Connexin Degradation Research Articles

Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis

A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMV's role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

In vivo and in vitro cardiac responses to beta-adrenergic stimulation in volume-overload heart failure

Hearts in volume overload (VO) undergo progressive ventricular hypertrophy resulting in chronic heart failure that is unresponsive to β-adrenergic agonists. This study compared left ventricular (LV) and isolated cardiomyocyte contractility and β-adrenergic responsiveness in rats with end-stage VO heart failure (HF). Adult male Sprague-Dawley rats were studied 21weeks after aortocaval fistula (ACF) or sham surgery. Echocardiography revealed decreased fractional shortening accompanied by increased LV chamber diameter and decreased eccentric dilatation index at end-stage ACF compared to sham. Hemodynamic measurements showed a decrease in the slope of end-systolic pressure–volume relationship, indicating systolic dysfunction. Isolated LV myocytes from ACF exhibited decreased peak sarcomere shortening and kinetics. Both Ca2+ transient amplitude and kinetics were increased in ACF myocytes, with no change under the integrated Ca2+ curves relating to contraction and relaxation phases. Increases in ryanodine receptor and phospholamban phosphorylation, along with a decrease in SERCA2 levels, were observed in ACF. These changes were associated with decreased expression of β-myosin heavy chain, cardiac troponin I and cardiac myosin binding protein-C. In vivo inotropic responses to β-adrenergic stimulation were attenuated in ACF. Interestingly, ACF myocytes exhibited a similar peak shortening to those of sham in response to a β-adrenergic agonist. The protein expression of the gap junction protein connexin-43 was decreased, although its phosphorylation at Ser-368 increased. These changes were associated with alterations in Src and ZO-1. In summary, these data suggest that the disconnect in β-adrenergic responsiveness between in vivo and in vitro conditions may be associated with altered myofilament Ca2+ sensitivity and connexin-43 degradation.

Degradation of Connexins Through the Proteasomal, Endolysosomal and Phagolysosomal Pathways

Connexins comprise gap junction channels, which create a direct conduit between the cytoplasms of adjacent cells and provide for intercellular communication. Therefore, the level of total cellular connexin protein can have a direct influence on the level of intercellular communication. Control of connexin protein levels can occur through different mechanisms during the connexin life cycle, such as by regulation of connexin gene expression and turnover of existing protein. The degradation of connexins has been extensively studied, revealing proteasomal, endolysosomal and more recently autophagosomal degradation mechanisms that modulate connexin turnover and, subsequently, affect intercellular communication. Here, we review the current knowledge of connexin degradation pathways.

Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation

The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging.

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.

Smad ubiquitination regulatory factor-2 controls gap junction intercellular communication by modulating endocytosis and degradation of connexin43

Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junction channels are made of a family of integral membrane proteins called connexins, of which the best-studied member is connexin43. Gap junctions are dynamic plasma membrane domains, and connexin43 has a high turnover rate in most tissue types. However, the mechanisms involved in the regulation of connexin43 endocytosis and transport to lysosomes are still poorly understood. Here, we demonstrate by live-cell imaging analysis that treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) induces endocytosis of subdomains of connexin43 gap junctions. The internalized, connexin43-enriched vesicles were found to fuse with early endosomes, which was followed by transport of connexin43 to the lumen of early endosomes. The HECT E3 ubiquitin ligase smad ubiquitination regulatory factor-2 (Smurf2) was found to be recruited to connexin43 gap junctions in response to TPA treatment. Depletion of Smurf2 by small interfering RNA resulted in enhanced levels of connexin43 gap junctions between adjacent cells and increased gap junction intercellular communication. Smurf2 depletion also counteracted the TPA-induced endocytosis and degradation of connexin43. Collectively, these data identify Smurf2 as a novel regulator of connexin43 gap junctions.

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Gαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.

Inhibition of connexin43 gap junction channels by the endocrine disruptor ioxynil

Gap junctions are intercellular plasma membrane domains containing channels that mediate transport of ions, metabolites and small signaling molecules between adjacent cells. Gap junctions play important roles in a variety of cellular processes, including regulation of cell growth and differentiation, maintenance of tissue homeostasis and embryogenesis. The constituents of gap junction channels are a family of trans-membrane proteins called connexins, of which the best-studied is connexin43. Connexin43 functions as a tumor suppressor protein in various tissue types and is frequently dysregulated in human cancers. The pesticide ioxynil has previously been shown to act as an endocrine disrupting chemical and has multiple effects on the thyroid axis. Furthermore, both ioxynil and its derivative ioxynil octanoate have been reported to induce tumors in animal bioassays. However, the molecular mechanisms underlying the possible tumorigenic effects of these compounds are unknown. In the present study we show that ioxynil and ioxynil octanoate are strong inhibitors of connexin43 gap junction channels. Both compounds induced rapid loss of connexin43 gap junctions at the plasma membrane and increased connexin43 degradation. Ioxynil octanoate, but not ioxynil, was found to be a strong activator of ERK1/2. The compounds also had different effects on the phosphorylation status of connexin43. Taken together, the data show that ioxynil and ioxynil octanoate are potent inhibitors of intercellular communication via gap junctions.

Abstract 4448: Disruption of nedd4 and connexin 46 interaction by gap junction activator in breast cancer cells

Abstract Breast cancer is the second most common cancer among women; it is also the second leading cause of cancer death among women in the United States. Progress in understanding the etiology of this disease and developing therapies has been slow due to multiple deregulations of various genes. Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). Most normal cells have functional gap junctional intercellular communication (GJIC), while most, if not all, tumors cells have dysfunctional GJIC. GJIC consists of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, cell signaling and previous studies have been suggested their participation in tumor formation and progression. It is believed that restoring GJIC is linked to drug sensitivity and reduction of tumorigenicity. Thus, increasing gap junction activity or enhancing GJIC in tumor cells provides the targets to enhance anti-neoplastic therapies. Several GJIC enhancers have been reported; however, an effective clinical drug targeting gap junction is not available at this time. Recently, we synthesized a new class of substituted quinolines (code name: PQ) and found that they possess potent inhibitory activities against T47D breast cancer cells (IC50 value of PQ1 is 119 nM) through the enhancement of GJIC. Since the mechanism of action of GJIC enhancement caused by PQ is unknown, the current study is to determine the target proteins that PQ interacts with, which may lead to the mechanism of enhancement of GJIC and inhibition of cancer cell growth. Using surface plasmon resonance (SPR) spectroscopy, we have found that PQ1 has a strong binding with Nedd4, E3 ubiquitin ligase which previously been shown to involve in the degradation of gap junction. T47D breast cancer cells were treated with various concentrations of PQ1 for 24 hours. Whole cell extract was isolated and subjected to immunoprecipitation assay, followed by western blot analysis. The results show that 100 nM PQ1 can cause a 2-fold decrease in the interaction of Nedd4 and connexin 46 compared to control without treatment or solvent. Since gap junction turnover has been shown to be facilitated by lysosomal and proteasomal degradation, disruption of Nedd4 and connexin 46 suggests that PQ1 may inhibit the lysosomal and proteasomal degradation of connexin 46 in breast cancer cells. Future study will focus on whether PQ causes an increase in gap junction activity via inhibition of Nedd4-ubiquination of connexin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4448.

Cardiac Gap Junction Channels Are Upregulated by Metoprolol: An Unexpected Effect of Beta-Blockers

Background/Aims: Since β-adrenoceptors have been shown to affect cardiac gap junction channels, we wanted to elucidate the possible effect of metoprolol on the gap junction protein connexin-43, using racemic RS-metoprolol or the isomer R-metoprolol (no β-adrenoceptor blockade) or S-metoprolol (β₁-adrenoceptor blocker). Methods: Cultured neonatal rat cardiomyocytes were exposed to either RS-metoprolol or R-metoprolol or S-metoprolol (0.1 µmol/l each) without or with additional isoprenaline (0.1 µmol/l) treatment for 24 h. Results: The β-blocker treatment did not alter the frequency of spontaneously beating cardiomyocytes, whereas sole isoprenaline administration significantly enhanced the beating frequency by about 40%. This rise could be blocked by concomitant treatment with S- or RS-metoprolol but not with R-metoprolol. Connexin-43 protein was significantly enhanced by isoprenaline and by R-, S- or RS-metoprolol treatment alone as well as with the combined administration of isoprenaline and R-, S- or RS-metoprolol. Phospho-ERK1 and connexin-43 mRNA were significantly increased by isoprenaline application alone, whereas R-, S- or RS-metoprolol alone or in combination with isoprenaline exhibited no effect. Conclusion: Both isomers of metoprolol upregulate connexin-43 in cultured cardiomyocytes by a β-adrenoceptor-independent mechanism. Since the enhanced presence of connexin-43 in cell membranes under metoprolol was not accompanied by enhanced connexin-43 mRNA, we assume that the metoprolol effect involves reduced connexin-43 degradation.

Connexin43 phosphorylation: structural changes and biological effects

Vertebrate gap junctions, composed of proteins from the connexin gene family, play critical roles in embryonic development, co-ordinated contraction of excitable cells, tissue homoeostasis, normal cell growth and differentiation. Phosphorylation of connexin43, the most abundant and ubiquitously expressed connexin, has been implicated in the regulation of gap junctional communication at several stages of the connexin 'life cycle', including hemichannel oligomerization, export of the protein to the plasma membrane, hemichannel activity, gap junction assembly, gap junction channel gating and connexin degradation. Consistent with a short (1-5 h) protein half-life, connexin43 phosphorylation is dynamic and changes in response to activation of many different kinases. The present review assesses our current understanding of the effects of phosphorylation on connexin43 structure and function that in turn regulate gap junction biology, with an emphasis on events occurring in heart and skin.

The 2,2′,4,4′,5,5′-Hexachlorobiphenyl–Enhanced Degradation of Connexin 43 Involves Both Proteasomal and Lysosomal Activities

One of the toxic effects of non-dioxin-like polychlorinated biphenyls (NDL-PCBs) is the acute inhibition of gap junctional intercellular communication (GJIC), an event possibly associated with tumor promotion. The model NDL-PCB-2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-induces a sustained GJIC inhibition in rat liver epithelial WB-F344 cells. As this effect might be related to deregulation of connexin 43 (Cx43) synthesis, trafficking, or degradation, we investigated the impact of PCB 153 on these events. Although PCB 153 had no effect on Cx43 mRNA levels, it induced a gradual loss of Cx43 protein and significantly decreased the amount of gap junction plaques in plasma membrane. PCB 153 contributed to extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent accumulation of hyperphosphorylated Cx43-P3 form, thus indicating that ERK1/2 activation by PCB 153 might contribute to its effects on Cx43 internalization or degradation. Inhibition of either proteasomes or lysosomes with their specific inhibitors largely restored total Cx43 protein levels, thus suggesting that both proteasomes and lysosomes may participate in the PCB 153-enhanced Cx43 internalization and degradation. However, neither the proteasomal nor the lysosomal inhibitors restored normal GJIC or number/size of gap junction plaques. Finally, PCB 153 also interfered with restoration of gap junction plaques following the inhibition of Cx43 transport to plasma membrane. Taken together, multiple modes of action seem to contribute to downregulation of Cx43 in PCB 153-treated rat liver epithelial cells. The enhanced degradation of Cx43, together with persistent inhibition of GJIC, might contribute to tumor-promoting effects of NDL-PCBs.

A Novel Connexin43-interacting Protein, CIP75, Which Belongs to the UbL-UBA Protein Family, Regulates the Turnover of Connexin43

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys 264 and Asn 302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.

Modulation of osteoblast gap junction connectivity by serum, TNFα, and TRAIL

We studied the effects of serum growth factors and of TNF family proteins on osteoblast gap junction connectivity. Serum starvation of human MG63 osteosarcoma cells or nontransformed osteoblasts decreased connexin43 protein. TNFα or TRAIL reduced connexin43 further. Serum starvation redistributed gap junctions but did not reduce intercellular diffusion. In contrast, TNFα or TRAIL reduced gap junctions on cell processes and decreased intercellular diffusion. Effects of TNFs on connexin43 were mediated by lysosomal proteolysis. Activating analogs of cAMP increased connexin43 protein, but did not block effects of serum starvation, TNFα, or TRAIL on connexin43 protein. Connexin43 and connectivity recovered overnight if stimuli were withdrawn. Surprisingly, connexin43 mRNA increased in serum starvation and with TNFα or TRAIL. Since β-catenin is a binding partner of connexin43, when connexin43 is degraded, β-catenin activation may contribute to a reflexive increase in connexin43 transcription. We conclude that osteoblast connectivity is regulated by a multifactorial system that maintains intercellular connections. Serum starvation, TNFα and TRAIL augmented connexin43 degradation and connexin43 transcription. Cell–cell communication was maintained in serum starvation, which may model response to acute injury, but was sensitive to TNFs. These inflammatory agents mediated selective, reversible removal of connexin43 from cell processes.

Changes in gap junction organization and decreased coupling during induced apoptosis in lens epithelial and NIH-3T3 cells

We demonstrate that global induction of apoptosis in primary bovine lens epithelial (LEC) or fibroblastic mouse NIH-3T3 cells by staurosporine, puromycin, cycloheximide, or etoposide is accompanied by a decrease in coupling by gap junctions. Cell coupling as tested by neurobiotin spreading was maintained when the LEC or NIH-3T3 cells were pre-incubated with the pan-caspase inhibitor zVAD or the caspase-3 inhibiting tetrapeptide DEVD. Immunohistochemistry using anti-connexin-43 antibodies showed a reduction of plasma membrane integrated connexin-43 in both cell lines when undergoing apoptosis. Western blotting indicated degradation of connexin-43 that was inhibited by zVAD or DEVD. Cell coupling at single cell level was tested by direct microinjecting into LEC apoptosis-inducing agents of low molecular mass like staurosporine, etoposide and puromycin or the high molecular mass proteins caspase-3 and -8 in activated state. Microinjection of puromycin or etoposide induced apoptotic morphological changes of only the injected cell within 90 or 180 min, but did not affect adjacent cells. In contrast, microinjection of staurosporine led to a rapid induction of apoptosis of the injected and a number of adjacent cells suggesting spreading of staurosporine most probably through gap junction pores held open by dephosphorylation of connexin-43 as verified by immunoblotting and staining using a phospho-serine368-specific anti-connexin-43 antibody. Microinjection of active caspase-8 led after 3 h to morphological apoptotic alterations of only the injected cell, but did not inhibit spreading of co-injected neurobiotin to neighboring cells during the first hour. In contrast, microinjection of active caspase-3-induced apoptosis only of the injected cell after 60 min and rapidly and completely suppressed coupling to neighboring cells.

Degradation of connexin 43 induced by phosphorylation and dephosphorylation

Degradation of connexin 43 induced by phosphorylation and dephosphorylation

Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.

Cardiomyocyte hypertrophy and degradation of connexin43 through spatially restricted autocrine/paracrine heparin-binding EGF

Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling was demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away. Thus, HB-EGF acts as an autocrine and local paracrine cardiac growth factor that leads to loss of gap junction proteins within a spatially confined microenvironment. These findings demonstrate how cells can coordinate remodeling with their immediate neighboring cells with highly localized extracellular EGF signaling.

Intercellular communication via gap junctions in activated rat hepatic stellate cells

Gap junctional communication was studied in quiescent and activated hepatic stellate cells. Connexin expression and intercellular dye transfer were studied in rat hepatic stellate cells in culture and in vivo. Protein expression of connexin 43 was up-regulated in activated hepatic stellate cells in vivo and in vitro and was mainly localized on the cell surface, whereas connexin 26 was found intracellularly. In contrast to hepatocytes, hepatic stellate cells do not express connexin 32. Confluent hepatic stellate cells in culture communicate via gap junctions, resulting in lucifer yellow transfer and propagation of intracellular calcium signals. Phorbol ester induces a protein kinase C-dependent hyperphosphorylation and degradation of connexin 43 and inhibits intercellular communication on a short-term time scale. At the long-term level, vitamin D(3) , lipopolysaccharide, thyroid hormone T(3), dexamethasone, platelet-derived growth factor, endothelin 1, and interleukin 1beta up-regulate connexin 43 protein and messenger RNA expression and enhance intercellular communication. Slight down-regulation of connexin 43 is observed in response to vitamin A. Connexin 43 induction by endothelin 1 is inhibited by both endothelin A and endothelin B receptor antagonists. In coculture systems, hepatic stellate cells communicate with each other, which is suggestive of a syncytial organization, but no communication was found between hepatic stellate cells and other liver cell types. As shown by immunohistochemistry and electron microscopy, gap junctions are formed between activated hepatic stellate cells in vivo. Gap junctional communication occurs between hepatic stellate cells, is enhanced after activation, and underlies complex regulation by cytokines, hormones, and vitamins.

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.