Role of connexin (gap junction) genes in cell growth control and carcinogenesis

Gap junctional intercellular communication (GJIC) is considered to play a key role in the maintenance of tissue independence and homeostasis in multicellular organisms by controlling the growth of GJIC-connected cells. Gap junction channels are composed of connexin molecules and, so far, more than a dozen different connexin genes have been shown to be expressed in mammals. Reflecting the importance of GJIC in various physiological functions, deletion of different connexin genes from mice results in various disorders, including cancers, heart malformation or conduction abnormality, cataract, etc. The possible involvement of aberrant GJIC in abnormal cell growth and carcinogenesis has long been postulated and recent studies in our own and other laboratories have confirmed that expression and function of connexin genes play an important role in cell growth control. Thus, almost all malignant cells show altered homologous and/or heterologous GJIC and are often associated with aberrant expression or localization of connexins. Aberrant localization of connexins in some tumour cells is associated with lack of function of cell adhesion molecules, suggesting the importance of cell-cell recognition for GJIC. Transfection of connexin genes into tumorigenic cells restores normal cell growth, supporting the idea that connexins form a family of tumour-suppressor genes. Some studies also show that specific connexins may be necessary to control growth of specific cell types. We have produced various dominant-negative mutants of Cx26, Cx32 and Cx43 and showed that some of them prevent the growth control exerted by the corresponding wild-type genes. However, we have found that connexins 32, 37 and 43 genes are rarely mutated in tumours. In some of these studies, we noted that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function. We have recently created a transgenic mouse strain in which a mutant Cx32 is specifically overexpressed in the liver. Studies with such mice indicate that Cx32 plays a key role in liver regeneration after partial hepatectomy. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through GJIC. Recently, we and others have shown that GJIC is responsible for the bystander effect seen in HSV-tk/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.     

DU-145 prostate carcinoma cells that selectively transmigrate narrow obstacles express elevated levels of Cx43

The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the “leading front” formation during cancer invasion.     

Up-regulation of connexin 43 and gap junctional intercellular communication by Coleusin Factor is associated with growth inhibition in rat osteosarcoma UMR106 cells

Gap junctions, formed by connexin (Cx) family proteins, permit direct exchange of regulatory ions and small signal molecules between neighbouring cells. Gap junctional intercellular communication (GJIC) plays an important role in maintaining the homeostasis and preventing cell transformation. Most of the tumour cells feature deficient or aberrant connexin expression and GJIC level, and restoration of connexin expression and GJIC is correlated with cell growth control. Numerous researches has suggested the possibility of connexins as potential anti-tumour targets for chemoprevention and chemotherapy. We investigated the ability of Coleusin Factor (CF, also named FSK88) to regulate the Cx43 expression and GJIC level in rat osteosarcoma UMR106 cells. The results have demonstrated that CF increased the mRNA and protein expression of Cx43 in both in a dose- and timedependent manner, and concomitant with up-regulation of Cx43, CF treatment up-regulated the diminished GJIC level in UMR106 cells as assayed by dye transfer experiments. In addition, Cx43 distribution at the plasma membrane was also enhanced dramatically by CF treatment. Furthermore, we discovered that CF was potent to inhibit the growth and proliferation of UMR106 cells. These results provide the first evidence that CF can regulate connexin and GJIC, indicating that Cx43 may be a target of CF to exert its anti-tumour effects.     

Growth inhibition by connexin26 expression in cultured rodent tumor cells

The Connexin (Cx) gene family acts as a tumor suppressor. However, it is unclear whether the tumor-suppressing activity acquired by Cx gene transfection is mainly due to the recovery of the gap junction-mediated intercellular communication (GJIC) or to other unknown mechanisms. In order to elucidate the mechanism of the Cx-induced tumor-suppressing activity, we transfected Cx26 cDNA into a rodent mammary tumor cell-line (BICR-M1Rk) in which Cx43 had been normally expressed and a typical pattern of GJIC had been observed. The exogenous Cx26 was mainly localized on the nuclear envelope, whereas most of the endogenous Cx43 resided at the plasma membrane of the transfected BICR-M1Rk. Consistent with the localization of Cx26, GJIC was not increased upon the transfection of Cx26 when it was assessed by a scrape-loading dye transfer technique. A conventional [3H]-thymidine incorporation study showed that the growth rate of the Cx26-transfected cells was significantly decreased (70%), compared to that of the control BICR-M1Rk. Therefore, our results clearly demonstrate that the exogenously expressed Cx26 in the BICR-M1Rk cancer cell-line exerts an anti-proliferate activity in a GJIC-independent manner.     

Pathological Significance of Intracytoplasmic Connexin Proteins: Implication in Tumor Progression

A considerable amount of evidence has established that gap junctional intercellular communication (GJIC) suppresses tumor development by halting the stage of tumor promotion. Consistently, GJIC is downregulated in tumors. The downregulation of GJIC is caused by not only the reduced expression level of connexin proteins but also their aberrant cytoplasmic localization. Although it has long been thought that cytoplasmic localization of connexin proteins is merely one of the mechanisms of the downregulation of GJIC, careful studies with human tumor samples have indicated that the expression level of intracytoplasmic connexin proteins correlates well with the grade of malignancy and the progression stage of tumors. Hypothesizing that intracytoplasmic connexin proteins should have their proper functions and that their increase should facilitate tumor progression such as cell migration, invasion and metastasis, we examined the effects of overexpressed connexin32 (Cx32) protein on the phenotype of human HuH7 hepatoma cells, which express a basal level of endogenous Cx32 only in cytoplasm. The cells were retrovirally transduced with the Tet-off Cx32 construct so that withdrawal of doxycycline from the culture medium could induce overexpression of Cx32 protein in cytoplasm. Even when overexpressed, Cx32 protein was retained in cytoplasm, i.e., Golgi apparatuses, and did not induce GJIC. However, overexpression of Cx32 protein in cytoplasm enhanced both the motility and the invasiveness of HuH7 cells and induced metastasis when the cells were xenografted into SCID mice. Taken together, cytoplasmic accumulation of connexin proteins may exert effects favorable for tumor progression.     

A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

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.     

Connexins and Gap Junctions in Mammary Gland Development and Breast Cancer Progression

The development and function of the mammary gland require precise control of gap junctional intercellular communication (GJIC). Here, we review the expression and function of gap junction proteins, connexins, in the normal mouse and human mammary gland. We then discuss the possible tumor-suppressive role of Cx26 and Cx43 in primary breast tumors and through the various stages of breast cancer metastasis and consider whether connexins or GJIC may actually promote tumorigenesis at some stages. Finally, we present in vitro data on the impact of connexin expression on breast cancer cell metastasis to the bone. We observed that Cx43 expression inhibited the invasive and migratory potentials of MDA-MB-231 breast cancer cells in a bone microenvironment, provided by the MC3T3-E1 mouse osteoblastic cell line. Expression of either Cx26 or Cx43 had no effect on MDA-MB-231 growth and adhesion under the influence of osteoblasts and did not result in regulation of osteogenic gene expression in these breast cancer cells. Furthermore, connexin-expressing MDA-MB-231 cells did not have an effect on the growth or differentiation of MC3T3-E1 cells. In summary, we conclude that connexin expression and GJIC are integral to the development and differentiation of the mammary gland. In breast cancer, connexins generally act as tumor suppressors in the primary tumor; however, in advanced breast tumors, connexins appear to act as both context-dependent tumor suppressors and facilitators of disease progression.     

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

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

Localisation aberrante de la connexine 43 dans le cancer du testicule

Most cells can communicate directly via gap junction channels. Gap junction intercellular communication (GJIC) participates in the control of cell proliferation. Abnormal expression of connexins (Cx), the constitutive proteins of gap junctions, has been associated with a transformed phenotype. In the seminiferous tubules, connexin Cx43 is predominantly expressed by Sertoli cell and germinal cell membranes. We studied Cx43 expression in four testicular cancers (pure seminoma). Cx43 mRNA and protein characterized by RT PCR and Western blot were found to be similar to controls (normal testes) in each case. However, immunofluorscence study of Cx43 protein indicated a cytoplasmic localization with no membrane expression, excluding the participation of Cx43 in GJIC. The significance of this aberrant localization will be discussed in relation to carcinogenesis.     

The connexin 43 C-terminus: A tail of many tales

Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

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

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

Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma. LPS- plus IFN-gamma-induced dye-coupling was mediated by DC-derived TNF-alpha. In addition, CpG plus IFN-gamma induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-alpha. LPS- plus IFN-gamma-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-gamma-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.     

Exogenous Cx43 expression decrease cell proliferation rate in rat hepatocarcinoma cells independently of functional gap junction

Background  

Gap junction intercellular communication (GJIC) is considered to play a role in the regulation of homeostasis because it regulates important processes, such as cell proliferation and cell differentiation. A reduced or lost GJIC capacity has been observed in solid tumors and studies have demonstrated that GJIC restoration in tumor cells contribute to reversion of the transformed phenotype. This observation supports the idea that restoration of the functional channel is essential in this process. However, in the last years, reports have proposed that just the increase in the expression of specific connexins can contribute to reversion of the malign phenotype in some tumor cells. In the present work, we studied the effects of exogenous Connexin 43 (Cx43) expression on the proliferative behavior and phenotype of rat hepatocarcinoma cells.     

Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

Gefitinib efficiency in non-small-cell lung cancer (NSCLC) therapy is limited due to development of drug resistance. The molecular mechanisms of gefitinib resistance remain still unclear. In this study, we first found that connexin 26 (Cx26) is the predominant Cx isoform expressed in various NSCLC cell lines. Then, two gefitinib-resistant (GR) NSCLC cell lines, HCC827 GR and PC9 GR, from their parental cells were established. In these GR cells, the results showed that gefitinib resistance correlated with changes in cellular EMT phenotypes and upregulation of Cx26. Cx26 was detected to be accumulated in the cytoplasm and failed to establish functional gap-junctional intercellular communication (GJIC) either in GR cells or their parental cells. Ectopic expression of GJIC-deficient chimeric Cx26 was sufficient to induce EMT and gefitinib insensitivity in HCC827 and PC9 cells, while knockdown of Cx26 reversed EMT and gefitinib resistance in their GR cells both in vitro and in vivo. Furthermore, Cx26 overexpression could activate PI3K/Akt signaling in these cells. Cx26-mediated EMT and gefitinib resistance were significantly blocked by inhibition of PI3K/Akt pathway. Specifically, inhibition of the constitutive activation of PI3K/Akt pathway substantially suppressed Cx26 expression, and Cx26 was confirmed to functionally interplay with PI3K/Akt signaling to promote EMT and gefitinib resistance in NSCLC cells. In conclusion, the reciprocal positive regulation between Cx26 and PI3K/Akt signaling contributes to acquired gefitinib resistance in NSCLC cells by promoting EMT via a GJIC-independent manner.Lung cancer, of which non-small-cell lung cancer (NSCLC) is the most common form, remains the leading cause of cancer-related deaths worldwide.¹ Currently, gefitinib, as the first epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is one of the most accepted therapies against NSCLC carrying EGFR mutations. However, almost all NSCLC patients who initially respond well to EGFR-TKIs eventually develop acquired resistance.² Development of effective therapeutic interventions to overcome gefitinib resistance is an urgent need.Epithelial-mesenchymal transition (EMT), during which cancer cells lose epithelial markers such as E-cadherin but gain mesenchymal markers such as vimentin, is known to be deeply involved in cancer progression and chemotherapy resistance. Specially in NSCLC, EMT plays pivotal roles in the acquired resistance to EGFR-TKIs such as gefitinib.^{3, 4} For example, restoring E-cadherin expression or silencing EMT regulator Slug increases gefitinib sensitivity in NSCLC cells with a mesenchymal phenotype.^{5, 6} Accumulating evidences indicate that constitutively activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling is a central feature of EMT in many cancers including NSCLC.^{7, 8} However, the exact mechanism for the acquired gefitinib resistance of NSCLC remains unclear.Connexins (Cxs) are a family of transmembrane proteins, which compose the intercellular gap junctions between the neighboring cells.⁹ Gap junctions directly connect the cytoplasms of adjacent cells, thereby mediating direct exchange of signaling molecules smaller than 1 kDa, such as ions, small metabolites, and second messengers. This process is termed gap-junctional intercellular communication (GJIC). Cx expression and/or GJIC are frequently reduced or loss in malignant cell lines and cancers, while restoration of Cx expression and/or GJIC retarded tumor growth and increased cytotoxicities of chemotherapeutics such as cisplatin and docetaxel.^{10, 11, 12, 13} Therefore, Cxs have long been deemed tumor suppressors. However, increasing new observations were apparently contradicting the ''dogma'' and became clear that Cxs and GJIC also contribute to cancer progression and chemoresistance. For example, Cx32 expression was detected in breast cancer and significantly increased in lymph node metastases compared with primary tumors, suggesting Cx32 may be a sign of more malignant phenotype of breast cancer.¹⁴ Besides, cytoplasmic accumulation of Cx32 exerted favorable effects for hepatocellular carcinoma (HCC) progression including invasion and metastasis by Cx linked, but GJIC-independent mechanism.¹⁵ Recently, Gielen et al.¹⁶ reported that increasing the level of Cx43 confers temozolomide resistance in human glioma cells whereas knockdown of Cx43 sensitizes them to temozolomide treatment via both GJIC-dependent and -independent mechanisms.Up to now, there are ~21 isoforms of Cxs that distribute in almost all human organs in tissue-specific patterns.¹⁷ Cx26, one of the most common isoforms of Cxs, is predominantly expressed in lung tissue.^{18, 19} Despite Cx26 has been considered as a potential tumor suppressor or chemotherapy sensitizer in some types of tumors,^{20, 21} Ito et al.²² found that Cx26 helps lung squamous cell carcinoma (SCC, one histological type of NSCLC), acquire aggressive phenotypes, lymph node metastasis, and poor prognosis, indicating that a potential role of Cx26 on the malignant development of SCC. However, the roles of Cx26 and its derived GJIC in the development of gefitinib resistance in NSCLC have not been explored.In this study, to clarify the potential role of Cx26 and its derived GJIC in gefitinib resistance in NSCLC, we first surveyed the expression of four major Cxs in different gefitinib-sensitive NSCLC cell lines and found a positive correlation between high level of Cx26 and gefitinib insensitivity in NSCLC cells. Such an association was further confirmed in established gefitinib-resistant (GR) HCC827 and PC9 cell lines both in vitro and in vivo. Importantly, we find a positive mutual regulation between Cx26 and PI3K/Akt pathway, which confers acquired gefitinib resistance in NSCLC cells by GJIC-independent induction of EMT.     

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

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

Growth inhibition in G(1) and altered expression of cyclin D1 and p27(kip-1 )after forced connexin expression in lung and liver carcinoma cells

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.     

Downregulation of gap junction expression and function by endoplasmic reticulum stress

Gap junctional intercellular communication (GJIC) plays a critical role in the control of multiple cell behavior as well as in the maintenance of tissue and organ homeostasis. However, mechanisms involved in the regulation of gap junctions (GJs) have not been fully understood. Given endoplasmic reticulum (ER) stress and dysfunction of GJs coexist in several pathological situations, we asked whether GJs could be regulated by ER stress. Incubation of mesangial cells with ER stress‐inducing agents (thapsigargin, tunicamycin, and AB₅ subtilase cytotoxin) resulted in a decrease in connexin 43 (Cx43) expression at both protein and mRNA levels. This was accompanied by a loss of GJIC, as evidenced by the reduced numbers of dye‐coupled cells after single cell microinjection or scrape loading dye transfer. Further studies demonstrated that ER stress significantly inhibited the promoter activity of the Cx43 gene, reduced [³⁵S]‐methionine incorporation into Cx43 protein and accelerated degradation of Cx43. ER stress also decreased the Cx43 protein levels in several different cell types, including human umbilical vein endothelial cells, mouse‐derived renin‐secreting cells and human hepatoma cells. Furthermore, induction of ER stress by hypoxic chemicals thenoyltrifluoroacetone and cobalt chloride was found to be associated with a reduction in Cx43. Our findings thus reveal a close link between ER stress and GJs. ER stress may represent a novel mechanism underlying the altered GJs in a variety of pathological situations. J. Cell. Biochem. 107: 973–983, 2009. © 2009 Wiley‐Liss, Inc.