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/métodosRESUMO
The HSP70 co-chaperone BAG3 targets unfolded proteins to degradation via chaperone assisted selective autophagy (CASA), thereby playing pivotal roles in the proteostasis of adult cardiomyocytes (CMs). However, the complex functions of BAG3 for regulating autophagy in cardiac disease are not completely understood. Here, we demonstrate that conditional inactivation of Bag3 in murine CMs leads to age-dependent dysregulation of autophagy, associated with progressive cardiomyopathy. Surprisingly, Bag3-deficient CMs show increased canonical and non-canonical autophagic flux in the juvenile period when first signs of cardiac dysfunction appear, but reduced autophagy during later stages of the disease. Juvenile Bag3-deficient CMs are characterized by decreased levels of soluble proteins involved in synchronous contraction of the heart, including the gap junction protein Connexin 43 (CX43). Reiterative administration of chloroquine (CQ), an inhibitor of canonical and non-canonical autophagy, but not inactivation of Atg5, restores normal concentrations of soluble cardiac proteins in juvenile Bag3-deficient CMs without an increase of detergent-insoluble proteins, leading to complete recovery of early-stage cardiac dysfunction in Bag3-deficient mice. We conclude that loss of Bag3 in CMs leads to age-dependent differences in autophagy and cardiac dysfunction. Increased non-canonical autophagic flux in the juvenile period removes soluble proteins involved in cardiac contraction, leading to early-stage cardiomyopathy, which is prevented by reiterative CQ treatment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Autofagia , Cardiomiopatias , Miócitos Cardíacos , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/deficiência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Cloroquina/farmacologia , Camundongos KnockoutRESUMO
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/metabolismoRESUMO
Human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Though the underlying mechanisms remain poorly characterized, mouse models of congenital CMV infection have demonstrated that the neuronal migration process is damaged. In this study, we evaluated the effects of HCMV infection on connexin 43 (Cx43), a crucial adhesion molecule mediating neuronal migration. We show in multiple cellular models that HCMV infection downregulated Cx43 posttranslationally. Further analysis identified the immediate early protein IE1 as the viral protein responsible for the reduction of Cx43. IE1 was found to bind the Cx43 C terminus and promote Cx43 degradation through the ubiquitin-proteasome pathway. Deletion of the Cx43-binding site in IE1 rendered it incapable of inducing Cx43 degradation. We validated the IE1-induced loss of Cx43 in vivo by introducing IE1 into the fetal mouse brain. Noteworthily, ectopic IE1 expression induced cortical atrophy and neuronal migration defects. Several lines of evidence suggest that these damages result from decreased Cx43, and restoration of Cx43 levels partially rescued IE1-induced interruption of neuronal migration. Taken together, the results of our investigation reveal a novel mechanism of HCMV-induced neural maldevelopment and identify a potential intervention target. IMPORTANCE Congenital CMV (cCMV) infection causes neurological sequelae in newborns. Recent studies of cCMV pathogenesis in animal models reveal ventriculomegaly and cortical atrophy associated with impaired neural progenitor cell (NPC) proliferation and migration. In this study, we investigated the mechanisms underlying these NPC abnormalities. We show that Cx43, a critical adhesion molecule mediating NPC migration, is downregulated by HCMV infection in vitro and HCMV-IE1 in vivo. We provide evidence that IE1 interacts with the C terminus of Cx43 to promote its ubiquitination and consequent degradation through the proteasome. Moreover, we demonstrate that introducing IE1 into mouse fetal brains led to neuronal migration defects, which was associated with Cx43 reduction. Deletion of the Cx43-binding region in IE1 or ectopic expression of Cx43 rescued the IE1-induced migration defects in vivo. Our study provides insight into how cCMV infection impairs neuronal migration and reveals a target for therapeutic interventions.
Assuntos
Conexina 43 , Infecções por Citomegalovirus , Citomegalovirus , Proteínas Imediatamente Precoces , Animais , Humanos , Recém-Nascido , Camundongos , Conexina 43/genética , Conexina 43/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
This study investigated Cerium oxide nanoparticles (CeONPs) effect on central neuropathic pain (CNP). The compressive method of spinal cord injury (SCI) model was used for pain induction. Three groups were formed by a random allocation of 24 rats. In the treatment group, CeONPs were injected above and below the lesion site immediately after inducing SCI. pain symptoms were evaluated using acetone, Radian Heat, and Von Frey tests weekly for six weeks. Finally, we counted fibroblasts using H&E staining. We evaluated the expression of Cx43, GAD65 and HDAC2 proteins using the western blot method. The analysis of results was done by PRISM software. At the end of the study, we found that CeONPs reduced pain symptoms to levels similar to those observed in normal animals. CeONPs also increased the expression of GAD65 and Cx43 proteins but did not affect HDAC2 inhibition. CeONPs probably have a pain-relieving effect on chronic pain by potentially preserving GAD65 and Cx43 protein expression and hindering fibroblast infiltration.
RESUMO
OBJECTIVE: The present study aimed to evaluate the effects of reuterin, a bioactive isolated from the probiotic Lactobacillus reuteri (L. reuteri) on periodontal tissue regeneration, and provide a new strategy for periodontitis treatment in the future. BACKGROUND: Data discussing the present state of the field: Probiotics are essential for maintaining oral microecological balance. Our previous study confirmed that probiotic L. reuteri extracts could rescue the function of mesenchymal stem cells (MSCs) and promote soft tissue wound healing by neutralizing inflammatory Porphyromonas gingivalis-LPS. Periodontitis is a chronic inflammatory disease caused by bacteria seriously leading to tooth loss. In this study, we isolated and purified reuterin from an extract of L. reuteri to characterize from the extracts of L. reuteri to characterize its role in promoting periodontal tissue regeneration and controlling inflammation in periodontitis. METHODS: Chromatographic analysis was used to isolate and purify reuterin from an extract of L. reuteri, and HNMR was used to characterize its structure. The inflammatory cytokine TNFα was used to simulate the inflammatory environment. Periodontal ligament stem cells (PDLSCs) were treated with TNFα and reuterin after which their effects were characterized using scratch wound cell migration assays to determine the concentration of reuterin, an experimental periodontitis model in rats was used to investigate the function of reuterin in periodontal regeneration and inflammation control in vivo. Real-time PCR, dye transfer experiments, image analysis, alkaline phosphatase activity, Alizarin red staining, cell proliferation, RNA-sequencing and Western Blot assays were used to detect the function of PDLSCs. RESULTS: In vivo, local injection of reuterin promoted periodontal tissue regeneration of experimental periodontitis in rats and reduced local inflammatory response. Moreover, we found that TNFα stimulation caused endoplasmic reticulum (ER) stress in PDLSCs, which resulted in decreased osteogenic differentiation. Treatment with reuterin inhibited the ER stress state of PDLSCs caused by the inflammatory environment and restored the osteogenic differentiation and cell proliferation functions of inflammatory PDLSCs. Mechanistically, we found that reuterin restored the functions of inflammatory PDLSCs by inhibiting the intercellular transmission of ER stress mediated by Cx43 in inflammatory PDLSCs and regulated osteogenic differentiation capacity. CONCLUSION: Our findings identified reuterin isolated from extracts of the probiotic L. reuteri, which improves tissue regeneration and controls inflammation, thus providing a new therapeutic method for treating periodontitis.
Assuntos
Estresse do Retículo Endoplasmático , Gliceraldeído , Limosilactobacillus reuteri , Probióticos , Propano , Regeneração , Animais , Propano/análogos & derivados , Propano/farmacologia , Propano/uso terapêutico , Probióticos/uso terapêutico , Probióticos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Gliceraldeído/análogos & derivados , Gliceraldeído/farmacologia , Ratos , Regeneração/efeitos dos fármacos , Periodontite/microbiologia , Ligamento Periodontal/efeitos dos fármacos , Humanos , Masculino , Fator de Necrose Tumoral alfa , Ratos Sprague-Dawley , Proliferação de Células/efeitos dos fármacos , Células-Tronco/efeitos dos fármacosRESUMO
Adult male Sertoli cell-specific Connexin43 knockout mice (SCCx43KO) exhibit higher Sertoli cell (SC) numbers per seminiferous tubule compared to their wild type (WT) littermates. Thus, deletion of this testicular gap junction protein seems to affect the proliferative potential and differentiation of "younger" SC. Although SC have so far mostly been characterised as postmitotic cells that cease to divide and become an adult, terminally differentiated cell population at around puberty, there is rising evidence that there exist exceptions from this for a very long time accepted paradigm. Aim of this study was to investigate postnatal SC development and to figure out underlying causes for observed higher SC numbers in adult KO mice. Therefore, the amount of SC mitotic figures was compared, resulting in slightly more and prolonged detection of SC mitotic figures in KO mice compared to WT. SC counting per tubular cross section revealed significantly different time curves, and comparing proliferation rates using Bromodesoxyuridine and Sox9 showed higher proliferation rates in 8-day old KO mice. SC proliferation was further investigated by Ki67 immunohistochemistry. SC in KO mice displayed a delayed initiation of cell-cycle-inhibitor p27Kip1 synthesis and prolonged synthesis of the phosphorylated tumour suppressor pRb and proliferation marker Ki67. Thus, the higher SC numbers in adult male SCCx43KO mice may arise due to two different reasons: Firstly, in prepubertal KO mice, the proliferation rate of SC was higher. Secondly, there were differences in their ability to cease proliferation as shown by the delayed initiation of p27Kip1 synthesis and the prolonged production of phosphorylated pRb and Ki67. Immunohistochemical results indicating a prolonged period of SC proliferation in SCCx43KO were confirmed by detection of proliferating SC in 17-days-old KO mice. In conclusion, deletion of the testicular gap junction protein Cx43 might prevent normal SC maturation and might even alter also the proliferation potential of adult SC.
Assuntos
Conexina 43 , Células de Sertoli , Masculino , Animais , Camundongos , Conexina 43/genética , Conexina 43/metabolismo , Antígeno Ki-67/genética , Testículo , Camundongos Knockout , Conexinas/metabolismo , Proliferação de Células/genética , EspermatogêneseRESUMO
AS602801 has been reported as a potential drug candidate against brain metastasis by suppressing the gap-junction communication between lung cancer stem cells and astrocytes. In this study, we aimed to study the molecular mechanism underlying the role of AS602801 in the treatment of brain metastasis in breast cancer. We utilized female athymic BALB/c nude mice and MDA-MB-231/BT-474BR cells to establish experimental models. Polymerase chain reaction assays were performed to observe changes in the connexin 43 (Cx43) messenger RNA (mRNA) and c-Jun N-terminal kinase (JNK) mRNA levels. Dye transfer assay was used to observe the effect of AS602801 on cell-cell communication. An organotypic blood-brain barrier (BBB) model was utilized to observe the effect of AS602801 on transmigration through the BBB barrier. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and flow cytometry were performed to evaluate the proliferation and apoptosis of breast cancer cells co-cultivated with astrocytes. AS602801 inhibited the upregulation of Cx43 and JNK in brain metastasized breast cancer cells in a dose-dependent manner. Also, AS602801 significantly decreased the dye transfer rate from astrocytes to breast cancer cells, indicating the inhibitory effect of AS602801 on cell-cell communication. The transmigration ability of breast cancer cells co-cultured with astrocytes was decreased by AS602801. Furthermore, AS602801 reduced the elevated Cx43/JNK mRNA expression in the co-astrocyte group while suppressing the increased proliferation and promoting the decreased apoptosis of breast cancer cells co-cultivated with astrocytes. AS602801 also suppressed the brain metastasis of breast cancer cells and increased mouse survival. AS602801 downregulates the expressions of JNK and Cx43 to suppress the gap-junction activity. AS602801 also inhibits the communication between breast cancer cells and astrocytes, thus contributing to the treatment of brain metastasis in breast cancer.
Assuntos
Benzotiazóis , Neoplasias Encefálicas , Conexina 43 , Pirimidinas , Animais , Camundongos , Feminino , Conexina 43/genética , Conexina 43/metabolismo , Camundongos Nus , Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/metabolismo , RNA Mensageiro/metabolismoRESUMO
Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.
Assuntos
Criopreservação , Células do Cúmulo , Junções Comunicantes , Oócitos , Animais , Oócitos/metabolismo , Oócitos/citologia , Criopreservação/métodos , Junções Comunicantes/metabolismo , Células do Cúmulo/metabolismo , Células do Cúmulo/citologia , Bovinos , Feminino , Conexina 43/metabolismo , Conexina 43/genética , Conexinas/metabolismo , Conexinas/genética , Vitrificação , Técnicas de Cocultura/métodos , Sobrevivência Celular , Técnicas de Maturação in Vitro de Oócitos/métodosRESUMO
Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future.
Assuntos
Conexina 43 , Junções Comunicantes , Humanos , Conexina 43/química , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Proteínas de Membrana/metabolismo , Células CACO-2 , Peptídeos/metabolismo , Fosfoproteínas/metabolismoRESUMO
BACKGROUND: Previous studies by our group have demonstrated chronic intermittent hypoxia (CIH) can decrease connexin 43 (Cx43) protein expression and thus increase atrial fibrillation (AF) inducibility. Cardiac sympathetic denervation (CSD) can reduce AF and increase Cx43 expression, however, the underlying molecular mechanisms and signaling pathways are still unclear. METHODS AND RESULTS: An obstructive sleep apnea (OSA) rat model in vivo experiments and CIH H9c2 cells model in vitro experiments were used to figure out the roles and underlying mechanisms of Cx43 on OSA-associated AF. In this study, we examined the expression of Cx43, CaMK⠡γ, Bax, Caspase 3, HIF-1 Bcl-2, Tunel, and CPB/p300, to discover the association between proteins and the mechanism of regulatory changes. The downstream proteins of Cx43 were calculated by gene sequencing and data analysis. We found Cx43 expression was significantly downregulated after CIH exposure in rat and H9c2 cells. Active caspase-3 and Bax at CIH+8 h group are high, but decreased at OE+8 h group. The Bcl-2 expression was higher in the N and OE+8 h group than CIH+8 h group. TUNEL-positive cells from the CIH+8 h group was markedly higher. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated Cx43 overexpression inhibited the CaMKIIγ expression, and CaMKIIγ was involved in the HIF-1 signaling pathway. In addition, we also found Cx43 overexpression remarkably decreased the HIF-1 protein and p300 mRNA expression, which inhibits the CaMKIIγ/HIF-1 signaling pathway. CONCLUSIONS: Taken together, these results suggested Cx43 overexpression inhibits the expression of calcium/calmodulin dependent protein CaMK⠡γ via the Cx43/CaMKIIγ/HIF-1 axis, which finally reduces the myocardial apoptosis and incidence of AF.
Assuntos
Fibrilação Atrial , Apneia Obstrutiva do Sono , Animais , Ratos , Fibrilação Atrial/genética , Proteína X Associada a bcl-2 , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Conexina 43/genética , Modelos Animais de Doenças , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia , Incidência , Apneia Obstrutiva do Sono/complicações , Apneia Obstrutiva do Sono/genética , Apneia Obstrutiva do Sono/metabolismoRESUMO
Zika virus (ZIKV) is transmitted mostly via mosquito bites and no vaccine is available, so it may reemerge. We and others previously demonstrated that neonatal infection of ZIKV results in heart failure and can be fatal. Animal models implicated ZIKV involvement in viral heart diseases. It is unknown whether and how ZIKV causes heart failure in adults. Herein, we studied the effects of ZIKV infection on the heart function of adult A129 mice. First, we found that ZIKV productively infects the rat-, mouse-, or human-originated heart cell lines and caused ubiquitination-mediated degradation of and distortive effects on connexin 43 (Cx43) protein that is important for communications between cardiomyocytes. Second, ZIKV infection caused 100% death of the A129 mice with decreasing body weight, worsening health score, shrugging fur, and paralysis. The viral replication was detected in multiple organs. In searching for the viral effects on heart of the A129 mice, we found that ZIKV infection resulted in the increase of cardiac muscle enzymes, implicating a viral acute myocardial injury. ZIKV-caused heart injury was also demonstrated by electrocardiogram (ECG) showing widened and fragmented QRS waves, prolonged PR interval, and slower heart rate. The intercalated disc (ICD) between two cardiomyocytes was destroyed, as shown by the electronic microscopy, and the Cx43 distribution in the ICDs was less organized in the ZIKV-infected mice compared to that in the phosphate-buffered saline (PBS)-treated mice. Consistently, ZIKV productively infected the heart of A129 mice and decreased Cx43 protein. Therefore, we demonstrated that ZIKV infection caused heart failure, which might lead to fatal sequelae in ZIKV-infected A129 mice. IMPORTANCE Zika virus (ZIKV) is a teratogen causing devastating sequelae to the newborns who suffer a congenital ZIKV infection while it brings about only mild symptoms to the health-competent older children or adults. Mouse models have played an important role in mechanistic and pathogenic studies of ZIKV. In this study, we employed 3 to 4 week-old A129 mice for ZIKV infection. RT-qPCR assays discovered that ZIKV replicated in multiple organs, including the heart. As a result of ZIKV infection, the A129 mice experienced weight loss, health score worsening, paralysis, and deaths. We revealed that the ZIKV infection caused abnormal electrocardiogram presentations, increased cardiac muscle enzymes, downregulated Cx43, and destroyed the gap junction and the intercalated disc between the cardiomyocytes, implicating that ZIKV may cause an acute myocardial injury in A129 mice. Therefore, our data imply that ZIKV infection may jeopardize the immunocompromised population with a severe clinical consequence, such as heart defect.
Assuntos
Cardiopatias , Insuficiência Cardíaca , Infecção por Zika virus , Zika virus , Recém-Nascido , Criança , Animais , Camundongos , Humanos , Ratos , Adolescente , Conexina 43 , Miócitos Cardíacos/patologia , Modelos Animais de Doenças , Junções Comunicantes/patologia , ParalisiaRESUMO
OBJECTIVES: This study aimed to determine the function of Cx43 in the endothelial-to-mesenchymal transition (EndMT) process of endothelial cells (ECs) and to explore the potential signaling pathways underlying these functions. METHODS: ECs were extracted from rat aorta. ECs were transfected with Cx43 cDNA and Cx43 siRNA and then exposed to 5 or 12 dyne/cm2. Immunofluorescence staining was used to detect the expression of SM22α, Cx43, and acetylated α-tubulin in ECs. Western blotting was used to detect the protein expression of α-SMA, CD31, Cx43, H1-calponin, Ift88, and p-smad3 in ECs. RESULTS: The expression of αSMA, SM22α, and Cx43 was significantly increased, and CD31 was markedly decreased in ECs treated with laminar shear stress at 5 dyn/cm2. The Cx43 cDNA transfection could induce the expression of SM22α or H1-calponin and attenuate CD31 expression in ECs. Also, Cx43 overexpression harms cilia formation in ECs exposed to 5 dyn/cm2, accompanied with the regulated Ift88 and smad signaling. CONCLUSIONS: This study found that laminar shear stress at 5 dyn/cm2 would increase the expression of Cx43 to facilitate the EndMT process of ECs, associated with morphological changes in primary cilia and the decreased expression of Ift88 in ECs.
Assuntos
Conexina 43 , Células Endoteliais , Animais , Ratos , Células Cultivadas , Conexina 43/genética , DNA Complementar , Transdução de Sinais , Estresse MecânicoRESUMO
The development of the vertebrate retina relies on complex regulatory mechanisms to achieve its characteristic layered morphology containing multiple neuronal cell types. While connexin 43 (CX43) is not expressed by mature retinal neurons, mutations in its gene GJA1 are associated with microphthalmia and low vision in patients. To delineate how lack of CX43 affects retinal development, GJA1 was disrupted in human induced pluripotent stem cells (hiPSCs) (GJA1-/-) using CRISPR/Cas9 editing, and these were subsequently differentiated into retinal organoids. GJA1-/- hiPSCs do not display defects in self-renewal and pluripotency, but the resulting organoids are smaller with a thinner neural retina and decreased abundance of many retinal cell types. CX43-deficient organoids express lower levels of the neural marker PAX6 and the retinal progenitor cell (RPC) markers PAX6, SIX3, and SIX6. Conversely, expression of the early neuroectoderm markers SOX1 and SOX2 remains high in GJA1-/- organoids throughout their development. The lack of CX43 results in an increased population of CHX10-positive RPCs that are smaller, disorganized, do not become polarized, and possess a limited ability to commit to retinal fate specification. Our data indicate that lack of CX43 causes a developmental arrest in RPCs that subsequently leads to pan-retinal defects and stunted ocular growth.
Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Diferenciação Celular/genética , Conexina 43/genética , Conexina 43/metabolismo , Humanos , RetinaRESUMO
Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.
Assuntos
Astrócitos/metabolismo , Complexo Nuclear Basolateral da Amígdala/metabolismo , Conexina 43/metabolismo , Medo/fisiologia , Memória de Curto Prazo/fisiologia , Neurotransmissores/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Ácido Glutâmico/metabolismo , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Serina/metabolismoRESUMO
Connexin 43 (Cx43, also known as Gja1) is the most abundant testicular gap junction protein. It has a crucial role in the support of spermatogenesis by Sertoli cells in the seminiferous tubules as well as in androgen synthesis by Leydig cells. The multifunctional family of Ca2+/calmodulin-dependent protein kinases (CaMK) is composed of CaMK I, II, and IV and each can serve as a mediator of nuclear Ca2+ signals. These kinases can control gene expression by phosphorylation of key regulatory sites on transcription factors. Among these, AP-1 members cFos and cJun are interesting candidates that seem to cooperate with CaMKs to regulate Cx43 expression in Leydig cells. In this study, the Cx43 promoter region important for CaMK-dependent activation is characterized using co-transfection of plasmid reporter-constructs with different plasmids coding for CaMKs and/or AP-1 members in MA-10 Leydig cells. Here we report that the activation of Cx43 expression by cFos and cJun is increased by CaMKI. Furthermore, results from chromatin immunoprecipitation suggest that the recruitment of AP-1 family members to the proximal region of the Cx43 promoter may involve another uncharacterized AP-1 DNA regulatory element and/or protein-protein interactions with other partners. Thus, our data provide new insights into the molecular regulatory mechanisms that control mouse Cx43 transcription in testicular Leydig cells.
Assuntos
Células Intersticiais do Testículo , Neoplasias , Masculino , Camundongos , Animais , Células Intersticiais do Testículo/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Neoplasias/metabolismoRESUMO
As a kind of environmental noise, infrasonic noise has negative effects on various human organs. To date, research has shown that infrasound impairs cognitive function, especially the ability for learning and memory. Previously, we demonstrated that impaired learning and memory induced by infrasound was closely related with glia activation; however, the underlying mechanisms remain unclear. Connexin 43 hemichannels (Cx43 HCs), which are mainly expressed in hippocampal astrocytes, are activated under pathological conditions, lending support to the hypothesis that Cx43 HCs might function in the impaired learning and memory induced by infrasound. This study revealed that that blocking hippocampal Cx43 HCs or downregulating hippocampal Cx43 expression significantly alleviated impaired learning and memory induced by infrasound. We also observed that infrasound exposure led to the abundant release of glutamate and ATP through Cx43 HCs. In addition, the abundant release of glutamate and ATP depended on proinflammatory cytokines. Our finds suggested that the enhanced release of ATP and glutamate by astroglial Cx43 HCs may be involved in the learning and memory deficits caused by infrasound exposure.
Assuntos
Astrócitos , Conexina 43 , Humanos , Astrócitos/metabolismo , Conexina 43/metabolismo , Transtornos da Memória/etiologia , Transtornos da Memória/metabolismo , Glutamatos/metabolismo , Glutamatos/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologiaRESUMO
Early life stress such as maternal separation (MS), is a major risk factor for developing psychiatric disorders in adulthood. Connexin 43 (CX43), the main type of connexins expressed in astrocytes, has been indicated to participate in depression disorders. Nevertheless, the role of CX43 in MS-induced cognitive impairment and astrocyte dysfunction is unclear. Neonatal C57BL/6 mice were exposed to MS to mimic early life stress. Adeno-associated virus carrying CX43 was inoculated into mice for CX43 overexpression. Sucrose preference test, forced swim test and Morris water maze were performed for evaluating depression-like behaviors and spatial learning and memory of mice in adulthood. Real time quantitative polymerase chain reaction was conducted to detect CX43 mRNA expression in mouse brain. Immunofluorescence staining and western blotting were used for measuring expression levels of astrocytic markers in murine hippocampal dentate gyrus. The results showed that overexpressing CX43 attenuated MS exposure-induced depression-like behaviors and decrease in spatial learning and memory in mice. Upregulating CX43 alleviated MS exposure-induced downregulation of astrocytic markers. Collectively, CX43 overexpression attenuates cognitive deficits and astrocyte dysfunction in mice exposed to MS.
Assuntos
Disfunção Cognitiva , Conexina 43 , Animais , Camundongos , Astrócitos/metabolismo , Cognição , Disfunção Cognitiva/metabolismo , Conexina 43/metabolismo , Privação Materna , Camundongos Endogâmicos C57BL , Estresse PsicológicoRESUMO
Gap-junction-forming connexins are exquisitely regulated by post-translational modifications (PTMs). In particular, the PTM of connexin 43 (Cx43), a tumor suppressor protein, regulates its turnover and activity. Here, we investigated the interaction of Cx43 with the ubiquitin-related modifier 1 (URM-1) protein and its impact on tumor progression in two breast cancer cell lines, highly metastatic triple-negative MDA-MB-231 and luminal breast cancer MCF-7 cell lines. To evaluate the subsequent modulation of Cx43 levels, URM-1 was downregulated in these cells. The transcriptional levels of epithelial-to-mesenchymal transition (EMT) markers and the metastatic phenotype were assessed. We demonstrated that Cx43 co-localizes and interacts with URM-1, and URMylated Cx43 was accentuated upon cellular stress. The significant upregulation of small ubiquitin-like modifier-1 (SUMO-1) was also observed. In cells with downregulated URM-1, Cx43 expression significantly decreased, and SUMOylation by SUMO-1 was affected. The concomitant expression of EMT markers increased, leading to increased proliferation, migration, and invasion potential. Inversely, the upregulation of URM-1 increased Cx43 expression and reversed EMT-induced processes, underpinning a role for this PTM in the observed phenotypes. This study proposes that the URMylation of Cx43 in breast cancer cells regulates its tumor suppression properties and contributes to breast cancer cell malignancy.
Assuntos
Neoplasias da Mama , Conexina 43 , Feminino , Humanos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/metabolismo , Transição Epitelial-Mesenquimal/genética , Junções Comunicantes/metabolismo , Células MCF-7 , Ubiquitina/metabolismoRESUMO
The heart is one of the major organs affected by microwave radiation, and these effects have been extensively studied. Previous studies have shown that microwave-radiation-induced heart injury might be related to the abnormal expression and distribution of Cx43. In order to make the research model closer to humans, we used iPSC-CMs as the cell injury model to investigate the biological effect and mechanism of iPSC-CM injury after microwave radiation. To model the damage, iPSC-CMs were separated into four groups and exposed to single or composite S-band (2.856 GHz) and X-band (9.375 GHz) microwave radiation sources with an average power density of 30 mW/cm2. After that, FCM was used to detect cell activity, and ELISA was used to detect the contents of myocardial enzymes and injury markers in the culture medium, and it was discovered that cell activity decreased and the contents increased after radiation. TEM and SEM showed that the ultrastructure of the cell membrane, mitochondria, and ID was damaged. Mitochondrial function was aberrant, and glycolytic capacity decreased after exposure. The electrical conduction function of iPSC-CM was abnormal; the conduction velocity was decreased, and the pulsation amplitude was reduced. Wb, qRT-PCR, and IF detections showed that the expression of Cx43 was decreased and the distribution of Cx43 at the gap junction was disordered. Single or composite exposure to S- and X-band microwave radiation caused damage to the structure and function of iPSC-CMs, primarily affecting the cell membrane, mitochondria, and ID. The composite exposure group was more severely harmed than the single exposure group. These abnormalities in structure and function were related to the decreased expression and disordered distribution of Cx43.