Role Of Cx43 Research Articles

Connexin 43 in osteogenesis

Skeleton formation and its proper functioning is possible thanks to specialized bone tissue cells: bone forming osteoblasts, bone resorbing osteoclasts and osteocytes located in bone cavities. Gap junctions are transmembrane channels connecting neighboring cell. Thanks to gap junctions it is possible for signals to be directly transmitted by cells. Gap junction type channels, and more specifically the connexin proteins that build them, have a key impacton the bone turnover process, and thus on both bone building and remodeling. A particularly important connexin in bone tissue is connexin43 (Cx43), which is necessary in the proper course of the bone formation process and in maintaining bone homeostasis. The importance of the presence of Cx43 in bones is showed by skeletal defects in diseases such as ODD syndrome and craniometaphyseal dysplasia caused by mutations in GJA1, the gene encoding Cx43. The role of Cx43 in the differentiation of stem cells into bone cells, anti-apoptotic action of bisphosphonates and bone responses to hormonal and mechanical stimuli have also been demonstrated. In addition to connexin43, the presence of other connexins such as connexin45, 46 and 37 was also noted in bone tissue.

NGF induces pulmonary arterial hyperreactivity through Connexin 43 increased expression

We have previously shown that the nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. To characterize mechanisms activated by NGF to induce PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Expression of NGF and its TrkA receptor were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from controls or from patients suffering from idiopathic pulmonary arterial hypertension (IPAH). In cells treated or not with NGF (0.1-100 ng/ml, 24 h), Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and cell surface biotinylation. Gap junction activity was assessed by Lucifer yellow (LY) dye transfer assay. Contractions of control rat PA were induced by phenylephrine (PHE, 10-10-10-4 M) in absence or presence of NGF (100 ng/ml, 24 h). The role of Cx43 in NGF-induced rat PA reactivity was evaluated by use of 43Gap26 (Cx43 blocking peptide, 300 μM), or an anti-Cx43 siRNA (10nmol IV). NGF and TrkA expression were increased in hPASMC from IPAH patients compared to controls. NGF increased Cx43 expression in hPASMC from both controls and IPAH patients, but with a higher sensitivity in IPAH cells (maximum at 1 ng/ml vs. 100 ng/ml). This increase was abolished after treatment with K252a (TrkA kinase inhibitor). In control hPASMC, NGF increased Cx43 plasma membrane localisation and LY diffusion speed through Gap junctions. NGF-induced PA hyperreactivity to PHE was inhibited ex vivo by treatment with 43Gap26 and in vivo after administration of Cx43 siRNA. NGF pathway is unregulated in IPAH cells. NGF increases Cx43 PA expression through activation of its TrkA receptor. Cx43 increased expression at hPASMC plasma membrane upregulates gap junction activity, thus promoting NGF-induced PA hyperreactivity.

Cx43 promotes SHF-DPCs proliferation in the hair follicle of Albas cashmere goats from anagen to telogen

Connexin 43 (Cx43), known to form gap junction transmembrane channels between the cytoplasm of two adjacent cells, plays a key role in physiological functions, such as regulating cell growth, differentiation, and maintaining tissue homeostasis. Cashmere goat is an important farm animal that provides cashmere, which was produced by secondary hair follicles (SHF), for human consumption; however, there is no report about the role of Cx43 on the growth and development of SHF in cashmere goat. In this study, we investigated the effect of Cx43 on proliferation secondary hair follicle dermal papilla cells (SHF-DPCs) in Albas cashmere goat. In SHF-DPCs, Cx43 overexpression promoted cell proliferation and upregulated the expression of IGF-1, whereas Cx43 knockdown was associated with the opposite effects. These results suggested that Cx43 may promote cell proliferation by inducing IGF-1. Overall, our research not only contributes to a better understanding of the mechanism of the growth and development of SHF in cashmere goat, but also shed light on cashmere quality control in the future.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Disease-Linked MLC1 Protein Favors Gap-Junction Intercellular Communication by Regulating Connexin 43 Trafficking in Astrocytes.

Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.

Cisplatin-induced ototoxicity in organotypic cochlear cultures occurs independent of gap junctional intercellular communication

Cisplatin is a very effective chemotherapeutic, but severe and permanent hearing loss remains a prevalent side effect. The processes underpinning cisplatin-induced ototoxicity are not well understood. Gap junction channels composed of connexin (Cx) subunits allow for the passage of small molecules and ions between contacting neighboring cells. These specialized channels have been postulated to enhance cisplatin-induced cell death by spreading “death signals” throughout the supporting cells of the organ of Corti. This study sought to investigate the role of Cx43 in cisplatin-induced ototoxicity using organotypic cochlear cultures from control and two Cx43-mutant mouse strains harboring either a moderate (Cx43I130T/+) or severe (Cx43G60S/+) reduction of Cx43 function. Cochlear cultures from Cx43-mutant mice with a severe reduction in Cx43-based gap junctional intercellular communication (GJIC) had an enhanced number of hair cells that were positive for cleaved caspase 3, a marker of active apoptosis, after cisplatin treatment. In cisplatin-treated organotypic cochlear cultures, there was a decrease in the co-localization of Cx26 and Cx30 compared with untreated cultures, suggesting that cisplatin causes reorganization of connexin composition in supporting cells. Both Cx26 and Cx30 protein expression as well as GJIC were decreased in organotypic cochlear cultures treated with the gap-junction blocker carbenoxolone. When cisplatin and carbenoxolone were co-administered, there were no differences in hair cell loss compared with cisplatin treatment alone. Using cisplatin-treated control and Cx43-ablated organ of Corti derived HEI-OC1 mouse cells, we found that greatly reducing GJIC led to preferential induction of an ER stress pathway. Taken together, this study strongly suggests that inhibition of GJIC in organ of Corti cells does not lead to differential susceptibility to cisplatin-induced ototoxicity. Although cisplatin causes the same degree of cell death in gap junction competent and incompetent cochlear cells, the engagement of the mitochondrial dysregulation and ER stress differs.

Establishment and functional characterization of a murine primary Sertoli cell line deficient of connexin43

The Sertoli cell (SC) specific connexin43 (Cx43) knockout (SCCx43KO) mouse line is ideal to gain insight into the mechanistic gap junction formation in SC and the seminiferous epithelium. A method for developing primary SC cultures from these mice was established, validated and successfully characterized via polymerase chain reaction, immunohistochemistry, immunofluorescence (IF), and Western blots (WB). It was evident that both knockout (KO) and wild-type (WT) primary cell cultures were similar in morphology. These highly pure SC cultures were subjected to cell proliferation assays indicating no notable proliferation in cultures of both genotypes. Measurements of cell monolayer integrity indicated significant increases in transepithelial electrical resistance and consequently in tight junction expression of the KO cultures. Using semi-quantitative WB and IF, tight junction protein claudin-11 was analyzed. These results support a role for Cx43 in regulating blood-testis barrier (BTB) function, composition, and dynamics in vitro. Thus, the SC deficient Cx43 cell cultures may provide a valuable in vitro tool for a better understanding of the mechanistic role of Cx43 in spermatogenesis and BTB assembly.

Characterizing the Craniofacial Morphological Changes in Neural Crest Cell Specific Conditional Connexin‐43 Knock‐Out Mice

The craniofacial skeleton is an intricate structure that forms through complex cell‐cell interactions across neural crest and mesoderm derived cell populations. Proper cell‐cell communication during the migration and differentiation of cranial neural crest cells is critical for normal craniofacial development and this intercellular communication is thought to be facilitated through gap junctions – membrane channels composed of connexin proteins. In particular, connexin‐43 (Cx43) has been shown to be expressed in pre‐migratory and migratory cranial neural crest cells and plays a role in neural crest epithelial‐to‐mesenchyme transition. Additionally, within the skull, neural crest‐derived regions show the greatest morphological changes in mice wherein Cx43 has been globally ablated providing further evidence that Cx43 is important for proper neural crest function and ultimately proper development of the skull. To date, there have been no studies that have directly looked at the role of Cx43 in cranial neural crest cells in a mouse model. Thus, we have developed a neural crest‐specific Cx43 knockout mouse to address this gap in knowledge. In this study we (1) verify that Cx43 ablation is limited to neural crest cell and (2) characterize and compare craniofacial size and shape among wildtype, heterozygous and homozygous knockout mice.Heterozygous Wnt1‐Cre2 recombinase mice were bred with homozygous floxed Cx43 mice to create offspring which were bred to express the genotype, Wnt1‐Cre2/+:Cx43fl/+. These mice were bred to create: control mice (Wnt‐1Cre2/+:Cx43+/+), heterozygous conditional knock‐outs (Wnt1‐Cre2/+:Cx43fl/fl) and homozygous conditional knock‐outs (Wnt1‐Cre2+/+:Cx43fl/fl). To verify the knockout, paraffin embedded sagittal sections of embryonic and post‐natal mouse hearts and skulls were tagged with Wnt1 and Cx43 antibodies. RT‐PCR, and Western blotting were performed on neural crest and mesoderm derived newborn skull bones, to verify the expression levels of Cx43 mRNA and protein respectively. Newborn skulls from each genotype were imaged using high resolution micro‐CT and these images were used to quantify and compare skull morphology among genotypes using statistical analyses of shape.We have confirmed that Cx43 ablation is limited to neural crest cells in our mice. Analyses of skull shape comparisons among genotypes is ongoing with our initial findings indicating that mice homozygous for the knockout demonstrate overt changes to skull morphology, heterozygous mouse skulls are qualitatively similar to control mouse skulls. We predict that statistical analyses will uncover that only the bones derived from neural crest cells will display morphological differences among genotypes.Our study addresses a fundamental gap of the role connexin‐43 plays in cranial neural crest function and subsequent development of much of the skull.Support or Funding InformationFunding: Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (R5211A02)

Connexin 32 deficiency protects the liver against ischemia/reperfusion injury

Hepatic ischemia/reperfusion (I/R) injury is a common complication in the clinical setting. Our previous study has shown that connexin 32 (Cx32) plays a major role in renal I/R injury; however, the role of Cx32 in hepatic I/R injury remains unknown. Liver tissue and serum samples from patients undergoing orthotopic liver transplantation (OLT) were used to evaluate the function of Cx32 in OLT post-reperfusion injury. Then, partial hepatic ischemia was established in global Cx32 knockout mice and wild-type mice followed by reperfusion. Hepatic injury markers were examined. Cx32 small interfering RNA and the p53 inhibitor, pifithrin-α, tenovin-1 were used to examine the relationship between Cx32 and the p53/puma pathways in the BRL-3A and murine primary hepatocytes hypoxia/reoxygenation (H/R) model. Corresponding to liver damage, Cx32 was significantly induced both during OLT in human patients and partial hepatic I/R in mice. Cx32 KO mice exhibited less liver injury than controls. Cx32 deficiency significantly suppressed the p53/puma pathways and hepatocyte apoptosis. Similar results were observed in the BRL-3A and murine primary hepatocytes H/R model. Propofol protected against OLT post-reperfusion injury and hepatocyte apoptosis by inhibiting Cx32. In conclusion Cx32 is a novel regulator of hepatic I/R injury through the modulation of hepatocyte apoptosis and damage, largely via the p53/puma signaling pathway.

Expression of connexin 43 in peripheral blood monocytes from patients with acute coronary syndrome

To investigate the expression of connexin 43 (Cx43) in peripheral blood monocytes (PBMCs) from patients with acute coronary syndrome (ACS) and its clinical implications. We prospectively collected the clinical data from 40 patients with ACS including 20 with unstable angina pectoris (UAP) and 20 with acute myocardial infarction (AMI) admitted in our department between January, 2018 and June, 2018, with 20 healthy subjects undergoing routine physical examinations serving as the control group. Peripheral blood samples were obtained from all the participants and plasma and PBMCs were separated. Enzyme-linked immunosorbent assay (ELISA) and turbidimetric inhibition immunoassay (TIIA) were used for analysis of plasma levels of interleukin (IL)-1β and high sensitive C-reactive protein (hs-CRP), respectively; real-time quantitative RT-PCR and Western blotting were used to detect the mRNA and protein levels of Cx43 in the PBMCs. Compared with the control group, the patients with UAP showed significantly increased plasma levels of IL-1β and hs-CRP (P < 0.001) and obviously elevated expressions of Cx43 at both mRNA and protein levels in the PBMCs (P < 0.001). Compared with the patients with UAP, the patients with AMI had significantly higher plasma IL-1β and hs-CRP levels (P < 0.001 and P < 0.01) but lower expression levels of Cx43 in the PBMCs (P < 0.05). Patients with UAP and AMI have activated inflammatory responses and reverse changes in Cx43 expression in the PBMCs, suggesting the different roles of Cx43 in the pathogenic mechanisms of different types of ACS.

Cx32 exerts anti-apoptotic and pro-tumor effects via the epidermal growth factor receptor pathway in hepatocellular carcinoma

BackgroundAbnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear.MethodsThe expression level of Cx32 in 96 hepatocellular carcinoma (HCC) specimens was determined using western blotting and immunohistochemistry. The correlation between Cx32 expression and clinicopathological parameters was analyzed. The cell apoptosis rate was examined using flow cytometry and western blotting. The role of Cx32 in the Src kinase and epidermal growth factor receptor (EGFR) signaling pathways was measured by quantitative real-time PCR, western blotting and coimmunoprecipitation (CO-IP). The effect of Cx32 overexpression on the streptonigrin (SN)-induced tumor growth suppression and apoptosis was assessed in nude mice.ResultsOur study showed that overexpressed Cx32 accumulated in the cytoplasm and that Cx32-containing gap junctions (GJs) were nearly absent in HCC specimens. Upregulated Cx32 expression was highly correlated with advanced tumor-node-metastasis (TNM) stage and poor tumor differentiation and was an independent predictive marker for poor prognosis in HCC. Overexpression of Cx32 significantly inhibited SN-induced apoptosis by activating the EGFR signaling pathway in vitro and in vivo. Moreover, the expression levels of Cx32 and EGFR were positively correlated in HCC specimens. The CO-IP experiments demonstrated that Cx32 could bind to Src kinase, and the western blotting results revealed that Cx32 increased the levels of EGFR and p-EGFR by upregulating Src expression.ConclusionThe present study demonstrated that overexpressed and internalized Cx32 was associated with advanced TNM stage and poor tumor differentiation and predicted poor prognosis in HCC. Cx32 facilitated HCC progression by blocking chemotherapy-induced apoptosis in vitro and in vivo via interacting with Src and thus promoting the phosphorylation of EGFR, subsequently activating the EGFR signaling pathway. Cx32 may be a potential biomarker and a new therapeutic target for HCC.

Mechanical stretch upregulates connexin43 in human trabecular meshwork cells.

Primary open angle glaucoma (POAG) patients have hallmark increases in intraocular pressure (IOP) and noted dysfunction of the trabecular meshwork (TM). Connexin43 (Cx43) is a gap junction widely expressed on the TM that is important for intercellular communication. The human gene is known as gap junction alpha-1 (GJA1). Since the role of Cx43 in the TM is not fully understood, we set out to determine the effect of excess mechanical stretch on cultured human trabecular meshwork cells (hTMCs) and to specifically investigate the effect of stretch on Cx43 expression and function. Primary hTMCs were cultured and subjected to 48 hours of 15% cyclic mechanical stretch at a frequency of 1 Hz. Levels of apoptosis and necrosis secondary to stretch were investigated using colorimetric assays. The effect of stretch on gap junction Cx43 and GJA1 was investigated by RT-PCR, immunoblotting and immunofluorescence. The migration of Lucifer Yellow dye was used to assess intercellular communication. Stretch significantly increased the rates of apoptosis and necrosis in hTMCs. The increased rate of injury in stretched hTMCs was further associated with significant upregulation of GJA1 mRNA and Cx43 protein. Upregulation of Cx43 protein was concomitant to increased intercellular communication. We have shown stretch to increase GJA1 gene and Cx43 protein expression, as well as intercellular communication. We have further shown stretch to be injurious to hTMCs. Upregulation of Cx43 in the hTM subsequent to stretch is a novel finding, which may be useful in elucidating the mechanism of TM injury in POAG patients.

Abstract 21374: Adenovirus Targets Cardiac Gap Junctions to Facilitate Viral Replication

Myocarditis is a leading cause of sudden cardiac death in young adults, accounting for 20% of cases under 40 years of age in the United States. Adenovirus is a major etiological agent in this disease yet much remains unknown regarding pathogenesis and predisposition to infection. During adenoviral infection, viral proteins uncouple cell cycle checkpoints for viral DNA genome replication to occur. Connexin43 (Cx43) gap junctions play an essential role in propagation of action potentials to precipitate each heartbeat. Cx43 has also been implicated in cell cycle regulation, leading us to hypothesize that adenovirus targets Cx43 to facilitate viral replication, resulting in arrhythmogenic alterations in cardiac intercellular communication. We find reduced Cx43 expression at the mRNA and protein level within 24 hours of infection with adenovirus type 5 (Ad5) in human epithelial cells. Studies in human induced pluripotent stem cell-derived cardiomyocytes reveal alterations in calcium transient propagation and reduced Cx43 protein levels. Growth factor signaling is activated by early Ad5 proteins and is known to negatively regulate gap junction coupling. We performed genetic screens and have identified the viral protein E4-ORF1 as sufficient in suppression of Cx43 expression and modulation of cardiac transcription factors. siRNA-mediated knockdown of Cx43 protein enhances viral genome replication while transient overexpression of Cx43 significantly inhibits late viral protein synthesis and genome replication. Finally, using dominant-negative trafficking-dead and closed-channel Cx43 mutants, we conclude that inhibition of Ad5 replication by Cx43 is gap junction dependent. Together, these data provide novel insight into molecular mechanisms underlying arrhythmogenic viral myocarditis in addition to understanding the role of Cx43 in adenoviral pathogenesis. Revealing viral mechanisms of gap junction dissolution will contribute not just to the development of novel antiviral therapeutics, but will also provide insight into fundamental Cx43 biology relevant to other forms of heart disease.

Abstract 235: GJA1-11k Localizes to the Nucleus and Inhibits Cell Cycle Progression

Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), are involved in the exchange of ions and small molecules to maintain homeostasis and also have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Numerous studies have found that Cx43 (predominantly the C-domain) is associated with control of cell proliferation, yet it is unclear how Cx43-based membrane channels effect these processes. We recently identified the existence of six endogenous Cx43 isoforms which are produced from the same full-length mRNA molecule in human cells by means of alternative translation and many of the known non-canonical roles of Cx43 can be attributed to these recently identified isoforms. Here we report that the smaller, non-channel related, alternatively translated isoform of Cx43 can directly affect cell growth. We found that 11KDa isoform (GJA1-11k) is localized in the nucleus of HEK293FT cells. GJA1-11k is a more potent growth suppressor than the wild type and full length isoform GJA1-43k. GJA1-11k-mediated growth suppression was achieved by limiting cell cycle progression from G0/G1 to S phase, in part, through regulation of internal translation of GJA1-11k. Nuclear localization experiments indicate that the cell growth-suppressive properties of GJA1-11k is linked to nuclear activity. RNA-seq based genome-wide mRNA transcriptome analysis revealed that the heat shock protein HSPA6, which is normally upregulated in certain cancer cells, was significantly downregulated in GJA1-11k overexpressing cells compared to cells overexpressing the full length isoform, suggesting a potential mechanism and/or new binding protein for the Cx43 C-terminal domain. In general, further understanding of the role of small Cx43 isoforms will provide a way not only to understand the mechanism controlling myocyte proliferation to improve the repair of the myocardium, but also to understand the pathobiology of cell growth associated with tumor suppressive properties of Cx43. It may be possible to develop new therapeutics by regulating alternative translation of Cx43.

A novel role for Connexin 43 in IFN beta expression during Chlamydia trachomatis infection and cytosolic DNA-sensing

Abstract Background The DNA sensor cGAS is essential for IFNβ expression in cells infected with Chlamydia. cGAMP generated during DNA sensing can also transfer via intercellular gap-junction proteins, connexins CX43/45 leading to IFNβ expression in adjacent uninfected cells. Present study highlights a novel role for CX43 in IFNβ expression during DNA-sensing and during Chlamydia infection, independent of its gap junction function. Methods Total IFNβ transcripts were measured by qRT-PCR following CX43 or CX45 siRNA mediated knockdown (KD). Single cell RNA-ISH method view RNA™ was used to visualize IFNβ mRNA in infected HeLa cells. CX43 localization was studied using IF and confocal microscopy. Results CX43-KD in HeLa cells reduced IFNβ expression by >90% (p<0.001) compared to control or CX45 KD, as measured by qRT-PCR. CX43-KD also reduced IFNβ mRNA in dsDNA transfected cells (p<0.001) but not with poly IC RNA. RNA-ISH showed IFNβ mRNA transcripts in infected cells (>90%) and in adjacent uninfected (~10%) cells, but not in distal uninfected cells, suggesting cell contact is required for adjacent-cell effect. Surprisingly, RNA-ISH of CX43-KD cells showed >85% reduction in IFNβ transcripts (p<0.0001) in infected cells, in addition to the loss of transcripts in cells adjacent to infected cells. CX43 localized proximal to the chlamydial inclusion membrane. Co-localization of CX43 was also observed with transfected dsDNA, but not dsRNA. Conclusion CX43 depletion has been shown to increase autophagy in cells. Here we show that CX43 depletion decreases IFNβ induction during DNA sensing. Our results suggest that like STING, CX43 could be a key player in both DNA sensing and autophagy, but with opposing roles. (Funded by NIAID R01-AI067678 to UN).

Role of connexin 43 in different forms of intercellular communication - gap junctions, extracellular vesicles and tunnelling nanotubes.

Communication is important to ensure the correct and efficient flow of information, which is required to sustain active social networks. A fine-tuned communication between cells is vital to maintain the homeostasis and function of multicellular or unicellular organisms in a community environment. Although there are different levels of complexity, intercellular communication, in prokaryotes to mammalians, can occur through secreted molecules (either soluble or encapsulated in vesicles), tubular structures connecting close cells or intercellular channels that link the cytoplasm of adjacent cells. In mammals, these different types of communication serve different purposes, may involve distinct factors and are mediated by extracellular vesicles, tunnelling nanotubes or gap junctions. Recent studies have shown that connexin 43 (Cx43, also known as GJA1), a transmembrane protein initially described as a gap junction protein, participates in all these forms of communication; this emphasizes the concept of adopting strategies to maximize the potential of available resources by reutilizing the same factor in different scenarios. In this Review, we provide an overview of the most recent advances regarding the role of Cx43 in intercellular communication mediated by extracellular vesicles, tunnelling nanotubes and gap junctions.

Connexin43 and AMPK Have Essential Role in Resistance to Oxidative Stress Induced Necrosis

Reactive oxygen species (ROS) induced oxidative stress leads to cell damage and neurological disorders in astrocytes. The gap junction protein connexin43 (Cx43) could form intercellular channels in astrocytes and the expression of Cx43 plays an important role in protecting the cells from damage. In the present study, we investigated the contribution of Cx43 to astrocytic necrosis induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which AMPK was involved in this process. Fluorescence microscopy, flow cytometry, and western blot were used quantitatively and qualitatively to determine the cell apoptosis, necrosis, and protein expression. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased H2O2-induced astrocytic necrosis, supporting a cell protective effect of functional Cx43 channels. Our data suggest that AMPK is important for Cx43-mediated ROS resistance. Inhibition of AMPK activation results in reduction of necrosis and ROS production. Taken together, our findings suggest that the role of Cx43 in response to H2O2 stress is dependent on the activation of AMPK signaling pathways and regulates ROS production and cell necrosis.

Connexin 43 and Mitochondria in Cardiovascular Health and Disease.

Connexin 43 (Cx43) is the major connexin protein in ventricular cardiomyocytes. Six Cx43 proteins assemble into so-called hemichannels at the sarcolemma and opposing hemichannels form gap junctions, which allow the passage of small molecules and electrical current flow between adjacent cells. Apart from its localization at the plasma membrane, Cx43 is also present in cardiomyocyte mitochondria, where it is important for mitochondrial function in terms of oxygen consumption and potassium fluxes. The expression of gap junctional and mitochondrial Cx43 is altered under several pathophysiological conditions among them are hypertension, hypertrophy, hypercholesterolemia, ischemia/reperfusion injury, post-infarction remodeling, and heart failure. The present review will focus on the role of Cx43 in cardiovascular diseases and will highlight the importance of mitochondrial Cx43 in cardioprotection.

Connexin 43 expression is associated with poor survival in patients with esophageal squamous cell carcinoma.

Connexin 43 (Cx43) is an important gap junction protein in vertebrates, which has been reported to function as a tumor suppressor in a number of organs. However, the mechanism underlying the effect of Cx43 on tumor progression remains unknown, with only a limited number of studies reporting on the role of Cx43 in esophageal cancer. In the present study, Cx43 expression was analyzed by immunohistochemical staining and the associations between Cx43 expression and clinicopathological characteristics or prognosis were evaluated. Cx43 was expressed at a high frequency in patients with esophageal squamous cell carcinoma (ESCC). Of the 98 ESCC cases investigated, positivity for Cx43 was observed in 62 cases (63.26%). In patients with high Cx43 expression, the survival rates were significantly reduced compared with those in patients with low Cx43 expression. Moreover, the overexpression of Cx43, as measured by immunohistochemistry, was an independent prognostic indicator of ESCC. Thus, our data indicated that Cx43 may be a candidate molecular prognostic marker and molecular target for the development of an effective therapeutic intervention for patients with esophageal cancer.

Role of connexin 43 in vascular hyperpermeability and relationship to Rock1-MLC20pathway in septic rats

Connexin (Cx)43 has been shown to participate in several cardiovascular diseases. Increased vascular permeability is a common and severe complication in sepsis or septic shock. Whether or not Cx43 takes part in the regulation of vascular permeability in severe sepsis is not known, and the underlying mechanism has not been described. With cecal ligation and puncture-induced sepsis in rats and lipopolysaccharide (LPS)-treated vascular endothelial cells (VECs) from pulmonary veins, the role of Cx43 in increased vascular permeability and its relationship to the RhoA/Rock1 pathway were studied. It was shown that vascular permeability in the lungs, kidneys, and mesentery in sepsis rats and LPS-stimulated monolayer pulmonary vein VECs was significantly increased and positively correlated with the increased expression of Cx43 and Rock1 in these organs and cultured pulmonary vein VECs. The connexin inhibitor carbenoxolone (10 mg/kg iv) and the Rock1 inhibitor Y-27632 (2 mg/kg iv) alleviated the vascular leakage of lung, mesentery, and kidney in sepsis rats. Overexpressed Cx43 increased the phosphorylation of 20-kDa myosin light chain (MLC20) and the expression of Rock1 and increased the vascular permeability and decreased the transendothelial electrical resistance of pulmonary vein VECs. Cx43 RNA interference decreased the phosphorylation of MLC20 and the expression of Rock1 and decreased LPS-stimulated hyperpermeability of cultured pulmonary vein VECs. The Rock1 inhibitor Y-27632 alleviated LPS- and overexpressed Cx43-induced hyperpermeability of monolayer pulmonary vein VECs. This report shows that Cx43 participates in the regulation of vascular permeability in sepsis and that the mechanism is related to the Rock1-MLC20 phosphorylation pathway.

Astrocytes promote glioma invasion via the gap junction protein connexin43.

Reactive astrocytes are integral to the glioma microenvironment. Connexin43 (Cx43) is a major gap junction protein in astrocytes and its expression is enhanced significantly in glioma-associated astrocytes, especially at the peri-tumoral region. Although downregulation of Cx43-mediated intercellular communication is associated with increased malignancy in tumor cells, the role of Cx43 in stromal cells in glioma progression is not defined. Using a mouse model consisting of syngeneic intracranial implantation of GL261 glioma cells into Nestin-Cre:Cx43(fl/fl) mice where Cx43 was eliminated in astrocytes, we demonstrate a role of astrocytic Cx43 in the dissemination of glioma cells from the tumor core. To determine whether heterocellular communication between astrocytes and glioma cells is essential for reduced invasion in the absence of astrocytic Cx43, we abolished channel formation between glioma cells and astrocytes by either knocking down Cx43 in glioma cells with short hairpin RNA (shRNA) or overexpressing a dominant-negative channel-defective Cx43-T154A mutant in these cells. Although Cx43shRNA in glioma cells reduced invasion, expression of Cx43-T154A had no effect on glioma invasion, suggesting tumoral Cx43 may influence motility independently from its channel function. Alteration in astrocytic Cx43 function, such as by replacing the wild-type allele with a C-terminal truncated Cx43 mutant exhibiting reduced intercellular coupling, is sufficient to reduce glioma spreading into the brain parenchyma. Our results reveal a novel role of astrocytic Cx43 in the formation of an invasive niche and raise the possibility to control glioma progression by manipulating the microenvironment.