Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.     

Connexin 26 expression prevents down-regulation of barrier and fence functions of tight junctions by Na+/K+-ATPase inhibitor ouabain in human airway epithelial cell line Calu-3

Gap junctions are considered to play a crucial role in differentiation of epithelial cells and to be associated with tight junction proteins. In this study, to investigate the role of gap junctions in regulation of the barrier function and fence function on the tight junctions, we introduced the Cx26 gene into human airway epithelial cell line Clau-3 and used a disruption model of tight junctions employing the Na(+)/K(+)-ATPase inhibitor ouabain. In parental Calu-3 cells, gap junction proteins Cx32 and Cx43, but not Cx26, and tight junction proteins occludin, JAM-1, ZO-1, claudin-1, -2, -3, -4, -5, -6, -7, -8, -9, and -14 were detected by RT-PCR. The barrier function and fence function of tight junctions were well maintained, whereas the GJIC was low level. Treatment with ouabain caused disruption of the barrier function and fence function of tight junctions together with down-regulation of occludin, JAM-1, claudin-2, and -4 and up-regulation of ZO-1 and claudin-14. In Cx26 transfectants, Cx26 protein was detected by Western blotting and immunocytochemistry, and many gap junction plaques were observed with well-developed tight junction strands. Expression of claudin-14 was significantly increased in Cx26 transfectants compared to parental cells, and in some cells, Cx26 was co-localized with claudin-14. Interestingly, transfection with Cx26 prevented disruption of both functions of tight junctions by treatment with ouabain without changes in the tight junction proteins. Pretreatment with the GJIC blockers 18beta-glycyrrhetinic acid and oleamide did not affect the changes induced by Cx26 transfection. These results suggest that Cx26 expression, but not the mediated intercellular communication, may regulate tight junction barrier and fence functions in human airway epithelial cell line Calu-3.     

Tight junction protein MAGI-1 is up-regulated by transfection with connexin 32 in an immortalized mouse hepatic cell line: cDNA microarray analysis

Gap junctions are considered to play a crucial role in differentiation of epithelial cells, including hepatocytes. Recently, we found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Kojima et al., Exp Cell Res 263:193–201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Kojima et al., Exp Cell Res 276:40–51, 2002). In this study, to investigate the mechanisms of induction of tight junctions by transfection with Cx32, we performed cDNA microarray analysis of Cx32 transfectants, compared with parental cells derived from Cx32-deficient hepatocytes. In cDNA microarray analysis, a 2.5-fold increase in expression of membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed. High expression of MAGI-1 in Cx32 transfectants was confirmed by Western blotting and RT-PCR. MAGI-1 was colocalized with occludin, claudin-2, ZO-1, and F-actin, but not with E-cadherin in the apical-most regions at cell borders of Cx32 transfectants, similar to junctional adhesion molecule-1 (JAM-1), which may play a crucial role in formation and assembly of tight junctions. Treatment with the gap junction blocker 18-glycyrrhetinic acid did not affect expression of MAGI-1 and JAM-1 in Cx32 transfectants. These results suggest that Cx32 expression is in part related to induction of tight junctions through modulation of MAGI-1 expression in an immortalized mouse hepatic cell line.This work was supported in by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science Technology and the Ministry of Health, Labour and Welfare of Japan, and by the Kato Memorial Bioscience Foundation, the Uehara Memorial Foundation, the Suhara Memorial Foundation, the Smoking Research Foundation, and the Long-Range Research Initiative Project of the Japan Chemical Industry Association.     

Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where?

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-junction proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser microscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most subapical plasma membrane at cell borders, while on the basolateral membrane both Cx32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most subapical plasma membrane and were colocalized with only Cx32-immunoreactive lines. In freeze-fracture analysis, many small gap-junction plaques were observed within a well-developed tight-junction strand network. The fence function of tight junctions in the cells, as examined by diffusion of labeled sphingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or anti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These results suggest that Cx32 gap junctions, but not those with Cx26, are closely coordinated with the expression and function of tight junctions in hepatocytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression.     

Occludin and claudin-1 concentrate in the midbody of immortalized mouse hepatocytes during cell division.

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. In late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.     

Connexins Induce and Maintain Tight Junctions in Epithelial Cells

Connexins (Cx) are considered to play a crucial role in the differentiation of epithelial cells and to be associated with adherens and tight junctions. This review describes how connexins contribute to the induction and maintenance of tight junctions in epithelial cells, hepatic cells and airway epithelial cells. Endogenous Cx32 expression and mediated intercellular communication are associated with the expression of tight junction proteins of primary cultured rat hepatocytes. We introduced the human Cx32 gene into immortalized mouse hepatic cells derived from Cx32-deficient mice. Exogenous Cx32 expression and the mediated intercellular communication by transfection could induce the expression and function of tight junctions. Transfection also induced expression of MAGI-1, which localized at adherens and tight junction areas in a gap junctional intercellular communication (GJIC)–independent manner. Furthermore, expression of Cx32 was related to the formation of single epithelial cell polarity of the hepatic cells. On the other hand, Cx26 expression, but not mediated intercellular communication, contributed to the expression and function of tight junctions in human airway epithelial cells. We introduced the human Cx26 gene into the human airway epithelial cell line Calu-3 and used a model of tight junction disruption by the Na⁺/K⁺-ATPase inhibitor ouabain. Transfection with Cx26 prevented disruption of both tight junction functions, the fence and barrier, and the changes of tight junction proteins by treatment with ouabain in a GJIC–independent manner. These results suggest that connexins can induce and maintain tight junctions in both GJIC-dependent and –independent manners in epithelial cells.     

Role of the p38 MAP-kinase signaling pathway for Cx32 and claudin-1 in the rat liver

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivo and in vitro, including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivo were studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.     

Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects.

We have characterized the function of connexin (Cx) 32 gene mutations found in X-linked dominant Charcot-Marie-Tooth disease with respect to their ability to form functional gap junctions among themselves and to inactivate wild-type Cx32 by a dominant negative mechanism. We prepared four types of Cx32 mutant cDNAs and transfected them into HeLa cells, which do not show detectable levels of gap junctional intercellular communication (GJIC), nor expression of any connexins examined. Cells transfected with the wild-type Cx32 gene, but not those transfected with three different base substitution mutations (i.e. Cys 60 to Phe, Val 139 to Met, and Arg 215 to Trp), restored GJIC. Unexpectedly, in cells transfected with a nonsense mutant at codon 220, there was also restored GJIC. When we double-transfected these mutant constructs into the HeLa cells that had already been transfected with the wild-type Cx32 gene and thus were GJIC proficient, three base substitution mutants inhibited GJIC, suggesting that these three mutants can eliminate the function of wild-type Cx32 in a dominant negative manner. The nonsense mutation at codon 220 did not show such a dominant negative effect. Since both mutant and wild-type Cx32 mRNAs were detected, but only poor Cx32 protein expression at cell-cell contact areas was observed in the double transfectants, it is suggested that certain mutants form nonfunctional chimeric connexons with wild-type connexins, which are not properly inserted into the cytoplasmic membrane.     

Role of the p38 MAP-Kinase Signaling Pathway for Cx32 and Claudin-1 in the Rat Liver

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivoand in vitro, including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivowere studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.     

Changes of Gap and Tight Junctions during Differentiation of Human Nasal Epithelial Cells Using Primary Human Nasal Epithelial Cells and Primary Human Nasal Fibroblast Cells in a Noncontact Coculture System

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.     

Changes in Cellular Distribution of Connexins 32 and 26 during Formation of Gap Junctions in Primary Cultures of Rat Hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sectionsin vivo.Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.     

Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells

Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed.     

A novel function of connexin 32: marked enhancement of liver function in a hepatoma cell line

Connexin 32 (Cx32) is the main gap junction protein in hepatocytes and plays an important role in the regulation of signal transfer and growth control in the liver by constructing gap junction channels and gap junctional intercellular communication (GJIC). In this study, the human Cx32 gene was transfected into a hepatoma cell line (HepG2) that showed aberrant expression of Cx32 and was deficient in GJIC. Cx32-transfected HepG2 not only expressed a higher level of Cx32 mRNA, but also showed increased GJIC compared with HepG2 and vector-transfected HepG2. Furthermore, the liver functions of ammonia removal and albumin secretion of HepG2 were markedly enhanced with Cx32 gene transfection. It may be expected to improve the cellular functions of the hepatoma cell line by Cx32 gene transfection and serve to develop an efficacious bioartificial liver.     

Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice

Accumulating evidence indicates that gap junctions play an important role in the maintenance of normal cell growth, so that genes for the connexin gap junction proteins form a family of tumor-suppressor genes. Although mice from which nine types of connexin gene are deleted have been established, little information from carcinogenesis experiments with these mice is available. We have previously found several mutant forms of connexin 32 (Cx32) to be able to inhibit, in a dominant-negative manner, gap junctional intercellular communication (GJIC) exerted by wild-type Cx32. By introducing a gene for such a dominant-negative Cx32 mutant expressed under the control of a liver-specific albumin gene promoter, we have generated transgenic mouse lines in which the function of Cx32 is down-regulated only in the liver. Although GJIC was diminished in the transgenic liver as expected, the reduced GJIC did not affect viability nor the number of spontaneous liver tumors. Although susceptibility to diethylnitrosamine-induced hepatocarcinogenesis was significantly elevated in the transgenic mice, liver regeneration after partial hepatectomy was delayed compared with wild-type mice, suggesting that gap junctions function not only to suppress excessive cell growth but also to promote cell proliferation when necessary for normal function of tissues. Although the phenotype of Cx32-deficient mice was similar to that of the transgenic mice, the former showed more drastically altered phenotypes, i.e. increased BrdU incorporation in the quiescent liver and development of spontaneous liver tumors. We also established 3T3 fibroblasts from embryos lacking the Cx43 gene and characterized their growth. These fibroblasts showed no difference from the wild type in growth characteristics. From these and other studies, we suggest that gap junctions do not necessarily suppress cell growth but support an optimal growth rate.     

Astrocyte and oligodendrocyte connexins of the glial syncytium in relation to astrocyte anatomical domains and spatial buffering

Astroctyes express a set of three connexins (Cx26, Cx30, and Cx43) that are contained in astrocyte-to-astrocyte (A/A) gap junctions; oligodendrocytes express a different set of three connexins (Cx29, Cx32, and Cx47) that are contained in the oligodendrocyte side of necessarily heterotypic astrocyte-to-oligodendrocyte (A/O) gap junctions, and there is little ultrastructural evidence for gap junction formation between individual oligodendrocytes. In addition, primarily Cx29 and Cx32 are contained deeper in myelin sheaths, where they form autologous gap junctions at sites of uncompacted myelin. The presence of six connexins in macroglial cell populations has revealed unprecedented complexity of potential connexin coupling partners, and with restricted deployment of gap junctional intercellular communication (GJIC) within the "pan-glial" syncytium. New implications for the organization and regulation of spatial buffering mediated by glial GJIC are derived from recent observations of the existence of separate astrocyte anatomical domains, with only narrow regions of overlap between astrocyte processes at domain borders. Thus, widespread spatial buffering in the CNS may occur not successively through a multitude of processes arising from different astrocytes, but rather in a more orderly fashion from one astrocyte domain to another via intercellular coupling that occurs only at restricted regions of overlap between astrocyte domains, augmented by autocellular coupling that occurs within each domain.     

Astrocyte and Oligodendrocyte Connexins of the Glial Syncytium in Relation to Astrocyte Anatomical Domains and Spatial Buffering

Astroctyes express a set of three connexins (Cx26, Cx30, and Cx43) that are contained in astrocyte-to-astrocyte (A/A) gap junctions; oligodendrocytes express a different set of three connexins (Cx29, Cx32, and Cx47) that are contained in the oligodendrocyte side of necessarily heterotypic astrocyte-to-oligodendrocyte (A/O) gap junctions, and there is little ultrastructural evidence for gap junction formation between individual oligodendrocytes. In addition, primarily Cx29 and Cx32 are contained deeper in myelin sheaths, where they form autologous gap junctions at sites of uncompacted myelin. The presence of six connexins in macroglial cell populations has revealed unprecedented complexity of potential connexin coupling partners, and with restricted deployment of gap junctional intercellular communication (GJIC) within the “pan-glial” syncytium. New implications for the organization and regulation of spatial buffering mediated by glial GJIC are derived from recent observations of the existence of separate astrocyte anatomical domains, with only narrow regions of overlap between astrocyte processes at domain borders. Thus, widespread spatial buffering in the CNS may occur not successively through a multitude of processes arising from different astrocytes, but rather in a more orderly fashion from one astrocyte domain to another via intercellular coupling that occurs only at restricted regions of overlap between astrocyte domains, augmented by autocellular coupling that occurs within each domain.     

Disruption of circumferential actin filament causes disappearance of occludin from the cell borders of rat hepatocytes in primary culture without distinct changes of tight junction strands.

We investigated the relationship of actin filament organization to occludin and tight junction strands in primary cultured rat hepatocytes using an actin depolymerizing agent, mycalolide B. In control cultures, well-developed circumferential actin filaments and occludin immunoreactivity were observed on the most subapical plasma membrane of the cells, and tight junction strands formed well-developed networks in freeze-fracture replicas. In hepatocytes treated with 3 microM mycalolide B for 6 h, circumferential actin filaments and occludin immunoreactivity disappeared from the cell borders. However, there were no marked abnormalities of tight junction strands in freeze fracture replicas. Similar results were obtained from cells cultured in medium with 0.05 mM Ca2+ for 6 h. The close association of occludin with actin and the existence of intact tight junction strands that are virtually free of both occludin and actin suggest a physiological role of occludin, but not the other proteins forming the tight junction strands, in the linkage between actin cytoskeleton and tight junction.     

Dynamic changes in protein components of the tight junction during liver regeneration

The construction of the hepatocyte tight junction is one of the most important events during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division. To understand this event at the molecular level, we examined the expression of tight junction proteins by Western blot analysis and their cellular localization by immunofluorescence microscopy in regenerating rat liver after two-thirds hepatectomy. The levels of tight junction components such as claudin-3, ZO-1 and atypical protein kinase C (PKC)-specific interacting protein (ASIP) increased two- to three-fold over control levels in coordination with a peak 2-3 days after partial hepatectomy, whereas occludin levels remained unchanged. The bile canaliculi outlined by tight junction components and actin filaments reveal significant morphological changes from 2-3 days after partial hepatectomy. During this period, claudin-3/ZO-1 and ASIP/ZO-1 were nearly co-localized, whereas occludin was locally reduced or almost absent on the bile canaliculi outlined by ZO-1 staining. The uncoupled localization of F-actin and tight junction components was often observed. The function of hepatocytes, as revealed by the serum bile acids level, was distorted temporally at an early stage of regeneration but mostly restored 3 days after partial hepatectomy. These observations suggest that the de novo construction of tight junctions proceeds mainly 2-3 days after partial hepatectomy in parallel with the cell polarization required for hepatocyte function. However, the complete normalization of the composition of the tight junction components, such as occludin and the association with F-actin, requires additional time, which may support the regeneration of fully polarized normal hepatocytes.     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.