Upregulation of Cx43 Expression Under Stretch Condition is Mediated by TGF Beta1 and Cytoskeletal Network

2021 ◽  
Author(s):  
Yumeng Shi ◽  
Xinbo Li ◽  
Jin Yang
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Gap Junction ◽  
Umbilical Vein ◽  
Specific Inhibitor ◽  
Cx43 Expression ◽  
Cx43 Protein ◽  
Protein Ubiquitination ◽  
Cytoskeletal Network ◽  
Stretch Condition

Abstract Many physiological and pathophysiological processes in cells or tissues are involving mechanical stretch, which is inducing gap junction gene expression and cytokine TGF beta changes. However, the underlying mechanisms of gap junction gene expression changes remain unknown. Here, we showed that the expression of Cx43 at mRNA and protein level in Human umbilical-vein endothelial cells (HUVECs) is significantly increased after 24 h stretch stimulation, and TGF beta1, but not TGF beta2 expression is also upregulated. Administration of TGF betal into HUVECs without stretch also induced upregulation of Cx43 mRNA and protein expression. While simultaneously administration of TGF beta1 with SB431542, a specific inhibitor of TGF beta1 receptor, blocked the Cx43 protein upregulation by TGF beta1. Further, the increase of Cx43 protein expression under stretch condition can be partially blocked by SB431542; moreover, it also can be partially blocked by simultaneously administration of anti-TGF beta1 monoclonal neutralization antibody. Importantly, the stretch induced upregulation of Cx43 can be blocked by administration of actin and microtubule inhibitors, while NEDD4, a key element in mediating Cx43 protein ubiquitination and degradation, is not changed under stretch condition. Therefore, we conclude that upregulation of Cx43 expression under 24 h stretch condition is mediated by TGF beta1 via TGF beta1 receptor signaling pathway, and it also involves the actin and microtubule cytoskeletal network.

scholarly journals Sphingosine 1-Phosphate Induces Myoblast Differentiation through Cx43 Protein Expression: A Role for a Gap Junction-dependent and -independent Function

2006 ◽  
Vol 17 (11) ◽  
pp. 4896-4910 ◽  
Author(s):  
R. Squecco ◽  
C. Sassoli ◽  
F. Nuti ◽  
M. Martinesi ◽  
F. Chellini ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gap Junction ◽  
P38 Mapk ◽  
Myoblast Differentiation ◽  
Dependent Manner ◽  
Cx43 Expression ◽  
Cx43 Protein ◽  
Junctional Communication ◽  
Sphingosine 1 Phosphate ◽  
Gap Junctional

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43Δ130–136 reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

Effects of Gingko biloba Extract on Gap Junction Changes Induced by Reperfusion/Reoxygenation After Ischemia/Hypoxia in Rat Brain

2005 ◽  
Vol 33 (06) ◽  
pp. 923-934 ◽  
Author(s):  
Zhen Li ◽  
Xian-Ming Lin ◽  
Pei-Li Gong ◽  
Fan-Dian Zeng ◽  
Guan-Hua Du
Keyword(s):  
Protein Expression ◽  
Gap Junction ◽  
Neurological Deficit ◽  
Ischemia Reperfusion ◽  
Cx43 Expression ◽  
Expression Levels ◽  
Gingko Biloba ◽  
Mrna And Protein Expression ◽  
Cx43 Mrna ◽  
Hypoxia Reoxygenation

Gap junction communication between astrocytes plays an important role in the brain. The purpose of this study was to investigate the effects of Gingko biloba extract (GBE) on the changes of connexin 43 (Cx43) mRNA and protein expression levels of rat cortex and hippocampus induced by ischemia-reperfusion and astrocyte gap junction intercellular communication (GJIC) induced by hypoxia-reoxygenation. After 2 hours of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion, there was obvious neurological deficit in rats. Cx43 mRNA and protein expression levels of rat cortex and hippocampus in the ischemia hemisphere were decreased significantly. When GBE at doses of 50 and 100 mg/kg body weight was administrated by p.o. daily for 7 days, the neurological deficit was improved, and lower Cx43 mRNA and protein expression levels induced by ischemia-reperfusion were recovered to normal. The i.p. injection of nimodipine (0.7 mg/kg weight body) also showed improvement on neurological deficit and Cx43 expression levels. Astrocyte GJIC was measured by the fluorescence recovery after photobleaching (FRAP). Hypoxia-reoxygenation induced a significant decrease in GJIC. Pretreatment with GBE (100 mg/l) and nimodipine (1.6 mg/l) significantly prevented the hypoxia-reoxygenation inhibition of GJIC. These results suggest that GBE could exert its neuroprotective effects by improvement of Cx43 expression and GJIC induced by hypoxia/ischemia-reoxygenation/ reperfusion injury.

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells

2001 ◽  
Vol 280 (4) ◽  
pp. L680-L688 ◽  
Author(s):  
Andrea I. Alford ◽  
D. Eugene Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Gap Junction ◽  
Primary Culture ◽  
Alveolar Epithelial Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Cx43 Expression ◽  
Cx43 Protein ◽  
Alveolar Epithelial

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8–10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.

scholarly journals 14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel

2020 ◽  
Vol 319 (1) ◽  
pp. F52-F62
Author(s):  
Shan Chen ◽  
Xiuyan Feng ◽  
Xinxin Chen ◽  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Specific Inhibitor ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Protein Ubiquitination ◽  
293 Cells

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

Bortezomib Improves Endothelial Thromboresistance Via Induction of KLF Transcription Factors.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1890-1890
Author(s):  
Toyoko Hiroi ◽  
Clayton B Deming ◽  
Haige Zhao ◽  
Baranda S Hansen ◽  
Elisabeth K Arkenbout ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Protein Expression ◽  
Tissue Factor ◽  
Lung Tissue ◽  
Protein C ◽  
Umbilical Vein ◽  
Small Interfering Rnas ◽  
Gene And Protein Expression

Abstract Background: Patients with multiple myeloma (MM) are at high risk for venothromboembolic events (VTE). Recent studies, however, suggest that MM patients treated with bortezomib, an approved proteasome inhibitor with potent NF-kB inhibitory effects, appear to have a lower risk of VTE compared to those treated with other therapies. We hypothesize that this could be due to a beneficial effect of bortezomib on endothelial thromboresistance. Methods and Results: Human umbilical vein endothelial cells (HUVECs) were incubated with bortezomib for 20 hours and changes in the expression of a panel of coagulation and inflammation-related genes measured by qPCR. Bortezomib stimulated baseline expression of anticoagulant genes (thrombomodulin (TM), eNOS and tissue factor pathway inhibitor), suppressed baseline expression of pro-coagulant genes (vWF and protease activated receptor-1) and suppressed cytokine-mediated induction of E-selectin, VCAM-1 and tissue factor. Most pronounced, was the dose-dependent upregulation of TM, a member of the protein C anticoagulant pathway (229 ± 15% and 341 ± 7% of control, at 5 nM and 10 nM bortezomib, respectively, p <0.0001). Induction of TM gene expression was paralleled by a significant upregulation of TM protein expression, assessed by Western blot analysis, and by an increased capacity to generate activated protein C (205 ± 5% of control with 5 nM bortezomib, p <0.0001). Bortezomib-induced TM upregulation was blocked by cycloheximide, suggesting that induction of a transcriptional pathway, and not simply inhibition of the NF-kB pathway, was required. We therefore examined the effects of bortezomib on the expression of several Krüppel like transcription factors (KLFs) that are known to be important regulators of TM expression and endothelial thromboresistance. Bortezomib significantly upregulated the expression of KLF2, KLF4 and KLF6 in HUVECs (18 ± 1, 8 ± 1 and 2 ± 0.1-fold of control, respectively, p <0.0001 for each) following a 5 nM exposure for 20 hours. Knock-down experiments using small interfering RNAs revealed that KLF2 and KLF4, but not KLF6, play critical and synergistic roles in mediating bortezomib-induced TM upregulation. To determine the in vivo significance of these findings, mice were administered ascending doses of bortezomib for 7 days and TM expression measured in selected tissue. Compared to controls, a dose of 0.8 mg/kg bortezomib ip increased TM gene and protein expression in the liver by 7.0 ± 1.1 and 9.5 ± 2.9-fold, respectively (p <0.0001 for each) and TM gene expression in the kidney by 2.5 ± 0.2-fold (p <0.0001). There was no significant change in TM expression observed in heart and lung tissue. Paralleling changes in TM expression, expression of the KLF2 and KLF4 genes was also increased in the liver (2.1 ± 0.3 and 6.2 ± 1.5-fold of controls, respectively, p <0.001 for each) and kidney (1.9 ± 0.2 and 2.9 ± 0.3-fold of controls, respectively, p <0.01 for each), but not in heart or lung tissue. Conclusions: Bortezomib markedly stimulates endothelial TM expression, both in vitro and in vivo in a tissue-specific manner. TM upregulation appears dependent on the induction of KLF2 and KLF4 transcription factors rather than by inhibition of the NFkB pathways. Our findings provide a rationale for further studies of bortezomib-induced enhancement of endothelial thromboresistance in patients with MM and may help explain why these patients are reduced risk for VTE.

Liver X receptor-α and miR-130a-3p regulate expression of sphingosine 1-phosphate receptor 2 in human umbilical vein endothelial cells

2016 ◽  
Vol 310 (3) ◽  
pp. C216-C226 ◽  
Author(s):  
Aihui Fan ◽  
Qian Wang ◽  
Yongjun Yuan ◽  
Jilun Cheng ◽  
Lixian Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Inflammatory Diseases ◽  
Umbilical Vein ◽  
Specific Inhibitor ◽  
Barrier Dysfunction ◽  
Human Umbilical Vein ◽  
Tnf Α ◽  
Sphingosine 1 Phosphate ◽  
Umbilical Vein Endothelial Cells

Recent studies have shown that activation of liver X receptors (LXRs) attenuates the development of atherosclerosis, not only by regulating lipid metabolism but also by suppressing inflammatory signaling. Sphingosine 1-phosphate receptor 2 (S1PR2), an important inflammatory gene product, plays a role in the development of various inflammatory diseases. It was proposed that S1PR2 might be regulated by LXR-α. In the present study, the effect of LXR-α on tumor necrosis factor-α (TNF-α)-induced S1PR2 expression in human umbilical vein endothelial cells (HUVECs) was investigated and the underlying mechanism was explored. The results demonstrated that TNF-α led to an increase in S1PR2 expression and triggered a downregulation of LXR-α expression in HUVECs as well. Downregulation of LXR-α with specific small interfering RNA (siRNA) remarkably enhanced the primary as well as TNF-α-induced expression of S1PR2 in HUVECs. Activation of LXR-α by agonist GW3965 inhibited both primary and TNF-α-induced S1PR2 expression. GW3965 also attenuated S1PR2-induced endothelial barrier dysfunction. The data further showed that TNF-α induced a significant decrease in miR-130a-3p expression. Overexpression of miR-130a-3p with mimic product reduced S1PR2 protein expression, and inhibition of miR-130a-3p by specific inhibitor resulted in an increase in S1PR2 protein expression. Furthermore, activation of LXRs with agonist enhanced the expression of miR-130a-3p, and knockdown of LXR-α by siRNA suppressed miR-130a-3p expression. These results suggest that LXR-α might downregulate S1PR2 expression via miR-130a-3p in quiescent HUVECs. Stimulation of TNF-α attenuates the activity of LXR-α and results in enhanced S1PR2 expression.

Negative Transcriptional Regulation of Connexin 43 by Tbx2 in Rat Immature Coronal Sutures and ROS 17/2.8 Cells in Culture

2003 ◽  
Vol 40 (3) ◽  
pp. 284-290 ◽  
Author(s):  
James L. Borke ◽  
Jung-Ren Chen ◽  
Jack C. Yu ◽  
Roni J. Bollag ◽  
Maria F. Orellana ◽  
...  
Keyword(s):  
Protein Expression ◽  
Connexin 43 ◽  
Protein Localization ◽  
Cell Communication ◽  
Negative Regulator ◽  
Cranial Sutures ◽  
Transcriptional Level ◽  
Cx43 Expression ◽  
Cx43 Protein

Background Tbx2 is a member of the T-box family of transcriptional regulatory genes with an extensive but not yet fully understood role in embryonic development. This study explores the potential role of Tbx2 in calvarial morphogenesis. Objectives To explore the hypothesis that Tbx2 has a negative regulatory effect on the expression of connexin 43 (Cx43), a protein necessary for cell-to-cell communication; document the presence of Tbx2 protein in the developing cranial sutures; and determine the spatial pattern of expression of this developmentally regulated transcription factor in calvariae. Design The osteoblast-like cell line ROS 17/2.8 was stably transfected with sense or antisense Tbx2. Immunohistochemistry and Western blotting was used to study Tbx2 and Cx43 expression in these cells and sections of embedded developing coronal sutures. Results The ROS 17/2.8 cells transfected with antisense Tbx2 showed a decrease in expression of Tbx2 protein and an increase in expression of endogenous Cx43. The reverse is seen with sense-transfected cells. Both of these proteins are expressed in rat developing coronal sutures. The pattern of Tbx2 expression in the developing was also reciprocal to the pattern of Cx43 expression. Tbx2 protein is concentrated in the center of the sutural blastema, an area devoid of Cx43 protein localization. Conversely, Tbx2 protein expression is low in the periphery of the sutures, in which there is high Cx43 protein expression. Conclusions Taken together, these studies suggest that Tbx2 protein is a negative regulator of Cx43 expression at the transcriptional level in cranial sutures in vivo.

scholarly journals Ginkgolide B Inhibits JAM-A, Cx43, and VE-Cadherin Expression and Reduces Monocyte Transmigration in Oxidized LDL-Stimulated Human Umbilical Vein Endothelial Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Xueqing Liu ◽  
Wenjia Sun ◽  
Yanyang Zhao ◽  
Beidong Chen ◽  
Wei Wu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Umbilical Vein ◽  
Ginkgolide B ◽  
Cx43 Expression ◽  
Human Umbilical Vein ◽  
Monocyte Migration ◽  
Junction Protein ◽  
Umbilical Vein Endothelial Cells ◽  
Junction Proteins

Aim. To investigate the effect of ginkgolide B on junction proteins and the reduction of monocyte migration in oxidized low-density lipoprotein- (ox-LDL-) treated endothelial cells.Methods. Human umbilical vein endothelial cells (HUVECs) were used in the present study. Immunofluorescence and Western blot were performed to determine the expression of junctional adhesion molecule-A (JAM-A), connexin 43 (Cx43), and vascular endothelial cadherin (VE-cadherin). Monocyte migration was detected by the Transwell assay.Results. ox-LDL stimulation increased JAM-A expression by 35%, Cx43 expression by 24%, and VE-cadherin expression by 37% in HUVECs. Ginkgolide B (0.2, 0.4, and 0.6 mg/mL) dose-dependently abolished the expression of these junction proteins. The monocyte transmigration experiments showed that the level of monocyte migration was sixfold higher in the ox-LDL-treated group than in the control group. Ginkgolide B (0.6 mg/mL) nearly completely abolished monocyte migration. Both ginkgolide B and LY294002 suppressed Akt phosphorylation and the expression of these junction proteins in ox-LDL-treated endothelial cells. These results suggest that the ginkgolide B-induced inhibition of junction protein expression is associated with blockade of the PI3K/Akt pathway.Conclusion. Ginkgolide B suppressed junction protein expression and reduced monocyte transmigration that was induced by ox-LDL. Ginkgolide B may improve vascular permeability in atherosclerosis.

Asymmetric dimethylarginine damages connexin43-mediated endothelial gap junction intercellular communication

2009 ◽  
Vol 87 (6) ◽  
pp. 867-874 ◽  
Author(s):  
Su-Jie Jia ◽  
Zhi Zhou ◽  
Bi-Kui Zhang ◽  
Zhuo-Wei Hu ◽  
Han-Wu Deng ◽  
...  
Keyword(s):  
Gap Junction ◽  
P38 Mapk ◽  
Asymmetric Dimethylarginine ◽  
Umbilical Vein ◽  
Inhibitory Effect ◽  
Oxidase Inhibitor ◽  
Cx43 Expression ◽  
P38 Mapk Inhibitor ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase

Asymmetric dimethylarginine (ADMA), a major endogenous inhibitor of nitric oxide synthase, is recently defined as a novel atherogenic factor. Communication via gap junction (GJIC) is involved in the regulation of a variety of endothelial activities, such as cell differentiation and senescence. The aim of this study is to explore the effects of ADMA on connexin43 (Cx43) mediated endothelial GJIC. Lysophosphatidylcholine (LPC) caused the downregulation of Cx43 expression and GJIC dysfunction in cultured human umbilical vein endothelial cells (HUVECs), which were significantly ameliorated by decreasing ADMA accumulation. Furthermore, we found that ADMA (10 µmol·L–1, 24 h) markedly downregulated Cx43 expression and damaged GJIC function in HUVECs. ADMA also increased production of intracellular reactive oxygen species (ROS) and induced phosphorylation of p38 MAPK. Furthermore, the inhibitory effect of ADMA on Cx43-mediated GJIC could be attenuated by NADPH oxidase inhibitor diphenyleneiodonium and apocynin as well as p38 MAPK inhibitor SB203580, respectively. In conclusion, our present results suggest that ADMA inhibits endothelial GJIC function via downregulating Cx43 expression, which suggesting a novel mechanism linking between elevated ADMA level and progression of atherosclerosis.

scholarly journals Inhibition of gap junction communication in alveolar epithelial cells by 18α-glycyrrhetinic acid

1999 ◽  
Vol 276 (6) ◽  
pp. L1018-L1026 ◽  
Author(s):  
Yihe Guo ◽  
Cara Martinez-Williams ◽  
Kirk A. Gilbert ◽  
D. Eugene Rannels
Keyword(s):  
Epithelial Cells ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Glycyrrhetinic Acid ◽  
Alveolar Epithelial Cells ◽  
Cx43 Expression ◽  
Cx43 Protein ◽  
Alveolar Epithelial ◽  
Gap Junction Communication ◽  
Mammalian Lung

Cultured alveolar epithelial cells exhibit gap junction intercellular communication (GJIC) and express regulated levels of connexin (Cx) 43 mRNA and protein. Newly synthesized radiolabeled Cx43 protein equilibrates with phosphorylated Cx43 isoforms; these species assemble to form both connexons and functional gap junction plaques. The saponin 18α-glycyrrhetinic acid (GA) rapidly and reversibly blocks GJIC at low concentrations (5 μM). Extended exposure to 18α-GA at higher concentrations causes inhibition of GJIC and time- and dose-dependent reductions in both Cx43 protein and mRNA expression. The latter toxic effects are paralleled by disassembly of gap junction plaques and are reversed less readily than acute effects on GJIC. These observations demonstrate 18α-GA-sensitive regulation of intercellular communication in epithelial cells from the mammalian lung and suggest a role for Cx43 expression and phosphorylation in acute and chronic regulation of GJIC between alveolar epithelial cells.

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