Microinjected anti-actin antibodies decrease gap junctional intercellular commmunication in cultured astrocytes

The purpose of the present study was to investigate the influence of the actin cytoskeleton on gap junctional intercellular communication (GJIC) in cultured astrocytes. This report describes an decrease of GJIC following microinjection of anti-actin antibodies or cytochalasin D treatment. Dye transfer of microinjected neurobiotin was used to assay gap junctional permeability in cultured astrocytes. Besides this translocation of connexins to the plasma membrane we investigated subsequent anti-actin antibody injection. While control cultures exhibited intensive dye spreading of microinjected neurobiotin, GJIC was impaired by microinjection of anti-actin antibodies. Additionally, impaired GJIC was observed after cytochalasin D treatment for 15 min. After the drug had been washed out, a recovery of GJIC was achieved. Cultured astrocytes exhibited a prominent actin cytoskeleton, with strong staining of actin filaments at the plasma membrane. Confocal laser scanning microscopy revealed an impaired translocation of Cx 43 from the Golgi apparatus to the cell membrane of cell processes following anti-actin antibody injection. These results suggest that the morphological integrity of microfilaments seems to be fundamental for GJIC, probably by means of associations among actin filaments, actin binding proteins, and Cx 43 at the plasma membrane or indirectly through the transport of connexins from the cytoplasm to the cell membrane.     

Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication

Gap junctional channelsfacilitate intercellular communication and in doing so maycontribute to cellular differentiation. To test this hypothesis, weexamined gap junction expression and function in atemperature-sensitive human fetal osteoblastic cell line (hFOB 1.19)that when cultured at 37°C proliferates rapidly but when culturedat 39.5°C proliferates slowly and displays increased alkalinephosphatase activity and osteocalcin synthesis. We found that hFOB 1.19 cells express abundant connexin 43 (Cx43) protein and mRNA. Incontrast, Cx45 mRNA was expressed to a lesser degree, and Cx26 and Cx32mRNA were not detected. Culturing hFOB 1.19 cells at 39.5°C,relative to 37°C, inhibited proliferation, increased Cx43 mRNA andprotein expression, and increased gap junctional intercellularcommunication (GJIC). Blocking GJIC with 18-glycyrrhetinic acid prevented the increase in alkaline phosphataseactivity resulting from culture at 39.5°C but did not affectosteocalcin levels. These results suggest that gap junction functionand expression parallel osteoblastic differentiation and contribute tothe expression of alkaline phosphatase activity, a marker for fullydifferentiated osteoblastic cells.

     

Patch-clamp study reveals that the importance of connexin43-mediated gap junctional communication for ovarian folliculogenesis is strain specific in the mouse

Genetic ablation of connexin37 (Cx37) or connexin43 (Cx43), the two gap junction proteins expressed by mouse ovarian granulosa cells, has been shown to result in impaired follicle development. We used patch-clamp techniques to evaluate quantitatively the contribution of these connexins to gap junctional intercellular communication (GJIC) among granulosa cells. The coupling conductance derived from a voltage step-induced capacitive current transient was used as a measure of GJIC in cultured granulosa cells. Using this method, we determined that the conductance of wild-type (84.1 ± 28.6 nS; n = 6) and Cx37-deficient granulosa cells (83.7 ± 6.4 nS; n = 11) does not differ significantly (P = 0.35), suggesting a limited contribution, if any, of Cx37 to granulosa cell coupling. In contrast, the conductance between granulosa cells of Cx43-deficient mice (2.6 ± 0.8 nS; n = 5) was not significantly different from that of single, isolated wild-type granulosa cells (2.5 ± 0.7 nS, n = 5; P = 0.83), indicating that Cx43-deficient granulosa cells were not electrically coupled. A direct measurement of transjunctional conductance between isolated granulosa cell pairs using a dual patch-clamp technique confirmed this conclusion. Interestingly, a partial rescue of folliculogenesis was observed when the Cx43-null mutation in C57BL/6 mice was crossed into the CD1 strain, and capacitive current measurement demonstrated that this rescue was not due to reestablishment of GJIC. These results demonstrate that folliculogenesis is impaired in the absence of GJIC between granulosa cells, but they also indicate that the severity is dependent on genetic background, a phenomenon that cannot be attributed to the expression of additional connexins. ovarian follicle; oogenesis; connexin37; intercellular communication     

Caveolin-1 and -2 interact with connexin43 and regulate gap junctional intercellular communication in keratinocytes

Connexin43 (Cx43) has been reported to interact with caveolin (Cav)-1, but the role of this association and whether other members of the caveolin family bind Cx43 had yet to be established. In this study, we show that Cx43 coimmunoprecipitates and colocalizes with Cav-1 and Cav-2 in rat epidermal keratinocytes. The colocalization of Cx43 with Cav-1 was confirmed in keratinocytes from human epidermis in vivo. Our mutation and Far Western analyses revealed that the C-terminal tail of Cx43 is required for its association with Cavs and that the Cx43/Cav-1 interaction is direct. Our results indicate that newly synthesized Cx43 interacts with Cavs in the Golgi apparatus and that the Cx43/Cavs complex also exists at the plasma membrane in lipid rafts. Using overexpression and small interfering RNA approaches, we demonstrated that caveolins regulate gap junctional intercellular communication (GJIC) and that the presence of Cx43 in lipid raft domains may contribute to the mechanism modulating GJIC. Our results suggest that the Cx43/Cavs association occurs during exocytic transport, and they clearly indicate that caveolin regulates GJIC.     

The carboxy‐terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C‐tail) to demonstrate that the C‐tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C‐tail in mediating Cx43‐dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1–242) and a construct encompassing only the C‐tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C‐tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C‐tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time‐lapse imaging to examine the behavior of migrating cells and it was apparent that the C‐tail was associated with a lamellipodia‐based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C‐tail is sufficient to induce Cx43‐dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton. J. Cell. Biochem. 110: 589–597, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Modulation of connexin43 alters expression of osteoblastic differentiation markers

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43^–). hFOB/Cx43^– cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43^– cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43^– cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor 1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43^–. Osteopontin mRNA levels were increased in hFOB/Cx43^– relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43^– and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19     

An analysis of Ca2+ release by DGEA: mobilization of two functionally distinct internal stores in Saos-2 cells

Osteoblasts can be activated by their collagen matrix and inparticular the DGEA peptide motif. We have reported that DGEA is ableto activate Ca²⁺ signalingpathways in the human osteoblast-like cell line, Saos-2, by a tyrosinekinase-dependent pathway (T. J. McCann, W. T. Mason, M. C. Meikle, andF. McDonald. Matrix Biol. 16:271-280, 1997). In the present study, we show that this activityis due to coupling of the signal to intracellularCa²⁺ stores, since the DGEA actionis not blocked by La³⁺ but is lostwhen Ca²⁺ stores are depleted with2 µM and blocked by 10 µM ryanodine. The activated stores alsodiffer functionally from those activated by thrombin, as blockade withU-73122 obstructs only thrombin-activated Ca²⁺ release. We have shown thatthe DGEA activity was not due to its high-charge density, since the twoacidic residues can be substituted with their uncharged homologues(asparagine and glutamine) without significant loss of activity. Thiswas in turn measured by an adhesion assay that also demonstrated thislevel of specificity. Furthermore, by constructing DGEA bound to FITC,we have shown that DGEA binding was dependent on divalent cations. Wehave also demonstrated that an intact actin cytoskeleton is notrequired for Ca²⁺ activation byinhibiting actin polymerization with the addition of cytochalasin B. These data strengthen the argument that collagen has a significant rolein regulating osteoblast function via this peptide motif.

     

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.     

RhoA GTPase and F-actin dynamically regulate the permeability of Cx43-made channels in rat cardiac myocytes

Gap junctions are clusters of transmembrane channels allowing a passive diffusion of ions and small molecules between adjacent cells. Connexin43, the main channel-forming protein expressed in ventricular myocytes, can associate with zonula occludens-1, a scaffolding protein linked to the actin cytoskeleton and to signal transduction molecules. The possible influence of Rho GTPases, major regulators of cellular junctions and of the actin cytoskeleton, in the modulation of gap junctional intercellular communication (GJIC) was examined. The activation of RhoA by cytoxic necrotizing factor 1 markedly enhanced GJIC, whereas its specific inhibition by the Clostridium botulinum C3 exoenzyme significantly reduced it. RhoA activity affects GJIC without major cellular redistribution of junctional plaques or changes in the Cx43 phosphorylation pattern. As these GTPases frequently act via the cortical cytoskeleton, the importance of F-actin in the modulation of GJIC was investigated by means of agents interfering with actin polymerization. Cytoskeleton stabilization by phalloidin slowed down the kinetics of channel rundown in the absence of ATP, whereas its disruption by cytochalasin D rapidly and markedly reduced GJIC despite ATP presence. Cytoskeleton stabilization by phalloidin markedly reduced the consequences of RhoA activation or inactivation. This mechanism appears to be the first described capable to both up- or down-regulate GJIC through RhoA activation or, conversely, inhibition. The inhibition of Rho downstream kinase effectors had no effect on GJIC. The present results provide further insight into the gating and regulation of junctional channels and identify a new downstream target for the small G-protein RhoA.     

The Response of Bean Plants to UV-B Radiation Under Different Irradiances of Background Visible Light

Plants of Phaseolus vulgaris L. (cv. Stella) were grown in controlledconditions under three different irradiances of visible lightwith or without UV-B (280–320nm) radiation. The biologicallyeffective UV-B radiation (UV-B_BE) was 6.17 kJ m^–2 d^–1,and simulated a c. 5% decrease in stratospheric ozone at 55.7?N,13.4?E. The photon flux densities of the photosyntheticallyactive radiation (PAR, 400–700 nm) were either 700 µmolm^–2–1 (HL), 500, µmol m^–2 s^–1(ML) or 230 µmol m^–2 s^–1 PAR (LL). Under highlight (HL) conditions plus UV-B radiation, bean plants appearedmost resistant to the enhanced levels of UV-B radiation, andresponded only by increasing leaf thickness by c. 18%. A smallincrease in UV screening pigments was also observed. Both thelower irradiances (ML and LL) increased the sensitivity of theplants to UV-B radiation. Changes in leaf structure were alsoobserved. Photosystem II was inhibited under ML and LL togetherwith UV-B radiation, as determined by Chi fluorescence inductionand calculation of the fluorescence half-rise times. Leaf reflectivitymeasurements showed that the amount of PAR able to penetrateleaves of UV-B treated plants was reduced, and that a possiblecorrelation may exist between the reduced PAR levels, loss ofChi and lowered photosynthetic activity, especially for LL +UV-Bgrown plants, where surface reflection from leaves was highest.Changes in leaf chlorophyll content were mostly confined toplants grown under LL + UV-B, where a decrease of c. 20% wasfound. With regard to protective pigments (the carotenoids andUV screening pigments) plants subjected to different visiblelight conditions responded differently. Among the growth parametersmeasured, there was a substantial decrease in leaf area, particularlyunder LL + UV-B (c. 47% relative to controls), where leaf dryweight was also reduced by c. 25%. Key words: Chlorophyll fluorescence induction, bean, flavonoids, Phaseolus vulgaris, reflectance, UV-B radiation     

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells

ANa⁺ current is present in human jejunal circular smoothmuscle cells. The aim of the present study was to determine the role ofthe cytoskeleton in the regulation of the Na⁺ current.Whole cell currents were recorded by using standard patch-clamptechniques with Cs⁺ in the pipette to block K⁺currents. Cytochalasin D and gelsolin were used to disrupt the actincytoskeleton and phalloidin to stabilize it. Colchicine was used todisassemble the microtubule cytoskeleton (and intermediate filaments)and paclitaxel to stabilize it. Acrylamide was used to disrupt theintermediate filament cytoskeleton. Perfusion of the recording chamberat 10 ml/min increased peak Na⁺ current recorded fromjejunal smooth muscle cells by 27 ± 3%. Cytochalasin D andgelsolin abolished the perfusion-induced increase in Na⁺current, whereas incubation with phalloidin, colchicine, paclitaxel, oracrylamide had no effect. In conclusion, the Na⁺ currentexpressed in human jejunal circular smooth muscle cells appears to beregulated by the cytoskeleton. An intact actin cytoskeleton is requiredfor perfusion-induced activation of the Na⁺ current.

     

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.     

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED₅₀ of 15, 31, and 28 nM, respectively, with maximal response at 10^–7 M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC₅₀ for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis. human; cell activation     

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.     

Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells

We tested the hypothesis that fluidshear stress () modifies the expression, function, and distributionof junctional proteins [connexin (Cx)43, Cx45, and zona occludens(ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to-values of 5 or 20 dyn/cm² for 1-3 h.Immunostaining indicated that at 5 dyn/cm², thedistribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cellmembranes; at 20 dyn/cm², disruption was more severe.Intercellular coupling was significantly decreased at both shear stresslevels. Western blots showed the downregulation of membrane-bound Cx43and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at differentlevels of shear stress. Similarly, Northern blots revealed thatexpression of Cx43, Cx45, and ZO-1 was selectively up- anddownregulated in response to different shear stress levels. Theseresults indicate that in cultured bone cells, fluid shear stressdisrupts junctional communication, rearranges junctional proteins, anddetermines de novo synthesis of specific connexins to an extent thatdepends on the magnitude of the shear stress. Such disconnection fromthe bone cell network may provide part of the signal whereby thedisconnected cells or the remaining network initiate focal bone remodeling.

     

High glucose down-regulates intercellular communication in retinal endothelial cells by enhancing degradation of connexin 43 by a proteasome-dependent mechanism

Intercellular communication through gap junctions (GJIC) is most likely relevant to maintaining the integrity of the blood-retinal barrier. In this study, we investigated the mechanism whereby high glucose enhances degradation of connexin 43 (Cx43), thus contributing to a decrease in GJIC. The levels of Cx43 in bovine retinal endothelial cells exposed to high glucose (25 mm) decreased about 50% as compared with controls (5.5 mm glucose). Consistently, the half-life of the protein decreased from 2.3 to 1.9 h. The proteasome inhibitors MG132 and lactacystin prevented the loss of Cx43 induced by high glucose and extended Cx43 half-life. The amount of phosphorylated Cx43 increased in high glucose and after proteasome inhibition. Scrape-loading dye transfer experiments show that high glucose is associated to a decrease of 40% in GJIC. Significantly, this reduction can be reversed by proteasome inhibitors. The decrease in GJIC in cells exposed to high glucose is associated with a loss of Cx43 from the plasma membrane, as demonstrated by immunofluorescence and biotinylation of cell-surface proteins. Results indicate that increased phosphorylation of Cx43 under high glucose is the mechanism targeting Cx43 for degradation by a proteasome-dependent mechanism. Increased degradation of Cx43 and reduction of GJIC in high glucose may be of physiological importance by contributing to endothelial cell dysfunction associated with the breakdown of the blood-retinal barrier in diabetic retinopathy.     

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).