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The gap junction protein plays an important role in the bone formation and alteration of these proteins leading to cause bone development. Aim to determine the effects of different concentration of fluoride on gap-junctional intercellular communication (GJIC) related genes and proteins in the rats' osteoblast cells. We treated the osteoblast cells with various concentrations (0, 0.01, 0.1, 0.5, and 1.0 mM) NaF for 24 and 72 h. We used the scrape loading and dye transfer technique to research the intracellular connectivity. Moreover, the mRNA expression levels of connexin 43 (Cx43), connexin45 (Cx45), collagen I, and osteocalcin (OCN) were analyzed by qRT-PCR, the protein expression levels of connexin43 (Cx43) were analyzed by western blotting and immunofluorescence. Our results suggested that the osteoblast proliferations were decreased in the 0.5 and 1 mM NaF groups, after 24 and 72 treatments. The scrape loading and dye transfer experiment showed that the GJIC were increased in the 0.01 mM NaF group and decreased in the 0.5 and 1 mM NaF groups. In addition, the mRNA expressions of Cx43, Cx45, and OCN, and the protein expressions of Cx43 were increased in the 0.01 mM NaF group and decreased in the 0.5 and 1 mM NaF groups. In summary, these results suggest that the low concentration NaF is good for the GJIC, but the high concentration NaF damages the GJIC.

Within the testis, connexin43 encoded by Gja1 plays an important role in cell-to-cell communication between Leydig cells as well as between Sertoli cells and spermatogonia. In the adult male, Leydig cells are the principal producers of testosterone sustaining spermatogenesis, while Sertoli cells nourish, protect and support the differentiating germ cells. It has been shown previously that members of the AP-1 family regulate Gja1 expression in myometrial cells, suggesting that such regulatory mechanism may also be relevant within the testis. Thus, we performed cotransfections of AP-1 expression plasmids with different mouse Gja1 promoter/luciferase reporter constructs within TM3 Leydig and TM4 Sertoli cells. We showed that a functional cooperation between cJun and cFos activates Gja1 expression and requires an AP-1 DNA regulatory element located between -132 and -26 bp. In addition, such synergy relies on the recruitment of cFos to this region of the mouse Gja1 promoter. Hence, our data indicate that AP-1 members are important for optimal expression of Gja1 within Sertoli and Leydig cells from the testis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Type 1 diabetes is one of the most extensively studied autoimmune diseases, but the cellular and molecular mechanisms leading to T cell-mediated destruction of insulin-producing β cells are still not well understood. In this study, we show that regulatory T cells (T(regs)) in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate T(regs) in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory functions can be corrected, even in T cells isolated from aged, diabetic mice, by a synergistic activity of retinoic acid, TGF-β, and IL-2, which enhance connexin 43 and Foxp3 expression in T(regs) and restore the ability of conventional CD4(+) T cells to upregulate Foxp3 and generate peripherally derived T(regs). Moreover, we demonstrate that suppression mediated by T(regs) from diabetic mice is enhanced by a novel reagent, which facilitates gap junction aggregation. In summary, our report identifies gap junction-mediated intercellular communication as an important component of the T(reg) suppression mechanism compromised in NOD mice and suggests how T(reg) mediated immune regulation can be improved., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Objective: All-trans retinoic acid (ATRA) has been demonstrated to inhibit tumor growth by restoration of gap junctional intercellular communication (GJIC) via upregulation of connexin (Cx) expression in some solid tumors. However, the relationship between ATRA and GJIC remains unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the effect of ATRA on the GJIC function of OSCC., Study Design: We measured the effects of ATRA on the viability and cell cycle distribution of SCC9 and Tca8113 OSCC cells. The GJIC function was observed using the scrape-loading dye transfer technique, and the mRNA and protein levels of Cx32 and Cx43 were detected by qRT-PCR, Western blot, and immunofluorescence assays., Results: ATRA inhibited the growth of OSCC cells in a dose- and time-dependent manner (P <0.05) and caused cell cycle arrest. ATRA-treated cells showed a 2.69-fold and 2.06-fold enhancement of GJIC in SCC9 and Tca8113 cells, respectively (P <0.05). Moreover, ATRA induced upregulation of Cx32 and Cx43 at both the mRNA and protein levels in OSCC cells., Conclusion: Our results indicated that restoration of GJIC via enhanced Cx32 and Cx43 expression might serve as a novel mechanism for the anti-tumor effect of ATRA in OSCC.

Spinal astrocytes have important mechanistic contributions to the initiation and maintenance of neurodegenerative diseases and chronic pain. Under inflammatory conditions, spinal astrocytes are exposed to cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and these cytokines could alter astrocytic function by modulating connexin (Cx43), subunits that form channels that modulate intercellular communication in astrocytes. The current study investigated the alteration of Cx43-gap junction in rat primary cultured spinal astrocytes stimulated with cytokines by real-time PCR and Western blotting. The transcriptional and translational levels of Cx43 were significantly but partially reduced 24 and 48 hr treatment with either TNF-α (10 ng/ml) or IFN-γ (5 ng/ml). A mixture of TNF-α and IFN-γ led to a robust decrease of Cx43 expression and, moreover, a moderate reduction of gap junction intercellular communication (GJIC), which was evaluated by a scrap loading/dye transfer assay. Both the decrease of Cx43 expression and the reduction in GJIC induced by the mixture of TNF-α and IFN-γ were prevented by blocking c-jun terminal kinase (JNK) but not by blocking extracellular signaling molecules ERK and p38 kinase, indicating a specific role of astrocytic JNK in the response to cytokines. In addition, treatment with cytokines potently induced the phosphorylation of JNK and c-jun in a time-dependent manner. These results indicate that intercellular communication of astrocytes is significantly disrupted in the inflammatory state and that stimulation of spinal astrocytes with inflammatory cytokines leads to significant inhibition of Cx43-GJIC through activation of the JNK signaling pathway., (Copyright © 2013 Wiley Periodicals, Inc.)

HYS-32 [4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone] is a new analogue of the anti-tumor compound combretastatin A-4 containing a cis-stilbene moiety. In this study, we investigated its effects on Cx43 gap junction intercellular communication (GJIC) and the signaling pathway involved in rat primary astrocytes. Western blot analyses showed that HYS-32 dose- and time-dependently upregulated Cx43 expression. A confocal microscopic study and scrape-loading/dye transfer analyses demonstrated that HYS-32 (5μM) induced microtubule coiling, accumulation of Cx43 in gap junction plaques, and increased GJIC in astrocytes. The HYS-32-induced microtubule coiling and Cx43 accumulation in gap junction plaques was reversed when HYS-32 was removed. Treatment of astrocytes with cycloheximide resulted in time-dependent degradation of by co-treatment with HYS-32 by increasing the half-life of Cx43. Co-treatment with HYS-32 also prevented the LPS-induced downregulation of Cx43 and inhibition of GJIC in astrocytes. HYS-32 induced activation of PKC, ERK, and JNK, and co-treatment with the PKC inhibitor Go6976 or the ERK inhibitor PD98059, but not the JNK inhibitor SP600125, prevented the HYS-32-induced increase in Cx43 expression and GJIC. Go6976 suppressed the HYS-32-induced PKC phosphorylation and increase in phospho-ERK levels, while PD98059 did not prevent the HYS-32-induced increase in phospho-PKC levels, suggesting that PKC is an upstream effector of ERK. In conclusion, our results show that HYS-32 increases the half-life of Cx43 and enhances Cx43 expression and GJIC in astrocytes via a PKC-ERK signaling cascade. These novel biological effects of HYS-32 on astrocyte gap junctions support its potential for therapeutic use as a protective agent for the central nervous system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the α(2) isoform of the Na,K-pump (α(2)), Na(+), Ca(2+)-exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating α(2) in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca(2+)](i) was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 μM ouabain uncoupled them. Downregulation of α(2) reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of α(2) in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endothelium-dependent hyperpolarizing factor-like component of the response was significantly diminished in α(2)-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the α(2)-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on α(2)-downregulated arteries. In the presence of a low NE concentration the α(2)-downregulated arteries had higher [Ca(2+)](i) and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca(2+)-sensitivity. The α(2)-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K(+) was not affected. Our results demonstrate the importance of α(2) for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone.

Background: Gap junctional intercellular communication decreases with HSV-2 infection. To determine the importance of functional gap junctions for infectivity, we compared HSV-2 growth in communication-competent and -deficient cell lines., Methods: HSV-2 infectivity was tested in five cell lines: WB rat liver epithelial cells (communication-competent), WB-aB1 (communication-deficient), WB-a/32-10 (communication-rescued), HeLa (communication-deficient), and Cx43-transfected HeLa (communication-rescued) cells. HSV-2 growth curves and indirect immunofluorescence labeling of viral and cell proteins were performed in wild-type and mutant WB cells., Results: Although wild-type WB cells were highly permissive for HSV-2 infection, virus production was significantly attenuated in communication-deficient and -rescued mutant WB cells. HeLa exhibited no difference in infectivity between communication-competent and -deficient cell lines. Tight and adherens junction proteins, including zonula occludens-1 and nectin-1, were not different in the WB cell lines. However, E-cadherin levels were elevated and β-catenin was found to co-localize with glycoprotein E, a viral glycoprotein associated with cell-to-cell spread, in the mutant WB cells., Conclusions: These results suggest that attenuated viral production in mutant WB cells is due to viral protein co-localization with adherens junction proteins rather than the loss or restoration of functional gap junctions., (Copyright © 2012 S. Karger AG, Basel.)

Silver nanoparticles (Ag NPs) are increasingly being used in wound dressings, medical settings, and various household products due to their unique properties and antimicrobial activity. Despite the widespread use of Ag NP products, the molecular mechanisms underlying the biological effects of Ag NPs remain unclear. Gap junctional intercellular communication (GJIC), formed by the connexin protein family, plays a critical role in the maintenance of tissue and organ homeostasis. This study was undertaken to investigate the effects of well characterized, PVP-coated Ag NPs (69 +/- 3 nm) and silver nitrate on GJIC and connexin43 (Cx43) expression in human lung adenocarcinoma cell line A549. Our results showed that Ag NPs increased GJIC in A549 cells as assayed by dye transfer method. Western blot analysis showed that incubation of cells with Ag NPs significantly increased the expression of Cx43 protein. In addition, Ag NPs up-regulated expression of Cx43 mRNA in a dose-dependent manner. Silver nitrate failed to increase GJIC and the expression of Cx43 protein. It, however, increased Cx43 mRNA expression in A549 cells. Taken together, our results provide the first evidence that Ag NPs induced the increase of GJIC activity in A549 cells through up-regulation of Cx43 protein, suggesting that Cx43 and GJIC may be one of the targets for Ag NPs biological effects.

Intercellular communication is an important tool used by the cells to effectively regulate concerted responses. Hepatic stellate cells (HSCs) communicate to each other through functional gap junctions composed of connexin 43 (Cx43) proteins. We show that exogenous human TGF-beta1 (hTGF-beta1), a pro-fibrotic stimulus, decreases Cx43 mRNA and protein in a rat HSC cell line and primary HSCs. Furthermore, hTGF-beta1 increases the phosphorylation of Cx43 at serine 368. These effects lead to a decrease in the gap junction intercellular communication between the HSCs, as shown by gap-FRAP analysis. We also observe the binding of Snai1, from the nuclear extract of HSCs, to a Snai1 consensus sequence in the Cx43 promoter. In the same context, Snai1 siRNA transfection results in an up-regulation of Cx43 suggesting that TGF-beta1 may regulate Cx43 via Snai1. In addition, we demonstrate that the knockdown of Cx43 by siRNA transfection results in a slower proliferation of HSCs. These findings illuminate a new effect of TGF-beta1 in HSCs, namely the regulation of intercellular communication by affecting the expression level and the phosphorylation state of Cx43 through Snai1 signaling.

Cisplatin-induced cell death can be triggered by cell-to-cell communication through gap junctions. Here, we show that activated src produces tyrosine phosphorylation of the gap junction protein connexin 43, decreases gap junction communication, and increases cell survival in response to cisplatin. Experiments with mixed cell populations show that src activity in one cell can confer increased cisplatin survival on neighboring cells, even when the neighboring cells lack such src activity. This work is the first demonstration that expression of an oncogene in one cell can affect the survival of a neighboring cell not expressing the oncogene in response to a chemotherapeutic drug. The trans-acting effect of activated src on neighboring cells can be blocked by inhibitors of src kinase or by siRNA-mediated knockdown of src expression, and it can be counteracted by forced up-regulation of connexin 43, via either gene transfer or proteasome inhibition. These results identify a novel pathway of cisplatin resistance that may be amenable to therapeutic intervention.

Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector-mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.

Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (chi2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 micromol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a "hyperphosphorylated" connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.

Ochratoxin A (OTA) is one of the most potent renal carcinogens studied to date, but the mechanism of tumor formation by ochratoxin A remains largely unknown. Cell adhesion and cell-cell communication participate in the regulation of signaling pathways involved in cell proliferation and growth control and it is therefore not surprising that modulation of cell-cell signaling has been implicated in cancer development. Several nephrotoxicants and renal carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercell communication (GJIC) and/or cell adhesion, and the aim of this study was to determine if disruption of cell-cell interactions occurs in kidney epithelial cells in response to OTA treatment. MDCK cells were treated with OTA (0-50 microM) for up to 24h and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, expression and intracellular localization of C x 43, E-cadherin and beta-catenin were determined by immunoblot and immunofluorescence analysis. A clear decrease in the distance of dye transfer was evident following treatment with OTA at concentrations/incubation times which did not affect cell viability. Consistent with the functional inhibition of GJIC, treatment with OTA resulted in a dose-dependent decrease in C x 43 expression. In contrast to C x 43, OTA did not alter total amount of the adherens junction proteins E-cadherin and beta-catenin. Moreover, Western blot analysis of Triton X-100 soluble and insoluble protein fractions did not indicate translocation of cell adhesion molecules from the membrane to the cytoplasm. However, a approximately 78 kDa fragment of beta-catenin was detected in the detergent soluble fraction, indicating proteolytic cleavage of beta-catenin. Immunofluorescence analysis also revealed changes in the pattern of both beta-catenin and E-cadherin labeling, suggesting that OTA may alter cell-adhesion. Taken together, these data support the hypothesis that disruption of cell-cell signaling may contribute to OTA toxicity and carcinogenicity.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) promote foci formation in the two-stage cell transformation assay in vitro. These chlorohydroxyfuranones (CHFs) and their structural congener 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) inhibit gap junctional intercellular communication (GJIC) in Balb/c 3T3 mouse fibroblast cells. In the present study, the effects of MX, MCA, CMCF, and MCF on GJIC were evaluated in liver cells (WB-F344 rat liver epithelial cells), the target cells of MX-induced carcinogenicity, using the scrape-loading dye transfer technique. The CHFs inhibited GJIC after 1 h exposure in a concentration-dependent fashion. The order of potency was MX>CMCF approximately MCA>MCF. In terms of the lowest observed effective concentrations, the difference in the potency was about 27-fold (MX 1.875 microM, MCF 50 microM). After a prolonged exposure period (12 h), the inhibition of GJIC by MX and CMCF remained stable, but MCA and MCF exhibited increasing inhibitory effects. After removal of the CHFs, the GJIC slowly recovered. At the transcriptional level, CHFs caused essentially no change in the level of connexin43 (Cx43) mRNA. Preincubation of cells with the protein kinase C (PKC) inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 decreased substantially the inhibition of GJIC by all four CHFs. Activation of the mitogen-activated protein kinases (MAPKs) signaling pathway was necessary for inhibition of GJIC. CHFs did not increase the basal phosphorylation state of the Cx43 protein, but all CHFs caused a concentration-dependent degradation of the Cx43 protein. The results indicate that all the studied CHFs inhibit GJIC in WB-F344 cells by altering Cx43 expression.

The transplantation of bioartificial pancreas has the potential to restore endogenous insulin secretion in type I diabetes. The bioartificial pancreas is constructed in vitro from cells and a support matrix. Hyaluronic acid (HA) is an extremely ubiquitous polysaccharide of extracellular matrix in the body and plays various biological roles. It has been suggested that high molecular weight (HMW) HA increases in the function of gap-junctional intercellular communications (GJIC) and the expression of connexin-43 (Cx43). To determine whether the function of pancreatic beta-cells is affected by gap junctions after HMW HA-treatment, we exposed HIT-T15, a clonal pancreatic beta-cell line, in various concentrations of HA for 24h, and then detected the insulin secretion and content, using an insulin assay kit by ELISA technique. The cellular functions of GJIC were assayed by dye-transfer method using the dye solution of Lucifer yellow. HA-treatment resulted in the enhancement of GJIC function, the increase of insulin release and insulin content. The results obtained in this study suggest that HA-coating increases the insulin secretion of HIT-T15 cells by the enhancement of Cx43-mediated GJIC. The results give useful information on design biocompatibility of HA when is used as a biomaterial for bioartificial pancreas.

A histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induces apoptosis in neoplastic cells, but its effect on gap junctional intercellular communication in relation to apoptosis was unclear. Therefore, we carried out a comparative study of the effects of two HDAC inhibitors, SAHA and trichostatin-A, on gap junctional intercellular communication in nonmalignant human peritoneal mesothelial cells (HPMC) and tumorigenic ras oncogene-transformed rat liver epithelial cells (WB-ras) that showed a significantly lower level of gap junctional intercellular communication than did HPMC. Gap junctional intercellular communication was assessed by recovery rate of fluorescence recovery after photobleaching. Treatment of HPMC with SAHA at nanomolar concentrations caused a dose-dependent increase of recovery rate without inducing apoptosis. This effect was accompanied by enhanced connexin 43 (Cx43) mRNA and protein expression and increased presence of Cx43 protein on cell membrane. Trichostatin-A induced apoptosis in HPMC but was less potent than SAHA in enhancing the recovery rate. In contrast, treatment of WB-ras cells with SAHA or trichostatin-A induced apoptosis at low concentrations, in spite of smaller increases in recovery rate, Cx43 mRNA, and protein than in HPMC. Chromatin immunoprecipitation analysis revealed that SAHA enhanced acetylated histones H3 and H4 in the chromatin fragments associated with Cx43 gene in HPMC. These results indicate that SAHA at low concentrations selectively up-regulates Cx43 expression in normal human cells without induction of apoptosis, as a result of histone acetylation in selective chromatin fragments, in contrast to the apoptotic effect observed in tumorigenic WB-ras cells. These results support a cancer therapeutic and preventive role for specific HDAC inhibitors.

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.

Objective: To further understand the roles of gap junctional intercellular communication in carcinogenesis and progression of human lung cancer., Methods: The heterologous communication was characterized among human lung epithelial cells HLEC, human lung fibroblasts HLF, human lung giant carcinoma PG cells and its Cx43 transfectants PG/C4 by using a preloading assay. Cx43 immunofluorescent staining and Northern blot were performed to examine Cx43 gene expression., Results: Although both human lung fibroblasts HLF and epithelial cells HLEC expressed Cx43 and had very strong homologous communication, HLEC cells were unable to communicate with HLF cells. The human lung carcinoma cell line PG was defective of both homologous communication, and heterologous communication with its epithelial origin HLEC. Transfection of Cx43 into PG cells, which rescued PG cells' homologous communication and enhanced heterologous communication with HLF cells, could restore heterologous coupling with HLEC cells., Conclusion: Selective heterologous communication exists among different kinds of human lung cells, when human lung carcinoma cells lost coupling with its epithelial origin, Cx43 gene expression is not enough to establish heterologous communication between them.

Objective: Clinical studies imply that (-)-epigallocatechin-3-gallate (EGCG), a main ingredient of green tea catechins, has a chemopreventive action against cancers and suppresses the proliferation of cancer cells. However, there is no report about its chemopreventive effect for renal cancer. We previously determined that renal carcinogens suppressed the gap junction intercellular communication (GJIC) of renal epithelial cells. In this study, we investigated the effect of EGCG on the GJIC of renal epithelial cells treated with a renal carcinogen., Materials and Methods: Mardin-Darby canine kidney (MDCK) cells were used to determine the protective effects of EGCG on dimethylnitrosamine-induced alteration of GJIC and connexin 43 (Cx 43). The maximum concentration of EGCG was determined by the lactate dehydrogenase assay method. The scrape-loading dye transfer method was used to assess the expression and cellular localization of Cx 43. The phosphorylation status of Cx 43 was determined by Western blot analysis., Results: The optimal noncytotoxic concentration of EGCG was determined to be 10 microg/ml. The levels of GJIC and Cx 43 expression were markedly decreased in MDCK cells exposed to dimethylnitrosamine. A 12-h pretreatment with EGCG greatly ameliorated the GJIC-inhibitory effects of dimethylnitrosamine., Conclusion: These results suggest that the preservation of GJIC may indicate the chemopreventive effect of green tea on renal epithelial cells treated with a renal carcinogen in vitro.

During pregnancy, the villous trophoblast develops from the fusion of cytotrophoblastic cells (CT) into a syncytiotrophoblast (ST), supporting the main physiological functions of the human placenta. Connexin43 (Cx43) is demonstrated in situ and in vitro in the villous trophoblast between CT and between CT and ST. Moreover, the presence of a gap junctional intercellular communication (GJIC) during in vitro trophoblast differentiation was previously demonstrated. Because the exchange of molecules through gap junctions is considered to play a major role in the control of cell and tissue differentiation, we studied the effects of a gap junctional uncoupler, heptanol, on morphological and functional trophoblast differentiation and on GJIC measured by the fluorescence recovery after photobleaching method. We found that when the GJIC was interrupted, CT still aggregated but fused poorly. This morphological effect was associated with a significant decrease of trophoblastic-specific gene expression (beta human chorionic gonadotropin and human chorionic somatomammotropin). This blocking action was reversible as demonstrated by recovery of GJIC and trophoblast differentiation process after heptanol removal. Moreover, the inhibition of the trophoblast differentiation did not affect Cx43 transcript expression and Cx43 protein expression. These data suggest that the molecular exchanges through gap junctions preceding cellular fusion are essential for trophoblast differentiation generating the multifunctional syncytiotrophoblast.

Okadaic acid (OA) is produced by several types of dinoflagellates (marine plankton) and has been implicated as the causative agent of diarrhetic shellfish syndrome. Previous studies have shown that okadaic acid is a tumor promoter and a specific potent inhibitor of protein phosphatases and protein synthesis. These effects in turn affect intracellular processes such as metabolism, contractility, gene transcription, and the maintenance of cytoskeletal structure. Gap junctional intercellular communication (GJIC) is a means of maintaining cellular homeostasis in organs, the disruption of which favors tumor cell growth. The GJIC involves the transfer of small water-soluble molecules through intercellular channels (gap junctions), composed of proteins called connexins. OA disrupts cellular homeostasis in Caco-2 cells through several mechanisms including protein synthesis inhibition, apoptosis, and clastogenic effects. The aim of this study was then to evaluate the expression of the connexin 43 (Cx 43) mRNA in relation with the cytotoxicity induced by OA (3.75-60 ng/ml) in a human colonic epithelial cell line in culture (Caco-2 cells). OA produced a dose-dependent inhibition of GJIC in Caco-2 cells, along with a parallel decrease in the expression of Cx 43 as shown by immunohistochemistry using anti-Cx 43 antibody. Since Cx 43 is implicated in the suppression of tumors and OA is a tumor promoter, the inhibition of GJIC may play an important role in its carcinogenesis. These data are discussed in relation to the toxicity of OA, total RNA synthesis, and possible specificity of Cx 43 inhibition in the GJIC.

Connexin43 (cx43) forms gap junctions in astrocytes, and these gap junctions mediate intercellular communication by providing transport of low-molecular-weight metabolites and ions. We have recently shown that systemic growth hormone increases cx43 in the brain. One possibility was that local brain insulin-like growth factor-I (IGF-I) could mediate the effect by acting directly on astrocytes. In the present study, we examined the effects of direct application of recombinant human IGF-I (rhIGF-I) on astrocytes in primary culture concerning cx43 protein expression and gap junctional communication (GJC). After 24 hr of stimulation with rhIGF-I under serum-free conditions, the GJC and cx43 protein were analyzed. Administration of 30 ng/ml rhIGF-I increased the GJC and the abundance of cx43 protein. Cell proliferation of the astrocytes was not significantly increased by rhIGF-I at this concentration. However, a higher concentration of rhIGF-I (150 ng/ml) had no effect on GJC/cx43 but increased cell proliferation. Because of the important modulatory role of IGF binding proteins (IGFBPs) on IGF-I action, we analyzed IGFBPs in conditioned media. In cultures with a low abundance of IGFBPs (especially IGFBP-2), the GJC response to 30 ng/ml rhIGF-I was 81%, compared with the average of 25%. Finally, as a control, insulin was given in equimolar concentrations. However, GJC was not affected, which suggests that rhIGF-I acted via IGF-I receptors. In summary, the data show that rhIGF-I may increase GJC/cx43, whereas a higher concentration of rhIGF-I--at which stimulation of proliferation occurred--did not affect GJC/cx43. Furthermore, IGFBP-2 appeared to modulate the action of rhIGF-I on GJC in astrocytes by a paracrine mechanism., (Copyright 2003 Wiley-Liss, Inc.)

Dimethylnitrosamine(DMN) is an alkylating agent and a known renal carcinogen. A short exposure of renal epithelial cells to cytotoxic concentrations of DMN influences the expression of gap junction proteins. In this study, we examined gap junctional intercellular communication and connexin 43 expression in renal epithelial cells treated with 1% DMN and also examined the effects of dibutyryl-cAMP on preventing gap junctional disturbances. Connexin 43 becomes hypophosphorylated after treatment with 1% DMN for 15 minutes, but this hypophosphorylation is inhibited by pretreatment with dibutyryl-cAMP. These results suggest that changes in gap junction protein expression are early events associated with 1% DMN treatment of renal epithelial cells, and such changes are prevented by dibutyryl-cAMP pretreatment.

In this work, the effects of bacterial LPS, TNF-alpha, and IFN-gamma on gap junctional communication (dye coupling) and on the expression of connexin43 (immunofluorescence, immunoblotting, and RT-PCR) in monocytes/macrophages were studied. Freshly isolated human monocytes plated at high density and treated either with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma became transiently dye coupled (Lucifer yellow) within 24 h. Cells treated with LPS, TNF-alpha, or IFN-gamma alone remained dye uncoupled. In dye-coupled cells, the spread of Lucifer yellow to neighboring cells was reversibly blocked with 18 alpha-glycyrrhetinic acid, a gap junction blocker, but it was unaffected by oxidized ATP or probenecid, which block ionotropic ATP-activated channels and organic anion transporters, respectively. Abs against TNF-alpha significantly reduced the LPS plus IFN-gamma-induced increase in dye coupling. In dye-coupled monocytes/macrophages, but not in control cells, both connexin43 protein and mRNA were detected, and their levels were higher in cells with an elevated incidence of dye coupling. In dye-coupled cells, the localization of connexin43 immunoreactivity was diffuse at perinuclear regions and thin cell processes. The addition of 18-alpha-glycyrrhetinic acid induced a profound reduction of monocyte/macrophage transmigration across a blood brain barrier model. It also induced a significant reduction in the secretion of metalloproteinase-2 in cells treated with TNF-alpha plus IFN-gamma. We propose that some monocyte/macrophage responses are coordinated by connexin-formed membrane channels expressed transiently at inflammatory sites in which these cells form aggregates.

Zinc released from a subpopulation of glutamatergic synapses, mainly localized in the cerebral cortex and the hippocampus, facilitates or reduces glutamatergic transmission by acting on neuronal AMPA and NMDA receptors, respectively. However, neurons are not the only targets of zinc. In the present study, we provide evidence that zinc inhibits protein synthesis in cultured astrocytes from the cerebral cortex of embryonic mice. This inhibition, which reached 85% in the presence of 100 micro m zinc, was partially and slowly reversible and resulted from the successive inhibition of the elongation and the initiation steps of the protein translation process. This was assessed by measuring the phosphorylation level of the elongation factor eEF-2 and of the alpha subunit of the initiation factor eIF-2. Due to the rapid turnover of connexin-43 that forms junction channels in cultured astrocytes, the zinc-induced decrease of protein synthesis led to a partial disappearance of connexin-43, which was associated with an inhibition of the cellular coupling in the astrocytic syncitium. In conclusion, zinc not only inhibits protein synthesis in neurons, as previously demonstrated, but also in astrocytes. The resulting decrease in the intercellular communication between astrocytes should alter the function of surrounding neurons as well as their survival.

Objective: To study the effects of extremely low frequency magnetic fields(ELF MF) on the amount and localization of connexin 43(Cx43) gap-junction protein in the Chinese hamster lung(CHL) cells, and to explore the mechanism of ELF MF suppression on gap-junctional intercellular communication(GJIC)., Methods: The cells were irradiated for 24 h with 50 Hz sinusoidal magnetic field at 0.8 mT without or with 12-O-tetrade-canoylphorbol-3-acetate(TPA), 5 ng/ml for 1 h. The localization of Cx43 proteins were performed by indirect immunofluorescence histochemical analysis and detected by confocal microscopy. The second experiment was conducted to examine the quantity of Cx43 proteins level in nuclei or cytoplasm and detected by Western blotting analysis., Results: The cells exposed to TPA for 1 h displayed less bright labelled spots in the regions of intercellular junction than the normal cells. Most of Cx43 labelled spots occurred in the cytoplasm and aggregated near the nuclei. At the same time, the amount of Cx43 protein in cytoplasm were increased[(2.03 +/- 0.89) in ELF group, (2.43 +/- 0.82) in TPA group] as compared to normal control(1.04 +/- 0.17) (P < 0.01)., Conclusion: Inhibition on GJIC function by ELF MF alone or combined with TPA may be related with the shift of Cx43 from the regions of intercellular junction to the cytoplasm.

The loss of gap junctional intercellular communication has been proposedas playing a major role in the process of carcinogenesis. Most neoplastic cells, including C6 gliomas, express less connexins and have fewer gap junctions, reduced gap junctional intercellular communication, and increased growth rates compared with their nonneoplastic counterparts. The purpose of this study was to determine whether ciliary neurotrophic factor (CNTF) can be used to increase endogenous connexin43 levels, increase intercellular coupling, and retard the growth rate of C6 glioma cells. C6 cells were grown in serum-reduced medium (1% serum) and exposed to the following agents: vehicle (PBS), CNTF (20 ng/ml), CNTF soluble receptor (CNTFRalpha; 200 ng/ml), or Complex (CNTF + CNTFRalpha). Reverse transcription-PCR analysis indicated that C6 cells express CNTF mRNA but not CNTFRalpha mRNA. When cells were exposed to the above agents, only Complex caused an up-regulation of connexin43 protein (based on immunocytochemical and immunoblot analysis). Furthermore, Complex increased gap junctional coupling in C6 cells as noted by the passage of the gap junction permeable dye calcein. Finally, it was demonstrated that Complex-treatment reduces the growth rate of C6 cells compared with all of the other agents tested. Taken together, this study has demonstrated that CNTF in combination with its soluble receptor can increase connexin43 expression, increase gap junctional coupling, and reduce the in vitro proliferation of C6 glioma cells.

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.

Objective: To investigate the relationship between intercellular communication and three human rhabdomyosarcoma (RMS) cell lines of different metastatic potential., Methods: Indirect immunofluorescent staining and laser scanning confocal microscope (ISCM) were used to detect connexin 43 (CX43), a molecule related with gap junctional intercellular communication (GJIC). Fluorescence redistribution after photobleaching (FRAP) was used to detect function of GJIC., Results: A high level of CX43 protein expression was revealed in normal human myoblasts. CX43 protein was mainly localized on the surface of cells and sometimes in cytoplasm. CX43 expression was decreased in RMS cells. Both the detection rates and fluorescent intensity of CX43 decreased when the metastatic potential of RMS increased (P < 0.05). In contrast to normal, the fluorescence recovery rates of the RMS cells decreased and there was a negative correlation between the function of GJIC and malignancy of RMS cell lines (P < 0.05)., Conclusion: Varying degrees of GJIC inhibition may correlate with different metastatic potential of RMS. This may help to determine the malignant behavior of RMS and be used as a prognostic index of RMS.

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells. We have recently shown that 7-day-old cultures of AT2 cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca(2+) changes via gap junctions (25). In this study, we cultured AT2 cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of keratinocyte growth factor. Under these conditions, cultured AT2 cells display AT2 type morphology, express the AT2-specific marker surfactant protein C, and do not express AT1-specific cell marker aquaporin 5, all consistent with maintenance of AT2 phenotype. These AT2-like cells also coordinate mechanically induced intercellular Ca(2+) signaling, but, unlike AT1-like cells, do so by using extracellular nucleotide triphosphate release. Additionally, cultured cells that retain AT2 cell-specific markers express connexin profiles different from cultured cells with AT1 characteristics. The parallel changes in intercellular Ca(2+) signaling with cell differentiation suggest that cell signaling mechanisms are an intrinsic component of lung alveolar cell phenotype. Because lung epithelial injury is accompanied by extracellular matrix and growth factor changes, followed by extensive cell division, differentiation, and migration of AT2 progenitor cells, we suggest that similar changes may be vital to the lung recovery and repair process in vivo.

Lindane (gamma-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell-cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 microM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.

Oestradiol increases the protein expression of connexin43 (Cx43) gap junctions in myometrium but the effect of oestriol on gap junction expression has not been described previously. Oestriol is the most abundant free oestrogen in pregnant women and there is a marked surge in oestriol concentrations before term and idiopathic preterm labour. In order to determine whether oestriol may have a physiological action on the myometrium, cultured human myometrial cells obtained from non-pregnant hysterectomy specimens were exposed to 10 nmol/l oestradiol or oestriol. Intercellular communication between myometrial cells was investigated by microinjection of confluent cultured cells with the gap junction-permeant tracer Cascade Blue. There was a progressive increase in coupling after exposure to oestradiol or oestriol (P < 0.0005). An increase in Cx43 protein expression was demonstrated by immunocytochemistry after 1 h (P < 0.01) and 3 days (P < 0.01) exposure, and by Western blotting after 1 h (P < 0.01) and 3 days (P < 0.05) exposure, to both oestradiol and to oestriol. We conclude that oestriol increases gap junction communication in human myometrium by increasing gap junction expression. Elevated oestriol concentrations may thus play a role in the initiation of labour in women, by increasing cell-cell communication in the myometrium.

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 microM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 microM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.

Previous studies indicated that transforming RNA, derived from the 3' half of the U5 small nuclear RNA first stem structure, suppressed the secretory protein translation in vitro. Gap junctions facilitate homeostatic control of cell growth and differentiation and their dysfunction has been correlated with carcinogenesis. Here, we reported that transforming RNA directly suppressed the gap junction protein, connexin 43, translation and thereby inhibited functional gap junction function in rat epithelial cells. Together with previous data, this implies that altered expression of transforming RNA itself is a potential mechanism in inhibiting gap junction function during carcinogenesis.

Purpose: Downregulation of gap junctional intercellular communication (GJIC) has been implicated in carcinogenesis. This is a result of altered expression of connexins, the proteins that mediate GJIC, including connexin 43 (Cx43). Our aim was to evaluate the effect of known inducers of Cx43 on the chemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherapeutic agents., Methods: We examined the effect of dibutyryl-cyclic AMP (db-cAMP) and all-trans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma-glutamyl-cysteine-synthetase (gamma-GCS) levels, and glutathione S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxorubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db-cAMP and/or tRA., Results: Exposure to db-cAMP led to the upregulation of GJIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylation were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Treatment with db-cAMP, but not with tRA, was associated with a significant increase in the cytotoxic effects of the anticancer drugs doxorubicin, melphalan and BCNU as shown by a decrease in their IC50 values. Concomitant exposure to db-cAMP and tRA, however, had a more pronounced effect on cell sensitization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma-GCS activities remained unchanged., Conclusions: This study suggests that GJIC plays a role in cellular drug resistance, and highlights the potential use of GJIC modulators in combination with chemotherapy. Also, this is the first study exploring the ability of both db-cAMP and tRA to enhance cell chemosensitivity.

We examined whether a suicide gene/prodrug system using the uracil phosphoribosyltransferase (UPRT) of E. coli origin and 5-fluorouracil (5-FU) could achieve a bystander effect in two rodent tumor cell lines, murine colon carcinoma (Colon 26) and rat gliosarcoma (9L) cells. Cytotoxicity tests of mixed populations consisting of parent and transduced cells showed that the bystander effect was not produced in Colon 26 cells in either the UPRT/5-FU system or the herpes simplex virus-thymidine kinase/ganciclovir system but a strong bystander effect was evidenced by both suicide gene systems in 9L cells. The expression level of connexin 43, a protein that constitutes gap junctions, was high in 9L but low in Colon 26 cells. A gap junction-permeable fluorescein dye could be transferred among 9L cells but hardly at all among Colon 26 cells. Taken together, the efficacy of the bystander effect in the UPRT/5-FU system can be affected by gap junction-mediated intercellular communication.

Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.

Caffeic acid phenethyl ester (CAPE), an active ingredient of honeybee propolis, has been identified as having anti-inflammatory, anti-viral and anti-cancer properties. Since the deficiency of gap junctional intercellular communication (GJIC) has been shown to be a characteristic of most cancer cells, this study was designed to test the hypothesis that the anti-carcinogenic activity of CAPE might be related to its ability to restore GJIC in tumorigenic GJIC-deficient cells (WB-ras2 cells). The results showed that CAPE restored GJIC, phosphorylation of connexin 43 (Cx43) and its normal localization on the plasma membrane in WB-ras2 cells after 3 days at 5 microg/ml concentration. Additionally, CAPE inhibited growth in soft agar and decreased the protein level of p21(ras). The results are consistent with the hypothesis that the anti-cancer mechanism of CAPE may be mediated by its ability to restore GJIC.

Mechanical stretch is thought to play an important role in remodeling atrial and ventricular myocardium and may produce substrates that promote arrhythmogenesis. In the present work, neonatal rat ventricular myocytes were cultured for 4 days as confluent monolayers on thin silicone membranes and then subjected to linear pulsatile stretch for up to 6 hours. Action potential upstrokes and propagation velocity (theta) were measured with multisite optical recording of transmembrane voltage of the cells stained with the voltage-sensitive dye RH237. Expression of the gap junction protein connexin43 (Cx43) and the fascia adherens junction protein N-cadherin was measured immunohistochemically in the same preparations. Pulsatile stretch caused dramatic upregulation of intercellular junction proteins after only 1 hour and a further increase after 6 hours (Cx43 signal increased from 0.73 to 1.86 and 2.02% cell area, and N-cadherin signal increased from 1.21 to 2.11 and 2.74% cell area after 1 and 6 hours, respectively). This was paralleled by an increase in theta from 27 to 35 cm/s after 1 hour and 37 cm/s after 6 hours. No significant change in the upstroke velocity of the action potential or cell size was observed. Increased theta and protein expression were not reversible after 24 hours of relaxation. Nonpulsatile (static) stretch produced qualitatively similar but significantly smaller changes than pulsatile stretch. Thus, pulsatile linear stretch in vitro causes marked upregulation of proteins that form electrical and mechanical junctions, as well as a concomitant increase in propagation velocity. These changes may contribute to arrhythmogenesis in myocardium exposed to acute stretch.

Gap junctional intercellular communication (GJIC) may contribute to cellular differentiation. To examine this possibility in bone cells we examined markers of cellular differentiation, including alkaline phosphatase, osteocalcin, and osteopontin, in ROS17/2.8 cells (ROS), a rat osteoblastic cell line expressing phenotypic characteristics of fully differentiated osteoblasts. We utilized ROS rendered communication deficient either by stable transfection with antisense cDNA to connexin 43 (Cx43), the predominant gap junction protein in bone (RCx16 cells), or by overexpression of Cx45, a gap junction protein not normally expressed in ROS (ROS/Cx45 cells). Both RCx16 and ROS/Cx45 cells displayed reduced dye coupling and Cx43 protein expression relative to ROS, control transfectants, and ROS/Cx45tr, ROS cells expressing carboxylterminal truncated Cx45. Steady-state mRNA levels for osteocalcin as well as alkaline phosphatase activity, two markers of osteoblastic differentiation, were also reduced in poorly coupled RCx16 and ROS/Cx45 cells. On the other hand, steady-state mRNA levels for osteopontin increased slightly in RCx16 and ROS/Cx45 cells. These results suggest that GJIC at least partly contributes to the regulation of expression of markers of osteoblastic differentiation.

Gliomas are lethal because of local invasion into brain parenchyma. Glioma cells were isolated from different regions (white matter, gray matter and tumor core) of a glioma-bearing dog brain. Individual clonal cell lines were established from each area, and characterized for growth, migration and gap junctions. The regional clonal cell lines differed in rates and preferred substrate for migration. Cell lines generated from invaded white matter showed stimulated migration on collagen and variable migration on merosin, whereas migration of cell lines derived from invaded gray matter showed the reciprocal responses: stimulation on merosin and inhibition on collagen. Gap junctional communication showed significant degrees of variation between the different clones. A direct inverse relationship between the number of cells demonstrating gap junctional communication and migration rate of cells away from multicellular spheroids was evident. Glioma cells which have a reduced capacity to connect to each other have an accelerated migration rate onto autologous, glioma-derived matrix. These results suggest that invasive glioma cells suppress autologous cell-to-cell cohesion, partly evident as reduced formation of gap junctions. In addition, glioma cells were stimulated to migrate in a dose-dependant manner in response to epidermal growth factor (EGF) coincident with the reduction of Cx43 levels and increased serine phosphorylation. We speculate that in order for glioma cells to invade locally into brain parenchyma they must first detach from neighboring cells ("let go...let's go" paradigm of invasion).

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) are frequently decreased in neoplastic cells and have been increased by cAMP and retinoids. GJIC and connexin expression were investigated in early passage normal human ovarian surface epithelial (HOSE) cells, human ovarian adenocarcinoma cell lines (CaOV-3, NIH:OVCAR-3, SK-OV-3 and SW626) and surgical specimens of human serous cystadenocarcinomas. We hypothesized that GJIC and connexin expression would be decreased in neoplastic cells and would be increased by cAMP and retinoic acid. Cultured HOSE cells exhibited extensive fluorescent dye-coupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate and all-trans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.

Communication through gap junctions was first suggested to have a role in the social control of cell growth over 30 years ago. However, despite extensive experimentation, the importance of gap junctions as a general mechanism of growth control remains to be established. A number of different studies have shown that a common early response of cells in culture to polypeptide growth factors such as PDGF is a rapid and transient inhibition of cell communication suggesting that a cell may have to lose communication with its neighbors before it can undergo cell division. Here we show that 3T3 A31 fibroblasts exposed to PDGF exhibit a 50% decrease in cell communication as measured by dye transfer in the absence of significant changes in the cellular content and distribution of Cx43. Likewise, PDGF inhibited cell communication in cells transfected either with a vector which did not contain a cDNA or with an expression vector encoding full-length Cx43 fused to a c-myc tag (Cx43-M). In contrast, 3T3 A31 fibroblasts transfected with an expression construct encoding a deletion mutant of Cx43 (Cx43-256M) consisting of amino acids 1-256 of Cx43 fused to a c-myc tag maintain high levels of gap junction activity following exposure to PDGF. These results suggest that sites which trigger loss of cell communication in response to PDGF are located within amino acids 257 to 382 of the Cx43 molecule. Cells transfected with an expression vector encoding full-length Cx43 fused to a c-myc tail exhibited a reduced basal growth rate compared to both parent cells and cells transfected with a control vector but maintained a strong mitogenic response to PDGF. In contrast, both the basal growth rate and the mitogenic response to PDGF was markedly reduced in cells which expressed Cx43-256M consistent with the hypothesis that loss of cell communication is required before a cell can respond to mitogenic stimuli., (Copyright 1999 Academic Press.)

Rats chronically exposed to acrylonitrile (ACN) have shown a dose-dependent increase in the incidence of astrocytomas in the brain. The mechanism(s) by which ACN induces cancer in rodents has not been established. ACN does not appear to be directly genotoxic in the brain and thus a nongenotoxic mode of action has been proposed. Inhibition of gap junctional intercellular communication (GJIC) has been shown to be a property of many nongenotoxic carcinogens. The present study examined the effects of ACN on GJIC in a rat astrocyte transformed cell line, DI TNC1 cells (a target cell for ACN carcinogenicity) and primary cultured hepatocytes (a nontarget cell for ACN carcinogenicity). ACN inhibited GJIC in rat astrocytes in a dose-dependent manner. Inhibition of GJIC was observed following 2 h treatment with 0.10 mmol/L and 1.00 mmol/L ACN. However, in primary cultured hepatocytes, ACN exposed did not result in inhibition of GJIC even after 48 h of continued treatment. In the astrocytes, GJIC inhibition plateaued after 4 h of treatment and remained blocked throughout the entire experimental period examined. Inhibition of GJIC in DI TNC1 cells was reversed by removal of ACN from the culture medium after 4 or 24 h of treatment. Cotreatment of astrocytes with vitamin E reduced the effect of ACN-induced inhibition of GJIC. Similarly, inhibition of GJIC was prevented by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC), a precursor of glutathione synthesis. Decreasing cellular glutathione by treatment with buthionine sulfoxamine alone (without ACN) did not affect GJIC in astrocytes. Collectively, these results demonstrate that treatment with ACN caused a selective inhibition of GJIC in rat DI TNC1 astrocytes (the target cell type), but not in rat hepatocytes (a nontarget tissue). Inhibition of GJIC in astrocytes was reversed by treatment with antioxidants and suggests a potential role for oxidative stress in ACN-induced carcinogenesis.

The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.

Decreased connexin gene expression and loss of the capacity for either homologous or heterologous intercellular communication has been associated with neoplastic transformation. We tested the hypothesis that loss of gap junctional intercellular communication (GJIC) correlates with tumorigenic potential in the HeLa x skin fibroblast human hybrid cell system. Connexin gene expression, gap junction function and tumorigenicity were determined for the non-tumorigenic somatic hybrid cell line (CGL1) and a series of UVC-induced tumorigenic cell lines derived from CGL1. CGL1 and the parental skin fibroblasts express connexin43 (alpha1 gap junction gene) mRNA and protein, form gap junctional plaques and have functional gap junctions. UVC-irradiation of CGL1 cells produced a cell line (UV12) with an aggressive tumorigenic phenotype, which lost connexin43 expression as well as both homologous and heterologous GJIC and was in this respect similar to HeLa cells. However, the phenotype of UV12 cells exhibited some instability and revertants to a less aggressive tumorigenic phenotype were isolated. These cells expressed connexin43 mRNA and protein, and demonstrated homologous GJIC. Furthermore, cells reconstituted from a tumor derived from this revertant cell line retained significant connexin43 expression and homologous GJIC, although they exhibited an aggressive tumorigenic phenotype. Thus, functional homologous GJIC cannot be dissociated from tumorigenicity in this system. However, heterologous GJIC between these same UVC-induced tumorigenic cell lines and normal human skin fibroblasts was reduced, whereas the non-tumorigenic hybrid cells showed extensive heterologous GJIC. In summary, re-acquisition of connexin43 expression and homologous GJIC does not restore the non-tumorigenic phenotype in UVC-induced tumorigenic HeLa skin fibroblast human hybrid cells. However, reduction of heterologous GJIC does correlate with tumorigenicity in this cell system.