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Query: UNIPROT:P06889 (Mol)
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We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.
Am J Physiol Lung Cell Mol Physiol 2001 Jun
PMID:Heterocellular gap junctional communication between alveolar epithelial cells. 1135 Jul 86

We previously reported that connexin (Cx) 26 expression is involved in negative growth control of HepG2 cells established from a human hepatoma. We also found that induction of E-cadherin and subsequent formation of a cell adhesion complex were induced in HepG2 cells by Cx 26 expression. To examine the exact role of Cx 26-induced E-cadherin junctions in regulating appearance of malignant phenotypes of HepG2 cells, we expressed a Cx 26 antisense oligodeoxynucleotide (AS-ODN) in an established HepG2 cell clone that has stable expression of Cx 26 genes. We investigated changes in the expression of E-cadherin, the localization of beta-catenin, and some malignant phenotypes of HepG2 clone after the suppression of Cx 26 expression by AS-ODN treatment. The AS-ODN treatment prevented the expression of Cx 26 and E-cadherin, and the localization of beta-catenin was changed from cytoplasmic membrane to the cytoplasm. In parallel, a morphological change from a monolayer of polygonal cells to multilayered colonies was induced by the treatment, indicating a change of a malignant phenotype of HepG2 cells. The activity of matrix metalloproteinase 9 (MMP-9) was elevated by the AS-ODN treatment. A concomitant increase in invasiveness of the Cx 26-expressing cells by the treatment was also observed in an in vitro assay with Matrigel matrix. These results suggest that the induction of E-cadherin and formation of the cell adhesion complex by Cx 26 expression contribute to the reversal of some malignant phenotypes of HepG2 cells. Furthermore, the Cx 26-dependent expression of E-cadherin leads to reduction of the invasiveness of the cells through suppression of MMP-9 activity.
Mol Carcinog 2001 Jun
PMID:Regulation of cellular invasion and matrix metalloproteinase activity in HepG2 cell by connexin 26 transfection. 1142 87

This study examined connexin (Cx) gene activity in relation to oocyte maturation in the red seabream (Pagrus major) ovary. Mixed primers for the polymerase chain reaction (PCR) were designed based on the high sequence homology of selected regions of known Cx genes. PCR-amplified cDNA fragments generated by 3' and 5' rapid amplication of cDNA ends (RACE) were combined to generate full-length cDNA sequences. The 1212-bp cDNA has an open reading frame encoding 282 amino acids, with a molecular mass of 32.3 kDa (red seabream Cx32.3). Hydropathy plots of red seabream Cx32.3 show the four typical major hydrophobic and four major hydrophilic regions of Cx proteins. Typical Cx consensus sequences are observed in the first and second extracellular loops. The ovarian follicles of matured female seabream were incubated in the presence of 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP, 10 ng/ml), gonadotropin (GtH)-I (300 ng/ml) and GtH-II (300 ng/ml). Northern blot analysis of poly(A)(+) RNA extracted from the ovarian follicles were hybridized with red seabream Cx32.3 and beta-actin probes. The transcription level of PmCx32.3 in the presence of DHP, PmGtH-I and PmGtH-II was significantly higher than in the control.
Comp Biochem Physiol B Biochem Mol Biol 2001 Jul
PMID:Molecular cloning and expression of connexin 32.3 cDNA in the ovary from the red seabream (Pagrus major). 1143 31

Cell transplantation has been proposed as a future therapy for various myocardial diseases. It is unknown, however, whether the encouraging results obtained in animal models of ischemia and reperfusion, cryoinjury or cardiomyopathy can be reproduced in the setting of permanent coronary artery occlusion and extensive myocardial infarction (MI). Embryonic cardiac cells were isolated and cultured for 3 days to confirm viability, morphology and to label cells with BrdU or the reporter gene LacZ. Seven days after extensive MI, rats were randomized to cell (1.5x10(6)) transplantation (n=11) or culture medium injection (n=16) into the myocardial scar. Echocardiography study was performed before and 53+/-3 days after implantation to assess left ventricular (LV) remodeling and function. During follow-up, there was no mortality among cell-treated rats v 4 of 16 control rats (P=0.12). X-gal staining, BrdU and alpha -SMA immunohistochemistry identified the engrafted cells 1 week, 4 weeks and 8 weeks after transplantation, respectively. Antibodies against alpha -SMA, connexin-43, fast and slow myosin heavy chain revealed grafts in various stages of differentiation in 10 of 11 cell-treated hearts. Many of them, however, kept their embryonic phenotype and were isolated from the host myocardium by scar tissue. Serial echocardiography studies revealed that cell transplantation prevented scar thinning, LV dilatation and dysfunction while control animals developed scar thinning, significant LV dilatation accompanied by progressive deterioration in LV contractility. Transplantation of embryonic cardiomyocytes after extensive MI in a rat model attenuate LV dilatation, infarct thinning, and myocardial dysfunction. Still, many grafts remain isolated and do not differentiate into an adult phenotype, even when studied 2 months after grafting.
J Mol Cell Cardiol 2001 Jul
PMID:Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. 1143 38

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na(+) and Ca(2+) accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca(2+)]i and [Na(+)]i in myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4 microM fluo-3 AM for 30 min, or with 5 microM sodium green AM for 60 min at 37 degrees C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 mM NaCN, 20 mM 2-deoxy-D-glucose and 0-glucose); or (3) MI+H (0.95 mM, 4.7 mM) for 60 min. Propidium iodide (PI, 25 microM) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca(2+)]i and [Na(+)]i were then calculated by calibration. In some experiments, the effect of 10 microM tetrodotoxin (TTX) and 20 microM nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca(2+)]i and [Na(+)]i in myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca(2+)]i and [Na(+)]i in myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca(2+)]i and [Na(+)]i overload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na(+) and Ca(2+) channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.
J Mol Cell Cardiol 2001 Dec
PMID:Activation of connexin-43 hemichannels can elevate [Ca(2+)]i and [Na(+)]i in rabbit ventricular myocytes during metabolic inhibition. 1173 61

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.
Hum Mol Genet 2001 Dec 01
PMID:Mutations in GJA1 (connexin 43) are associated with non-syndromic autosomal recessive deafness. 1174 37

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.
J Mol Biol 2002 Jan 25
PMID:Isolation and characterization of gap junctions from tissue culture cells. 1181 32

Mutations of connexin alpha 8 (GJA8 or Cx50) and connexin alpha 3 (GJA3 or Cx46) in humans have been reported to cause cataracts with semi-dominant inheritance patterns. Targeted null mutations in Gja8 and Gja3 in mice cause cataracts with recessive inheritance. The molecular bases for these differences in inheritance patterns and the mechanism for cataractogenesis in these mutants are poorly understood. We recently mapped an autosomal semi-dominant cataract [lens opacity 10 (Lop10)] mutation to mouse chromosome 3 and identified a missense mutation (G-->C) in the Gja8 gene, which causes glycine at codon 22 to be replaced with arginine (G22R). Moreover, we demonstrated that the alpha 8 G22R isoform is a loss-of-function mutant for alpha 8, as well as a dominant mutation for reducing the phosphorylated forms of alpha 3 connexin in vivo. To test the hypothesis that the alteration of endogenous alpha 3 connexin in Lop10 mice led to a unique lens phenotype, we generated double mutant offspring between Lop10 and the Gja3(tm1) (alpha 3(-/-)) mice. The double homozygous mutant mice (Lop10/Lop10 alpha 3(-/-)) showed relatively normal lens cortical fibers compared to the Lop10 mice. A functional impairment of endogenous alpha 3 connexin is therefore partly responsible for cellular phenotypes in the Lop10 mice. This study has provided some novel molecular insights into mouse and human cataractogenesis caused by alpha 8 and alpha 3 mutations. These mouse models will be useful for investigating the mechanistic relationship between gap junction impairment and cataract formation.
Hum Mol Genet 2002 Mar 01
PMID:A Gja8 (Cx50) point mutation causes an alteration of alpha 3 connexin (Cx46) in semi-dominant cataracts of Lop10 mice. 1187 45

Erythrokeratodermia variabilis (EKV) is a skin disorder characterized by variable (transient) erythemas and fixed keratosis. The disorder maps to chromosome 1p34-35, a location that contains the GJB3 gene encoding the gap junction protein connexin 31. Until now, only heterozygote mutations in the form of dominant inheritance have been described in this gene associated with EKV. We report here a homozygote mutation in the connexin 31 gene, found in a family that shows recessive inheritance of the disorder, thus providing the first molecular support for a recessive variant of EKV. The entire GJB3 coding sequence was scanned for mutations by sequencing. We detected a T-->C transition at position 101 of the coding sequence, which replaces a leucine with a proline at residue 34 of the protein (L34P). Evolutionary analysis shows that this mutation is located at a highly conserved region of connexin in the first putative transmembrane helix (TMH). In transfected keratinocytes, L34P connexin 31 had a cytoplasmic distribution, suggesting that the mutant form of this protein will not form normal gap junctions between adjacent cells. The change of leucine to proline is likely to alter the structure of the first TMH of connexin by inducing a kink, thus influencing connexon structure and function.
Hum Mol Genet 2002 May 15
PMID:A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective trafficking of the connexin 31 protein. 1201 12

Gap junctions, clusters of transmembrane channels that link adjoining cells, mediate myocyte-to-myocyte electrical coupling and communication. The component proteins of gap junction channels are termed connexins and, in in vitro expression systems, gap-junctional channels composed of different connexin types exhibit different biophysical properties. In common with other tissues, the heart expresses multiple connexin isoforms. Spatially defined patterns of expression of three connexin isoforms - connexin43, connexin40 and connexin45 - form the cell-to-cell conduction pathways responsible for the orderly spread of current flow that governs the normal cardiac rhythm. Remodeling of gap junction organization and connexin expression is a common feature of human heart disease conditions in which there is an arrhythmic tendency. This remodeling may take the form of disturbances in the distribution of gap junctions and/or quantitative alterations in connexin expression, notably reduced ventricular connexin43 levels. The idea that such changes may contribute to the development of a pro-arrhythmic substrate in the diseased heart has gained ground over the last decade. Recent studies using transgenic mice models have raised new opportunities to explore the significance of gap junction remodeling in the diseased heart.
J Cell Mol Med
PMID:Gap junction remodeling and cardiac arrhythmogenesis: cause or coincidence? 1206 69


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