A potential role of connexin 43 in epidermal growth factor-induced proliferation of mouse embryonic stem cells: involvement of Ca2+/PKC, p44/42 and p38 MAPKs pathways

Abstract. Objectives: The gap junction protein, connexin (Cx), plays an important role in maintaining cellular homeostasis and cell proliferation by allowing communication between adjacent cells. Therefore, this study has examined the effect of epidermal growth factor (EGF) on Cx43 and its relationship to proliferation of mouse embryonic stem cells. Materials and methods: Expressions of Cx43, mitogen‐activated protein kinases (MAPKs) and cell cycle regulatory proteins were assessed by Western blot analysis. Cell proliferation was assayed with [³H]thymidine incorporation. Intercellular communication level was measured by a scrape loading/dye transfer method. Results: The results showed that EGF increased the level of Cx43 phosphorylation in a time‐ (≥5 min) and dose‐ (≥10 ng/mL) dependent manner. Indeed, EGF‐induced increase in phospho‐Cx43 level was significantly blocked by either AG 1478 or herbimycin A (tyrosine kinase inhibitors). EGF increased Ca²⁺ influx and protein kinase C (PKC) translocation from the cytosolic compartment to the membrane compartment. Moreover, pre‐treatment with BAPTA‐AM (an intracellular Ca²⁺ chelator), EGTA (an extracellular Ca²⁺ chelator), bisindolylmaleimide I or staurosporine (PKC inhibitors) inhibited the EGF‐induced phosphorylation of Cx43. EGF induced phosphorylation of p38 and p44/42 MAPKs, and this was blocked by SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a p44/42 MAPK inhibitor), respectively. EGF or 18α‐glycyrrhetinic acid (GA; a gap junction inhibitor) increased expression levels of the protooncogenes (c‐fos, c‐jun and c‐myc), cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin‐dependent kinase 2 (CDK2), CDK4 and p‐Rb], [³H]thymidine incorporation and cell number, but decreased expression levels of the p21^WAF1/Cip1 and p27^Kip1, CDK inhibitory proteins. Transfection of Cx43 siRNA also increased the level of [³H]thymidine incorporation and cell number. EGF, 18α‐GA or transfection of Cx43 siRNA increased 2‐DG uptake and GLUT‐1 protein expression. Conclusions: EGF‐induced phosphorylation of Cx43, which was mediated by the Ca²⁺/PKC, p44/42 and p38 MAPKs pathways, partially contributed to regulation of mouse embryonic stem cell proliferation.     

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.     

A 14‐3‐3 Mode‐1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43^Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43^S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43^S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43^Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.      

The antioxidant edaravone attenuates ER-stress-mediated cardiac apoptosis and dysfunction in rats with autoimmune myocarditis

Abstract

Experimental autoimmune myocarditis (EAM) is mediated by myocardial infiltration by myosin-specific T-cells secreting inflammatory cytokines. In this study, rat models of EAM were prepared by injection with porcine cardiac myosin. One week after immunization, edaravone was administered intraperitoneally at 3 or 10 mg/kg/day to rats for 2 weeks. Cardiac function was measured by haemodynamic and echocardiographic studies and TUNEL assay was performed. Left ventricular (LV) expression of NADPH oxidase sub-units (p47^phox and p67^phox), pro-inflammatory cytokines (TNF-α), endoplasmic reticulum (ER) stress signalling proteins (GRP78, caspase-12 and GADD153) and mitogen-activated protein kinase (MAPK) family proteins (phospho-p38 MAPK and phospho-JNK) were measured by western blotting. Edaravone improved LV function in a dose-dependent manner. Central venous pressure was significantly low and LV ejection fraction and fractional shortening was significantly high in edaravone groups compared with those in the vehicle group. In addition, edaravone treatment down-regulated LV expressions of p47^phox, TNF-α, GADD153, phospho-p38 MAPK and phospho-JNK. Furthermore, the LV expressions of p67^phox, GRP78, caspase-12 and TUNEL-positive cells of rats with EAM treated with edaravone were significantly low compared with those of the vehicle group. These findings suggest that edaravone ameliorated the progression of EAM by inhibiting oxidative and ER stress and, subsequently, cardiac apoptosis.     

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid

Human lung neoplasms frequently express mutations that down‐regulate expression of various tumor suppressor molecules, including mitogen‐activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti‐tumor therapies. We now report that 4‐phenyl‐3‐butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non‐cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell‐cell communication. H2009 human lung carcinoma cells and ras‐transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras‐transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell–cell communication and connexin 43 phosphorylation in ras‐transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up‐regulation of p38 MAPK activity, altered connexin 43 expression, and down‐regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down‐regulated p38 MAPK activity and/or activated JNK and altered cell–cell communication. J. Cell. Biochem. 113: 269–281, 2012. © 2011 Wiley Periodicals, Inc.     

Enhanced connexin 43 expression delays intra‐mitoitc duration and cell cycle traverse independently of gap junction channel function

Connexins (Cxs) and gap junction (GJ)‐mediated communication have been linked with the regulation of cell cycle traverse. However, it is not clear whether Cx expression or GJ channel function are the key mediators in this process or at what stage this regulation may occur. We therefore tested the hypothesis that enhanced Cx expression could alter the rate of cell cycle traverse independently of GJ channel function. Sodium butyrate (NaBu) or anti‐arrhythmic peptide (AAP10) were used to enhance Cx expression in HeLa cells stably expressing Cx43 (HeLa‐43) and primary cultures of human fibroblasts (HFF) that predominantly express Cx43. To reduce GJ‐mediated communication, 18‐α‐glycyrrhetinic acid (GA) was used. In HeLa‐43 and HFF cells, NaBu and AAP10 enhanced Cx43 expression and increased channel function, while GA reduced GJ‐mediated communication but did not significantly alter Cx43 expression levels. Timelapse microscopy and flow cytometry of HeLa‐WT (wild‐type, Cx deficient) and HeLa‐43 cells dissected cell cycle traverse and enabled measurements of intra‐mitotic time and determined levels of G1 arrest. Enhanced Cx43 expression increased mitotic durations corresponding with a G1 delay in cell cycle, which was linked to an increase in expression of the cell cycle inhibitor p21^waf1/cip1 in both HeLa‐43 and HFF cells. Reductions in Cx43 channel function did not abrogate these responses, indicating that GJ channel function was not a critical factor in reducing cell proliferation in either cell type. We conclude that enhanced Cx43 expression and not GJ‐mediated communication, is involved in regulating cell cycle traverse. J. Cell. Biochem. 110: 772–782, 2010. © 2010 Wiley‐Liss, Inc.     

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non‐diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.     

Cardiac β‐adrenergic receptor activation mediates distinct and cell type‐dependent changes in the expression and distribution of connexin 43

Activation of the sympatho‐β‐adrenergic receptors (β‐ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type‐dependent changes in Cx43 remodelling due to β‐AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of β₂‐AR were used, which exhibited cardiac fibrosis and up‐regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of β₂‐AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the β₂‐antagonist ICI‐118551 or protein kinase A inhibitor H‐89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that β‐AR activation‐induced production of IL‐18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of β‐AR in mediating distinct and cell type‐dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.     

EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.     

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

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

HIF-1α promotes the proliferation and migration of pulmonary arterial smooth muscle cells via activation of Cx43

The proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important cause of pulmonary vascular remodelling in hypoxia-induced pulmonary hypertension (HPH). However, its underlying mechanism has not been well elucidated. Connexin 43 (Cx43) plays crucial roles in vascular smooth muscle cell proliferation in various cardiovascular diseases. Here, the male Sprague-Dawley (SD) rats were exposed to hypoxia (10% O₂) for 21 days to induce rat HPH model. PASMCs were treated with CoCl₂ (200 µM) for 24 h to establish the HPH cell model. It was found that hypoxia up-regulated the expression of Cx43 and phosphorylation of Cx43 at Ser 368 in rat pulmonary arteries and PASMCs, and stimulated the proliferation and migration of PASMCs. HIF-1α inhibitor echinomycin attenuated the CoCl₂-induced Cx43 expression and phosphorylation of Cx43 at Ser 368 in PASMCs. The interaction between HIF-1α and Cx43 promotor was also identified using chromatin immunoprecipitation assay. Moreover, Cx43 specific blocker (^37,43Gap27) or knockdown of Cx43 efficiently alleviated the proliferation and migration of PASMCs under chemically induced hypoxia. Therefore, the results above suggest that HIF-1α, as an upstream regulator, promotes the expression of Cx43, and the HIF-1α/Cx43 axis regulates the proliferation and migration of PASMCs in HPH.     

Ischaemia alters the effects of cardiomyocyte‐derived extracellular vesicles on macrophage activation

Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell‐cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte‐derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro‐inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL‐1β and IL‐6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL‐1β, IL‐6, IL‐10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum‐circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte‐derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post‐infarction remodelling.     

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43‐silenced MLO‐Y4 osteocytic (Cx43^def) cells undergo spontaneous cell death in culture through caspase‐3 activation and exhibit increased levels of apoptosis‐related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43^def cell death. Cx43^def cells and bones from old mice exhibit reduced levels of the pro‐survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43^def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43‐expressing cells and miR21 deletion in miR21^fl/fl bones increases apoptosis‐related gene expression, whereas a miR21 mimic prevents Cx43^def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43^def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high‐mobility group box‐1 (HMGB1)], and caspase‐3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43^def cells, which is blocked by antagonizing HMGB1‐RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

Rapid disruption of gap junctional communication and phosphorylation of connexin43 by platelet-derived growth factor in T51B rat liver epithelial cells expressing platelet-derived growth factor receptor

Gap junctional communication (GJC) between contacting cells has been postulated to be involved in the regulation of cell proliferation. This suggestion stems from numerous studies showing modulation of GJC by agents that influence cellular proliferation. Platelet-derived growth factor (PDGF), a strong mitogen, inhibits GJC in many cell types. To understand the molecular nature of the signal transduction pathway responsible for the GJC blockade, T51B rat liver epithelial cells, which lack endogenous PDGF receptor (PDGFr), were infected with a retrovirus containing either wild-type full-length cDNA of human PDGFrβ (Kin⁺) or a mutant PDGFrβ lacking receptor tyrosine kinase activity (Kin⁻). PDGF caused a complete but transient interruption of cell communication in Kin⁺ cells within 15–20 min of addition. This interruption of GJC was not associated with a gross destabilization of gap junction plaques but with the phosphorylation of connexin43 (Cx43), the only known gap junction protein expressed in these cells. These effects were not exhibited in either control T51B cells or in Kin⁻ cells, indicating a requirement of the receptor tyrosine kinase activity. Further examination revealed that the newly phosphorylated Cx43 then undergoes a rapid degradation utilizing the lysosomal pathway resulting in a decreased total Cx43 protein level. The re-establishment of GJC following PDGF treatment was dependent on protein synthesis. This report describes a suitable cell system which is currently being utilized for the characterization of the PDGF signaling pathway responsible for the inhibition of GJC. J. Cell. Physiol. 174:66–77, 1998. © 1998 Wiley-Liss, Inc.