Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes

In the present study, we have analyzed the direct effects of cytokines, which mediate the acute-phase response in liver, on connexin expression and gap-junctional intercellular communication in immortalized MHSV12 mouse hepatocytes. When these cells were stimulated for 24 h with interleukin 1 and interleukin 6, the amount of connexin26 (Cx26) mRNA increased together with β?fibrinogen mRNA, as expected for this positive acute-phase gene. In contrast, connexin32 (Cx32) mRNA expression was not affected under these conditions. Indirect immunfluorescence revealed a drastic decrease in Cx32 signals, whereas slightly more Cx26 signals were found. Stronger stimulation with interleukin 1 and tumor necrosis factor α gave a dose-dependent increase in steady state levels of Cx26 and β-fibrinogen mRNA, but no further change in Cx32 mRNA level was seen. However, when Cx32 protein was analyzed on immunoblots, we found a 5-fold decrease in expression even at low cytokine doses that did not affect Cx32 mRNA expression. Under these conditions, cell to cell transfer of Lucifer yellow, microinjected into immortalized hepatocytes, was decreased by 70%, suggesting that intercellular communication through Cx32 channels was partially inhibited earlier than other genetic alterations characteristic of the acute-phase response. Thus, the major hepatic gap junction protein was largely downregulated at the beginning of the experimental inflammatory reaction, but about 30% of gap-junctional intercellular communication was maintained. This suggests that, during the acute-phase response, the second hepatic Cx26 protein may compensate in part for the downregulation of the Cx32 protein.     

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.     

Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation (β-casein expression) was evaluated. Heterocellular interaction is critical for β-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components (α-catenin, β-catenin and ZO−2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although β-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and β-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear β-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of β-catenin in GJ complexes.     

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.     

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.     

β-Catenin mediates cyclic strain-stimulated cardiomyogenesis in mouse embryonic stem cells through ROS-dependent and integrin-mediated PI3K/Akt pathways

Wnt/β-catenin signaling regulates various cellular events involved in the proliferation and differentiation and these events are affected sensitively by applying to mechanical stimuli. However, the mechanisms by which mechanical force stimulates cardiomyogenesis are not extensively explored. In this study we investigated the cellular mechanisms by which β-catenin signaling regulates cardiac differentiation of strain-subjected embryonic stem (ES) cells. The application of cells to cyclic strain increased beating cardiomyocyte foci with the attendant increases of Cx 43 and Nkx 2.5 proteins. Anti-oxidants such as vitamin C or N-acetyl cysteine (NAC) blocked the strain-mediated increases of Cx 43, Nkx 2.5, and α5/β1 integrins. These anti-oxidants also suppressed the activation of phosphoinositide 3-kinase (PI3K) and Akt in cyclic strain-subjected cells. Western blot analysis revealed that PI3K is a critical downstream effector of β1 integrin signaling and mediates Cx 43 and Nkx 2.5 expression in cyclic strain-applied ES cells. Cyclic strain increased the expression of β-catenin and stimulated its nuclear translocation from the cytosol, which was prevented by anti-oxidant treatment. In addition, the application to cyclic strain increased mRNA expression of β-catenin target genes, Axin2 and c-myc, as well as the phosphorylation of glycogen synthase kinase-3β. Furthermore, the blockage of β-catenin by its specific siRNA transfection diminished the cellular levels of Cx 43 and Nkx 2.5 proteins and the number of beating cardiomyocyte foci. Collectively, these results suggest that β-catenin-mediated signaling is required for cyclic strain-stimulated cardiomyogenesis through ROS-dependent and integrin-mediated PI3K-Akt signaling cascades.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.     

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus.     

PDGF-mediated PI3K/AKT/β-catenin signaling regulates gap junctions in corpus cavernosum smooth muscle cells

Erectile dysfunction (ED) is the most common sexual disorder that men report to healthcare providers. Gap junctions (GJs) are thought to be responsible for synchronous shrinkage of corpus cavernosum smooth muscle cells (CCSMCs), and play thus an important role in the maintenance of an erection. Hypoxia has been suggested as a pathological mechanism underlying ED. Here we demonstrate that hypoxia increased the expression of platelet-derived growth factor (PDGF) and the main GJ component connexin (Cx)43 in CCSMCs. Inhibiting PDGF receptor (PDGFR) activity decreased Cx43 expression. Treatment with different concentrations of PDGF increased the levels of phosphorylated protein kinase B (AKT), β-catenin, and Cx43, whereas inhibition of PDGFR or activation of phosphatidylinositol 3 kinase (PI3K)/AKT signaling altered β-catenin and Cx43 expression. Meanwhile, silencing β-catenin resulted in the downregulation of Cx43. These results demonstrate that PDGF secretion by CCSMCs and vascular endothelial cells is enhanced under hypoxic conditions, leading to increased Cx43 expression through PI3K/AKT/β-catenin signaling and ultimately affecting GJ function in ED. Thus, targeting this pathway is a potential therapeutic strategy for the treatment of ED.     

Expression of gap junctional connexins 26, 32, and 43 mRNA in ovarian preovulatory follicles and corpora lutea in sheep

The objective of the current study was to evaluate the expression of connexins (Cx)26, Cx32, and Cx43 mRNA in granulosa and theca cells during the peri-ovulatory period (experiment 1) and in the corpus luteum (CL) during the estrous cycle (experiment 2) and during prostaglandin F2alpha (PGF)-induced luteal regression (experiment 3) in FSH-treated ewes. In experiment 1, Cx26, Cx32, and Cx43 mRNA was expressed in granulosa and theca cells, and expression of Cx32 and Cx43 mRNA, but not Cx26, was greater (p<0.001) in granulosa than in theca cells throughout the peri-ovulatory period. Expression of Cx43 mRNA in granulosa and theca cells decreased (p<0.01) 24 h after hCG treatment. In experiment 2, expression of Cx26 mRNA in the CL tended to be greater (p<0.06) on day 10 than on days 5 or 15, but expression of Cx43 mRNA was greater (p<0.01) on day 5 than on days 10 and 15 of the estrous cycle. In experiment 3, expression of Cx26, but not Cx32 or Cx43 mRNA decreased (p<0.001) during PGF-induced luteal regression. In all 3 experiments, expression of Cx32 mRNA was much less than Cx26 and Cx43 mRNA. Moreover, Cx32 mRNA expression was unchanged during the peri-ovulatory period or during several stages of luteal development and PGF-induced regression of the CL. Thus, we have shown that the mRNA expression pattern of Cx26 and Cx43 changes during peri-ovulatory period and during several stages of the luteal development. This suggests that Cx26 and Cx43 play a role in ovarian tissue remodeling during the critical time around ovulation and throughout luteal tissue growth, differentiation, and regression in sheep.     

Effects of a murine germ cell-specific knockout of Connexin 43 on Connexin expression in testis and fertility

Connexin 43 (Cx 43)—expressed by germ cells (GC), Sertoli cells (SC) and Leydig cells—is one of at least eleven Cx in the murine testis. A general knockout (KO) of Cx 43 in mice results in perinatal death and a SC-specific KO of Cx 43 (SCCx43KO) causes infertility of male mice by preventing the initiation of spermatogenesis. To further elucidate the role of Cx 43 in the testis, a new mouse model with a GC-specific KO of Cx 43 (GCCx43KO) was created by using the Cre/loxP recombination system. A transgenic mouse line expressing the Cre gene under the tissue non-specific alkaline phosphatase promoter and a transgenic floxed Cx 43-LacZ mouse line were mated. The resulting F1-generation was backcrossed with homozygous Cx 43 floxed mice, and offspring was genotyped. Immunohistochemical analysis of testes of different aged homozygous mice revealed normal spermatogenesis and reduced Cx 43 immunoreactions. RT-qPCR and Western blots showed a downregulation of Cx 43 mRNA and protein, and a nearly unchanged mRNA expression of Cx 26, Cx 33 and Cx 45 in pubertal and adult KO mice. Western blots revealed considerable immunoreactive bands for Cx 26 and Cx 45. Male and female homozygous GCCx43KO mice were viable and fertile. Our data suggest, in contrast to inter SC and inter SC–GC cross talk in SCCx43KO mice which depends selectively on Cx 43 expression, that Cx 43 in GC seems not to be essential in GC–SC communication, when other Cx persist to be expressed.     

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

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

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

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.     

Effects of Hyperglycemia and Protein Kinase C on Connexin43 Expression in Cultured Rat Retinal Pigment Epithelial Cells

Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial (RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons (RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46 were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively. Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 μM phorbol 12-myristate 13-acetate (PMA). The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions. Received: 21 July 2000/Revised: 19 January 2001     

Phenotype and growth behavior of residual β-catenin-positive hepatocytes in livers of β-catenin-deficient mice

Signaling through the Wnt/β-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of β-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.     

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.     

Decreased oocyte-granulosa cell gap junction communication and connexin expression in a type 1 diabetic mouse model

In women, type 1 diabetes is associated with an increased risk of poor prenatal outcomes such as congenital anomalies and early miscarriage. In murine models of type 1 diabetes, impaired oocyte meiotic maturation, abnormal oocyte metabolism, and increased granulosa cell apoptosis have been noted. because gap junction communication is critical for the regulation of oocyte growth and meiotic maturation, we investigated the level of communication between the oocyte and surrounding cumulus cells in a streptozotocin-induced type 1 diabetic B6SJL/F1 mouse model and the expression of gap junction proteins known as connexins. Fluorescence recovery after photobleaching analyses of cumulus cell-enclosed oocytes (CEOs) from diabetic mice showed a 60% decrease in communication as compared with CEOs from nondiabetic mice. Real-time RT-PCR analyses confirmed the presence of Cx26, Cx37, and Cx57 mRNA and revealed a significant decrease in Cx37 mRNA expression in oocytes from diabetic mice compared with nondiabetic mice. Western analyses detected Cx26 expression in CEO but not denuded oocyte (DO) samples, and Cx37 in DO samples. Cx26 protein levels were decreased by 78% in CEOs from diabetic mice, and Cx37 protein levels were decreased 36% in DOs from diabetic mice. This decrease in connexin expression and gap junction communication in CEOs from diabetic mice may be responsible for the impaired oocyte meiotic maturation and poor pregnancy outcomes.