Changes in cytosolic CA2+ are not involved in DDT-induced loss of gap junctional communication in WB-F344 cells

Recent studies have demonstrated that the insecticide DDT is a tumor promoting agent. Similar to many other tumor promoting agents, DDT has been shown to inhibit gap junctional intercellular communication (GJIC) between cells in culture, and it has been suggested that DDT-induced loss of communication between adjacent cells may depend on changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). In the present study, the role of[Ca²⁺]_i in DDT-induced loss of GJIC was investigated in WB-F344 rat liver cells using the scrape-loading/dye transfer assay (SLDT) and the Ca²⁺ fourescent indicator, furà-2. Our results show that DDT at non-cytotoxic concentrations caused a reversible loss of GJIC. Inhibition of GJIC was not associated with detectable increases in [Ca²⁺]_i, and was not prevented by loading cells with the intracellular Ca²⁺ chelator, BAPTA. In addition, the hydroquinone, tBuBHQ, which caused a 2+3 fold sustained increase in [Ca²⁺]_i, did not inhibit GJIC. Conversely, when untreated cells were loaded with increasing BAPTA concentrations, GJIC were lost. These results indicate that increases in [Ca²⁺]_i are not responsible for DDT-induced loss of communication and that, in general an increase in [Ca²⁺]_i, within physiological levels is not sufficient to abolish GJIC. However, Ca²⁺-dependent processes that are active at normal resting [Ca²⁺ _i appear to be required for the maintenance of GJIC.Abbreviations [Ca²⁺] cytosolic free Ca²⁺ concentration - GJIC gap junctional intercellular communication - SLDT scrape-loading/dye transfer assay - DDT 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane - tBuBHQ 2,5-di(tert-butyl)-1,4-benzohydroquinone - LDH lactate dehydrogenase - ER endoplasmic reticulum - Fura-2 1-[2-(5carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid - BAPTA bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraaceticacid - Fura-2/AM and BAPTA/AM are the cell permeant acetoxymethyl ester forms of fura-2 and BAPTA, respectively     

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

Background

We investigated the effects of the signaling molecules, cyclic AMP (cAMP) and protein-kinase C (PKC), on gap junctional intercellular communication (GJIC) between thymic epithelial cells (TEC).

Results

Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC.

Conclusions

Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.     

Phosphatidylcholine Specific PLC-Induced Dysregulation of Gap Junctions,a Robust Cellular Response to Environmental Toxicants,and Prevention by Resveratrol in a Rat Liver Cell Model

Dysregulation of gap junctional intercellular communication (GJIC) has been associated with different pathologies, including cancer; however, molecular mechanisms regulating GJIC are not fully understood. Mitogen Activated Protein Kinase (MAPK)-dependent mechanisms of GJIC-dysregulation have been well-established, however recent discoveries have implicated phosphatidylcholine-specific phospholipase C (PC-PLC) in the regulation of GJIC. What is not known is how prevalent these two signaling mechanisms are in toxicant/toxin-induced dysregulation of GJIC, and do toxicants/toxins work through either signaling mechanisms or both, or through alternative signaling mechanisms. Different chemical toxicants were used to assess whether they dysregulate GJIC via MEK or PC-PLC, or both Mek and PC-PLC, or through other signaling pathways, using a pluripotent rat liver epithelial oval-cell line, WB-F344. Epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, thrombin receptor activating peptide-6 and lindane regulated GJIC through a MEK1/2-dependent mechanism that was independent of PC-PLC; whereas PAHs, DDT, PCB 153, dicumylperoxide and perfluorodecanoic acid inhibited GJIC through PC-PLC independent of Mek. Dysregulation of GJIC by perfluorooctanoic acid and {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 required both MEK1/2 and PC-PLC; while benzoylperoxide, arachidonic acid, 18β-glycyrrhetinic acid, perfluorooctane sulfonic acid, 1-monolaurin, pentachlorophenol and alachlor required neither MEK1/2 nor PC-PLC. Resveratrol prevented dysregulation of GJIC by toxicants that acted either through MEK1/2 or PC-PLC. Except for alachlor, resveratrol did not prevent dysregulation of GJIC by toxicants that worked through PC-PLC-independent and MEK1/2-independent pathways, which indicated at least two other, yet unidentified, pathways that are involved in the regulation of GJIC. In conclusion: the dysregulation of GJIC is a contributing factor to the cancer process; however the underlying mechanisms by which gap junction channels are closed by toxicants vary. Thus, accurate assessments of risk posed by toxic agents, and the role of dietary phytochemicals play in preventing or reversing the effects of these agents must take into account the specific mechanisms involved in the cancer process.     

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.     

Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SLIDT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37° C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SLIDT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC. Another assay (e.g., electrical coupling, microinjection, metabolic cooperation, radioactive metabolite transfer, or fluorescence redistribution after photobleaching) should be considered to quantify changes in GJIC and construct chemical concentration-response curves.Abbreviations FBS, fetal bovine serum - GJIC, gap junctional intercellular communication - HBSS, Hank's balanced saline solution - SL/DT, scrape-loading/dye transfer - TPA, 12-O-tetradecanoylphorbol-13-acetate.     

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.     

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.     

CTGF facilitates cell‐cell communication in chondrocytes via PI3K/Akt signalling pathway

PurposesGap junction intercellular communication (GJIC) is essential for articular cartilage to respond appropriately to physical or biological stimuli and maintain homeostasis. Connective tissue growth factor (CTGF), identified as an endochondral ossification genetic factor, plays a vital role in cell proliferation, migration and adhesion. However, how CTGF regulates GJIC in chondrocytes is still unknown. This study aims to explore the effects of CTGF on GJIC in chondrocytes and its potential biomechanism.Materials and methodsqPCR was performed to determine the expression of gene profile in the CCN family in chondrocytes. After CTGF treatment, CCK‐8 assay and scratch assay were performed to explore cell proliferation and migration. A scrape loading/dye transfer assay was adopted to visualize GJIC in living chondrocytes. Western blot analysis was done to detect the expression of Cx43 and PI3K/Akt signalling. Immunofluorescence staining was used to show protein distribution. siRNA targeting CTGF was used to detect the influence on cell‐cell communication.ResultsThe CTGF (CCN2) was shown to be the highest expressed member of the CCN family in chondrocytes. CTGF facilitated functional gap junction intercellular communication in chondrocytes through up‐regulation of Cx43 expressions. CTGF activated PI3K/Akt signalling to promote Akt phosphorylation and translocation. Suppressing CTGF also reduced the expression of Cx43. The inhibition of PI3K/Akt signalling decreased the expressions of Cx43 and thus impaired gap junction intercellular communication enhanced by CTGF.ConclusionsFor the first time, we provide evidence to show CTGF facilitates cell communication in chondrocytes via PI3K/Akt signalling pathway.     

GJIC Enhances the phototoxicity of photofrin‐mediated photodynamic treatment by the mechanisms related with ROS and Calcium pathways

Despite initially positive responses, recurrences after Photodynamic treatment (PDT) can occur and there is need for improvement in the effectiveness of PDT. Our study uniquely showed that there was a significantly gap junctional intercellular communication (GJIC)‐dependent PDT cytotoxicity. The presence of GJIC composed of Connexin 32 increased the PDT phototoxicity in transfected HeLa cells and in the xenograft tumors, and the enhanced phototoxicity of Photofrin‐mediated PDT by GJIC was related with ROS and calcium pathways. Our study indicates the possibility that up‐regulation or maintenance of gap junction functionality may be used to increase the efficacy of PDT.

The phototoxicity effect of Photofrin was substantially greater in Dox‐treated cells, which expressed the Cx32 and formed the GJ, than Dox‐untreated.     

Loss of gap junctional intercellular communication in rat lung epithelial cells exposed to quartz particles

Chronic inhalation of quartz particles has been implicated in lung diseases including silicosis and cancer. The aim of this study was to investigate whether quartz particles affect gap junctional intercellular communication (GJIC) in rat lung epithelial cells (RLE-6TN). Here, we demonstrate that exposure of RLE-6TN cells to subtoxic doses of DQ12 standard quartz resulted in an up to 55% reduction of GJIC, as determined in a dye transfer assay. We show that connexin-43 (Cx43) is the major connexin responsible for intercellular communication in these lung epithelial cells and that exposure to quartz particles induces a significant internalization of Cx43. Downregulation of GJIC was attenuated by N-acetyl cysteine, suggesting the involvement of reactive oxygen species and/or cellular thiol homeostasis in the regulation of GJIC. Furthermore, an inhibitor of activation of extracellular signal-regulated kinases prevented the loss of GJIC in cells exposed to DQ12 quartz, although no direct phosphorylation of Cx43 upon exposure to DQ12 was detected.     

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.     

Effects of pyrethroid insecticides on gap junctional intercellular communications in Balb/c3T3 cells by dye-transfer assay

The effects of fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin, p-chlorophenylisovaleric acid (CPIA, major metabolite of fenvalerate) and DDT, a liver tumor promoter, on gap junctional intercellular communication (GJIC) were examined in Balb/c3T3 cells by dye-transfer assay. Separate groups of Balb/c3T3 cells were exposed to the chemicals for 1 day. On the following day, GJIC was measured by counting the number of dye-transferring cells per injection of Lucifer Yellow under a fluorescent microscope. Fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin and DDT inhibited GJIC at noncytotoxic concentrations, while CPIA did not inhibit GJIC even, at a cytotoxic concentration. It is concluded that the examined pyrethroid insecticides, but not a metabolite, have inhibitory effects on GJIC in Balb/c3T3 cells.Abbreviations DDT 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane - DMSO dimethyl sulfoxide     

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.

     

STUDY ON GAP JUNCTIONAL INTERCELLULAR COMMUNICATION INHIBITION BY ELF MAGNETIC FIELDS USING FRAP METHOD

Inhibition of gap junctional intercellular communication (GJIC) is an important event in the multistage process of carcinogenesis. Our previous study showed that extremely low frequency (ELF) magnetic fields (MFs) inhibit GJIC, and enhance the suppression of GJIC induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) using a microinjection technique. In the present study, the inhibition of GJIC by ELF MFs and its threshold were further studied with fluorescence recovery after photobleaching (FRAP) technique. The results indicated that the FRAP technique is more sensitive in detecting the changes of GJIC than microinjection, and the threshold level is 0.4 mT for GJIC suppression by 50 Hz MFs. In addition, 0.2 mT, or more than 0.2 mT ELF can enhance the inhibition of GJIC induced by TPA. We concluded that MFs thus might act as a cancer promoter or work in synergy with other cancer promoters. The data also provide grounds to revise the reference standard of ELF MFs exposure.     

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.     

Gap junctions and fluid flow response in MC3T3-E1 cells

In thecurrent study, we examined the role of gap junctions in oscillatoryfluid flow-induced changes in intracellular Ca²⁺concentration and prostaglandin release in osteoblastic cells. Thiswork was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Ourresults demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E₂ (PGE₂) release, whereas cellswith diminished gap junctional communication do not. Furthermore, wefound that cytosolic Ca²⁺ (Ca) responsewas unaltered by the disruption in gap junctional communication and wasnot significantly different among the cell lines. Thus our resultssuggest that gap junctions contribute to the PGE₂ but notto the Ca response to oscillatory fluid flow. Thesefindings implicate gap junctional intercellular communication (GJIC) inbone cell ensemble responsiveness to oscillatory fluid flow and suggestthat gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

     

Lipopolysaccharide-induced inhibition of connexin43 gap junction communication in astrocytes is mediated by downregulation of caveolin-3

Astrocytes play a crucial role in maintaining the homeostasis of the brain. Changes to gap junctional intercellular communication (GJIC) in astrocytes and excessive inflammation may trigger brain damage and neurodegenerative diseases. In this study, we investigated the effect of lipopolysaccharide (LPS) on connexin43 (Cx43) gap junctions in rat primary astrocytes. Following LPS treatment, dose- and time-dependent inhibition of Cx43 expression was seen. Moreover, LPS induced a reduction in Cx43 immunoreactivity at cell–cell contacts and significantly inhibited GJIC, as revealed by the fluorescent dye scrape loading assay. Toll-like receptor 4 (TLR4) protein expression was increased 2–3-fold following LPS treatment. To study the pathways underlying these LPS-induced effects, we examined downstream effectors of TLR4 signaling and found that LPS induced a significant increase in phosphorylated extracellular signal-regulated kinase (pERK) levels up to 6 h, followed by signal attenuation and downregulation of caveolin-3 expression. Interestingly, LPS treatment also induced a dramatic increase in inducible nitric oxide synthase (iNOS) levels at 6 h, which were sustained up to 18–24 h. The LPS-induced downregulation of Cx43 and caveolin-3 was prevented by co-treatment of astrocytes with the iNOS cofactor inhibitor 1400W, but not the ERK inhibitor PD98059. Specific knockdown of caveolin-3 using siRNA had a significant inhibitory effect on GJIC and resulted in a downregulation of Cx43. Our results suggest that long-term LPS treatment of astrocytes leads to inhibition of Cx43 gap junction communication by the activation of iNOS and downregulation of caveolin-3 via a TLR4-mediated signaling pathway.     

Deletion of a single allele of Cx43 is associated with a reduction in the gap junctional intercellular communication and increased cell proliferation of mouse lung pneumocytes type II

OBJECTIVES: Connexins (Cx) are proteins that form the gap junctional channels at neighbouring plasma membranes between adjacent cells. Cxs are involved in cell communication, which is reportedly correlated with cell proliferation and differentiation. Alterations in connexin expression and/or gap junctional intercellular communication (GJIC) capacity have long been postulated to be important in a number of pathological conditions including cancer. This study was performed to determine the consequences of the deletion of a single allele of Gja1 (Cx43 gene) in Alveolar Type II cells (APTIIs), and its impact on GJIC and cell proliferation. MATERIAL AND METHODS: In order to do so, APTIIs from wild type (Cx43(+/+)) and heterozygous (Cx43(+/-)) mice were harvested and cultured for 4 days. The GJIC capacity was evaluated by scrape-loading method, with the transfer of lucifer yellow dye. The expression of Cx43 was evaluated by immunofluorescence method and Western blotting. Cell proliferation was evaluated by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: It was observed that GJIC capacity was significantly reduced and cell proliferation index was significantly higher in Cx43(+/-) cells compared to Cx43(+/+) cells. CONCLUSIONS: These results show that knocking out one allele of Cx43 leads to a lower cell to cell communication capacity, and consequently induces a higher cell proliferation. Because chemically induced lung adenomas in mice are known to originate from APTIIs, these alterations may play a critical role in their susceptibility to lung carcinogenesis.     

Quantitative Determination of Gap Junction Intercellular Communication by Scrape Loading and Image Analysis

Gap junction intercellular communication (GJIC) consists of intercellular exchange of low molecular weight molecules. Chemically induced alterations of this communication have been suggested to result in abnormal cell growth and tumour promotion. Several in vitro assays have been developed to determine the effect of chemicals on gap junction communication in cultured cells. The scrape loading dye transfer technique is based on studying the transfer of the fluorescent dye Lucifer Yellow in cells where the dye is loaded through a cut in the cell monolayer. This technique is rapid and relatively uncomplicated, but has only been used to qualitatively demonstrate communication, due to lack of an appropriate method for quantification of the dye spreading. We show here that analysis of digital fluorescence images of cells scrape loaded with Lucifer Yellow can be used for quantitative determination of GJIC. We have analysed the images both by means of distance of diffusion of the dye in the cell monolayer, as well as by area of dye-coupled cells. The results are consistent with that obtained using microinjection of Lucifer Yellow and the method offers a simple way for quantitative determination of GJIC.