Normalization of connexin 43 protein levels prevents cellular and functional signs of dystrophic cardiomyopathy in mice

Abstract The progesterone receptor (PR) plays important roles in the establishment and maintenance of pregnancy. By dynamic interactions with coregulators, PR represses the expression of genes that increase the contractile activity of myometrium and contribute to the initiation of labor. We have previously shown that PTB-associated RNA splicing factor (PSF) can function as a PR corepressor. In this report, we demonstrated that the PSF heterodimer partner, p54nrb (non-POU-domain-containing, octamer binding protein), can also function as a transcription corepressor, independent of PSF. p54nrb Interacts directly with PR independent of progesterone. In contrast to PSF, p54nrb neither enhances PR protein degradation nor blocks PR binding to DNA. Rather, p54nrb recruits mSin3A through its N terminus to the PR-DNA complex, resulting in an inhibition of PR-mediated transactivation of the progesterone-response element-luciferase reporter gene. PR also repressed transcription of the connexin 43 gene (Gja1), an effect dependent on the presence of an activator protein 1 site within the proximal Gja1 promoter. Mutation of this site abolished PR-mediated repression and decreased the recruitment of PR and p54nrb onto the Gja1 promoter. Furthermore, knockdown p54nrb expression by small interfering RNA alleviated PR-mediated repression on Gja1 transcription, whereas overexpression of p54nrb enhanced it. In the physiological context of pregnancy, p54nrb protein levels decrease with the approach of labor in the rat myometrium. We conclude that p54nrb is a transcriptional corepressor of PR. Decreased expression of p54nrb at the time of labor may act to derepress PR-mediated inhibition on connexin 43 expression and contribute to the initiation of labor.

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The ubiquitin-specific protease USP8 deubiquitinates and stabilizes Cx43

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.001315 ◽

2018 ◽

Vol 293 (21) ◽

pp. 8275-8284 ◽

Cited By ~ 12

Author(s):

Jian Sun ◽

Qianwen Hu ◽

Hong Peng ◽

Cheng Peng ◽

Liheng Zhou ◽

...

Keyword(s):

Connexin 43 ◽

Cultured Cells ◽

Deubiquitinating Enzyme ◽

Human Breast ◽

Dye Transfer ◽

Protein Levels ◽

Mammalian Tissues ◽

Specific Protease ◽

Bona Fide ◽

Ubiquitin Specific Protease

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.

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Mechanism of antrocytic connexin-43 autophagy degradation in oxygen-glucose deprivation and its effect on neuroinflammation

10.21203/rs.2.18795/v1 ◽

2019 ◽

Author(s):

Xinyu Wang ◽

Liangshu Feng ◽

Meiying Xin ◽

Yulei Hao ◽

Xu Wang ◽

...

Keyword(s):

Cerebral Ischemia ◽

Connexin 43 ◽

Cell Communication ◽

Glucose Deprivation ◽

In Vivo Model ◽

Oxygen Glucose Deprivation ◽

Cx43 Protein ◽

Protein Levels

Abstract Background : Connexin 43 (Cx43) are the most widely distributed gap junction proteins in the nervous system. Cx43 enables cell-to-cell communication and plays an important role in ion transport, substrate exchange and delivery of information , which have been implicated in cerebral ischemia injury. Our previous work revealed the relationships between Cx43 and glia-mediated neuroinflammation through the release of ATP in oxygen-glucose deprivation (OGD), which means degradation of Cx43 may improve neuroinflammatory damage during OGD injury . However, the roles of Cx43 degradation and neuroinflammation caused by OGD remain unclear. Methods: We used primary cultured astrocytes treated with OGD as an in vitro model of cerebral ischemia injury and we used middle cerebral artery occlusion (MCAO) model as an in vivo model of cerebral ischemia. HeLa cells were used in overexpression experiments. Cx43 protein levels were determined by western blotting. The interaction between Cx43 and related autophagy receptors was determined by co-immunoprecipitation and immunofluorescence. The gene knockdown (KD) of ATG5, OPTN, NDP52, PINK1 and Cx43 was applied by siRNA transfection. Related cytokines were detected by cytometric bead assay. Results: We found that Cx43 protein levels increased after ischemia in gene KD of ATG5, OPTN, NDP52 and PINK1 primary astrocytes. The interaction of Cx43 with OPTN, NDP52 and PINK1 was increased after cerebral ischemia injury in vitro and vivo. While the interaction was weakened after point mutation of Cx43 at Ser368, Tyr265 and Tyr247. Meanwhile, IL-10 upregulated during OGD after KD of ATG5, OPTN, NDP52 and PINK1 in astrocytes , while TNF downregulated during OGD after KD of ATG5, OPTN, NDP52 and PINK1 in astrocytes. Conclusions: Our results suggest that degradation of Cx43 is caused by selective autophagy during ischemia injury and the autophagy degradation of Cx43 plays important roles in neuroinflammation mediated by OGD injury. Treatment targeting Cx43 degradation pathway can improve neuroinflammation responses induced by OGD injury , which provide novel therapeutic strategies and crosstalk between autophagy and neuroinflammation.

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Antiarrhythmic Effects of Melatonin and Omega-3 Are Linked with Protection of Myocardial Cx43 Topology and Suppression of Fibrosis in Catecholamine Stressed Normotensive and Hypertensive Rats

Antioxidants ◽

10.3390/antiox9060546 ◽

2020 ◽

Vol 9 (6) ◽

pp. 546 ◽

Cited By ~ 3

Author(s):

Barbara Szeiffova Bacova ◽

Csilla Viczenczova ◽

Katarina Andelova ◽

Matus Sykora ◽

Kiranj Chaudagar ◽

...

Keyword(s):

Oxidative Stress ◽

Connexin 43 ◽

Electrical Coupling ◽

Threshold Current ◽

Prolonged Treatment ◽

Diffuse Fibrosis ◽

Omega 3 ◽

Hypertensive Rats ◽

Protein Levels ◽

Coupling Protein

Cardiac β-adrenergic overstimulation results in oxidative stress, hypertrophy, ischemia, lesion, and fibrosis rendering the heart vulnerable to malignant arrhythmias. We aimed to explore the anti-arrhythmic efficacy of the anti-oxidative and anti-inflammatory compounds, melatonin, and omega-3, and their mechanisms of actions in normotensive and hypertensive rats exposed to isoproterenol (ISO) induced β-adrenergic overdrive. Eight-month-old, male SHR, and Wistar rats were injected during 7 days with ISO (cumulative dose, 118 mg/kg). ISO rats were either untreated or concomitantly treated with melatonin (10 mg/kg/day) or omega-3 (Omacor, 1.68 g/kg/day) until 60 days of ISO withdrawal and compared to non-ISO controls. Findings showed that both melatonin and omega-3 increased threshold current to induce ventricular fibrillation (VF) in ISO rats regardless of the strain. Prolonged treatment with these compounds resulted in significant suppression of ISO-induced extracellular matrix alterations, as indicated by reduced areas of diffuse fibrosis and decline of hydroxyproline, collagen-1, SMAD2/3, and TGF-β1 protein levels. Importantly, the highly pro-arrhythmic ISO-induced disordered cardiomyocyte distribution of electrical coupling protein, connexin-43 (Cx43), and its remodeling (lateralization) were significantly attenuated by melatonin and omega-3 in Wistar as well as SHR hearts. In parallel, both compounds prevented the post-ISO-related increase in Cx43 variant phosphorylated at serine 368 along with PKCε, which are known to modulate Cx43 remodeling. Melatonin and omega-3 increased SOD1 or SOD2 protein levels in ISO-exposed rats of both strains. Altogether, the results indicate that anti-arrhythmic effects of melatonin and omega-3 might be attributed to the protection of myocardial Cx43 topology and suppression of fibrosis in the setting of oxidative stress induced by catecholamine overdrive in normotensive and hypertensive rats.

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Immunohistochemical Expression of RARα, RARβ, and Cx43 in Breast Tumor Cell Lines After Treatment With Lycopene and Correlation With RT-QPCR

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.7a7185.2007 ◽

2007 ◽

Vol 55 (9) ◽

pp. 877-883 ◽

Cited By ~ 20

Author(s):

Nasseéra Chalabi ◽

Laetitia Delort ◽

Samir Satih ◽

Pierre Déchelotte ◽

Yves-Jean Bignon ◽

...

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Connexin 43 ◽

Breast Cancer Cell Lines ◽

Pcr Analysis ◽

Cx43 Expression ◽

Real Time Quantitative Pcr ◽

Protein Levels ◽

Gap Junction Communication ◽

Mcf 7

Lycopene, the major carotenoid found in tomatoes, is a potent antioxidant associated with the prevention of degenerative diseases such as breast cancer. This effect could be due to the interaction between lycopene and retinoic acid receptors as well as the stimulation of gap junction communication and synthesis of connexin 43. The expression of the RARα, RARβ, and Cx43 proteins was analyzed using immunohistochemistry in two breast cancer cell lines, MCF-7 and MDA-MB-231, and in a fibrocystic dystrophy cell line, MCF-10a, after a 48-hr exposure to 10 μM lycopene. A real-time quantitative PCR analysis was then performed to measure mRNA expression. RARα and Cx43 expression were increased at both mRNA and protein levels in two breast cell lines.

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3,5,4′-tri-O-acetylresveratrol Ameliorates Seawater Exposure-Induced Lung Injury by Upregulating Connexin 43 Expression in Lung

Mediators of Inflammation ◽

10.1155/2013/182132 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 14

Author(s):

Lijie Ma ◽

Yanyan Li ◽

Yilin Zhao ◽

Qingwei Wang ◽

Yandong Nan ◽

...

Keyword(s):

Lung Injury ◽

Gap Junction ◽

Connexin 43 ◽

Umbilical Vein ◽

A549 Cells ◽

Inflammatory Factors ◽

Histological Changes ◽

Protein Levels ◽

Gap Junction Communication ◽

Umbilical Vein Endothelial Cells

The aim of the present study was to examine the effects of 3,5,4′-tri-O-acetylresveratrol on connexin 43 (Cx43) in acute lung injury (ALI) in rats induced by tracheal instillation of artificial seawater. Different doses (50, 150, and 450 mg/kg) of 3,5,4′-tri-O-acetylresveratrol were administered orally for 7 days before modeling. Four hours after seawater inhalation, histological changes, contents of TNF-α, IL-1βand IL-10, and the expression of Cx43 in lungs were detected. Besides, the gap junction communication in A549 cells and human umbilical vein endothelial cells (HUVECs) challenged by seawater was also evaluated. Histological changes, increased contents of inflammatory factors, upregulation in gene level, and deregulation in protein level of Cx43 in lungs stimulated by seawater were observed. On the other hand, pretreatment with 3,5,4′-tri-O-acetylresveratrol significantly inhibited infiltration of inflammation, development of pulmonary edema, and contents of inflammatory mediators in lungs. Above all, 3,5,4′-tri-O-acetylresveratrol upregulated the expression of Cx43 in both gene and protein levels, and its intermediate metabolite, resveratrol, also enhanced the gap junction communication in the two cell lines. The results of the present study suggested that administration of 3,5,4′-tri-O-acetylresveratrol may be beneficial for treatment of inflammatorycellsin lung.

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Omacor Protects Normotensive and Hypertensive Rats Exposed to Continuous Light from Increased Risk to Malignant Cardiac Arrhythmias

Marine Drugs ◽

10.3390/md19120659 ◽

2021 ◽

Vol 19 (12) ◽

pp. 659

Author(s):

Tamara Egan Benova ◽

Csilla Viczenczova ◽

Barbara Szeiffova Bacova ◽

Jitka Zurmanova ◽

Vladimir Knezl ◽

...

Keyword(s):

Connexin 43 ◽

Electrical Coupling ◽

Continuous Light ◽

Male Rat ◽

Human Populations ◽

Omega 3 ◽

Spontaneously Hypertensive ◽

Protein Levels ◽

Populations At Risk ◽

Increased Risk

Light pollution disturbs circadian rhythm, and this can also be deleterious to the heart by increased susceptibility to arrhythmias. Herein, we investigated if rats exposed to continuous light had altered myocardial gene transcripts and/or protein expression which affects arrhythmogenesis. We then assessed if Omacor® supplementation benefitted affected rats. Male and female spontaneously hypertensive (SHR) and normotensive Wistar rats (WR) were housed under standard 12 h/12 h light/dark cycles or exposed to 6-weeks continuous 300 lux light for 24 h. Half the rats were then treated with 200 mg/100 g b.w. Omacor®. Continuous light resulted in higher male rat vulnerability to malignant ventricular fibrillation (VF). This was linked with myocardial connexin-43 (Cx43) down-regulation and deteriorated intercellular electrical coupling, due in part to increased pro-inflammatory NF-κB and iNOS transcripts and decreased sarcoplasmic reticulum Ca2+ATPase transcripts. Omacor® treatment increased the electrical threshold to induce the VF linked with amelioration of myocardial Cx43 mRNA and Cx43 protein levels and the suppression of NF-κB and iNOS. This indicates that rat exposure to continuous light results in deleterious cardiac alterations jeopardizing intercellular Cx43 channel-mediated electrical communication, thereby increasing the risk of malignant arrhythmias. The adverse effects were attenuated by treatment with Omacor®, thus supporting its potential benefit and the relevance of monitoring omega-3 index in human populations at risk.

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Expression of connexin 43 in the human hair follicle: emphasis on the connexin 43 protein levels in the bulge and through the keratinization process

Journal of Cutaneous Pathology ◽

10.1111/cup.13050 ◽

2017 ◽

Vol 45 (1) ◽

pp. 8-15 ◽

Cited By ~ 3

Author(s):

Angel Fernandez Flores ◽

Adrian Varela-Vazquez ◽

Maria D. Mayan ◽

Eduardo Fonseca

Keyword(s):

Hair Follicle ◽

Human Hair ◽

Connexin 43 ◽

Human Hair Follicle ◽

Protein Levels

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Dexamethasone-induced insulin resistance is associated with increased connexin 36 mRNA and protein expression in pancreatic rat islets

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y07-037 ◽

2007 ◽

Vol 85 (5) ◽

pp. 536-545 ◽

Cited By ~ 41

Author(s):

A. Rafacho ◽

L.P. Roma ◽

S.R. Taboga ◽

A.C. Boschero ◽

J.R. Bosqueiro

Keyword(s):

Insulin Resistance ◽

Protein Expression ◽

Pancreatic Islets ◽

Connexin 43 ◽

Glucose Transporter ◽

Insulin Resistant ◽

Protein Levels ◽

Connexin 36 ◽

Peripheral Insulin Resistance ◽

Glucose Stimulated Insulin Secretion

Augmented glucose-stimulated insulin secretion (GSIS) is an adaptive mechanism exhibited by pancreatic islets from insulin-resistant animal models. Gap junction proteins have been proposed to contribute to islet function. As such, we investigated the expression of connexin 36 (Cx36), connexin 43 (Cx43), and the glucose transporter Glut2 at mRNA and protein levels in pancreatic islets of dexamethasone (DEX)-induced insulin-resistant rats. Study rats received daily injections of DEX (1 mg/kg body mass, i.p.) for 5 days, whereas control rats (CTL) received saline solution. DEX rats exhibited peripheral insulin resistance, as indicated by the significant postabsorptive insulin levels and by the constant rate for glucose disappearance (KITT). GSIS was significantly higher in DEX islets (1.8-fold in 16.7 mmol/L glucose vs. CTL, p < 0.05). A significant increase of 2.25-fold in islet area was observed in DEX vs. CTL islets (p < 0.05). Cx36 mRNA expression was significantly augmented, Cx43 diminished, and Glut2 mRNA was unaltered in islets of DEX vs. CTL (p < 0.05). Cx36 protein expression was 1.6-fold higher than that of CTL islets (p < 0.05). Glut2 protein expression was unaltered and Cx43 was not detected at the protein level. We conclude that DEX-induced insulin resistance is accompanied by increased GSIS and this may be associated with increase of Cx36 protein expression.

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Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01058.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. H806-H814 ◽

Cited By ~ 18

Author(s):

Tepmanas Bupha-Intr ◽

Kaylan M. Haizlip ◽

Paul M. L. Janssen

Keyword(s):

Connexin 43 ◽

Mechanical Load ◽

Contractile Function ◽

Underlying Mechanism ◽

Paracrine Factors ◽

Cx43 Expression ◽

Protein Levels ◽

Hypertrophied Myocardium ◽

Load Conditions

Upon remodeling of the ventricle after a provoking stimulus, such as hypertension, connections between adjacent myocytes may need to be “reformatted” to preserve a synchronization of excitation of the remodeling heart. In the mammalian heart, the protein connexin forms the gap junctions that allow electrical and chemical signaling communication between neighboring cells. We aim to elucidate whether mechanical load, in isolation, potentially changes the expression of connexin 43 (Cx43), the major isoform of the connexin family in the ventricle, and its phosphorylation. Cx43 expression levels and contractile function of multicellular rabbit cardiac preparations were assessed in a newly developed in vitro system that allows for the study of the transition of healthy multicellular rabbit myocardium to hypertrophied myocardium. We found that in mechanically loaded cardiac trabeculae, Cx43 levels remained stable for about 12 h and then rapidly declined. Phosphorylation at Ser368 declined much faster, being almost absent after 2 h of high-load conditions. No-load conditions did not affect Cx43 levels, nor did phosphorylation at Ser368. The downregulation of Cx43 under mechanical load did not correspond with the contractile changes that were observed. Furthermore, blocking paracrine activity of the muscle could only partially prevent the downregulation of Cx43. Additionally, no effect of mechanical loading on the expression of N-cadherin and zonula occludens-1 was observed, indicating a specificity of the connexin response. High mechanical load induced a rapid loss of Cx43 phosphorylation, followed by a decrease in Cx43 protein levels. Paracrine factors are partly responsible for the underlying mechanism of action, whereas no direct correlation to contractile ability was observed.

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