Cx43 Protein Levels Research Articles

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.

Human papillomavirus E6 regulates the trafficking of gap junction protein Cx43

High-risk human papillomavirus (HR-HPV) infects epithelial cells and is the major cause of anogenital and oropharyngeal cancers. HR-HPV oncogenic activity is through E6 control of p53, but E6 binds and degrades PDZ proteins such as the tumour suppress protein hDlg (human homologue of DrosophilaDiscs Large). The E6/hDlg complex also contains Connexin 43 (Cx43) (MacDonald, Sun et al. 2012), the major building block of gap junctions that allow intercellular molecular communication. In HPV16-positive non-tumour cervical keratinocytes (W12NT: low E6 levels) Cx43/hDlg is on the plasma membrane but in the cytoplasm in W12T tumour epithelial cells (W12T: high E6 levels) correlating with loss of gap junction cell-cell communication. E6 siRNA depletion in W12T cells restored Cx43 to the cell membrane, while overexpressing E6 in HPV-negative cervical cancer cells C33a resulted in Cx43 moving to the cytoplasm. E6 could control Cx43 trafficking through controlling hDlg or by altering cell signalling. In the absence of E6 in HEK293, HaCaT and normal immortalised keratinocytes (NIKS), Cx43 and hDlg could be co-immunoprecipitated and they co-localised on the plasma membrane. Thus, the Cx43/hDlg interaction is not carcinoma cell-specific, is not dependent on HR-HPV E6, and may have a functional role in non-cancer cells. siRNA depletion of hDlg, led to reduction in Cx43 protein levels and some relocation to the cytoplasm. This indicates that HPV E6 controls Cx43 through interaction with and degradation of hDlg. However, we cannot discount that E6 itself may have additional effects on Cx43 levels and trafficking besides via hDlg.

Upregulation of MMP-9 and CaMKII prompts cardiac electrophysiological changes that predispose denervated transplanted hearts to arrhythmogenesis after prolonged cold ischemic storage

BackgroundAlthough the importance of the potential mechanisms of arrhythmogenesis, including cardiac electrophysiology dysfunction and gap junction electrical uncoupling by Cx43 degradation under pathological conditions, has been widely and thoroughly established, the correlations between these mechanisms and biomarkers of cold ischemic insult have not yet been confirmed. Limited evidence suggests that these biomarkers are significantly associated with cardiac dysrhythmia. Thus, in this study, we analyzed such correlations in denervated transplanted hearts. Methods and resultsRat hearts were divided into two groups according to a random number table. The hearts in the first group were stored in histidine-tryptophan-ketoglutarate (HTK) solution for 6 h to produce cold ischemic insult (insult group, n = 8), while the hearts in the second group were left untreated (control group, n = 8). Ventricular arrhythmias (VAs) were successively observed in 8/8 hearts stored in HTK solution and 0/8 untreated hearts (P < 0.05). The analysis of the electrophysiological parameters by programmed electrical stimulation (PES) and monophasic action potential (MAP) recording demonstrated that compared with the control hearts, the insult hearts had increased transmural dispersion of repolarization (TDR) and effective refractory periods (ERPs) (P < 0.001), decreased conduction velocities (CVs) and excitation wavelengths (CV × ERP, λ) (P < 0.05), and longer times between peak MAP values and 90% repolarization (MAPD90) in the epicardium (P < 0.05). However, no significant difference in endocardial MAPD90 was observed between the two groups of hearts. The investigation of biomarkers and Cx43 proteins by a western blot analysis showed that the matrix metalloproteinase-9 (MMP-9) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) levels were strongly elevated and that the Cx43 protein levels were markedly reduced in the insult hearts. ConclusionMMP-9 overexpression mediates gap junction (GJ) electrical uncoupling induced by Cx43 decreases, while CaMKII upregulation induces transmural dispersion of repolarization (TDR) increase, both of which increase the risk of ventricular arrhythmias (VAs) in denervated transplanted hearts after prolonged cold ischemic storage.

Connexin 26 and 43 play a role in regulating proinflammatory events in the epidermis.

Dysregulation of Connexin (CX) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and nonhealing wounds. To mimic a proinflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/ml peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 min to 24 hr in the presence or absence of CX blockers and/or following CX26, CX43, PANX1 and TLR2 small interfering RNA (siRNA) knockdown (KD). Expression levels of IL-6, IL-8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by quantitative real-time polymerase chain reaction, western blot analysis and/or immunocytochemistry. Nuclear factor kappa β (NF-κβ) was blocked with BAY 11-7082, CX-channel function was determined by adenosine 5'-triphosphate (ATP) release assays. Enzyme-linked immunosorbent assay monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of CX or purinergic signalling. Exposure to PGN induced IL-6, IL-8, CX26 and TLR2 gene expression but it did not influence CX43, PANX1 or Ki67 messenger RNA expression levels. CX43 protein levels were reduced following 24 hr PGN exposure. PGN-induced CX26 and IL-6 expression were also aborted by TLR2-KD and inhibition of NF-κβ. ATP and IL-6 release were stimulated following 15 min and 1-24 hr challenge with PGN, respectively. Release of both agents was inhibited by coincubation with CX-channel blockers, CX26-, CX43- and TLR2-KD. The IL-6 response was also reduced by purinergic blockers. CX-signalling plays a role in the innate immune response in the epidermis. PGN is detected by TLR2, which via NF-κβ, directly activates CX26 and IL-6 expression. CX43 and CX26 maintain proinflammatory signalling by permitting ATP release, however, PANX1 does not participate.

Cx43 silencing inhibits mechanical stress-induced apoptosis in mouse articular chondrocyte

To explore the effect of connexin 43 (Cx43) silence on the apoptosis in mouse chondrocyte under mechanical stress. Methods: Mouse chondrocyte ATDC5 cells were divided into a control group, a mechanical stress group, a Cx43 siRNA transfection group, a scramble siRNA transfection group, a mechanical stress+scramble group, and a mechanical stress+siCx43 group. Flexcell FX-5000 system was used to produce mechanical stress on ATDC5 cells cultured in vitro. The mRNA and protein level of Cx43 was detected by quantitative RT-PCR (RT-qPCR) and Western blot. The cell activity and cell apoptosis was detected by cell counting kit-8 (CCK-8) method and flow cytometry, respectively. Caspase-3 activity was detected by colorimetric assay. The protein expression of Bcl-2, Bax, p-JNK and JNK was detected by Western blot. Results: Mechanical stress upregulated the mRNA and protein expression of Cx43 (both P<0.05). Transfection of Cx43 siRNA significantly decreased Cx43 mRNA and protein level (both P<0.05). After stimulation with mechanical stress, chondrocyte viability was significantly decreased, whereas cell apoptosis and caspase-3 activity were increased (both P<0.05). Mechanical stress obviously upregulated Bax protein level, and downregulated Bcl-2 protein expression and Bcl-2/Bax (both P<0.05). Cx43 siRNA transfection significantly increased cell viability, inhibited cell apoptosis and caspase-3 activity (both P<0.05). Cx43 siRNA also inhibited Bax expression, and increased the Bcl-2 protein expression and Bcl-2/Bax (both P<0.05). Furthermore, Cx43 siRNA significantly suppressed the p-JNK expression induced by mechanical stress (P<0.05). Conclusion: Cx43 silence inhibits mechanical stress-induced apoptosis in chondrocyte, which might be mediated by JNK signaling pathway.

Polycyclic Aromatic Hydrocarbons and Endocrine Disruption: Role of Testicular Gap Junctional Intercellular Communication and Connexins.

Ambient air pollution and smoking are well-documented risk factors for male infertility. Prevalent air pollutants and cigarette smoke components, polycyclic aromatic hydrocarbons (PAHs), are environmental and occupational toxicants that act as chemicals disrupting endocrine regulation and reproductive potential in males. Testicular gap junctional intercellular communication (GJIC) is critical for normal development and function of testicular tissue, thus we assessed GJIC as a process potentially targeted by PAHs in testes. Lower MW PAHs with a bay or bay-like region rapidly dysregulated GJIC in Leydig TM3 cells by relocalization of major testicular gap junctional protein connexin 43 (Cx43) from plasma membrane to cytoplasm. This was associated with colocalization between Cx43 and ubiquitin in intracellular compartments, but without any effect on Cx43 degradation rate or steady-state Cx43 mRNA levels. A longer exposure to active PAHs decreased steady-state levels of full-length Cx43 protein and its 2 N-truncated isoforms. Inhibition of GJIC by PAHs, similarly to a prototypic GJIC-inhibitor TPA, was mediated via the MAP kinase-Erk1/2 and PKC pathways. Polycyclic aromatic hydrocarbon-induced GJIC dysregulation in testes was cell-type-specific because neither PAH dysregulated GJIC in Sertoli TM4 cells, despite PAHs were rapidly taken up by both Leydig TM3 as well as Sertoli TM4 cells. Because TPA effectively dysregulated GJIC in both testicular cell types, a unique regulator of GJIC targeted by PAHs might exist in Leydig TM3 cells. Our results indicate that PAHs could be a potential etiological agent contributing to reproductive dysfunctions in males through an impairment of testicular GJIC and junctional and/or nonjunctional functions of Cx43.

Curcumin Promotes Connexin 43 Degradation and Temozolomide-Induced Apoptosis in Glioblastoma Cells

Glioblastoma (GBM) is the most commonly occurring tumor in the cerebral hemispheres. Currently, temozolomide (TMZ), an alkylating agent that induces DNA strand breaks, is considered the frontline chemotherapeutic agent for GBM.Despite its frontline status, GBM patients commonly exhibit resistance to TMZ treatment. We have recently established and characterized TMZ-resistant human glioma cells. The aim of this study is to investigate whether curcumin modulates cell apoptosis through the alternation of the connexin 43 (Cx43) protein level in TMZ-resistant GBM.Overexpression of Cx43, but not ATP-binding cassette transporters (ABC transporters), was observed (approximately 2.2-fold) in TMZ-resistant GBM cells compared to the Cx43 levels in parental GBM cells. Furthermore, at a concentration of 10 M, curcumin significantly reduced Cx43 protein expression by about 40%. In addition, curcumin did not affect the expression of other connexins like Cx26 or epithelial-to-mesenchymal transition (EMT) proteins such as -catenin or E-catenin. Curcumin treatment led to an increase in TMZ-induced cell apoptosis from 4% to 8%. Importantly, it did not affect the mRNA expression level of Cx43. Concomitant treatment with the translation inhibitor cycloheximide (CHX) exerted additional effects on Cx43 degradation. Treatment with the autophagy inhibitor 3-MA (methyladenine) did not affect the curcumin-induced Cx43 degradation. Interestingly, treatment with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) significantly negated the curcumin-induced Cx43 degradation, which suggests that curcumin-induced Cx43 degradation occurs through the ubiquitin-proteasome pathway.

Identification of downstream target genes regulated by CX43 in hepatocellular carcinoma.

The aim of study was to identify the downstream target genes of CX43 by Human Transcriptome Array. Therefore, a gene microarray was generated which consists of CX43-overexpressed hepatocellular carcinoma (HCC) cells transfected with the constructed plasmid and negative controls to identify candidate genes. Integrated bioinformatic analysis was used to clarify biological functions of the identified genes, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein-protein interaction network, and survival analysis. The candidate genes were further validated by qRT-PCR in liver cancer tissues and CX43-silenced HCC cells. We have found the mRNA and protein levels of CX43 significantly upregulated in HCC cells transfected with CX43 constructed plasmid. We identified 928 differentially expressed genes including 394 upregulated and 534 downregulated genes, enriched in the cancer related functions and pathways by GO and KEGG pathway analysis. The protein-protein interaction network revealed 9 hub genes in this study. Statistical analysis indicated that upregulation of RALA and SRC was associated with poor prognosis in liver cancer. The differential expression of 2 candidate genes were further validated in HCC cells and tissues. In conclusion, protein-coding genes RALA and SRC could be target genes of CX43 and therapeutic targets for hepatocellular carcinoma.

Hypoxic Preconditioning Maintains GLT-1 Against Transient Global Cerebral Ischemia Through Upregulating Cx43 and Inhibiting c-Src.

Transient global cerebral ischemia (tGCI) causes excessive release of glutamate from neurons. Astrocytic glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) together play a predominant role in maintaining glutamate at normal extracellular concentrations. Though our previous studies reported the alleviation of tGCI-induced neuronal death by hypoxic preconditioning (HPC) in hippocampal Cornu Ammonis 1 (CA1) of adult rats, the underlying mechanism has not yet been fully elaborated. In this study, we aimed to investigate the roles of GLT-1 and GS in the neuroprotection mediated by HPC against tGCI and to ascertain whether these roles can be regulated by connexin 43 (Cx43) and cellular-Src (c-Src) activity. We found that HPC decreased the level of extracellular glutamate in CA1 after tGCI via maintenance of GLT-1 expression and GS activity. Inhibition of GLT-1 expression with dihydrokainate (DHK) or inhibition of GS activity with methionine sulfoximine (MSO) abolished the neuroprotection induced by HPC. Also, HPC markedly upregulated Cx43 and inhibited p-c-Src expression in CA1 after tGCI, whereas inhibition of Cx43 with Gap26 dramatically reversed this effect. Furthermore, inhibition of p-c-Src with 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine (PP2) decreased c-Src activity, increased protein levels of GLT-1 and Cx43, enhanced GS activity, and thus reduced extracellular glutamate level in CA1 after tGCI. Collectively, our data demonstrated that reduced extracellular glutamate induced by HPC against tGCI through preventing the reduction of GLT-1 expression and maintaining GS activity in hippocampal CA1, which was mediated by upregulating Cx43 expression and inhibiting c-Src activity.

Astaxanthin ameliorates experimental diabetes-induced renal oxidative stress and fibronectin by upregulating connexin43 in glomerular mesangial cells and diabetic mice

Oxidative stress is the major cause of renal fibrosis in the progression of DN. Connexin43 (Cx43) exerts an anti-fibrosis effect on diabetic kidneys. The current study aimed to investigate whether astaxanthin (AST) could ameliorate the pathological progression of DN by upregulating Cx43 and activating the Nrf2/ARE signaling, which is a pivotal anti-oxidative stress system, to strengthen the cellular anti-oxidative capacity and diminish fibronectin (FN) accumulation in HG-induced glomerular mesangial cells (GMCs). Our hypothesis was verified in GMCs and the kidneys from db/db mice by western blot, immunofluorescence, immunohistochemistry, immunoprecipitation, dual luciferase reporter assay and reactive oxygen related detection kits. Results showed that AST simultaneously upregulated the Cx43 protein level and promoted the Nrf2/ARE signaling activity in the kidney of db/db mice and HG-treated GMCs. However, Cx43 depletion abrogated the Nrf2/ARE signaling activation induced by AST. AST reduced the interaction between c-Src and Nrf2 in the nuclei of GMCs cultured with HG, thereby enhancing the Nrf2 accumulation in the nuclei of GMCs. Our data suggested that AST promoted the Nrf2/ARE signaling by upregulating the Cx43 protein level to prevent renal fibrosis triggered by HG in GMCs and db/db mice. c-Src acted as a mediator in these processes.

Effects of bisphenol A on gap junctions in HaCaT cells as mediated by the estrogen receptor pathway.

Bisphenol A (BPA) is widely used as the raw material for the production of plastics and paper products. People can be exposed to BPA through dermal contact, particularly for cashiers in contact with thermal paper every day. BPA is a known endocrine disruptor that has been shown to be carcinogenic. Many tumors show weak gap junctional intercellular communication (GJIC) function. The aim of this study was to investigate the effects and possible mechanisms of BPA's action on GJIC of human HaCaT skin cells. The results showed that BPA increased cell proliferation rates, prolonged GJIC photobleaching fluorescence recovery times and reduced overall fluorescence recovery rates at 0.01, 0.1 and 1μm. BPA downregulated connexin (Cx)26 mRNA level at 0.1μm. Estrogen receptor (ER) antagonist ICI 182780 at 5nm partially blocked the above effects of BPA indicating involvement of the ER pathway with BPA exposure. However, BPA did not influence Cx43 mRNA and protein levels. Our immunofluorescence data showed that Cx26 was expressed in the cytoplasm and plasma membrane, and was involved in the formation of gap junctions between adjacent cells, while Cx43 was expressed only in the cytoplasm. Therefore, our data indicate that Cx26 gap junctions may be involved in the GJIC inhibition caused by BPA. In conclusion, our results indicate that BPA can promote human skin cell proliferation, inhibit skin cell GJIC function but not formation and downregulate Cx26 mRNA levels partially through the ER pathway. We hypothesize that BPA can exhibit carcinogenicity by inhibiting GJIC.

Specific deletion connexin43 in astrocyte ameliorates cognitive dysfunction in APP/PS1 mice

Emerging data indicate an important role for connexin43 (Cx43) in cognitive function, but there is a lack of direct evidence of the role of astroglial Cx43 in cognitive dysfunction in Alzheimer's disease (AD). Here we evaluated the expression pattern of Cx43 in AD and found progressive upregulation of the mRNA and protein levels of Cx43. Subsequently, we generated an astroglial Cx43 knockout (KO) AD mouse model by crossbreeding Gfap (glial fibrillary acidic protein)-Cx43 KO mice with APP/PS1 mice. Then we assessed the cognitive function of 12-month-old APP (amyloid precursor protein)/PS1 (presenilin 1)/Gfap-Cx43 KO mice, which demonstrated that the deletion of astroglial Cx43 significantly ameliorated cognitive dysfunction. To further investigate the underlying mechanisms, we evaluated amyloid plaque formation, astrogliosis, and synaptic function. The number and area of amyloid plaques were not altered, but GFAP expression was significantly decreased and the number of synapses was markedly upregulated. These results suggest that deletion of astroglial Cx43 in APP/PS1 mice did not affect the formation of amyloid plaques but depressed astrogliosis and upregulated synaptic function. Moreover, levels of critical modulators of astroglial activation were also notably reduced, but those of pro- and anti-inflammatory cytokines were not altered. Furthermore, Cx43 regulation of postsynaptic elements targets mainly NMDAR (N-methyl-d-aspartate). In addition, the prevention of AD pathology was reversed by Cx43 re-expression. In sum, specific deletion of astroglial Cx43 in APP/PS1 mice improved cognitive dysfunction by decreasing astrogliosis and increasing synaptic function without affecting amyloid plaque formation or the inflammatory response.

PO-131 Over expression of extracellular matrix protein 1 (ECM1) in muscle-invasive bladder cancer

IntroductionThe connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells to directly exchange ions and small molecules. The connexin channels assemble into distinct plasma membrane domains known as gap junctions. Intercellular communication via gap junctions has an important role in regulating cell growth, differentiation and in maintaining tissue homeostasis. The most ubiquitously expressed connexin isoform in human tissues, connexin 43 (Cx43), acts as a tumour suppressor in multiple tissue types and is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the regulation of Cx43 degradation remains poorly understood.Material and methodsThe cervical cancer cell lines HeLa and C33a were used as model systems. Silencing and ectopic overexpression of proteins were performed by siRNA and plasmid transfection, respectively. The Cx43 ubiquitination status was determined using immunoprecipitation and western blotting. Gap junction intercellular communication (GJIC) was measured using the scrape loading dye transfer assay. Cells were imaged using confocal microscopy.Results and discussionsHere, we identify a member of the NEDD4 (neural precursor cell-expressed developmentally downregulated gene 4) family of E3 ubiquitin ligases, termed ITCH, as a novel regulator of Cx43 degradation and GJIC. Depletion of ITCH resulted in increased Cx43 protein levels, gap junction size and GJIC. Ectopic overexpression of ITCH, but not a catalytically inactive ITCH mutant, led to decreased Cx43 protein levels and loss of gap junctions. The data further indicate that ITCH acts in concert with two other members of the NEDD4 family that previously have been shown to regulate Cx43 degradation, termed NEDD4 and SMURF2. Simultaneous depletion of NEDD4, SMURF2, and ITCH by siRNA was found to result in a significantly lower Cx43 ubiquitination level and reduced Cx43 degradation under basal conditions. The triple knock-down of these three E3 ubiquitin ligases was also found to strongly counteract the TPA (12-O-tetradecanoylphorbol 13-acetate)-induced degradation of Cx43.ConclusionThese data identify ITCH as a novel regulator of Cx43 degradation and GJIC. The data also indicate that ITCH acts together with NEDD4 and SMURF2 to mediate the basal and TPA-induced turnover of Cx43.

A study on anti-arrhythmia mechanisms of resveratrol on ischemia/reperfusion in rats by regulating PI3K/Akt signaling pathway

To investigate whether the phosphatidyl-inositol-3-kinase/protein kinase B(PI3K/Akt)pathway is involved in anti-arrhythmia of resveratrol on ischemia/reperfusion in rat hearts. Forty male rats of normal ECG were randomly divided into 4 groups (n=10):sham control (SC) group, ischemic/reperfusion (I/R) group, resveratrol (Res) group, PI3K inhibitor LY294002 (LY294002) group. The rat myocardial I/R injury model was established in vivo. The arrhythmia and left ventricular functional parameters including left ventricular pressure (LVP), left ventricular systolic pressure (LVSP) and its derivate (±dp/dtmax) were measured; the protein levels of total Akt, phosphorylated Akt and connexin 43 (Cx43) were measured by Western blot; the mRNA level of Cx43 was detected by Real-time PCR. The phosphorylated levels of Akt and myocardial Cx43 were significantly enhanced in Res group as compared with I/R group(P < 0.01); whereas the incidence rate of induced ventricular reperfusion arrhythmias was significantly lower, the left ventricular function was evident-ly enhanced. After addition of PI3K inhibitor LY294002, the protein and mRNA levels of Akt and Cx43 were decreased in LY294002 group, while the incidence rate of reperfusion arrhythmias was significantly higher and the left ventricular function were evidently damaged compared with Res group(P < 0.01). Resveratrol could prevent the occurrence of reperfusion arrhythmias by increasing the content and ac-tivity of myocardial Cx43 through the PI3K/Akt signaling pathway.

The ubiquitin-specific protease USP8 deubiquitinates and stabilizes Cx43

Connexin-43 (Cx43, also known as GJA1) is the most ubiquitously expressed connexin isoform in mammalian tissues. It forms intercellular gap junction (GJ) channels, enabling adjacent cells to communicate both electrically and metabolically. Cx43 is a short-lived protein which can be quickly degraded by the ubiquitin-dependent proteasomal, endolysosomal, and autophagosomal pathways. Here, we report that the ubiquitin-specific peptidase 8 (USP8) interacts with and deubiquitinates Cx43. USP8 reduces both multiple monoubiquitination and polyubiquitination of Cx43 to prevent autophagy-mediated degradation. Consistently, knockdown of USP8 results in decreased Cx43 protein levels in cultured cells and suppresses intercellular communication, revealed by the dye transfer assay. In human breast cancer specimens, the expression levels of USP8 and Cx43 proteins are positively correlated. Taken together, these results identified USP8 as a crucial and bona fide deubiquitinating enzyme involved in autophagy-mediated degradation of Cx43.

Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone

BackgroundGlia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown.MethodsPrimary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry.ResultsWe found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane’s Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane.ConclusionsWe propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

HoxA10 and HoxA11 Regulate the Expression of Contraction-Associated Proteins and Contribute to Regionalized Myometrium Phenotypes in Women.

A relaxed fundus (FUN) and a contracted lower uterine segment (LUS) of human myometrium are required for maintaining pregnancy. How this regional myometrium function is regulated remains unclear. We have previously reported that the homeobox protein A13 (HoxA13) is highly expressed in the LUS and can enhance the expression of contraction-associated proteins (CAPs). Here, we show that in contrast to HoxA13, HoxA10 and HoxA11 genes are expressed at significantly higher levels in myometrium tissues and primary myocytes from the FUN. When introduced exogenously into a human myometrial cell line, HoxA10 and HoxA11 suppress the messenger RNA (mRNA) levels of several CAP genes including interleukin-1 beta (IL-1β), IL-6, connexin 43 (Cx43), and cyclooxygenase 2 (Cox2). Consistently, enhanced HoxA10 and HoxA11 expressions strongly inhibited IL-1β and Cx43 protein levels. We further confirmed that higher expression of HoxA10 and HoxA11 genes in primary myocytes from the FUN compared to that from the LUS was associated with lower expression of IL-1β, IL-6, Cox2, and Cx43 genes. We conclude that the expression patterns of HoxA10, HoxA11, and HoxA13 and their actions in regulating CAP genes in FUN and LUS create regionalized myometrium phenotypes in women that may be important to control regionalized myometrium contractility for maintaining pregnancy.

Metoprolol protects cardiomyocytes in rabbit model of heart failure by regulating Cx43.

This study investigated the protective effect of metoprolol on cardiomyocytes in rabbits with heart failure and its possible mechanism. Sixty New Zealand white rabbits were randomly divided into infarction group and non-infarction group, 30 in each group. Myocardial infarction was constructed by ligation of anterior descending branch of coronary artery. Coronary artery threading without ligation after thoracotomy was performed for rabbits in non-infarction group. After model construction, rabbits in each group were further divided into control group (n=15) and metoprolol group (n=15), and fed with normal diet and normal diet + metoprolol. Animals were sacrificed 8 weeks later, and ventricular tissue around infarction area was collected. Expression of connexin 43 (Cx43) in myocardium was detected by immunohistochemistry. Expression of Cx43 protein and mRNA in each group was detected by western blot and reverse transcription PCR. The Cx43 protein was positively expressed in non-infarction group and was evenly distributed in intercellular space. Compared with non-infarction group, expression of Cx43 in infarction group was significantly decreased or even disappeared, while the decrease in expression level of Cx43 and the degree of dispersion were lower in metoprolol group than in control group. There was no significant difference in expression of level of Cx43 protein and mRNA between the subgroups of non-infarction group (P>0.05). In infarction group, expression level of Cx43 protein and mRNA in the metoprolol group were significantly higher than those in control group (P<0.05). The results showed that metoprolol can protect cardiomyocytes after myocardial infarction, and the possible mechanism is related to the regulation of Cx43 expression in cardiomyocytes.

Role of Cx43-Mediated NFкB Signaling Pathway in Ossification of Posterior Longitudinal Ligament: An In Vivo and In Vitro Study.

In vivo and in vitro experiments. To illustrate the further molecular mechanism of Cx43-mediated osteoblastic differentiation of ligament cells. Ossification of the posterior longitudinal ligament (OPLL) is one of the main causes of myelopathy in Asians, but its etiology has not been clarified. We have previously found the mechanical stress can upregulate Cx43 expression in ligament cells, which transduces mechanical signal to promote osteoblastic differentiation. The posterior longitudinal ligaments were collected intraoperatively. Ligament fibroblasts were isolated and cultured, and an in vitro mechanical loading model was established. In vivo and in vitro expression levels of Cx43 protein were compared between OPLL and non-OPLL patients. The activation of nuclear factor (NF)-κB (p65) signal and related inflammatory responses were detected in ligament cells under mechanical loading. The mechanical stress-induced inflammatory response and osteoblastic differentiation of OPLL cells were investigated after the treatment with Cx43 siRNA or NFкB (p65) inhibitor. We first confirmed higher Cx43 levels in both in vivo ligament tissue from OPLL patients and in vitro cultured OPLL cells. We also found NFκB (p65) signal and related inflammatory response were activated by mechanical stimulation. The activation of NFκB (p65) signal was dependent upon Cx43, as its knockdown reduced signal. Moreover, treatment with Cx43 siRNA or NFкB (p65) inhibitor significantly decreased the mechanical-induced inflammation response, but partly attenuated mechanical-stimulated osteoblastic differentiation of OPLL cells. Cx43-mediated NFкB (p65) signal played an important role in mechanical stress-induced OPLL by transduction of mechanical signal, while giving rise to the activation of inflammatory response in ligament fibroblastsLevel of Evidence: N/A.

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.