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1.
Cell ; 169(3): 510-522.e20, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28431249

RESUMO

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.


Assuntos
Sistema de Condução Cardíaco , Macrófagos/fisiologia , Animais , Conexina 43/metabolismo , Feminino , Átrios do Coração/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miócitos Cardíacos/fisiologia
2.
EMBO J ; 43(17): 3627-3649, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39044100

RESUMO

A robust and efficient cellular response to lysosomal membrane damage prevents leakage from the lysosome lumen into the cytoplasm. This response is understood to happen through either lysosomal membrane repair or lysophagy. Here we report exocytosis as a third response mechanism to lysosomal damage, which is further potentiated when membrane repair or lysosomal degradation mechanisms are impaired. We show that Connexin43 (Cx43), a protein canonically associated with gap junctions, is recruited from the plasma membrane to damaged lysosomes, promoting their secretion and accelerating cell recovery. The effects of Cx43 on lysosome exocytosis are mediated by a reorganization of the actin cytoskeleton that increases plasma membrane fluidity and decreases cell stiffness. Furthermore, we demonstrate that Cx43 interacts with the actin nucleator Arp2, the activity of which was shown to be necessary for Cx43-mediated actin rearrangement and lysosomal exocytosis following damage. These results define a novel mechanism of lysosomal quality control whereby Cx43-mediated actin remodelling potentiates the secretion of damaged lysosomes.


Assuntos
Actinas , Conexina 43 , Exocitose , Lisossomos , Lisossomos/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Actinas/metabolismo , Animais , Humanos , Membrana Celular/metabolismo , Camundongos
3.
J Cell Sci ; 137(7)2024 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-38533727

RESUMO

Connexins are channel-forming proteins that function to facilitate gap junctional intercellular communication. Here, we use dual cell voltage clamp and dye transfer studies to corroborate past findings showing that Cx31.1 (encoded by GJB5) is defective in gap junction channel formation, illustrating that Cx31.1 alone does not form functional gap junction channels in connexin-deficient mammalian cells. Rather Cx31.1 transiently localizes to the secretory pathway with a subpopulation reaching the cell surface, which is rarely seen in puncta reminiscent of gap junctions. Intracellular retained Cx31.1 was subject to degradation as Cx31.1 accumulated in the presence of proteasomal inhibition, had a faster turnover when Cx43 was present and ultimately reached lysosomes. Although intracellularly retained Cx31.1 was found to interact with Cx43, this interaction did not rescue its delivery to the cell surface. Conversely, the co-expression of Cx31 dramatically rescued the assembly of Cx31.1 into gap junctions where gap junction-mediated dye transfer was enhanced. Collectively, our results indicate that the localization and functional status of Cx31.1 is altered through selective interplay with co-expressed connexins, perhaps suggesting Cx31.1 is a key regulator of intercellular signaling in keratinocytes.


Assuntos
Conexinas , Animais , Comunicação Celular/fisiologia , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Queratinócitos/metabolismo , Mamíferos/metabolismo , Humanos
4.
PLoS Biol ; 21(4): e3002075, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37040348

RESUMO

Astrocytes crucially contribute to synaptic physiology and information processing. One of their key characteristics is to express high levels of connexins (Cxs), the gap junction-forming protein. Among them, Cx30 displays specific properties since it is postnatally expressed and dynamically upregulated by neuronal activity and modulates cognitive processes by shaping synaptic and network activities, as recently shown in knockout mice. However, it remains unknown whether local and selective upregulation of Cx30 in postnatal astrocytes within a physiological range modulates neuronal activities in the hippocampus. We here show in mice that, whereas Cx30 upregulation increases the connectivity of astroglial networks, it decreases spontaneous and evoked synaptic transmission. This effect results from a reduced neuronal excitability and translates into an alteration in the induction of synaptic plasticity and an in vivo impairment in learning processes. Altogether, these results suggest that astroglial networks have a physiologically optimized size to appropriately regulate neuronal functions.


Assuntos
Astrócitos , Conexina 43 , Camundongos , Animais , Conexina 30/metabolismo , Astrócitos/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Regulação para Cima , Conexinas/genética , Conexinas/metabolismo , Camundongos Knockout , Hipocampo/metabolismo
5.
Circ Res ; 134(10): 1306-1326, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38533639

RESUMO

BACKGROUND: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.


Assuntos
Ritmo Circadiano , Miócitos Cardíacos , Receptores de Glucocorticoides , Animais , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Camundongos , Miócitos Cardíacos/metabolismo , Masculino , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/genética , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Conexina 43/metabolismo , Conexina 43/genética , Camundongos Knockout , Potenciais de Ação
6.
J Biol Chem ; 300(7): 107484, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38897566

RESUMO

Progesterone (P4), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P4/PR partially exerts these effects by tethering to NF-κB bound to their promot-ers, thereby decreasing NF-κB transcriptional activity. However, the underlying mechanisms whereby P4/PR interaction blocks proinflammatory and CAP gene expression are not fully understood. Herein, we characterized CCR-NOT transcription complex subunit 1 (CNOT1) as a corepressor that also interacts within the same chromatin complex as PR-B. In mouse myome-trium increased expression of CAP genes Oxtr and Cx43 at term coincided with a marked decline in expression and binding of CNOT1 to NF-κB-response elements within the Oxtr and Cx43 promoters. Increased CAP gene expression was accompanied by a pronounced decrease in enrichment of repressive histone marks and increase in enrichment of active histone marks to this genomic region. These changes in histone modification were associated with changes in expression of corresponding histone modifying enzymes. Myometrial tissues from P4-treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. P4 treatment of PR-expressing hTERT-HM cells enhanced CNOT1 expression and its recruitment to PR bound NF-κB-response elements within the CX43 and OXTR promoters. Furthermore, knockdown of CNOT1 significantly increased expression of contractile genes. These novel findings suggest that decreased expression and DNA-binding of the P4/PR-regulated transcriptional corepressor CNOT1 near term and associated changes in histone modifications at the OXTR and CX43 promoters contribute to the induction of myometrial contractility leading to parturition.


Assuntos
Miométrio , Regiões Promotoras Genéticas , Receptores de Progesterona , Animais , Feminino , Humanos , Camundongos , Gravidez , Conexina 43/metabolismo , Conexina 43/genética , Regulação da Expressão Gênica , Miométrio/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Receptores de Progesterona/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Contração Uterina/metabolismo , Contração Uterina/genética
7.
J Cell Sci ; 136(11)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37288673

RESUMO

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.


Assuntos
Conexina 43 , Proteína 1 Homóloga a Discs-Large , Queratinócitos , Humanos , Comunicação Celular , Membrana Celular/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Guanilato Quinases/metabolismo , Queratinócitos/metabolismo , Proteína 1 Homóloga a Discs-Large/genética , Proteína 1 Homóloga a Discs-Large/metabolismo
8.
J Virol ; 98(7): e0047824, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38819132

RESUMO

ß-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human ß-coronavirus that induces mild clinical symptoms and can be safely studied in the BSL2 laboratory. Due to its low risk, OC43 can be a valuable and accessible model for understanding ß-coronavirus pathogenesis. One potential target for limiting virus infectivity could be gap junction-mediated communication. This study aims to unveil the status of cell-to-cell communications through gap junctions in human ß-coronavirus infection. Infection with OC43 leads to reduced expression of Cx43 in A549, a lung epithelial carcinoma cell line. Infection with this virus also shows a significant ER and oxidative stress increase. Internal localization of Cx43 is observed post-OC43 infection in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) region, which impairs the gap junction communication between two adjacent cells, confirmed by Lucifer yellow dye transfer assay. It also affects hemichannel formation, as depicted by the EtBr uptake assay. Impairment of Cx43 trafficking and the ability to form hemichannels and functional GJIC are hampered by virus-induced Golgi apparatus disruption. Altogether, these results suggest that several physiological changes accompany OC43 infection in A549 cells and can be considered an appropriate model system for understanding the differences in gap junction communication post-viral infections. This model system can provide valuable insights for developing therapies against human ß-coronavirus infections.IMPORTANCEThe enduring impact of the recent SARS-CoV-2 pandemic underscores the importance of studying human ß-coronaviruses, advancing our preparedness for future coronavirus infections. As SARS-CoV-2 is highly infectious, another human ß-coronavirus OC43 can be considered an experimental model. One of the crucial pathways that can be considered is gap junction communication, as it is vital for cellular homeostasis. Our study seeks to understand the changes in Cx43-mediated cell-to-cell communication during human ß-coronavirus OC43 infection. In vitro studies showed downregulation of the gap junction protein Cx43 and upregulation of the endoplasmic reticulum and oxidative stress markers post-OC43 infection. Furthermore, HCoV-OC43 infection causes reduced Cx43 trafficking, causing impairment of functional hemichannel and GJIC formation by virus-mediated Golgi apparatus disruption. Overall, this study infers that OC43 infection reshapes intercellular communication, suggesting that this pathway may be a promising target for designing highly effective therapeutics against human coronaviruses by regulating Cx43 expression.


Assuntos
Comunicação Celular , Conexina 43 , Coronavirus Humano OC43 , Retículo Endoplasmático , Junções Comunicantes , Humanos , Junções Comunicantes/metabolismo , Conexina 43/metabolismo , Células A549 , Coronavirus Humano OC43/fisiologia , Coronavirus Humano OC43/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Complexo de Golgi/metabolismo , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Infecções por Coronavirus/patologia , Estresse Oxidativo
9.
Am J Pathol ; 194(10): 1857-1878, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39069170

RESUMO

Remote ischemic preconditioning (RIPC) exerts a protective role on myocardial ischemia/reperfusion (I/R) injury by the release of various humoral factors. Lactate is a common metabolite in ischemic tissues. Nevertheless, little is known about the role lactate plays in myocardial I/R injury and its underlying mechanism. This investigation revealed that RIPC elevated the level of lactate in blood and myocardium. Furthermore, AZD3965, a selective monocarboxylate transporter 1 inhibitor, and 2-deoxy-d-glucose, a glycolysis inhibitor, mitigated the effects of RIPC-induced elevated lactate in the myocardium and prevented RIPC against myocardial I/R injury. In an in vitro hypoxia/reoxygenation model, lactate markedly mitigated hypoxia/reoxygenation-induced cell damage in H9c2 cells. Further studies suggested that lactate contributed to RIPC, rescuing I/R-induced autophagy deficiency by promoting transcription factor EB (TFEB) translocation to the nucleus through activating the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway without influencing the phosphatidylinositol 3-kinase-Akt pathway, thus reducing cardiomyocyte damage. Interestingly, lactate up-regulated the mRNA and protein expression of connexin 43 (CX43) by facilitating the binding of TFEB to CX43 promoter in the myocardium. Functionally, silencing of TFEB attenuated the protective effect of lactate on cell damage, which was reversed by overexpression of CX43. Further mechanistic studies suggested that lactate facilitated CX43-regulated autophagy via the AMPK-mTOR-TFEB signaling pathway. Collectively, this research demonstrates that RIPC protects against myocardial I/R injury through lactate-mediated myocardial autophagy via the AMPK-mTOR-TFEB-CX43 axis.


Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Conexina 43 , Traumatismo por Reperfusão Miocárdica , Serina-Treonina Quinases TOR , Animais , Masculino , Ratos , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Precondicionamento Isquêmico/métodos , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
10.
Nat Immunol ; 14(1): 72-81, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23202271

RESUMO

Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.


Assuntos
Conexina 43/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/metabolismo , Psoríase/imunologia , Células Th17/imunologia , Animais , Linhagem Celular , Conexina 43/genética , Conexina 43/imunologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Mutação/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Polimorfismo Genético , Ligação Proteica/genética , Ligação Proteica/imunologia , Psoríase/genética , Transdução de Sinais
11.
Circ Res ; 133(8): 658-673, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37681314

RESUMO

BACKGROUND: Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. METHODS: Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. RESULTS: CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. CONCLUSIONS: Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.


Assuntos
Conexina 43 , Dextranos , Animais , Cobaias , Dextranos/metabolismo , Conexina 43/metabolismo , Miócitos Cardíacos/metabolismo , Sódio/metabolismo , Junções Comunicantes/metabolismo , Albuminas/metabolismo , Manitol/farmacologia , Manitol/metabolismo , Potenciais de Ação
12.
Biochem J ; 481(12): 741-758, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38752978

RESUMO

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.


Assuntos
Conexinas , Junções Comunicantes , Técnicas de Patch-Clamp , Humanos , Células HEK293 , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Junções Comunicantes/genética , Conexina 43/genética , Conexina 43/metabolismo , Sistemas CRISPR-Cas , Engenharia Genética/métodos , Técnicas de Inativação de Genes/métodos
13.
Differentiation ; 138: 100789, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38896972

RESUMO

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.


Assuntos
Trifosfato de Adenosina , Diferenciação Celular , Osteoclastos , Osteoprotegerina , Osteoprotegerina/metabolismo , Osteoprotegerina/genética , Osteoclastos/metabolismo , Osteoclastos/citologia , Animais , Trifosfato de Adenosina/metabolismo , Camundongos , Conexina 43/metabolismo , Conexina 43/genética , Fusão Celular , Antígeno CD47/metabolismo , Antígeno CD47/genética , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Transdução de Sinais , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas do Tecido Nervoso
14.
J Mol Cell Cardiol ; 193: 1-10, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38789075

RESUMO

BACKGROUND: Hypothermic ischemia-reperfusion arrhythmia is a common complication of cardiothoracic surgery under cardiopulmonary bypass, but few studies have focused on this type of arrhythmia. Our prior study discovered reduced myocardial Cx43 protein levels may be linked to hypothermic reperfusion arrhythmias. However, more detailed molecular mechanism research is required. METHOD: The microRNA and mRNA expression levels in myocardial tissues were detected by real-time quantitative PCR (RT-qPCR). Besides, the occurrence of hypothermic reperfusion arrhythmias and changes in myocardial electrical conduction were assessed by electrocardiography and ventricular epicardial activation mapping. Furthermore, bioinformatics analysis, applying antagonists of miRNA, western blotting, immunohistochemistry, a dual luciferase assay, and pearson correlation analysis were performed to investigate the underlying molecular mechanisms. RESULTS: The expression level of novel-miR-17 was up-regulated in hypothermic ischemia-reperfusion myocardial tissues. Inhibition of novel-miR-17 upregulation ameliorated cardiomyocyte edema, reduced apoptosis, increased myocardial electrical conduction velocity, and shortened the duration of reperfusion arrhythmias. Mechanistic studies showed that novel-miR-17 reduced the expression of Cx43 by directly targeting Gja1 while mediating the activation of the PKC/c-Jun signaling pathway. CONCLUSION: Up-regulated novel-miR-17 is a newly discovered pro-arrhythmic microRNA that may serve as a potential therapeutic target and biomarker for hypothermic reperfusion arrhythmias.


Assuntos
Arritmias Cardíacas , Conexina 43 , MicroRNAs , Proteína Quinase C , Transdução de Sinais , Animais , Humanos , Masculino , Ratos , Apoptose/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/patologia , Conexina 43/metabolismo , Conexina 43/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/etiologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteína Quinase C/metabolismo , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Regulação para Cima
15.
Am J Physiol Cell Physiol ; 326(2): C414-C428, 2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38145302

RESUMO

The human lens is an avascular organ, and its transparency is dependent on gap junction (GJ)-mediated microcirculation. Lens GJs are composed of three connexins with Cx46 and Cx50 being expressed in lens fiber cells and Cx43 and Cx50 in the epithelial cells. Impairment of GJ communication by either Cx46 or Cx50 mutations has been shown to be one of the main molecular mechanisms of congenital cataracts in mutant carrier families. The docking compatibility and formation of functional heterotypic GJs for human lens connexins have not been studied. Previous study on rodent lens connexins revealed that Cx46 can form functional heterotypic GJs with Cx50 and Cx43, but Cx50 cannot form heterotypic GJ with Cx43 due to its second extracellular (EL2) domain. To study human lens connexin docking and formation of functional heterotypic GJs, we developed a genetically engineered HEK293 cell line with endogenously expressed Cx43 and Cx45 ablated. The human lens connexins showed docking compatibility identical to those found in the rodent connexins. To reveal the structural mechanisms of the docking incompatibility between Cx50 and Cx43, we designed eight variants based on the differences between the EL2 of Cx50 and Cx46. We found that Cx50I177L is sufficient to establish heterotypic docking with Cx43 with some interesting gating properties. Our structure models indicate this residue is important for interdomain interactions within a single connexin, Cx50 I177L showed an increased interdomain interaction which might alter the docking interface structure to be compatible with Cx43.NEW & NOTEWORTHY The human lens is an avascular organ, and its transparency is partially dependent on gap junction (GJ) network composed of Cx46, Cx50, and Cx43. We found that human Cx46 can dock and form functional heterotypic GJs with Cx50 and Cx43, but Cx50 is unable to form functional heterotypic GJs with Cx43. Through mutagenesis and patch-clamp study of several designed variants, we found that Cx50 I177L was sufficient to form functional heterotypic GJs with Cx43.


Assuntos
Conexina 43 , Cristalino , Humanos , Conexina 43/genética , Conexina 43/metabolismo , Células HEK293 , Junções Comunicantes/metabolismo , Conexinas/genética , Conexinas/metabolismo , Canais Iônicos/metabolismo , Cristalino/metabolismo
16.
Am J Physiol Cell Physiol ; 327(4): C1051-C1072, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39129489

RESUMO

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.


Assuntos
Sinalização do Cálcio , Movimento Celular , Cicatrização , Animais , Camundongos , Conexinas/metabolismo , Conexinas/genética , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Conexina 43/metabolismo , Conexina 43/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Camundongos Endogâmicos C57BL , Masculino , Cálcio/metabolismo
17.
Dev Biol ; 496: 73-86, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36805498

RESUMO

Glucocorticoids induced osteonecrosis of the femoral head (GIONFH) is a devastating orthopedic disease. Previous studies suggested that connexin43 is involved in the process of osteogenesis and angiogenesis. However, the role of Cx43 potentiates in the osteogenesis and angiogenesis of bone marrow-derived stromal stem cells (BMSCs) in GIONFH is still not investigated. In this study, BMSCs were isolated and transfected with green fluorescent protein or the fusion gene encoding GFP and Cx43. The osteogenic differentiation of BMSCs were detected after transfected with Cx43. In addition, the migration abilities and angiogenesis of human umbilical vein endothelial cells (HUVECs) were been detected after induced by transfected BMSCs supernatants in vitro. Finally, we established GC-ONFH rat model, then, a certain amount of transfected or controlled BMSCs were injected into the tibia of the rats. Immunohistological staining and micro-CT scanning results showed that the transplanted experiment group had significantly promoted more bone regeneration and vessel volume when compared with the effects of the negative or control groups. This study demonstrated for the first time that the Cx43 overexpression in BMSCs could promote bone regeneration as seen in the osteogenesis and angiogenesis process, suggesting that Cx43 may serve as a therapeutic gene target for GIONFH treatment.


Assuntos
Necrose da Cabeça do Fêmur , Glucocorticoides , Ratos , Humanos , Animais , Glucocorticoides/efeitos adversos , Glucocorticoides/metabolismo , Osteogênese , Conexina 43/metabolismo , Cabeça do Fêmur/metabolismo , Cabeça do Fêmur/patologia , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/patologia , Necrose da Cabeça do Fêmur/terapia , Ratos Sprague-Dawley , Regeneração Óssea , Diferenciação Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia
18.
J Biol Chem ; 299(2): 102836, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36572185

RESUMO

Gap junctional intercellular communication (GJIC) involving astrocytes is important for proper CNS homeostasis. As determined in our previous studies, trafficking of the predominant astrocyte GJ protein, Connexin43 (Cx43), is disrupted in response to infection with a neurotropic murine ß-coronavirus (MHV-A59). However, how host factors are involved in Cx43 trafficking and the infection response is not clear. Here, we show that Cx43 retention due to MHV-A59 infection was associated with increased ER stress and reduced expression of chaperone protein ERp29. Treatment of MHV-A59-infected astrocytes with the chemical chaperone 4-sodium phenylbutyrate increased ERp29 expression, rescued Cx43 transport to the cell surface, increased GJIC, and reduced ER stress. We obtained similar results using an astrocytoma cell line (delayed brain tumor) upon MHV-A59 infection. Critically, delayed brain tumor cells transfected to express exogenous ERp29 were less susceptible to MHV-A59 infection and showed increased Cx43-mediated GJIC. Treatment with Cx43 mimetic peptides inhibited GJIC and increased viral susceptibility, demonstrating a role for intercellular communication in reducing MHV-A59 infectivity. Taken together, these results support a therapeutically targetable ERp29-dependent mechanism where ß-coronavirus infectivity is modulated by reducing ER stress and rescuing Cx43 trafficking and function.


Assuntos
Suscetibilidade a Doenças , Retículo Endoplasmático , Interações entre Hospedeiro e Microrganismos , Chaperonas Moleculares , Vírus da Hepatite Murina , Animais , Camundongos , Astrocitoma/patologia , Astrocitoma/virologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/virologia , Comunicação Celular , Linhagem Celular Tumoral , Conexina 43/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Junções Comunicantes/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Vírus da Hepatite Murina/metabolismo , Transporte Proteico , Transfecção
19.
J Neurochem ; 168(6): 1045-1059, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38291613

RESUMO

Glial cells play relevant roles in neuroinflammation caused by epilepsy. Elevated hemichannel (HC) activity formed by connexins (Cxs) or pannexin1 (Panx1) largely explains brain dysfunctions commonly caused by neuroinflammation. Glia express HCs formed by Cxs 43, 30, or 26, while glia and neurons both express HCs formed by Panx1. Cx43 HCs allow for the influx of Ca2+, which promotes glial reactivity, enabling the release of the gliotransmitters that contribute to neuronal over-stimulation. Valproate (VPA), an antiseizure medication, has pleiotropic actions on neuronal molecular targets, and their action on glial cell HCs remains elusive. We used HeLa cells transfected with Cx43, Cx30, Cx26, or Panx1 to determine the effect of VPA on HC activity in the brain. VPA slightly increased HC activity under basal conditions, but significantly enhanced it in cells pre-exposed to conditions that promoted HC activity. Furthermore, VPA increased ATP release through Cx43 HCs. The increased HC activity caused by VPA was resistant to washout, being consistent with in silico studies, which predicted the binding site for VPA and Cx43, as well as for Panx1 HCs on the intracellular side, suggesting that VPA first enters through HCs, after which their activity increases.


Assuntos
Anticonvulsivantes , Conexinas , Ácido Valproico , Ácido Valproico/farmacologia , Humanos , Anticonvulsivantes/farmacologia , Conexinas/metabolismo , Células HeLa , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Conexina 43/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Trifosfato de Adenosina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Animais , Epilepsia/metabolismo , Epilepsia/tratamento farmacológico , Epilepsia/induzido quimicamente
20.
Eur J Neurosci ; 59(7): 1723-1742, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38326974

RESUMO

The circadian clock orchestrates many physiological and behavioural rhythms in mammals with 24-h periodicity, through a hierarchical organisation, with the central clock located in the suprachiasmatic nucleus (SCN) in the hypothalamus. The circuits of the SCN generate circadian rhythms with precision, relying on intrinsic coupling mechanisms, for example, neurotransmitters like arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), neuronal gamma-aminobutyric acid (GABA) signalling and astrocytes connected by gap junctions composed of connexins (Cx). In female rodents, the presence of estrogen receptors (ERs) in the dorsal SCN suggests an influence of estrogen (E2) on the circuit timekeeping that could regulate circadian rhythm and coupling. To investigate this, we used SCN explants together with hypothalamic neurons and astrocytes. First, we showed that E2 stabilised the circadian amplitude in the SCN when rAVPs (receptor-associated vasopressin peptides) were inhibited. However, the phase delay induced by VIPAC2 (VIP receptors) inhibition remained unaffected by E2. We then showed that E2 exerted its effects in the SCN via ERß (estrogen receptor beta), resulting in increased expression of Cx36 and Cx43. Notably, specific inhibition of both connexins resulted in a significant reduction in circadian amplitude within the SCN. Remarkably, E2 restored the period with inhibited Cx36 but not with Cx43 inhibition. This implies that the network between astrocytes and neurons, responsible for coupling in the SCN, can be reinforced through E2. In conclusion, these findings provide new insights into how E2 regulates circadian rhythms ex vivo in an ERß-dependent manner, underscoring its crucial role in fortifying the SCN's rhythm.


Assuntos
Conexina 43 , Receptor beta de Estrogênio , Animais , Feminino , Conexina 43/metabolismo , Receptor beta de Estrogênio/metabolismo , Núcleo Supraquiasmático/fisiologia , Ritmo Circadiano/fisiologia , Junções Comunicantes/metabolismo , Conexinas/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Peptídeo Intestinal Vasoativo/metabolismo , Estrogênios/farmacologia , Mamíferos/metabolismo
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