Myoendothelial gap junctions mediate regulation of angiopoietin-2-induced vascular hyporeactivity after hypoxia through connexin 43-gated cAMP transfer

Angiopoietin-2 (Ang-2) contributes to vascular hyporeactivity after hemorrhagic shock and hypoxia through upregulation of inducible nitric oxide synthase (iNOS) in a vascular endothelial cell (VEC)-specific and Ang-2/Tie2 receptor-dependent manner. While iNOS is primarily expressed in vascular smooth muscle cells (VSMCs), the mechanisms of signal transfer from VECs to VSMCs are unknown. A double-sided coculture model with VECs and VSMCs from Sprague-Dawley rats was used to investigate the role of myoendothelial gap junctions (MEGJs), the connexin (Cx) isoforms involved, and other relevant mechanisms. After hypoxia, VSMCs treated with exogenous Ang-2 showed increased iNOS expression and hyporeactivity, as well as MEGJ formation and communication. These Ang-2 effects were suppressed by the MEGJ inhibitor 18α-glycyrrhetic acid (18-GA), Tie2 siRNA, or Cx43 siRNA. Reagents antagonizing cAMP or protein kinase A (PKA) in VECs inhibited Cx43 expression in MEGJs, decreasing MEGJ formation and associated communication, after hypoxia following Ang-2 treatment. The increased cAMP levels in VSMCs and transfer of Alexa Fluor 488-labeled cAMP from VECs to VSMCs, after hypoxia following Ang-2 treatment, was antagonized by Cx43 siRNA. A cAMP antagonist added to VECs or VSMCs inhibited both increased iNOS expression and hyporeactivity in VSMCs subjected to hypoxia following Ang-2 treatment. Based on these findings, we propose that Cx43 was the Cx isoform involved in MEGJ-mediated VEC-dependent regulation of Ang-2, which induces iNOS protein expression and vascular hyporeactivity after hypoxia. Cx43 was upregulated by cAMP and PKA, permitting cAMP transfer between cells.

Download Full-text

CX43 expression, phosphorylation, and distribution in the normal and autoimmune orchitic testis with a look at gap junctions joining germ cell to germ cell

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00500.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. R121-R139 ◽

Cited By ~ 13

Author(s):

R.-Marc Pelletier ◽

Casimir D. Akpovi ◽

Li Chen ◽

Robert Day ◽

María L. Vitale

Keyword(s):

Cell Differentiation ◽

Gap Junctions ◽

Germ Cell ◽

Sertoli Cell ◽

Sertoli Cells ◽

Seminiferous Tubule ◽

Connexin 43 ◽

Cx43 Expression ◽

Cx43 Protein ◽

Cx43 Mrna

Spermatogenesis requires connexin 43 (Cx43).This study examines normal gene transcription, translation, and phosphorylation of Cx43 to define its role on germ cell growth and Sertoli cell's differentiation, and identifies abnormalities arising from spontaneous autoimmune orchitis (AIO) in mink, a seasonal breeder and a natural model for autoimmunity. Northern blot analysis detected 2.8- and a 3.7-kb Cx43 mRNA bands in seminiferous tubule-enriched fractions. Cx43 mRNA increased in seminiferous tubule-enriched fractions throughout development and then seasonally with the completion of spermatogenesis. Cx43 protein levels increased transiently during the colonization of the tubules by the early-stage spermatocytes. Cx43 phosphorylated (PCx43) and nonphosphorylated (NPCx43) in Ser368 decreased during the periods of completion of meiosis and Sertoli cell differentiation, while Cx43 mRNA remained elevated throughout. PCx43 labeled chiefly the plasma membrane except by stage VII when vesicles were also labeled in Sertoli cells. Vesicles and lysosomes in Sertoli cells and the Golgi apparatus in the round spermatids were NPCx43 positive. A decrease in Cx43 gene expression was matched by a Cx43 protein increase in the early, not the late, phase of AIO. Total Cx43 and PCx43 decreased with the advance of orchitis. The study makes a novel finding of gap junctions connecting germ cells. The data indicate that Cx43 protein expression and phosphorylation in Ser368 are stage-specific events that may locally influence the acquisition of meiotic competence and the Sertoli cell differentiation in normal testis. AIO modifies Cx43 levels, suggesting changes in Cx43-mediated intercommunication and spermatogenic activity in response to cytokines imbalances in Sertoli cells.

Download Full-text

Ischemia Alters the Expression of Connexins in the Aged Human Brain

Journal of Biomedicine and Biotechnology ◽

10.1155/2009/147946 ◽

2009 ◽

Vol 2009 ◽

pp. 1-8 ◽

Cited By ~ 20

Author(s):

Taizen Nakase ◽

Tetsuya Maeda ◽

Yasuji Yoshida ◽

Ken Nagata

Keyword(s):

Gap Junctions ◽

Connexin 43 ◽

Immunohistochemical Analysis ◽

Analysis Data ◽

Computer Assisted ◽

Ischemic Insult ◽

Cx43 Expression ◽

Neuronal Marker ◽

Neuronal Markers ◽

Human Stroke

Although the function of astrocytic gap junctions under ischemia is still under debate, increased expression of connexin 43 (Cx43) has been observed in ischemic brain lesions, suggesting that astrocytic gap junctions could provide neuronal protection against ischemic insult. Moreover, different connexin subtypes may play different roles in pathological conditions. We used immunohistochemical analysis to investigate alterations in the expression of connexin subtypes in human stroke brains. Seven samples, sectioned after brain embolic stroke, were used for the analysis. Data, evaluated semiquantitatively by computer-assisted densitometry, was compared between the intact hemisphere and ischemic lesions. The results showed that the coexpression of Cx32 and Cx45 with neuronal markers was significantly increased in ischemic lesions. Cx43 expression was significantly increased in the colocalization with astrocytes and relatively increased in the colocalization with neuronal marker in ischemic lesions. Therefore, Cx32, Cx43, and Cx45 may respond differently to ischemic insult in terms of neuroprotection.

Download Full-text

Expression of connexin 43 (Cx43) is critical for normal hematopoiesis

Blood ◽

10.1182/blood.v96.3.917 ◽

2000 ◽

Vol 96 (3) ◽

pp. 917-924 ◽

Cited By ~ 61

Author(s):

Encarnacion Montecino-Rodriguez ◽

Hyosuk Leathers ◽

Kenneth Dorshkind

Keyword(s):

Bone Marrow ◽

T Cell ◽

Blood Cell ◽

Gap Junctions ◽

Connexin 43 ◽

Cell Formation ◽

Cx43 Expression ◽

Hematopoietic System ◽

Blood Cell Formation

Abstract Gap junctions are intercellular channels, formed by individual structural units known as connexins (Cx), that allow the intercellular exchange of various messenger molecules. The finding that numbers of Cx43-type gap junctions in bone marrow are elevated during establishment and regeneration of the hematopoietic system has led to the hypothesis that expression of Cx43 is critical during the initiation of blood cell formation. To test this hypothesis, lymphoid and myeloid development were examined in mice with a targeted disruption of the gene encoding Cx43. Because Cx43−/− mice die perinatally, initial analyses were performed on Cx43−/−, Cx43+/−, and Cx43+/+ embryos and newborns. The data indicate that lack of Cx43 expression during embryogenesis compromises the terminal stages of primary T and B lymphopoiesis. Cx43−/− embryos and neonates had a reduced frequency of CD4+ and T-cell receptor-expressing thymocytes and surface IgM+cells compared to their Cx43+/+ littermates. Surprisingly, Cx43+/− embryos/neonates also showed defects in B- and T-cell development similar to those observed in Cx43−/− littermates, but their hematopoietic system was normal at 4 weeks of age. However, the regeneration of lymphoid and myeloid cells was severely impaired in the Cx43+/− mice after cytoablative treatment. Taken together, these data indicate that loss of a single Cx43 allele can affect blood cell formation. Finally, the results of reciprocal bone marrow transplants between Cx43+/+ and Cx43+/− mice and examination of hematopoietic progenitors and stromal cells in vitro indicates that the primary effects of Cx43 are mediated through its expression in the hematopoietic microenvironment.

Download Full-text

Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells

Cancers ◽

10.3390/cancers12102798 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2798

Author(s):

Alexander Tishchenko ◽

Daniel D. Azorín ◽

Laia Vidal-Brime ◽

María José Muñoz ◽

Pol Jiménez Arenas ◽

...

Keyword(s):

Breast Cancer ◽

Gap Junctions ◽

Small Molecules ◽

Cancer Cells ◽

Signaling Pathways ◽

Connexin 43 ◽

Breast Cancer Cell Lines ◽

Tunneling Nanotubes ◽

Cx43 Expression ◽

Cancer Signaling

Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that the loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by the inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, the drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type (WT) cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

Download Full-text

Connexin-43 dependent ATP release mediates macrophage activation during peritonitis

10.1101/424333 ◽

2018 ◽

Author(s):

Michel Dosch ◽

Joël Zindel ◽

Fadi Jebbawi ◽

Nicolas Melin ◽

Daniel Sanchez-Taltavull ◽

...

Keyword(s):

Connexin 43 ◽

Macrophage Activation ◽

Purinergic Receptor ◽

Atp Release ◽

Peritoneal Macrophages ◽

Dependent Manner ◽

Autocrine Signaling ◽

Cx43 Expression ◽

Pulmonary Macrophages ◽

And Control

ABSTRACTPeritonitis is the consequence of bacterial spillage into a sterile environment by gastrointestinal hollow-organ perforation that may lead to fulminant sepsis. Outcome of peritonitis-induced sepsis critically depends on macrophage activation by extracellular ATP release and associated para- and autocrine signaling via purinergic receptors. Mechanisms that mediate and control ATP release, however, are poorly understood. Here we show that TLR-2 and -4 agonists trigger ATP release via Connexin-43 (CX43) hemichannels in peritoneal macrophages leading to poor survival during sepsis. In humans, CX43 expression was upregulated on macrophages isolated from the peritoneal cavity in patients with intraperitoneal infection but not in healthy controls. Using a murine caecal ligation and puncture (CLP) model, we identified increased CX43 expression in activated infiltrating peritoneal, hepatic and pulmonary macrophages. Conditional MAC-CX43 KO Lyz2cre/creCx43flox/flox mice were developed to specifically assess the CX43 impact in macrophages. Both macrophage-specific CX43 deletion (using Lyz2cre/creCx43flox/flox mice) or pharmacological CX43 blockade were associated with reduced cytokine secretion by macrophages in response to LPS and CLP. This was ultimately resulting in increased survival in Lyz2cre/creCx43flox/flox mice and after pharmacological blockade. Specific inhibition of the purinergic receptor P2RY1 abrogated CX43 elicited cytokine responses. In conclusion, inhibition of autocrine ATP signaling via CX43 on macrophages and P2RY1 improves sepsis outcome in experimental peritonitis.Brief SummaryConnexin-43-mediated ATP release from macrophages in response to TLR-4 and -2 agonists modulates autocrine activation of macrophages in a P2Y1-dependent manner, ultimately determining sepsis survival.

Download Full-text

Connexin 43 Expression on Peripheral Blood Eosinophils: Role of Gap Junctions in Transendothelial Migration

BioMed Research International ◽

10.1155/2014/803257 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 10

Author(s):

Harissios Vliagoftis ◽

Cory Ebeling ◽

Ramses Ilarraza ◽

Salahaddin Mahmudi-Azer ◽

Melanie Abel ◽

...

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Peripheral Blood ◽

Connexin 43 ◽

Transendothelial Migration ◽

Glycyrrhetinic Acid ◽

Dependent Manner ◽

Dye Transfer ◽

Blood Eosinophils

Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

Download Full-text

Amyloid-β regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013705 ◽

2020 ◽

Vol 295 (44) ◽

pp. 15097-15111

Author(s):

Mahua Maulik ◽

Lakshmy Vasan ◽

Abhishek Bose ◽

Saikat Dutta Chowdhury ◽

Neelanjana Sengupta ◽

...

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Connexin 43 ◽

Cx43 Expression ◽

Chemical Chaperone ◽

Junction Protein ◽

Gap Junction Coupling ◽

Gap Junction Protein ◽

Junction Coupling ◽

And Function

Altered expression and function of astroglial gap junction protein connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). Although earlier studies have examined the effect of increased β-amyloid (Aβ) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aβ modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aβ can alter Cx43 gap junctions. We show that Aβ25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aβ25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aβ25-35-exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aβ-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aβ25-35-exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aβ25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aβ can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aβ modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

Download Full-text

Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells

10.20944/preprints202009.0192.v1 ◽

2020 ◽

Author(s):

Alexander Tishchenko ◽

Daniel Domínguez Azorín ◽

Laia Vidal-Brime ◽

María José Muñoz ◽

Pol Jiménez Arenas ◽

...

Keyword(s):

Breast Cancer ◽

Gap Junctions ◽

Small Molecules ◽

Cancer Cells ◽

Signaling Pathways ◽

Connexin 43 ◽

Breast Cancer Cell Lines ◽

Tunneling Nanotubes ◽

Cx43 Expression ◽

Cancer Signaling

Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

Download Full-text

Insight into the Role and Regulation of Gap Junction Genes in Lung Cancer and Identification of Nuclear Cx43 as a Putative Biomarker of Poor Prognosis

Cancers ◽

10.3390/cancers11030320 ◽

2019 ◽

Vol 11 (3) ◽

pp. 320 ◽

Cited By ~ 6

Author(s):

Trond Aasen ◽

Irene Sansano ◽

Maria Ángeles Montero ◽

Cleofé Romagosa ◽

Jordi Temprana-Salvador ◽

...

Keyword(s):

Lung Cancer ◽

Overall Survival ◽

Gap Junctions ◽

Mrna Expression ◽

Poor Prognosis ◽

Dependent Manner ◽

Cx43 Expression ◽

Lung Tumours ◽

Connexin Expression ◽

Independent Events

Direct intercellular communication, mediated by gap junctions formed by the connexin transmembrane protein family, is frequently dysregulated in cancer. Connexins have been described as tumour suppressors, but emerging evidence suggests that they can also act as tumour promoters. This feature is connexin- and tissue-specific and may be mediated by complex signalling pathways through gap junctions or hemichannels or by completely junction-independent events. Lung cancer is the number one cancer in terms of mortality worldwide, and novel biomarkers and therapeutic targets are urgently needed. Our objective was to gain a better understanding of connexins in this setting. We used several in silico tools to analyse TCGA data in order to compare connexin mRNA expression between healthy lung tissue and lung tumours and correlated these results with gene methylation patterns. Using Kaplan-Meier plotter tools, we analysed a microarray dataset and an RNA-seq dataset of non-small cell lung tumours in order to correlate connexin expression with patient prognosis. We found that connexin mRNA expression is frequently either upregulated or downregulated in lung tumours. This correlated with both good and poor prognosis (overall survival) in a clear connexin isoform-dependent manner. These associations were strongly influenced by the histological subtype (adenocarcinoma versus squamous cell carcinoma). We present an overview of all connexins but particularly focus on four isoforms implicated in lung cancer: Cx26, Cx30.3, Cx32 and Cx43. We further analysed the protein expression and localization of Cx43 in a series of 73 human lung tumours. We identified a subset of tumours that exhibited a unique strong nuclear Cx43 expression pattern that predicted worse overall survival (p = 0.014). Upon sub-stratification, the prognostic value remained highly significant in the adenocarcinoma subtype (p = 0.002) but not in the squamous carcinoma subtype (p = 0.578). This finding highlights the importance of analysis of connexin expression at the protein level, particularly the subcellular localization. Elucidation of the underlying pathways regulating Cx43 localization may provide for novel therapeutic opportunities.

Download Full-text

Astrocyte Networks and Epilepsy: When Stars Collide

Epilepsy Currents ◽

10.1111/j.1535-7511.2009.01310.x ◽

2009 ◽

Vol 9 (4) ◽

pp. 113-115 ◽

Cited By ~ 1

Author(s):

Michael Wong

Keyword(s):

Synaptic Transmission ◽

Gap Junctions ◽

Connexin 43 ◽

Molecular Mechanisms ◽

Epileptiform Activity ◽

Synaptic Activity ◽

Structural Basis ◽

Dependent Manner ◽

Domain Organization ◽

Activity Dependent

Loss of Astrocytic Domain Organization in the Epileptic Brain. Oberheim NA, Tian GF, Han X, Peng W, Takano T, Ransom B, Nedergaard M. J Neurosci 2008;28(13):3264–3276. Gliosis is a pathological hallmark of posttraumatic epileptic foci, but little is known about these reactive astrocytes beyond their high glial fibrillary acidic protein (GFAP) expression. Using diolistic labeling, we show that cortical astrocytes lost their nonoverlapping domain organization in three mouse models of epilepsy: posttraumatic injury, genetic susceptibility, and systemic kainate exposure. Neighboring astrocytes in epileptic mice showed a 10-fold increase in overlap of processes. Concurrently, spine density was increased on dendrites of excitatory neurons. Suppression of seizures by the common antiepileptic, valproate, reduced the overlap of astrocytic processes. Astrocytic domain organization was also preserved in APP transgenic mice expressing a mutant variant of human amyloid precursor protein despite a marked upregulation of GFAP. Our data suggest that loss of astrocytic domains was not universally associated with gliosis, but restricted to seizure pathologies. Reorganization of astrocytes may, in concert with dendritic sprouting and new synapse formation, form the structural basis for recurrent excitation in the epileptic brain. Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission. Rouach N, Koulakoff A, Abudara V, Willecke K, Giaume C. Science 2008;322(5907):1551–1555. Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.

Download Full-text