Benzalkonium Chloride Suppresses Rabbit Corneal Endothelium Intercellular Gap Junction Communication

Purpose

Gap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.

Methods

Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco''s Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity.

Results

Topical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.

Conclusions

The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.     

Cytokine effects on gap junction communication and connexin expression in human bladder smooth muscle cells and suburothelial myofibroblasts

Background

The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta1 (TGFβ1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF).

Methodology/Principal Findings

HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFβ1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC.

Conclusions/Significance

Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.     

Increased expression and functionality of the gap junction in peripheral blood lymphocytes is associated with hypertension-mediated inflammation in spontaneously hypertensive rats

Background

Imbalances in circulating T lymphocytes play critical roles in the pathogenesis of hypertension-mediated inflammation. Connexins (Cxs) in immune cells are involved in the maintenance of homeostasis of T lymphocytes. However, the association between Cxs in peripheral blood T lymphocytes and hypertension-mediated inflammation remains unknown. This study was designed to investigate the role of Cxs in T lymphocytes in hypertension-mediated inflammation in spontaneously hypertensive rats (SHRs).

Methods

The systolic blood pressure (SBP) in Wistar-Kyoto (WKY) rats and SHRs was monitored using the tail-cuff method. The serum cytokine level was determined using ELISA. The proportions of different T-lymphocyte subtypes in the peripheral blood, the expressions of Cx40/Cx43 in the T-cell subtypes, and the gap junctional intracellular communication (GJIC) of peripheral blood lymphocytes were measured using flow cytometry (FC). The accumulations of Cx40/Cx43 at the plasma membrane and/or in the cytoplasm were determined using immunofluorescence staining. The in vitro mRNA levels of cytokines and GJIC in the peripheral blood lymphocytes were respectively examined using real-time PCR and FC after treatment with Gap27 and/or concanavalin A (Con A).

Results

The percentage of CD4⁺ T cells and the CD4⁺/CD8⁺ ratio were high, and the accumulation or expressions of Cx40/Cx43 in the peripheral blood lymphocytes in SHRs were higher than in those of WKY rats. The percentage of CD8⁺ and CD4⁺CD25⁺ T cells was lower in SHRs. The serum levels of IL-2, IL-4 and IL-6 from SHRs were higher than those from WKY rats, and the serum levels of IL-2 and IL-6 positively correlated with the expression of Cx40/Cx43 in the peripheral blood T lymphocytes from SHRs. The peripheral blood lymphocytes of SHRs exhibited enhanced GJIC. Cx43-based channel inhibition, which was mediated by Gap27, remarkably reduced GJIC in lymphocytes, and suppressed IL-2 and IL-6 mRNA expressions in Con A stimulated peripheral blood lymphocytes.

Conclusions

Our data suggest that Cxs may be involved in the regulation of T-lymphocyte homeostasis and the production of cytokines. A clear association was found between alterations in Cxs expression or in Cx43-based GJIC and hypertension-mediated inflammation.

     

TLR2 Mediates Gap Junctional Intercellular Communication through Connexin-43 in Intestinal Epithelial Barrier Injury

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser³⁶⁸ to prevent spontaneous chronic colitis in MDR1α-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.The intestinal epithelial cell (IEC)³ barrier provides the front line of mucosal host defense in the intestine. The IEC barrier confers anatomic integrity and immunologic protection of the intestinal mucosal surface. Because the IEC barrier constantly faces diverse populations of lumenal microbes and other potential threats, it must exert a highly defined process of continuous discrimination: excluding harmful antigens while allowing host-beneficial substances to permeate (1, 2). Para- and intercellular transit of molecules is modulated by a complex network of closely arranged tight (TJ) and gap junctions (GJ) between juxtaposed IEC. Gap junctional intercellular communication (GJIC) is an essential, but not well understood, mechanism for cellular and tissue homeostasis that coordinates cell-cell passage of ions and small metabolites (<1 kDa). Thus, GJIC regulates cell proliferation, migration, and differentiation (3). GJ channels are formed by hexameric connexins at the plasma membrane. Cx43 is the major connexin and represents a key target in GJIC regulation (4). It is differentially phosphorylated at a dozen or more residues throughout its life cycle (5–9). Alteration of GJIC caused by changes in Cx43 has been proposed to be involved in the pathophysiology of diverse IEC barrier diseases, including inflammatory bowel diseases, necrotizing enterocolitis, cancer, and enteric infection (10–12). However, immune mediators that allow protective GJIC via Cx43 to sustain IEC barrier function during mucosal damage have not yet been identified.Toll-like receptor 2 (TLR2), a member of the TLR family that is constitutively expressed in IEC (13–15), recognizes conserved molecular patterns associated with both Gram-negative and -positive bacteria (16). We have previously shown that commensal-mediated TLR2 helps to maintain functional TJ barrier integrity of the intestinal epithelial layer. TLR2 enhances transepithelial resistance of the IEC barrier by apical redistribution of ZO-1 via protein kinase Cα/δ (17). Treatment with the TLR2 ligand PCSK protects ZO-1-associated IEC barrier integrity and decreases intestinal permeability in acute colitis (18). Previous studies in other cell types have demonstrated that the second PDZ domain of ZO-1 interacts with the carboxyl terminus of Cx43 (19, 20). ZO-1 binds to Cx43 preferentially during the G₀ phase, enhancing assembly and stabilization of GJIC (21, 22). Like TLR2, Cx43 and ZO-1 reside in caveolin-1-associated lipid raft microdomains (23–25). We therefore hypothesized that the binding between ZO-1 and Cx43 may allow TLR2 to control IEC barrier function by GJIC.In this study, we identified a new physiological mechanism of innate immune host defense in the injured intestine. Our findings indicated that Cx43 serves as an important component of the protective innate immune response of the intestinal epithelium. TLR2-induced GJIC via Cx43 appears to control IEC barrier function and restitution during acute and chronic inflammatory damage, enhancing mucosal homeostasis between commensals and host. UC-associated TLR2 mutant results in impaired GJIC by a proteasomal-dependent increase in Cx43 turnover.     

hESC Expansion and Stemness Are Independent of Connexin Forty-Three-Mediated Intercellular Communication between hESCs and hASC Feeder Cells

Background

Human embryonic stem cells (hESCs) are a promising and powerful source of cells for applications in regenerative medicine, tissue engineering, cell-based therapies, and drug discovery. Many researchers have employed conventional culture techniques using feeder cells to expand hESCs in significant numbers, although feeder-free culture techniques have recently been developed. In regard to stem cell expansion, gap junctional intercellular communication (GJIC) is thought to play an important role in hESC survival and differentiation. Indeed, it has been reported that hESC-hESC communication through connexin 43 (Cx43, one of the major gap junctional proteins) is crucial for the maintenance of hESC stemness during expansion. However, the role of GJIC between hESCs and feeder cells is unclear and has not yet been reported.

Methodology/Principal Findings

This study therefore examined whether a direct Cx43-mediated interaction between hESCs and human adipose-derived stem cells (hASCs) influences the maintenance of hESC stemness. Over 10 passages, hESCs cultured on a layer of Cx43-downregulated hASC feeder cells showed normal morphology, proliferation (colony growth), and stemness, as assessed by alkaline phosphatase (AP), OCT4 (POU5F1-Human gene Nomenclature Database), SOX2, and NANOG expression.

Conclusions/Significance

These results demonstrate that Cx43-mediated GJIC between hESCs and hASC feeder cells is not an important factor for the conservation of hESC stemness and expansion.     

HYS-32, a novel analogue of combretastatin A-4, enhances connexin43 expression and gap junction intercellular communication in rat astrocytes

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 (5 μM) 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.     

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

Background  

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

7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells

Background  

Connexin43 (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols) affect gap junction intercellular communication (GJIC).     

TGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells

Background

Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E₂) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.

Methodology/Principal Findings

Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E₂ as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E₂ secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E₂ and TGF-β1, and E₂ treatment in turn restored the inhibition of TGF-β1 on GJIC.

Conclusions

Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E₂ secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.     

Connexin 43 contributes to differentiation of retinal pigment epithelial cells via cyclic AMP signaling

Retinal pigment epithelium (RPE) cells play important roles in the visual system that supports neurosensory retina homeostasis. Connexin (Cx) 43-mediated gap-junctional intercellular communication (GJIC) participates in the regulation of retinal organogenesis, but much of the function of Cx43 on the differentiation of RPE cells is unclear. Here, we report the involvement of Cx43 in RPE differentiation. Knockdown of Cx43 in RPE cells dramatically inhibited the differentiation, whereas Cx43-overexpression successfully induced RPE cell differentiation under de-differentiation conditions. From the experiments using GJIC inhibitors and C-terminus-truncated mutant of Cx43, it was clearly demonstrated that the regulation of RPE cell differentiation by Cx43 did not result from Cx43-mediated GJIC. The RPE cell differentiation induced by Cx43-overexpression was abolished by a cAMP antagonist. In contrast, the treatment with forskolin and phosphodiesterase inhibitor rolipram induced RPE cell differentiation under de-differentiation conditions. These findings indicate that Cx43 contributes to RPE differentiation via cAMP signaling.     

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.     

Control of Intracellular Localization and Function of Cx43 by SEMA3F

Connexin genes are considered to form a family of tumor-suppressor genes. However, the mechanism of connexin-mediated growth control is not well understood. We now provide several lines of evidence which suggest that SEMA3F, a member of the class 3 semaphorin family, which is also reported to be a tumor suppressor, controls the intracellular localization and function of connexin 43 (Cx43). We employed a series of rat liver epithelial cell lines, among which we previously found that the level of expression of malignant phenotypes (IAR20 < IAR27E < IAR6-1 < IAR27F) is inversely related to that of gap junctional intercellular communication (GJIC). When we immunostained SEMA3F and Cx43 in these cell lines, the extent of immunostaining in the plasma membrane of both proteins decreased in the order of IAR20 > IAR27E > IAR6-1 > IAR27F, suggesting a close relationship between Cx43 and SEMA3F. Further studies revealed a partial colocalization of SEMA3F and Cx43 in the plasma membrane of IAR20 cells. We also found that both SEMA3F and Cx43 moved from the cytoplasm to the plasma membrane in a mouse papilloma cell line when E-cadherin became functional after transferring the cells from low- to high-calcium conditions. When SEMA3F gene expression was inhibited by siRNA in IAR20 cells, Cx43 localization in the plasma membrane and GJIC ability were reduced. Moreover, we found that SEMA3F binds with the cytoplasmic loop domain of Cx43, employing the yeast two-hybrid complementation and screening assays. Taken together, these results strongly suggest that SEMA3F directly associates with Cx43 and controls its intracellular localization and function.     

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

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

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.     

A 50% reduction of excitability but not of intercellular coupling affects conduction velocity restitution and activation delay in the mouse heart

Introduction

Computer simulations suggest that intercellular coupling is more robust than membrane excitability with regard to changes in and safety of conduction. Clinical studies indicate that SCN5A (excitability) and/or Connexin43 (Cx43, intercellular coupling) expression in heart disease is reduced by approximately 50%. In this retrospective study we assessed the effect of reduced membrane excitability or intercellular coupling on conduction in mouse models of reduced excitability or intercellular coupling.

Methods and Results

Epicardial activation mapping of LV and RV was performed on Langendorff-perfused mouse hearts having the following: 1) Reduced excitability: Scn5a haploinsufficient mice; and 2) reduced intercellular coupling: Cx43^CreER(T)/fl mice, uninduced (50% Cx43) or induced (10% Cx43) with Tamoxifen. Wild type (WT) littermates were used as control. Conduction velocity (CV) restitution and activation delay were determined longitudinal and transversal to fiber direction during S₁S₁ pacing and S₁S₂ premature stimulation until the effective refractory period. In both animal models, CV restitution and activation delay in LV were not changed compared to WT. In contrast, CV restitution decreased and activation delay increased in RV during conduction longitudinal but not transverse to fiber direction in Scn5a heterozygous animals compared to WT. In contrast, a 50% reduction of intercellular coupling did not affect either CV restitution or activation delay. A decrease of 90% Cx43, however, resulted in decreased CV restitution and increased activation delay in RV, but not LV.

Conclusion

Reducing excitability but not intercellular coupling by 50% affects CV restitution and activation delay in RV, indicating a higher safety factor for intercellular coupling than excitability in RV.     

Pivotal role of cAMP in the activation of liver glycogen breakdown in high-fat diet fed mice

Aims

Liver glycogen catabolism was evaluated in male Swiss mice fed a high-fat diet rich in saturated fatty acids (HFD) or normal fat diet (NFD) during one week.

Main methods

Liver glycogenolysis (LG) and liver glucose production (LGP) were measured either under basal or stimulated conditions (infusion of glycogenolytic agents). Thus, isolated perfused livers from HFD and NFD mice were infused with glycogenolytic agents, i.e., glucagon, epinephrine, phenylephrine, isoproterenol, adenosine-3′-5′-cyclic monophosphate (cAMP), N⁶,2′-O-dibutyryl-cAMP (DB-cAMP), 8-bromoadenosine-cAMP (8-Br-cAMP) or N⁶-monobutyryl-cAMP (N6-MB-cAMP). Moreover, glycemia and liver glycogen content were measured.

Key findings

Glycemia, liver glycogen content and basal rate of LGP and LG were not influenced by the HFD. However, LGP and LG were lower (p < 0.05) in HFD mice during the infusions of glucagon (1 nM), epinephrine (20 μM) or phenylephrine (20 μM). In contrast, the activation of LGP and LG during the infusion of isoproterenol (20 μM) was not different (HFD vs. NFD). Because glucagon showed the most prominent response, the effect of cAMP, its intracellular mediator, on LGP and LG was investigated. cAMP (150 μM) showed lower activation of LGP and LG in the HFD group. However, the activation of LGP and LG was not influenced by HFD whether DB-cAMP (3 μM), 8-Br-cAMP (3 μM) or N6-MB-cAMP (3 μM) were used.

Significance

The activation of LGP and LG depends on the intracellular availability of cAMP. It can be concluded that cAMP played a pivotal role on the activation of LG in high-fat diet fed mice.