Role of gap junctions in fluid secretion of lacrimal glands

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.     

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-7) M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.     

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.     

Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators

Hepatocyte gap junction proteins, connexins (Cxs) 26 and 32, are downregulated during obstructive cholestasis (OC) and lipopolysaccharide hepatocellular cholestasis (LPS-HC). We investigated rat hepatic Cxs during ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval fistula (CCF) and compared them with OC and LPS-HC. Levels (immunoblotting) and cellular distribution (immunofluorescence) of Cx26, -32, and -43, as well as macrophage infiltration, were studied in livers of rats under each condition. Cx26 and -32 were reduced in LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32 was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF, which was associated with increased levels of Cx43 in LPS-HC and OC but not CCF. No inflammation nor changes in Cx43 levels occurred during EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor necrosis factor-alpha and interleukins-1beta and -6, whereas reduction induced by LPS required coculture with Kupffer cells. Thus hepatocyte gap junctions are downregulated in forms of cholestasis associated with inflammation, and reduced intercellular communication might be induced in part by proinflammatory mediators.     

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.     

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.     

Hepatic gap junctions in the hepatocarcinogen-resistant DRH rat

Although the gap junction or connexin (Cx) is considered to be a tumor-suppressor, it is also required for tumor promotion. Therefore, we examined hepatic gap junctions in hepatocarcinogen-resistant (DRH) rats. Specifically, we investigated gap junction structure and Cx32 expression during normal conditions and in response to a hepatocarcinogen, 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). On a basal diet without 3'-MeDAB, hepatic gap junctions and Cx32 protein expression were greater in DRH rats than in control Donryu rats, as evidenced by morphometry, immunohistochemistry and immunoblotting. On a diet containing 3'-MeDAB, gap junctions and expressed Cx32 were increased significantly in Donryu rats, but not in DRH rats. In this condition, Donryu rats lost weight but DRH rats increased relative liver weight. After 3'-MeDAB treatment, cathepsin D expression in hepatocytes was significantly increased only in Donryu rats, indicating that DRH rats were less susceptible to 3'-MeDAB. The abundance of mitogen-activated protein kinase, some constituent of which might be associated with the degree of Cx protein phosphorylation, was reduced to a greater extent in Donryu than in DRH rats after 3'-MeDAB treatment. The resistance of DRH rats to carcinogenesis may be due partially to their stabilized gap junctions, which could coordinate metabolic coupling to evade 3'-MeDAB toxicity.     

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.     

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.     

The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog 1

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.     

Strongly decreased gap junctional permeability to inositol 1,4, 5-trisphosphate in connexin32 deficient hepatocytes

Livers from connexin32 (Cx32) deficient mice have been shown to be defective in hormonally induced glucose mobilization. In order to determine whether this effect is due to decreased diffusion of the second messenger inositol 1,4,5-trisphosphate (IP(3)) between hepatocytes, we injected iontophoretically different amounts of IP(3) in Fura-2 loaded hepatocyte doublets (i.e. cell pairs) from wild type or Cx32 deficient mice. Whereas 84% of wild type hepatocytes showed an intercellular Ca(2+) wave spreading from the injected cell to the neighboring cell, only 25% of Cx32 deficient hepatocyte doublets did so. The amount of IP(3) necessary to induce an intercellular Ca(2+) wave in Cx32 deficient hepatocyte doublets was estimated to be about 25-fold higher than in wild type doublets. This confirms the notion that the low hormonally or electrically induced glucose mobilization found in Cx32 deficient livers relative to wild type livers is due to largely hindered diffusion of IP(3) between Cx32 deficient hepatocytes.     

Downregulation of Connexin 32 Attenuates Hypoxia/Reoxygenation Injury in Liver Cells

Gap junction intercellular communication is involved in ischemia–reperfusion (IR) injury of organs. Connexins are proteins that are critical to the function of gap junctions. To clarify the role of gap junctions in IR injury in liver cells, the function of gap junctions was modulated in an in vitro hypoxia/reoxygenation (H/R) model. BRL‐3A rat liver cells, endogenously expressing connexins Cx32 and Cx43, were used to model the process of hepatic IR injury. Suppression of gap junction activity was achieved genetically, using Cx32‐specific small interfering RNA (siRNA), or chemically, with pharmacological inhibitors, oleamide, and 18‐α‐GA. BRL‐3A cells subjected to H/R exhibited reduced cell survival and pathologies indicative of IR injury. Cx32‐specific siRNA, oleamide, and 18‐α‐GA, respectively, decreased gap junction permeability, as assessed by the parachute assay. Pretreatment with Cx32‐specific siRNA increased cell survival. Pretreatment with oleamide or 18‐α‐GA did not improve cell survival. Modulating gap junction by Cx32 gene silencing protected BRL‐3A liver cells from H/R.     

Heterocellular gap junctional communication between alveolar epithelial cells

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.     

Molecular basis for pacemaker cells in epithelia

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca(2+) signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V(1a) vasopressin receptor (V(1a)R). Release of inositol 1,4,5-trisphosphate (InsP(3)) in wild type cells increased Ca(2+) in the injected cell but not in neighboring cells, while the Ca(2+) signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca(2+) rather than InsP(3) resulted in a small increase in Ca(2+) that did not spread to neighbors with or without gap junctions. However, photorelease of Ca(2+) in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca(2+), which spread to neighbors via gap junctions. Cells expressing tagged V(1a)R similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca(2+) signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V(1a)R. Thus, intercellular Ca(2+) signals in epithelia are organized by three factors: 1) InsP(3) must be generated in each cell to support a Ca(2+) signal in that cell; 2) gap junctions are necessary to synchronize Ca(2+) signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca(2+) signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.     

Neuroprotective role of astrocytic gap junctions in ischemic stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/-)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43(fl/ fl)/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43(fl/fl)] (here designated as Cre(-) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/-) and Cre(+) mice compared to Cx43(+/+) and Cre(-) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/-) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

Neuroprotective Role of Astrocytic Gap Junctions in Ischemic Stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/?)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43^{fl/ fl}/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43^fl/fl] (here designated as Cre(?) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/?) and Cre(+) mice compared to Cx43(+/+) and Cre(?) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/?) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

A Neuroprotective Role for Gap Junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.