Pretreatment with eicosapentaenoic acid prevented hypoxia/reoxygenation-induced abnormality in endothelial gap junctional intercellular communication through inhibiting the tyrosine kinase activity.

Eicosapentaenoic acid (EPA) may protect against atherosclerotic disease, and modulation of endothelium function is one possible mechanism. Hypoxia/reoxygenation (H/R) is a potential risk factor for the pathogenesis of atherosclerosis, and it causes endothelial dysfunction. To evaluate whether EPA may improve the endothelial dysfunction under the condition of H/R, we examined endothelial gap junctional intercellular communication (GJIC), which is said to be important for the endothelium to maintain its normal function. The results indicate that H/R induced a temporal reduction in GJIC after 2 h of reoxygenation in cultured human umbilical vein endothelial cells (HUVEC). This reduction in GJIC was not observed in cells pretreated with 3 microg/ml EPA for 2 days. The results of immunofluorescence show that 2 h reoxygenation caused an increased production of tyrosine-phosphorylated proteins, which was inhibited by EPA pretreatment. Immunoprecipitation demonstrated that tyrosine residues of connexin 43 (Cx43), an important gap junctional protein in HUVEC, were phosphorylated by H/R. However, pretreatment with EPA significantly suppressed this increased phosphorylation. The protective effect of EPA on the reduction in GJIC was also observed in cells treated with 1.5 mM vanadate, a tyrosine phosphatase inhibitor. These data suggest that EPA may ameliorate the H/R-induced GJIC abnormality via inhibition of the tyrosine kinase activation.     

Role of Cx43 Phosphorylation and MAP Kinase Activation in EGF Induced Enhancement of Cell Communication in Human Kidney Epithelial Cells

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.     

Down-regulation of connexin43 gap junction by serum deprivation in human endothelial cells was improved by (-)-Epigallocatechin gallate via ERK MAP kinase pathway

Intercellular communication through gap junctions (GJIC) plays an essential role in maintaining the functional integrity of vascular endothelium. Despite emerging evidence suggests that (−)-Epigallocatechin gallate (EGCG) may improve endothelial function. However, its effect on Cx43 gap junction in endothelial cells remains unexplored. Here we investigated the effect of EGCG on connexin43 (Cx43) gap junction in endothelial cells. The levels of Cx43 protein in human umbilical vein endothelial cells (HUVECs) cultured under serum-deprivation 48 h decreased about 50%, accompanied by decreased GJIC. This reduction can be reversed by treatments with EGCG. In addition, EGCG activated ERK, P38, and JNK mitogen-activated protein kinases (MAPKs), which were supposed to participate in the regulation of Cx43. A MEK inhibitor PD98059, but not SB203580 (a p38 kinase inhibitor) or SP600125 (a JNK kinase inhibitor), abolished the effects of EGCG on Cx43 expression and GJIC. Moreover, although both Akt and eNOS phosphorylation were time-dependently augmented by EGCG, neither PI3K inhibitor LY294002 nor eNOS inhibitor L-NAME blocked the effects of EGCG on Cx43 gap junctions. Thus, EGCG attenuated Cx43 down-regulation and impaired GJIC induced by serum deprivation, ERK MAPK Signal transduction pathway appears to be involved in these processes.     

Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

Inhibitory effects of eicosapentaenoic acid (EPA) on the hypoxia/reoxygenation-induced tyrosine kinase activation in cultured human umbilical vein endothelial cells

We have previously reported that the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) inhibited the abnormal gap junctional intercellular communication (GJIC) induced by hypoxia/reoxygenation (H/R) via suppressing tyrosine kinase (TK) activation (Zhang et al., Prostaglandins Leukot Essent Fatty Acids, 1999; 61: 33-40). However, the mechanisms by which EPA-inhibited TK activation remained unidentified. In this study we investigated whether reactive oxygen species (ROS) and growth factor-receptor systems would contribute to the H/R-induced TK activation or not. The results showed that H/R-induced ROS production, which reached the peak after 30 min of reoxygenation. Pretreatment with 10 microM EPA significantly inhibited this ROS production. However, the TK inhibitor genistein (10 microM) failed to inhibit the generation of ROS, although it completely inhibited TK activation. On the other hand, the ROS inhibitor DMSO (0.5% v/v) showed little effect on TK activation while it significantly blocked ROS production. Further EPA and genistein, but not DMSO and superoxide dismutase (SOD, 300 U/ml), prevented cells from GJIC injury induced by H/R. Moreover, EPA protected against VEGF-induced reduction in GJIC and phosphorylation of connexin 43. These data suggest that growth factor, but not ROS, might be involved in the EPA-inhibited TK activation induced by H/R.     

Lipopolysaccharide plus hypoxia and reoxygenation synergistically reduce electrical coupling between microvascular endothelial cells by dephosphorylating connexin40

We showed that lipopolysaccharide (LPS) or hypoxia and reoxygenation (H/R) decreases electrical coupling between microvascular endothelial cells by targeting the gap junction protein connexin40 (Cx40), tyrosine kinase-, ERK1/2-, and PKA-dependently. Since LPS can compromise microvascular blood flow, resulting in micro-regional H/R, the concurrent LPS + H/R could reduce coupling to a much greater extent than LPS or H/R alone. We examined this possibility in a model of cultured microvascular endothelial cells (mouse skeletal muscle origin) in terms of electrical coupling and the phosphorylation status of Cx40. To assess coupling, we measured the spread of electrical current injected into the cell monolayer and computed the intercellular resistance as an inversed measure of coupling. In wild type cells, but not in Cx40 null cells, concurrent LPS + H/R synergistically increased resistance by approximately 270%, well above the level observed for LPS or H/R alone. Cx37 and Cx43 protein expression did not differ between Cx40 null and wild type cells. LPS + H/R increased resistance PKA- and PKC-dependently. By immunoprecipitating Cx40, we found that LPS + H/R reduced serine phosphorylation to a much greater degree than that observed for LPS or H/R alone. Further, PKA-specific, but not PKC-specific serine phosphorylation of Cx40 was also significantly reduced following LPS + H/R. This reduction was prevented by tyrosine kinase and MEK1/2 inhibition, by PKA activation, and mimicked in control cells by PKA inhibition. We conclude that LPS + H/R initiates tyrosine kinase- and ERK1/2-sensitive signaling that synergistically reduces inter-endothelial electrical coupling by dephosphorylating PKA-specific serine residues of Cx40.     

Association of connexin43 with a receptor protein tyrosine phosphatase

Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase mu (RPTPmu) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPmu binds to Cx43. Our results support a model in which RPTPmu, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels.     

Association of Connexin43 with a Receptor Protein Tyrosine Phosphatase

Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase μ (RPTPμ) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPμ binds to Cx43. Our results support a model in which RPTPμ, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels.     

18beta-glycyrrhetinic acid promotes src interaction with connexin43 in rat cardiomyocytes

The mechanism by which 18beta-glycyrrhetinic acid regulates gap junction intercellular communication (GJIC) remains poorly understood. In this study, treatment of cultured rat neonatal cardiomyocytes with 18beta-glycyrrhetinic acid resulted in dose-dependent inhibition of GJIC as assessed by fluorescent dye transfer analysis. 18beta-Glycyrrhetinic acid induced time-dependent serine/threonine dephosphorylation and redistribution of connexin43 (Cx43) in cardiomyocytes and the induced Cx43 dephosphorylation was prevented by the protein phosphatase inhibitor, calyculin A. However, functional analyses showed that the inhibitory effect of 18beta-glycyrrhetinic acid on dye spreading among cardiomyocytes was not blocked by calyculin A, but was blocked by the Src-selective tyrosine kinase inhibitor, PP2. 18beta-Glycyrrhetinic acid also induced an increase in the levels of phosphorylated Src, and this effect was prevented by PP2. Immunoprecipitation using anti-Cx43 and anti-p-Src antibodies showed that 18beta-glycyrrhetinic acid increased the association between p-Src and Cx43 and induced tyrosine phosphorylation of Cx43. We conclude that the inhibitory effect of 18beta-glycyrrhetinic acid on GJIC in cardiomyocytes involves Src-mediated tyrosine phosphorylation of Cx43.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Connexin43 phosphorylation by PKC and MAPK signals VEGF-mediated gap junction internalization

Gap junctions (GJs) exhibit a complex modus of assembly and degradation to maintain balanced intercellular communication (GJIC). Several growth factors, including vascular endothelial growth factor (VEGF), have been reported to disrupt cell–cell junctions and abolish GJIC. VEGF directly stimulates VEGF-receptor tyrosine kinases on endothelial cell surfaces. Exposing primary porcine pulmonary artery endothelial cells (PAECs) to VEGF for 15 min resulted in a rapid and almost complete loss of connexin43 (Cx43) GJs at cell–cell appositions and a concomitant increase in cytoplasmic, vesicular Cx43. After prolonged incubation periods (60 min), Cx43 GJs reformed and intracellular Cx43 were restored to levels observed before treatment. GJ internalization correlated with efficient inhibition of GJIC, up to 2.8-fold increased phosphorylation of Cx43 serine residues 255, 262, 279/282, and 368, and appeared to be clathrin driven. Phosphorylation of serines 255, 262, and 279/282 was mediated by MAPK, whereas serine 368 phosphorylation was mediated by PKC. Pharmacological inhibition of both signaling pathways significantly reduced Cx43 phosphorylation and GJ internalization. Together, our results indicate that growth factors such as VEGF activate a hierarchical kinase program—including PKC and MAPK—that induces GJ internalization via phosphorylation of well-known regulatory amino acid residues located in the Cx43 C-terminal tail.     

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) alter connexin 43 phosphorylation in MC3T3-E1 Cells

Background  

Bone morphogenetic proteins (BMPs) and transforming growth factor-βs (TGF-βs) are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC) in MC3T3-E1 cells. Connexin 43 (Cx43) has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC.     

Association of Connexin43 with a Receptor Protein Tyrosine Phosphatase

Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase μ (RPTPμ) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPμ binds to Cx43. Our results support a model in which RPTPμ, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels.     

Gallic acid, a metabolite of the antioxidant propyl gallate, inhibits gap junctional intercellular communication via phosphorylation of connexin 43 and extracellular-signal-regulated kinase1/2 in rat liver epithelial cells

Propyl gallate and its metabolite, gallic acid, are widely used as antioxidants in the food industry, but they have been shown to exhibit liver toxicity and enhance carcinogenesis. In the present study, we investigated the possible undesirable effects of propyl gallate and gallic acid on gap junctional intercellular communication (GJIC), inhibition of which is closely linked to carcinogenesis. Gallic acid and propyl gallate exhibited dose-dependent free-radical-scavenging activities as determined by 1,1-diphenyl-2-picrylhydrazyl- or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-radical-scavenging assays, and the free-radical-scavenging activity of gallic acid was stronger than that of propyl gallate. However, using WB-F344 rat liver epithelial cells, gallic acid inhibited GJIC in a dose-dependent manner, while propyl gallate had no significant effect compared with untreated controls. The gallic-acid-induced inhibition of GJIC was reversible, with a recovery of nearly 65% after 120 min. Gallic acid induced the phosphorylation of connexin 43 (Cx43) and phosphorylation of extracellular-signal-regulated kinase1/2 (ERK1/2). The gallic-acid-induced inhibition of GJIC was attenuated by treatment with mitogen-activated protein kinase kinase inhibitors (U0126 and PD098059). U0126 blocked the gallic-acid-induced phosphorylation of Cx43 and ERK1/2, indicating that the gallic-acid-induced inhibition of GJIC is mediated by phosphorylation of Cx43 via activation of ERK1/2. In addition, gallic-acid-induced inhibition of GJIC was protected by ascorbic acid and quercetin, which might represent a simple example of the different effects of natural antioxidants in carcinogenesis.     

v-Src tyrosine phosphorylation of connexin43: regulation of gap junction communication and effects on cell transformation

The oncogenic tyrosine kinase, v-Src, phosphorylates connexin43 (Cx43) on Y247 and Y265 and inhibits Cx43 gap junctional communication (GJC), the process of intercellular exchange of ions and metabolites. To test the role of a negative charge on Cx43 induced by tyrosine phosphorylation, we expressed Cx43 with glutamic acid substitutions at Y247 or Y265. The Cx43Y247E or Cx43Y265E channels were functional in Cx43 knockout fibroblasts, indicating that introducing a negative charge on Cx43 was not likely the mechanism for v-Src disruption of GJC. Cells coexpressing v-Src and the triple serine to alanine mutant, Cx43S255/279/282A, confirmed that mitogen-activated protein (MAP) kinase phosphorylation of Cx43 was not required for v-Src-induced disruption of GJC and that tyrosine phosphorylation was sufficient. In addition, v-Src cells containing v-Src-resistant gap junctions, Cx43Y247/265F, displayed properties of cell migration, adhesion, and proliferation similar to Cx43wt/v-Src cells, suggesting that Cx43 tyrosine phosphorylation and disruption of GJC are not involved in these transformed cell properties.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.     

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet-derived growth factor (PDGF)-induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor beta (PDGFr beta). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF- or TPA-induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF- and TPA-induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2-mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N-acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC.     

Epidermal Growth Factor Inhibits Gap Junctional Communication and Stimulates Serine-Phosphorylation of Connexin43 in WB Cells by a Protein Kinase C-Independent Mechanism

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple irnmunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in ³²P_i incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.     

ELF magnetic field inhibits gap junctional intercellular communication and induces hyperphosphorylation of connexin43 in NIH3T3 cells.

The effects of extremely low frequency (ELF) magnetic field on gap junctional intercellular communication (GJIC), protein levels, and phosphorylation of connexin43 (Cx43) were studied in NIH3T3 cells. The suppression of GJIC by 24 h, 50 Hz, 0.8 mT ELF magnetic field, 2 h, 3 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or ELF combined with TPA treatment was confirmed by the fluorescence recovery after photobleaching (FRAP) analysis with a confocal microscope. The results showed that ELF or TPA exposure induced 50-60% inhibition of GJIC (P < 0.01). ELF combined with TPA enhanced the inhibition of GJIC. Western blot analysis using Cx43 specific antibodies showed obviously decreasing non phosphorylated Cx43 (P(0)) induced by ELF and/or TPA exposure. On the other hand, cells treated with ELF and/or TPA displayed a hyperphosphorylated Cx43 band (P(3)). However, there was no obvious changes in the level of Cx43 protein. The results implied that the P(3) band appeared to result from phosphorylation of P(0). But it remains possible that upon the ELF exposure P(0) is converted to P(1), P(2) or both and that P(3) is formed from P(1) or P(2) resulting in the observed hyperphosphorylation pattern. From the present study, we conclude that ELF magnetic field inhibits GJIC and the main mechanism is the hyperphosphorylation of Cx43.