extraction and repeated fractionation to isolate and sequence Lewis lung carcinoma (3LL)-specific peptide(s), which shows sequence homology to the connexin 37 protein. Synthetic octamers based on these sequences bind to 'empty' H-2Kb molecules on RMA-S cells, sensitize RMA-S cells to lysis by specific anti-3LL CTL, and induce anti-tumour CTL. The tumour-associated peptide originates from mutated connexin 37 expressed in 3LL.

Amino Acid Sequence↗
PubMed1994

Connexin37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities.

Gap junctions are thought to mediate the direct intercellular coupling of adjacent cells by the open-closed gating of an aqueous pore permeable to ions and molecules of up to 1 kDa or 10-14 A in diameter. We symmetrically altered the ionic composition or asymmetrically added 6-carboxyfluorescein (6-CF, M(r) = 376), a fluorescent tracer, to pairs of connexin37-transfected mouse neuro2A cells to examine the ionic and dye permeability of human connexin37 channels. We demonstrate that the 300-pS channel formed by connexin37 has an effective relative anion/cation permeability ratio of 0.43, directly converts to at least one intermediate (63 pS) subconductance state, and that 6-CF dye transfer is accompanied by a 24% decrease in unitary channel conductance. These observations favor a new interpretation of the gap junction pore consistent with direct ion-channel interactions or electrostatic charge effects common to more conventional multistate ion channels. These results have distinct implications about the different forms of intercellular signaling (cationic, ionic, and/or biochemical) that can occur depending on the expression and conformation of the connexin channel proteins.

Animals↗
PubMed1997

Connexin 37 mutations in rat hepatic angiosarcomas induced by vinyl chloride.

Connexin genes have been shown to restore normal cell growth when transfected into certain tumorigenic cells and thus are considered to form a family of tumor suppressor genes. In this study, we have analyzed mutations of the connexin 37 (Cx37) gene in rat hepatic angiosarcomas induced by vinyl chloride. A total of 25 rat liver tumors (22 hepatic angiosarcomas and 3 hepatocellular carcinomas) were analyzed by PCR-single-strand conformation polymorphism analysis and DNA sequencing. Four mutations were detected in three tumors: (a) one GGG(Gly) to GAG(Glu) mutation at codon 168; and (b) three silent mutations, CGA(Arg) to CGC(Arg), at codon 166. In addition, we found that codon 88 is polymorphic (GAG(Glu) to GAA(Glu)). Cx37 proteins are detectable in endothelial cells of normal liver by immunohistochemical analysis, but none of the angiosarcomas showed Cx37-positive spots. These results suggest that Cx37-mediated gap junctional intercellular communication may be disturbed in most of these angiosarcomas, but mutation of the Cx37 gene is rare.

Animals↗
PubMed1994

Expression of gap junction proteins Cx26, Cx31.1, Cx37, and Cx43 in developing and mature rat epidermis.

To elucidate mechanisms underlying gap junction-mediated intercellular communication in epidermal keratinocytes, we have examined the expression of four connexin genes, Cx26, Cx31.1, Cx37, and Cx43, in fetal (embryonic day 17-18), newborn (post-natal day 0), and mature rat skin. Northern analyses of total skin RNA showed that levels of Cx26, Cx37, and Cx43 mRNAs remained relatively constant throughout the three developmental stages examined, whereas Cx31.1 mRNA was 15-30 times more abundant in mature skin than in fetal skin. Antibodies specifically recognizing these connexin proteins were then used in conjunction with a recently described amplification technique to immunohistochemically stain sections of paraffin embedded rat tail epidermis. We show that Cx26, Cx31.1, Cx37, and Cx43 display overlapping but distinct patterns of expression within the keratinocyte cell layers of developing and mature epidermis.

Amino Acid Sequence↗
PubMed1997

Female infertility in mice lacking connexin 37.

The signals regulating ovarian follicle development and the mechanisms by which they are communicated are largely undefined. At birth, the ovary contains primordial follicles consisting of meiotically arrested oocytes surrounded by a single layer of supporting (granulosa) cells. Periodically, subsets of primordial follicles undergo further development during which the oocyte increases in size and the granulosa cells proliferate, stratify and develop a fluid-filled antrum. After ovulation, oocytes resume meiosis and granulosa cells retained in the follicle differentiate into steroidogenic cells, forming the corpus luteum. It has been proposed that intercellular signalling through gap junction channels may influence aspects of follicular development. Gap junctions are aggregations of intercellular channels composed of connexins, a family of at least 13 related proteins that directly connect adjacent cells allowing the diffusional movement of ions, metabolites, and other potential signalling molecules. Here we show that connexin 37 is present in gap junctions between oocyte and granulosa cells and that connexin 37-deficient mice lack mature (Graafian) follicles, fail to ovulate and develop numerous inappropriate corpora lutea. In addition, oocyte development arrests before meiotic competence is achieved. Thus, cell-cell signalling through intercellular channels critically regulates the highly coordinated set of cellular interactions required for successful oogenesis and ovulation.

Animals↗
PubMed1997

Linkage studies in erythrokeratodermias: fine mapping, genetic heterogeneity and analysis of candidate genes.

Erythrokeratodermias are a clinically heterogeneous group of rare autosomal dominant disorders of cornification with overlapping features including hyperkeratosis and erythema. We ascertained five extended pedigrees with different phenotypes for a linkage study. Three families presented with localized erythrokeratodermia variabilis, and one with erythrokeratodermia and ataxia. Another family had Greither disease associated with variable hyperkeratotic plaques. Despite their phenotypic differences, both erythrokeratodermia variabilis and erythrokeratodermia with ataxia map to a common region in 1p34-p35. Multipoint linkage and haplotype analyses place erythrokeratodermia variabilis between the marker D1S496 and D1S186 with a maximum LOD score of 12.88. Our linkage results provide compelling evidence for genetic homogeneity among families of mixed European and French-Canadian origin. In contrast, results excluded Greither's disease from the established erythrokeratodermia variabilis gene region indicating genetic heterogeneity of erythrokeratodermias. Based on recombinations, two genes assigned to 1p34-p35 were excluded: cartilage matrix protein and avian myelocytosis viral oncogene. Connexin-37 (GJA4), a member of the connexin gene family, maps within the erythrokeratodermia variabilis region and is an attractive candidate gene. Direct sequencing of the coding region of GJA4 in four patients revealed several variations, including a novel polymorphism within the 5' cytoplasmic domain, but no pathogenic mutations were found, thus excluding Connexin-37 as a candidate. There is evidence, however, that other epidermally expressed connexins cluster in this region, and one may yet be determined to play a role in the pathogenesis of erythrokeratodermia variabilis.

Chromosome Mapping↗
PubMed1996

Human connexin 37 is polymorphic but not mutated in tumours.

Connexins are phylogenetically conserved proteins responsible for gap junctional intercellular communication (GJIC). In tumours, GJIC is frequently disrupted. We have tested the hypothesis that the connexin 37 (Cx37) gene might be mutated in human tumours from tissues in which the Cx37 gene is known to be expressed. Eight lung adenocarcinomas and 18 sporadic breast carcinomas were analysed. While most tumours had GTA at codon 130, a base change GTA-->ATA converting valine into isoleucine was found in three breast cancers (one homozygous for ATA) and two lung tumour samples. However, screening of normal DNA from the same patients and DNA from 42 healthy donors revealed that such base change also exists in normal tissue. Thus, we conclude that there is polymorphism of the connexin 37 gene in the human population. This is the first finding of polymorphism in the connexin gene family.

Adenocarcinoma↗
PubMed1997

Identification of shared tumor-associated antigen peptides between two spontaneous lung carcinomas.

CTLs recognize antigenic peptides bound to MHC class I Ags on the cell surface of tumor cells. Tumor-associated Ag (TAA) peptides are 8 to 10 amino acids long and can be derived from normal, mutated, or viral proteins. The majority of T cell-defined Ags have been identified in human melanoma cells. These were shown to be commonly expressed by different allogeneic melanomas that share the same MHC molecule. We have recently isolated Kb-restricted TAA peptides, which are mutations of the gap junction protein connexin 37, from the spontaneous C57BL/6 Lewis lung carcinoma (3LL). These peptides, named MUT 1 and MUT 2, serve as CTL epitopes and can induce CTL activity in vivo. Using CTL cross-reaction assays, peptide extraction, HPLC fractionation, and reverse transcriptase-PCR amplification, we show that clones of another spontaneous C57BL/6 lung carcinoma, CMT 64, share TAA peptides with the 3LL carcinoma. Vaccination with synthetic MUT 1 or MUT 2 induces CTLs that efficiently lyse CMT 64-derived clones, protects mice from CMT 64 metastasis, and affords therapy of established CMT 64 metastases. Hence, shared CTL epitopes exist between two spontaneous murine lung carcinomas.

Animals↗
PubMed1997

Differential regulation of connexin43 and connexin37 in endothelial cells by cell density, growth, and TGF-beta1.

We studied the growth-related expression of the gap junction proteins connexin43 (Cx43) and connexin37 (Cx37) to characterize mechanisms of their differential regulation in cultured bovine aortic endothelial cells. During growth to confluency, Cx43 mRNA levels were high in subconfluent cells and decreased at confluency; Cx37 mRNA was weakly detectable until the cultures became confluent, when Cx37 levels became similar to those of Cx43. Immunoprecipitation, immunoblots, and immunostaining demonstrated that Cx43 synthesis and content paralleled the changes in mRNA levels. These data suggested regulation of connexin expression related to growth status or cell density. We tested this hypothesis by inhibiting growth with transforming growth factor-beta1 (TGF-beta1). TGF-beta1 treatment caused an upregulation of Cx43 synthesis, content, and apparent half-life and an upregulation of mRNA, independent of changes in cell density. Increases in Cx43 synthesis preceded increases in mRNA, suggesting both translational and transcriptional regulation, whereas the increased half-life suggested post-translational regulation, as well. Immunostaining revealed the development of intense vesicular staining in the treated cells, which may explain the increased half-life. TGF-beta1 treatment also suppressed the upregulation of Cx37 expression. These alterations in connexin expression may have implications for endothelial communication under conditions of elevated vascular TGF-beta1 concentrations such as in wound healing.

Animals↗