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1.
Neoplasma ; 20192019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31307202

RESUMO

The aim of study was to identify the downstream target genes of CX43 by Human Transcriptome Array. Therefore, a gene microarray was generated which consists of CX43-overexpressed hepatocellular carcinoma (HCC) cells transfected with the constructed plasmid and negative controls to identify candidate genes. Integrated bioinformatic analysis was used to clarify biological functions of the identified genes, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein-protein interaction network, and survival analysis. The candidate genes were further validated by qRT-PCR in liver cancer tissues and CX43-silenced HCC cells. We have found the mRNA and protein levels of CX43 significantly upregulated in HCC cells transfected with CX43 constructed plasmid. We identified 928 differentially expressed genes including 394 upregulated and 534 downregulated genes, enriched in the cancer related functions and pathways by GO and KEGG pathway analysis. The protein-protein interaction network revealed 9 hub genes in this study. Statistical analysis indicated that upregulation of RALA and SRC was associated with poor prognosis in liver cancer. The differential expression of 2 candidate genes were further validated in HCC cells and tissues. In conclusion, protein-coding genes RALA and SRC could be target genes of CX43 and therapeutic targets for hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular , Conexina 43 , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/fisiopatologia , Conexina 43/genética , Conexina 43/metabolismo , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatologia
2.
Life Sci ; 232: 116624, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276689

RESUMO

AIMS: Monocyte-endothelial adhesion is considered to be the primary initiator of inflammatory vascular diseases, such as atherosclerosis. Connexin 43 (Cx43) has been reported to play an important part in this process, however, the underlying mechanisms are not fully understood. Intravenous anesthetics, propofol is commonly used in the perioperative period and in the intensive care unit, and considered to have good anti-inflammatory and antioxidant effects. Thus, we speculate that propofol could influence monocyte-endothelial adhesion, and explore whether its possible mechanism is relative with Cx43 expression in U937 monocytes influencing cell adhesion of U937 monocytes to human umbilical vein endothelial cells (HUVEC). MAIN METHODS: Cx43-siRNAs or pc-DNA-Cx43 were used to alter Cx43 expression in U937 monocytes. Propofol was given as pretreatments to U937 monocytes. Then, cell adhesion, ZO-1, LFA-1, VLA-4, COX and MCP-1 were determined. PI3K/AKT/NF-κB signaling pathway was explored to clarify the possible mechanism. KEY FINDINGS: Alternation of Cx43 expression affects cell adhesion and adhesion molecules significantly, such as ZO-1, LFA-1, VLA-4, COX-2 and MCP-1, the mechanism of which is relative with Cx43 influencing the activation of PI3K/AKT/NF-κB signaling pathway. Preconditioning with propofol at its clinically relevant anesthesia concentration attenuates cell adhesion. Propofol not only decreases Cx43 expression in U937 monocytes, but also depresses the activation of PI3K/AKT/NF-κB signaling pathway. SIGNIFICANCE: Modulation Cx43 expression in U937 monocytes could affect cell adhesion via regulating the activation of PI3K/AKT/NF-κB signaling pathway. Propofol attenuates cell adhesion via inhibiting Cx43 and its downstream signaling pathway of PI3K/AKT/NF-κB.


Assuntos
Adesão Celular/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Propofol/farmacologia , Aterosclerose/metabolismo , Moléculas de Adesão Celular/metabolismo , Conexina 43/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Endotélio Vascular/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Monócitos/fisiologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Propofol/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células U937/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo
3.
J Food Sci ; 84(7): 1703-1711, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31218711

RESUMO

We evaluated the effect of krill oil (KO) supplement on seizures induced by pentylenetetrazole (PTZ) in animals with previous febrile seizures (FSs) induced by hyperthermia to determine its effectiveness in seizure susceptibility and as an anticonvulsant. Male Wistar rats with FS separated into water (W, 1 mL), palm oil (PO, 300 mg/kg, total volume 1 mL), or KO (300 mg/kg, total volume 1 mL) groups. All drugs were administered chronically via the intragastric route. Electrical activity was recorded by intracranial EEG simultaneously with convulsive behavior. All animals' brains were processed by immunofluorescence against GFAP, NeuN, and connexins (Cx); cellular quantification was performed in hippocampus and pyramidal or granular layer thickness was evaluated with cresyl violet (CV) staining. The results showed a significant delay in convulsive behavior and a slight increased survival time after PTZ administration in the group treated with KO compared with PO and W groups. The epileptiform activity showed high amplitude and frequency, with no significant differences between groups, nor were there differences in the number and duration of discharge trains. KO and PO increased the number of astrocytes and the number of neurons compared with the W group. KO and PO decreased the expression of Cx36 without affecting Cx43 expression or the thickness of layers. Based on these data, we consider it important to perform more experiments to determine the anticonvulsant role of KO, taking into account the partial effect found in this study. KO could be used as a coadjuvant of traditional anticonvulsive treatments. PRACTICAL APPLICATION: In this study was evaluated the anticonvulsive effect of a chronic krill oil (KO) supplement in animals with seizures. Results showed that KO had partial anticonvulsive effects measured by EEG activity and convulsive behavior analysis. These data justify further research that looks at KO supplementation as a prospective coadjuvant of pharmacologic management of seizure disorder.


Assuntos
Anticonvulsivantes/administração & dosagem , Euphausiacea/química , Hipocampo/efeitos dos fármacos , Óleos Vegetais/administração & dosagem , Convulsões Febris/tratamento farmacológico , Animais , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Suplementos Nutricionais/análise , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/metabolismo , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pentilenotetrazol/efeitos adversos , Ratos , Ratos Wistar , Convulsões Febris/induzido quimicamente , Convulsões Febris/genética , Convulsões Febris/metabolismo
4.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31072046

RESUMO

Chronic kidney disease (CKD) is characterized by an oxidative stress status, driving some CKD-associated complications, even at the gastrointestinal level. Indoxyl Sulfate (IS) is a protein-bound uremic toxin, poorly eliminated by dialysis. This toxin is able to affect the intestinal system, but its molecular mechanism/s in intestinal epithelial cells (IECs) remain poorly understood. This study's aim was to evaluate the effect of IS (31.2-250 µM) on oxidative stress in IEC-6 cells and on the intactness of IECs monolayers. Our results indicated that IS enhanced oxidative cell damage by inducing reactive oxygen species (ROS) release, reducing the antioxidant response and affecting Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation as well its related antioxidant enzymes. In the wound healing assay model, IS reduced IEC-6 migration, slightly impaired actin cytoskeleton rearrangement; this effect was associated with connexin 43 alteration. Moreover, we reported the effect of CKD patients' sera in IEC-6 cells. Our results indicated that patient sera induced ROS release in IEC-6 cells directly related to IS sera content and this effect was reduced by AST-120 serum treatment. Results highlighted the effect of IS in inducing oxidative stress in IECs and in impairing the intactness of the IECs cell monolayer, thus significantly contributing to CKD-associated intestinal alterations.


Assuntos
Antioxidantes/farmacologia , Indicã/farmacologia , Intestinos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Carbono/farmacologia , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Conexina 43/genética , Células Epiteliais/efeitos dos fármacos , Humanos , Intestinos/patologia , Fator 2 Relacionado a NF-E2/genética , Óxidos/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Uremia/tratamento farmacológico , Uremia/metabolismo , Uremia/patologia
5.
Cancer Sci ; 110(6): 1947-1958, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012516

RESUMO

MicroRNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of microRNA, which exerts biological effects on tumor cells. However, the length of microRNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to microRNAs consisting of 18-27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the microRNAs were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and shRNA significantly decreased the transfer of these microRNAs. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by microRNA-34a. Additionally, these effects of microRNA-34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of microRNA by transfer of microRNA to neighboring cells. Our data are the first to clarify the pattern of microRNA transmission through gap junctions and provide novel insights to show that antitumor microRNAs should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.


Assuntos
Comunicação Celular/genética , Proliferação de Células/genética , Junções Comunicantes/metabolismo , MicroRNAs/genética , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/genética , Técnicas de Cocultura , Conexina 43/genética , Conexinas/genética , Glioma/genética , Glioma/patologia , Células HeLa , Humanos , Interferência de RNA
6.
Biomed Environ Sci ; 32(3): 177-188, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30987692

RESUMO

OBJECTIVE: Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively. RESULTS: Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown. CONCLUSION: Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.


Assuntos
Caspase 1/genética , Conexina 43/genética , Regulação da Expressão Gênica/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Piroptose , Raios X/efeitos adversos , Caspase 1/metabolismo , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
7.
Mediators Inflamm ; 2019: 7854389, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948926

RESUMO

Intestinal injury has long been considered to play a crucial role in the pathophysiology of sepsis and has even been characterized as the "motor" of it. Thus, we explored the effects of connexin43 (Cx43) on sepsis-induced intestinal injury in order to provide potential therapeutic strategies. Rat cecal ligation and puncture (CLP) models in vivo and cell models (IEC-6 cells) pretreated with LPS in vitro were used in the current study. Firstly, different methods, such as Cx43 inhibitors (18-α-GA and oleamide) or siRNA targeting Cx43 and N-acetyl cysteine (NAC) (a kind of ROS scavenger), were used to observe the effects of Cx43 channels mediating ROS transfer on intestinal injury. Secondly, the influence of ROS content on the activity of the JNK1/Sirt1/FoxO3a signaling pathway was explored through the application of NAC, sp600125 (a JNK1 inhibitor), and nicotinamide (a Sirt1 inhibitor). Finally, luciferase assays and ChIP were used to determine the direct regulation of FoxO3a on proapoptotic proteins, Bim and Puma. The results showed that sepsis-induced intestinal injury presented a dynamic change, coincident with the alternation of Cx43 expression. The inhibition of Cx43 attenuated CLP-induced intestinal injury in vivo and LPS-induced IEC-6 injury in vitro. The changes of Cx43 channel function regulated ROS transfer between the neighboring cells, which mediated the activation of the JNK1/Sirt1/FoxO3a signaling pathway. FoxO3a directly affected its downstream target genes, Bim and Puma, which are responsible for cell or tissue apoptosis. In summary, our results suggest that Cx43 inhibition suppresses ROS transfer and inactivates the JNK1/Sirt1/FoxO3a signaling pathway to protect against sepsis-induced intestinal injury.


Assuntos
Conexina 43/metabolismo , Proteína Forkhead Box O3/metabolismo , Intestinos/lesões , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sepse/complicações , Sirtuína 1/metabolismo , Animais , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Ácidos Oleicos/farmacologia , Ratos , Ratos Sprague-Dawley , Sepse/tratamento farmacológico
8.
Pharmazie ; 74(4): 231-234, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30940307

RESUMO

Aims: Neuregulin-1 (NRG-1) is a member of the epidermal growth factor family, and has an important role in cardiomyocyte development and myocardial regeneration. The aim of this study was to determine the protective effect of NRG-1 on cardiac electrical conduction in a rat myocardial infarction (MI) model. Methods: Thirty-three adult male SPF SD rats were randomized into three groups: sham-operated (n=9), acute myocardial infarction (AMI, n=12), and the NRG-1-treated (NRG-1, n=12) groups. All rats were sacrificed on day 8 after inducing MI. The 6-lead electrocardiograms (ECG) were recorded pre-operatively and eight days after operation, and analyzed. The expression levels of matrix metalloproteinase (MMP)-9 and gap junction protein connexin 43 (Cx43) in the infarcted myocardium were measured by Western blotting, and its in-situ distribution was evaluated using immunohistochemistry. Results: The PR, QRS and QT intervals were significantly prolonged in the AMI group compared to the sham operated animals (P<0.05, P<0.01 and P<0.01 respectively), and the PR and QRS intervals were partially restored in the NRG-1-treated rats (P<0.01 and P<0.01 compared to AMI group). Similarly, the increased levels of MMP-9 in the AMI group was restored upon NRG-1 treatment. The myocardial expression of Cx43 was decreased significantly in the AMI group, and was upregulated by NRG-1 treatment. Conclusions: NRG-1 attenuates MI-induced dysfunctional cardiac electrical conduction by downregulating MMP-9 and upregulating Cx43.


Assuntos
Conexina 43/genética , Metaloproteinase 9 da Matriz/genética , Infarto do Miocárdio/fisiopatologia , Neuregulina-1/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Regulação para Baixo , Eletrocardiografia , Masculino , Neuregulina-1/administração & dosagem , Ratos , Ratos Sprague-Dawley , Regulação para Cima
9.
PLoS Negl Trop Dis ; 13(4): e0006843, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30943209

RESUMO

BACKGROUND: Clonorchis sinensis is a group I bio-carcinogen responsible for cholangiocarcinoma (CHCA) in humans. However, the mechanism by which C. sinensis promotes carcinogenesis is unclear. METHODOLOGY: Using the human cholangiocyte line H69, we investigated cell proliferation and gap junction protein expression after stimulation with the hepatotoxin N-nitrosodimethylamine (NDMA) and/or excretory-secretory products (ESP) of C. sinensis, which induce inflammation. NDMA and ESP treatment increased proliferation by 146% and the proportion of cells in the G2/M phase by 37%. Moreover, the expression of the cell proliferation-related proteins E2F1, Ki-67, and cancer related protein cytokeratin 19 and Cox-2 increased in response to combined treatment with NDMA and ESP. The gap-junction proteins connexin (Cx) 43 and Cx26 increased. In contrast, Cx32 expression decreased in cells treated with NDMA and ESP. Silencing of Cx43 reduced cell proliferation and significantly suppressed Cx26 and Cox-2 expression. CONCLUSIONS: These results suggest that Cx43 is an important factor in CHCA induced by C. sinensis ESP and NDMA and further investigations targeting this pathway may allow prevention of this deadly disease.


Assuntos
Carcinogênese/patologia , Carcinógenos/toxicidade , Clonorchis sinensis/metabolismo , Conexina 43/metabolismo , Dimetilnitrosamina/toxicidade , Células Epiteliais/efeitos dos fármacos , Animais , Carcinogênese/metabolismo , Carcinógenos/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Colangiocarcinoma/induzido quimicamente , Conexina 43/genética , Conexinas/metabolismo , Células Epiteliais/metabolismo , Inativação Gênica , Proteínas de Helminto/metabolismo , Humanos
10.
Mol Med Rep ; 19(5): 3743-3755, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896818

RESUMO

Gap junctions (GJs) formed by connexins (Cxs) in T lymphocytes have been reported to have important roles in the T lymphocyte­driven inflammatory response and hypertension­mediated inflammation. Estrogen has a protective effect on cardiovascular diseases, including hypertension and it attenuates excessive inflammatory responses in certain autoimmune diseases. However, the mechanisms involved in regulating the pro­inflammatory response are complex and poorly understood. The current study investigated whether ß­estradiol suppresses hypertension and pro­inflammatory stimuli­mediated inflammatory responses by regulating Cxs and Cx­mediated GJs in peripheral blood lymphocytes. Male, 16­week­old spontaneously hypertensive rats (SHR) and Wistar­Kyoto rats (WKY) rats were randomly divided into the following three groups: WKY rats, vehicle (saline)­treated SHRs, and ß­estradiol (20 µg/kg/day)­treated SHRs. ß­estradiol was administered subcutaneously for 5 weeks. Hematoxylin and eosin staining was performed to evaluate target organ injury. Flow cytometry and ELISA were used to measure the populations of T lymphocyte subtypes in the peripheral blood, and expression of Cx40/Cx43 in T cell subtypes, and pro­inflammation cytokines levels, respectively. ELISA, a dye transfer technique, immunofluorescence and immunoblotting were used to analyze the effect of ß­estradiol on pro­inflammatory cytokine secretion, Cx­mediated GJs and the expression of Cxs in concanavalin A (Con A)­stimulated peripheral blood lymphocytes isolated from WKY rat. ß­estradiol significantly decreased blood pressure and inhibited hypertension­induced target organ injury in SHRs. Additionally, ß­estradiol treatment significantly improved the immune homeostasis of SHRs, as demonstrated by the decreased percentage of cluster of differentiation (CD)4+/CD8+ T­cell subset ratio, reduced serum levels of pro­inflammatory cytokines and increased the percentage of CD4+CD25+ T cells. ß­estradiol also markedly reduced the expression of Cx40/Cx43 in T lymphocytes from SHRs. In vitro, ß­estradiol significantly suppressed the production of pro­inflammatory cytokines, reduced communication via Cx­mediated gap junctions and decreased the expression of Cx40/Cx43 in Con A­stimulated lymphocytes. These results indicate that ß­estradiol attenuates inflammation and end organ damage in hypertension, which may be partially mediated via downregulated expression of Cxs and reduced function of Cx­mediated GJ.


Assuntos
Concanavalina A/efeitos adversos , Conexinas/metabolismo , Estradiol/farmacologia , Hipertensão/complicações , Inflamação/etiologia , Inflamação/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Citocinas/sangue , Citocinas/metabolismo , Junções Comunicantes/metabolismo , Expressão Gênica , Hipertensão/fisiopatologia , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Ratos , Remodelação Vascular/efeitos dos fármacos
11.
Biochim Biophys Acta Mol Cell Res ; 1866(5): 828-838, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30769008

RESUMO

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.


Assuntos
Movimento Celular , Conexina 43/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Colforsina/farmacologia , Conexina 43/genética , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Células Endoteliais/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Células HeLa , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/genética
13.
Circ Res ; 124(1): 101-113, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30582443

RESUMO

RATIONALE: Cardiac lymphangiogenesis contributes to the reparative process post-myocardial infarction, but the factors and mechanisms regulating it are not well understood. OBJECTIVE: To determine if epicardial-secreted factor AM (adrenomedullin; Adm=gene) improves cardiac lymphangiogenesis post-myocardial infarction via lateralization of Cx43 (connexin 43) in cardiac lymphatic vasculature. METHODS AND RESULTS: Firstly, we identified sex-dependent differences in cardiac lymphatic numbers in uninjured mice using light-sheet microscopy. Using a mouse model of Adm hi/hi ( Adm overexpression) and permanent left anterior descending ligation to induce myocardial infarction, we investigated cardiac lymphatic structure, growth, and function in injured murine hearts. Overexpression of Adm increased lymphangiogenesis and cardiac function post-myocardial infarction while suppressing cardiac edema and correlated with changes in Cx43 localization. Lymphatic function in response to AM treatment was attenuated in mice with a lymphatic-specific Cx43 deletion. In vitro experiments in cultured human lymphatic endothelial cells identified a novel mechanism to improve gap junction coupling by pharmaceutically targeting Cx43 with verapamil. Finally, we show that connexin protein expression in cardiac lymphatics is conserved between mouse and human. CONCLUSIONS: AM is an endogenous, epicardial-derived factor that drives reparative cardiac lymphangiogenesis and function via Cx43, and this represents a new therapeutic pathway for improving myocardial edema after injury.


Assuntos
Adrenomedulina/metabolismo , Conexina 43/metabolismo , Edema Cardíaco/metabolismo , Linfangiogênese , Vasos Linfáticos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Pericárdio/metabolismo , Adrenomedulina/genética , Animais , Células Cultivadas , Conexina 43/genética , Modelos Animais de Doenças , Edema Cardíaco/genética , Edema Cardíaco/fisiopatologia , Edema Cardíaco/prevenção & controle , Feminino , Junções Comunicantes/metabolismo , Humanos , Vasos Linfáticos/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Pericárdio/fisiopatologia , Transdução de Sinais , Função Ventricular Esquerda
14.
Glia ; 67(4): 619-633, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30585358

RESUMO

Astrocytes support normal brain function, but may also contribute to neurodegeneration when they become reactive under pathological conditions such as stroke. However, the molecular underpinnings of this context-dependent interplay between beneficial and detrimental properties in reactive astrogliosis have remained incompletely understood. Therefore, using the RiboTag technique, we immunopurified translating mRNAs specifically from astrocytes 72 hr after transient middle cerebral artery occlusion in mice (tMCAO), thereby generating a stroke-specific astroglial translatome database. We found that compared to control brains, reactive astrocytes after tMCAO show an enrichment of transcripts linked to the A2 phenotype, which has been associated with neuroprotection. However, we found that astrocytes also upregulate a large number of potentially neurotoxic genes. In total, we identified the differential expression of 1,003 genes and 38 transcription factors, of which Stat3, Sp1, and Spi1 were the most prominent. To further explore the effects of Stat3-mediated pathways on stroke pathogenesis, we subjected mice with an astrocyte-specific conditional deletion of Stat3 to tMCAO, and found that these mice have reduced stroke volume and improved motor outcome 72 hr after focal ischemia. Taken together, our study extends the emerging database of novel astrocyte-specific targets for stroke therapy, and supports the role of astrocytes as critical safeguards of brain function in health and disease.


Assuntos
Astrócitos/metabolismo , Perfilação da Expressão Gênica/métodos , Infarto da Artéria Cerebral Média/patologia , Rombencéfalo/patologia , Animais , Biologia Computacional , Conexina 43/genética , Conexina 43/metabolismo , Modelos Animais de Doenças , Feminino , Galectina 3/genética , Galectina 3/metabolismo , Regulação da Expressão Gênica/genética , Imunoprecipitação , Infarto da Artéria Cerebral Média/fisiopatologia , Lipocalina-2/genética , Lipocalina-2/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Teste de Desempenho do Rota-Rod , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
15.
Acta Neuropathol Commun ; 6(1): 144, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30577786

RESUMO

GJA1 (connexin43) has been predicted as the top key driver of an astrocyte enriched subnetwork associated with Alzheimer's disease (AD). In this study, we comprehensively examined GJA1 expression across 29 transcriptomic and proteomic datasets from post-mortem AD and normal control brains. We demonstrated that GJA1 was strongly associated with AD amyloid and tau pathologies and cognitive functions. RNA sequencing analysis of Gja1-/- astrocytes validated that Gja1 regulated the subnetwork identified in AD, and many genes involved in Aß metabolism. Astrocytes lacking Gja1 showed reduced Apoe protein levels as well as impaired Aß phagocytosis. Consistent with this, wildtype neurons co-cultured with Gja1-/- astrocytes contained higher levels of Aß species than those with wildtype astrocytes. Moreover, Gja1-/- astrocytes was more neuroprotective under Aß stress. Our results underscore the importance of GJA1 in AD pathogenesis and its potential for further investigation as a promising pharmacological target in AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Conexina 43/metabolismo , Redes Reguladoras de Genes/fisiologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Apolipoproteínas E/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/patologia , Células Cultivadas , Estudos de Coortes , Conexina 43/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteômica
16.
J Biosci ; 43(5): 1055-1068, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30541963

RESUMO

The gap junctions (GJs), which form intercellular communicating channels between two apposing cells or form hemichannel with extracellular environment, perform crucial functions to maintain small molecule homeostasis. The central nervous system (CNS) GJs are important for maintenance of myelin sheath and neuronal activity. Connexin (Cx) proteins are building blocks of GJs. Recent cell-biological investigations show that amongst the CNS specific Cxs, the most abundant Cx protein, Cx43 and its oligodendrocytic coupling partner Cx47 primarily important for maintenance of CNS myelin. Recent investigations elucidate that the expression of Cx43 and Cx47 is very important to maintain K? buffering and nutrient homeostasis in oligodendrocytes, CNS myelin and oligodendrocyte function. The investigations on Multiple Sclerosis (MS) patient samples and EAE hypothesized that the functional loss of Cx43/Cx47 could be associated with spread of chronic MS lesions. Exploring the mechanism of initial GJ alteration and its effect on demyelination in this model of MS might play a primary role to understand the basis of altered CNS homeostasis, observed during MS. In this review, we mainly discuss the role of CNS GJs, specifically the Cx43/Cx47 axis in the perspective of demyelination.


Assuntos
Astrócitos/metabolismo , Conexina 43/genética , Conexinas/genética , Doenças Desmielinizantes/genética , Esclerose Múltipla/genética , Oligodendroglia/metabolismo , Animais , Astrócitos/patologia , Comunicação Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Conexina 43/metabolismo , Conexinas/metabolismo , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Junções Comunicantes/metabolismo , Junções Comunicantes/ultraestrutura , Regulação da Expressão Gênica , Humanos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Oligodendroglia/patologia , Remielinização/fisiologia , Transdução de Sinais
17.
Nat Commun ; 9(1): 5377, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560870

RESUMO

Collective cell migration mediates multiple tissue morphogenesis processes. Yet how multi-dimensional mesenchymal cell movements are coordinated remains mostly unknown. Here we report that coordinated mesenchymal cell migration during chicken feather elongation is accompanied by dynamic changes of bioelectric currents. Transcriptome profiling and functional assays implicate contributions from functional voltage-gated Ca2+ channels (VGCCs), Connexin-43 based gap junctions, and Ca2+ release activated Ca2+ (CRAC) channels. 4-Dimensional Ca2+ imaging reveals that the Sonic hedgehog-responsive mesenchymal cells display synchronized Ca2+ oscillations, which expand progressively in area during feather elongation. Inhibiting VGCCs, gap junctions, or Sonic hedgehog signaling alters the mesenchymal Ca2+ landscape, cell movement patterns and feather bud elongation. Ca2+ oscillations induced by cyclic activation of opto-cCRAC channels enhance feather bud elongation. Functional disruption experiments and promoter analysis implicate synergistic Hedgehog and WNT/ß-Catenin signaling in activating Connexin-43 expression, establishing gap junction networks synchronizing the Ca2+ profile among cells, thereby coordinating cell movement patterns.


Assuntos
Sinalização do Cálcio/fisiologia , Movimento Celular/fisiologia , Conexina 43/metabolismo , Plumas/crescimento & desenvolvimento , Proteínas Hedgehog/metabolismo , Animais , Células Cultivadas , Galinhas , Conexina 43/genética , Embrião não Mamífero , Plumas/citologia , Junções Comunicantes/metabolismo , Mesoderma/citologia , Mesoderma/fisiologia , Morfogênese/fisiologia , Regiões Promotoras Genéticas , Pele/citologia , Via de Sinalização Wnt/fisiologia
18.
Int J Mol Sci ; 20(1)2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30583492

RESUMO

It is becoming clear that in addition to gap junctions playing a role in cell⁻cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures-annular gap junctions-were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.


Assuntos
Vesículas Citoplasmáticas/metabolismo , Junções Comunicantes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias/metabolismo , Imagem Óptica , Comunicação Celular , Linhagem Celular Tumoral , Conexina 43/química , Conexina 43/genética , Conexina 43/metabolismo , Vesículas Citoplasmáticas/química , Junções Comunicantes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Imuno-Histoquímica , Citometria de Varredura a Laser , Lisossomos/química , Lisossomos/metabolismo , Mitocôndrias/química
19.
Cell Physiol Biochem ; 49(6): 2124-2137, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30257239

RESUMO

BACKGROUND/AIMS: Acute kidney injury (AKI) is a frequent and serious complication of sepsis; however, there is no effective treatment for it. FangJiFuling (FF) decoction is widely used to treat acute glomerulonephritis and nephritic syndrome in the clinical setting. METHODS: On the basis of its anti-inflammatory properties, the renoprotective effect of FF on a mouse model of lipopolysaccharide (LPS)-induced AKI was investigated. Major compounds were identified in FF with high-performance liquid chromatography. A bioinformatics analysis tool was used to predict target genes. Quantitative real-time PCR and western blot analyses were performed to validate the targets. Furthermore, the expression of a target gene was silenced by small interfering RNA-mediated knockdown in vitro. RESULTS: Bioinformatics analysis indicated that inflammation, apoptosis, and cell junction were closely related to the renoprotective effects of FF. Validation was confirmed by an in vivo test. A reduction of inflammatory cell infiltration and inflammatory cytokine mRNA expression (iNOS, NF-κB, MCP-1, and TNF-α) following the administration of FF (50 mg/kg) was observed in LPS-treated renal tissue. In addition, FF treatment suppressed mitochondrial-mediated apoptosis by regulating the Bax/Bcl-2 ratio in LPS-induced renal injury. Silencing Cx43, a cell-to-cell junction protein, was found to enhance the protective effect of FF against LPS-induced renal injury. CONCLUSION: Our study suggests that FF exhibits a renoprotective effect against LPS-induced inflammatory and apoptotic responses. In addition, Cx43 might be involved in these processes. These findings indicate the potential role of FF as a natural renoprotective product.


Assuntos
Lesão Renal Aguda/prevenção & controle , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Substâncias Protetoras/uso terapêutico , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/mortalidade , Animais , Linhagem Celular , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Conexina 43/metabolismo , Bases de Dados Factuais , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/uso terapêutico , Interações Ervas-Drogas/genética , Humanos , Lipopolissacarídeos/toxicidade , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Taxa de Sobrevida
20.
Cell Physiol Biochem ; 49(3): 985, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196298

RESUMO

Chronic kidney disease is an incurable to date pathology with a continuously growing incidence that contributes to the increase of the number of deaths worldwide. With currently no efficient prognostic or therapeutic options being available, the only possibility for treatment of end-stage renal disease is renal replacement therapy through dialysis or transplantation. Understanding the molecular mechanisms participating in the progression of renal diseases and uncovering the pathways implicated will permit the identification of novel and more efficient targets of therapy. Connexin43 was recently identified as a novel player in the development of chronic kidney disease. It was found de novo expressed and/or differentially localized in various renal cell populations during progression of renal disease, indicating an abnormal connexin signaling, both in patients and animal models. Subsequent in vivo studies demonstrated that connexin43 is involved in mediating inflammatory and fibrotic processes contributing to renal damage. Genetic, pharmaco-genetic or peptide-based inhibition of connexin43 in animal models and cell culture systems was successful in preventing the progression of the pathology and preserving the cell phenotypes. This review will summarize the recent advances on connexin43 in the field of kidney diseases and discuss the potential of future connexin43-based therapies against chronic kidney disease.


Assuntos
Conexina 43/metabolismo , Insuficiência Renal Crônica/patologia , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Humanos , Rim/metabolismo , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Podócitos/citologia , Podócitos/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo
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