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1.
Proc Natl Acad Sci U S A ; 117(31): 18810-18821, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690710

RESUMO

In the central nervous system, glycogen-derived bioenergetic resources in astrocytes help promote tissue survival in response to focal neuronal stress. However, our understanding of the extent to which these resources are mobilized and utilized during neurodegeneration, especially in nearby regions that are not actively degenerating, remains incomplete. Here we modeled neurodegeneration in glaucoma, the world's leading cause of irreversible blindness, and measured how metabolites mobilize through astrocyte gap junctions composed of connexin 43 (Cx43). We elevated intraocular pressure in one eye and determined how astrocyte-derived metabolites in the contralateral optic projection responded. Remarkably, astrocyte networks expand and redistribute metabolites along distances even 10 mm in length, donating resources from the unstressed to the stressed projection in response to intraocular pressure elevation. While resource donation improves axon function and visual acuity in the directly stressed region, it renders the donating tissue susceptible to bioenergetic, structural, and physiological degradation. Intriguingly, when both projections are stressed in a WT animal, axon function and visual acuity equilibrate between the two projections even when each projection is stressed for a different length of time. This equilibration does not occur when Cx43 is not present. Thus, Cx43-mediated astrocyte metabolic networks serve as an endogenous mechanism used to mitigate bioenergetic stress and distribute the impact of neurodegenerative disease processes. Redistribution ultimately renders the donating optic nerve vulnerable to further metabolic stress, which could explain why local neurodegeneration does not remain confined, but eventually impacts healthy regions of the brain more broadly.


Assuntos
Astrócitos , Glaucoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Junções Comunicantes/metabolismo , Pressão Intraocular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
2.
Nat Commun ; 11(1): 1860, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312952

RESUMO

Ependymal cells (ECs) are multiciliated neuroepithelial cells that line the ventricles of the brain and the central canal of the spinal cord (SC). How ependymal motile cilia are maintained remains largely unexplored. Here we show that zebrafish embryos deficient in Wnt signaling have defective motile cilia, yet harbor intact basal bodies. With respect to maintenance of ependymal motile cilia, plcδ3a is a target gene of Wnt signaling. Lack of Connexin43 (Cx43), especially its channel function, decreases motile cilia and intercellular Ca2+ wave (ICW) propagation. Genetic ablation of cx43 in zebrafish and mice diminished motile cilia. Finally, Cx43 is also expressed in ECs of the human SC. Taken together, our findings indicate that gap junction mediated ICWs play an important role in the maintenance of ependymal motile cilia, and suggest that the enhancement of functional gap junctions by pharmacological or genetic manipulations may be adopted to ameliorate motile ciliopathy.


Assuntos
Cílios/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Epêndima/metabolismo , Medula Espinal/metabolismo , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Cílios/genética , Conexina 43/genética , Epêndima/patologia , Junções Comunicantes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Via de Sinalização Wnt/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Am J Physiol Regul Integr Comp Physiol ; 318(6): R1078-R1090, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348681

RESUMO

Cx30.2 protein content and localization were assessed during development. An account of Cx30.2, Cx43, Cx46, and Cx50, and insulin receptor (IR) responses to Cx30.2, Cx46, or Cx50 deficiency in mouse interstitial tissue (ITf)- and seminiferous tubule-enriched fractions (STf) is given. The impact of high glucose/insulin on Cx30.2 was investigated in spontaneously diabetic and obese db/db and ob/ob mouse testis and anterior pituitary (AP). Cx30.2 labeled contacts in vascular endothelial and Leydig cells and Sertoli cell junctions in stage V-VII. Cx30.2 expression is regulated differently in the interstitium and tubules. Cx30.2 at 30-kDa levels peaked by 28 days in ITf and by 14 days in STf. In STf, deleting Cx30.2 decreased Cx43 and Cx50, whereas deleting Cx50 downregulated Cx30.2. The opposite occurred in ITf. In STf, deleting Cx30.2 upregulated Cx46 except the full-length reciprocally, deleting Cx46 upregulated Cx30.2. In ITf, Cx30.2 deficiency upregulated full-length and phosphorylated Cx46, whereas deleting Cx46 downregulated 48- to 50-kDa Cx30.2. The db/db and ob/ob mouse ITf, STf, and AP showed imbalanced Cx30.2 levels. IRα levels at 135 kDa declined in Cx30.2-/- and Cx50-/- mouse ITf and Cx46-/- and Cx50-/- STf. IRß at 98 to 110 kDa dropped in Cx30.2-/- and Cx46-/- mice STf suggesting that Cx30.2 deficiency decreases active IR sites. The results show the connexins interdependence and interaction and that altering a single connexin changes the remaining connexins expression, which can modify gap junction-mediated glucose exchanges in contacting cells. Data suggest that glucose/insulin influences Cx30.2 turnover in testis and AP and, reciprocally, that connexins modulate testis glucose uptake and response to insulin.


Assuntos
Conexina 43/genética , Conexinas/genética , Diabetes Mellitus Experimental/genética , Obesidade/genética , Receptor de Insulina/genética , Testículo/metabolismo , Animais , Conexina 43/metabolismo , Conexinas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Receptor de Insulina/metabolismo
4.
Invest Ophthalmol Vis Sci ; 61(3): 14, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32176265

RESUMO

Purpose: We used a human corneal epithelial cell (HCE) line to determine the involvement of the advanced glycation end products (AGEs) / receptor for AGEs (RAGE) couple in corneal epithelium wound healing. Methods: After wounding, HCE cells were exposed to two major RAGE ligands (HMGB1 and AGEs), and wound healing was evaluated using the in vitro scratch assay. Following wound healing, the HCE cells were used to study the influence of the RAGE ligands on HCE proliferation, invasion, and migration. Activation of the nuclear factor (NF)-κB signaling pathway by the AGEs/RAGE couple was tested using a luciferase reporter assay. Functional transcriptional regulation by this pathway was confirmed by quantification of expression of the connexin 43 target gene. For each experiment, specific RAGE involvement was confirmed by small interfering RNA treatments. Results: AGEs treatment at a dose of 100 µg/mL significantly improved the wound healing process in a RAGE-dependent manner by promoting cell migration, whereas HMGB1 had no effect. No significant influence of the AGEs/RAGE couple was observed on cell proliferation and invasion. However, this treatment induced an early activation of the NF-κB pathway and positively regulated the expression of the target gene, connexin 43, at both the mRNA and protein levels. Conclusions: Our results demonstrate that the RAGE pathway is activated by AGEs treatment and is involved in the promotion of corneal epithelial wound healing. This positive action is observed only during the early stages of wound healing, as illustrated by the quick activation of the NF-κB pathway and induction of connexin 43 expression.


Assuntos
Lesões da Córnea/fisiopatologia , Epitélio Anterior/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Receptor para Produtos Finais de Glicação Avançada/fisiologia , Cicatrização/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Lesões da Córnea/patologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio Anterior/citologia , Epitélio Anterior/lesões , Epitélio Anterior/fisiologia , Produtos Finais de Glicação Avançada/administração & dosagem , Produtos Finais de Glicação Avançada/fisiologia , Proteína HMGB1/administração & dosagem , Proteína HMGB1/farmacologia , Humanos , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptor para Produtos Finais de Glicação Avançada/genética , Transdução de Sinais/fisiologia , Cicatrização/fisiologia
5.
Toxicol Lett ; 318: 57-64, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31585160

RESUMO

3-Bromopyruvate (3-BrPA) is a promising agent that has been widely studied in the treatment of cancer and pulmonary hypertension. Rotenone is a pesticide commonly used on farms and was shown to have anti-cancer activity and delay fibrosis progression in chronic kidney disease in a recent study. However, there are few studies showing the toxicity of rotenone and 3-BrPA in the myocardium. To support further medical exploration, it is necessary to clarify the side effects of these compounds on the heart. This study was designed to examine the cardiotoxicity of 3-BrPA and rotenone by investigating electrical and structural cardiac remodeling in rats. Forty male rats were divided into 4 groups (n = 10 in each group) and injected intraperitoneally with 3-BrPA, rotenone or a combination of 3-BrPA and rotenone. The ventricular effective refractory period (VERP), corrected QT interval (QTc), and ventricular tachycardia/ventricular fibrillation (VT/VF) inducibility were measured. The expression of Cx43, Kir2.1, Kir6.2, DHPRα1, KCNH2, caspase3, caspase9, Bax, Bcl2, and P53 was detected. Masson's trichrome, TUNEL, HE, and PAS staining and transmission electron microscopy were used to detect pathological and ultrastructural changes. Our results showed that rotenone alone and rotenone combined with 3-BrPA significantly increased the risk of ventricular arrhythmias. Rotenone combined with 3-BrPA caused myocardial apoptosis, and rotenone alone and rotenone combined with 3-BrPA caused electrical and structural cardiac remodeling in rats.


Assuntos
Antineoplásicos/toxicidade , Ventrículos do Coração/efeitos dos fármacos , Inseticidas/toxicidade , Piruvatos/toxicidade , Rotenona/toxicidade , Taquicardia Ventricular/induzido quimicamente , Fibrilação Ventricular/induzido quimicamente , Remodelação Ventricular/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Cardiotoxicidade , Conexina 43/genética , Conexina 43/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Ventrículos do Coração/ultraestrutura , Masculino , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos Wistar , Período Refratário Eletrofisiológico/efeitos dos fármacos , Medição de Risco , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologia , Taquicardia Ventricular/fisiopatologia , Fibrilação Ventricular/metabolismo , Fibrilação Ventricular/patologia , Fibrilação Ventricular/fisiopatologia
6.
Cancer Lett ; 469: 238-245, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31678166

RESUMO

The therapy resistance of pancreatic cancer is associated with the loss of gap junction intercellular communication and connexin 43 expression. The broccoli isothiocyanate sulforaphane restored these features and therapy sensitivity. We investigated whether microRNA signaling is involved. Established cell lines and a patient tissue array (n = 96) were evaluated by miRNA and gene array, bioinformatics, expression studies, in situ hybridization and immunohistochemistry. Sulforaphane inhibited the expression of our top candidate miR30a-3p. Upon transfection of miR30a-3p inhibitors, the gemcitabine bystander effect, Cx43 expression and intercellular communication increased, whereas miR30a-3p mimics inhibited the luciferase activity of a Cx43 3'-UTR construct. miR30a-3p-overexpressing tumor xenografts had a decreased tumor volume and increased gemcitabine sensitivity. In patient tissues, a higher expression of miR30a-3p and a lower expression of Cx43 correlated with malignancy. These findings provide new knowledge and suggest sulforaphane as cotreatment for pancreatic cancer.


Assuntos
Conexina 43/genética , Isotiocianatos/farmacologia , MicroRNAs/genética , Neoplasias Pancreáticas/tratamento farmacológico , Adulto , Idoso , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Feminino , Junções Comunicantes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Hibridização In Situ , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos
7.
Invest Ophthalmol Vis Sci ; 60(15): 5104-5111, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31826237

RESUMO

Purpose: Cell-cell contact in retinal pigment epithelium (RPE) involves adherent junctions, gap junctions, and tight junctions, which are primarily composed by E-cadherin, zona occludens 1 (ZO-1), and connexin 43, respectively. Here, we aimed to explore the relationship and interplay between these junction-associated proteins. Methods: E-cadherin, connexin 43, and ZO-1 expression in human primary RPE in the early phase after TGF-ß1 stimulation was detected. The knockdown of E-cadherin, ZO-1, and connexin 43 was performed to characterize the regulatory network involving these three proteins. Dye transfer and FITC-dextran permeability assays were conducted to observe the epithelial functional alterations. Transmission electron microscopy (TEM) was used to observe the ultrastructure of the cell-cell junctions in mouse RPE. The immunofluorescence staining and coimmunoprecipitation were performed to observe the colocalization and the physical association of E-cadherin, ZO-1, and connexin 43. Results: Among these three components, E-cadherin appeared to be the first protein that was downregulated after TGF-ß1 treatment. The ultrastructures of adherent junctions, gap junctions, and tight junctions could be observed in mouse RPE by TEM. E-cadherin, ZO-1, and connexin 43 were colocalized and physically bound to each other. The knockdown of one of these three proteins led to downregulation of the other two proteins and compromised epithelial function. Conclusions: E-cadherin, ZO-1, and connexin 43 were physically associated with each other and were mutually regulated. To enhance the understanding of cell-cell contacts, a holistic view is needed. Our results provide new insights in RPE disorders such as proliferative vitreoretinopathy.


Assuntos
Caderinas/genética , Conexina 43/genética , Regulação da Expressão Gênica , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/genética , Proteína da Zônula de Oclusão-1/genética , Animais , Caderinas/biossíntese , Células Cultivadas , Conexina 43/biossíntese , Humanos , Junções Intercelulares , Camundongos , Microscopia Eletrônica de Transmissão , RNA/genética , Epitélio Pigmentado da Retina/ultraestrutura , Junções Íntimas , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Proteína da Zônula de Oclusão-1/biossíntese
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(12): 1191-1194, 2019 Dec 10.
Artigo em Chinês | MEDLINE | ID: mdl-31813145

RESUMO

OBJECTIVE: To explore the genetic basis of a pedigree affected with oculodentodigital dysplasia. METHODS: Genomic DNA was extracted from peripheral blood or amniotic fluid samples derived from the pedigree. Exon 2 of the GJA1 gene was amplified for sequencing. RESULTS: Two pedigree members were found to carry heterozygous missense variation of the GJA1 gene, c.221A>C (p.H74P). CONCLUSION: The missense c.221A>C variation of the GJA1 gene probably underlies the oculodentodigital dysplasia in this pedigree.


Assuntos
Conexina 43/genética , Anormalidades Craniofaciais/genética , Anormalidades do Olho/genética , Deformidades Congênitas do Pé/genética , Sindactilia/genética , Anormalidades Dentárias/genética , Humanos , Mutação , Linhagem
9.
Int J Mol Sci ; 20(24)2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31817921

RESUMO

Gap junctional channels are specialized components of the cellular membrane that allow the intercellular passage of small metabolites, ions, and second messengers to maintain homeostasis. They are comprised of members of the connexin gene family that encode a wide array of proteins that are expressed in nearly every tissue type. Cx43 is perceived to be the most broadly expressed connexin in humans, with several genetic skin diseases being linked to Cx43 mutations specifically. These mutations, in large, produce a gain of functional hemichannels that contribute to the phenotypes of Erythrokeratoderma Variabilis et Progressiva (EKVP), Palmoplantar Keratodemra Congenital Alopecia-1 (PPKCA1), and others that produce large conductance and increased permselectivity in otherwise quiescent structures. Gaining functional hemichannels can have adverse effects in the skin, inducing apoptosis via Ca2+ overload or increased ATP permeability. Here, we review the link between Cx43 and skin disease. We aim to provide insight into the mechanisms regulating the normal and pathophysiological gating of these essential proteins, as well as address current therapeutic strategies. We also demonstrate that transient transfection of neuro-2a (N2a) cells with mutant Cx43 cDNA resulted in increased hemichannel activity compared to wild-type Cx43 and untransfected cells, which is consistent with other studies in the current literature.


Assuntos
Conexina 43/genética , Canais Iônicos/fisiologia , Mutação , Dermatopatias/patologia , Animais , Conexina 43/metabolismo , Humanos , Dermatopatias/genética , Dermatopatias/metabolismo
10.
Mol Med Rep ; 20(6): 5091-5099, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638216

RESUMO

C­type natriuretic peptide (CNP), from the family of natriuretic peptides (NPs), has been shown to induce antihypertrophic and antifibrotic effects in cardiomyocytes. However, the roles of CNP in the atrial dysregulation of connexin (Cx)40 and Cx43 remain to be elucidated. The present study aimed to investigate the effects of CNP on angiotensin (Ang) II­induced Cx40 and Cx43 dysregulation in isolated perfused beating rat left atria. A rat isolated perfused beating atrial model was used and the protein levels were determined via western blotting. Ang II significantly upregulated NF­κB, activator protein­1, transforming growth factor­ß1 (TGF­ß1), collagen I and matrix metalloproteinase 2, leading to atrial fibrosis, and downregulated expression of Cx40 and Cx43. The changes in Cx40 and Cx43 induced by Ang II were abolished by CNP through upregulation of phosphorylated AMP­activated kinase a1 (AMPK) and downregulation of TGF­ß1. The effects of CNP on AMPK and TGF­ß1 levels were inhibited by KT5823 and pertussis toxin, inhibitors of protein kinase G (PKG) and NP receptor type C (NPR­C), respectively. Thus, CNP can prevent Ang II­induced dysregulation of Cx40 and Cx43 through activation of AMPK via the CNP­PKG and CNP­NPR­C pathways in isolated beating rat atria. The present findings suggested that CNP may be therapeutically useful for clinical conditions involving cardiac dysregulation of Cx expression­related diseases.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Angiotensina II/metabolismo , Conexina 43/genética , Conexinas/genética , Átrios do Coração/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Transdução de Sinais/genética , Angiotensina II/farmacologia , Animais , Biomarcadores , Conexina 43/metabolismo , Conexinas/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/patologia , Masculino , Peptídeo Natriurético Tipo C/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
11.
Genes Cells ; 24(12): 789-800, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31609038

RESUMO

The intercalated disk (ID), a highly organized adhesion structure connecting neighboring cardiomyocytes, fulfills mechanical and electrical signaling communication to ensure normal heart function. Lipoprotein receptor-related protein 6 (LRP6) is a co-receptor inducing canonical Wnt/ß-catenin signaling. It was recently reported that LRP6 deficiency in cardiomyocytes predisposes to arrhythmia independent of Wnt signaling. However, whether LRP6 directly regulates the structure of IDs requires further investigation. The aim of the present study was to explore the role of LRP6 in IDs and the potential underlying mechanisms by inducible cardiac-specific LRP6 knockout mice. The results revealed that LRP6 was predominately expressed in the cell membrane, including the IDs of cardiomyocytes. Tamoxifen-inducible cardiac-specific LRP6 knockout mice displayed overt cardiac dysfunction and disruption of ID structure. Further analysis revealed that cardiac LRP6 deficiency induced the imbalance of ID component proteins, characterized by the sharply decreased expression of connexin 43 (Cx43) and the significantly increased expression of N-cadherin, desmoplakin and γ-catenin in tissue lysates or membrane fraction from the left ventricle. STRING database analysis indicated that ß-catenin, but no other ID-associated proteins, interacted with LRP6. Our immunoprecipitation analysis demonstrated that LRP6 strongly interacted with Cx43, N-cadherin and γ-catenin, and weakly interacted with ß-catenin, whereas there was no association with desmoplakin. In response to LRP6 deficiency, the recruitment of ß- or γ-catenin to N-cadherin was increased, but they displayed little interaction with Cx43. In conclusion, LRP6 is required to maintain the integrity of ID structure and the balance of ID proteins, and the interaction between LRP6 and Cx43, N-cadherin and γ-catenin may be involved in this process.


Assuntos
Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Cateninas/genética , Cateninas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos , Ligação Proteica
12.
Eur J Endocrinol ; 181(6): 615-627, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31581124

RESUMO

Objective: Pituitary corticotroph adenomas commonly cause Cushing's disease (CD) but part of these tumours are hormonally inactive (silent corticotroph adenomas, SCA). USP8 mutations are well-known driver mutations in corticotrophinomas. Differences in transcriptomic profiles between functioning and silent tumours or tumours with different USP8 status have not been investigated. Design and methods: Forty-eight patients (28 CD, 20 SCA) were screened for USP8 mutations with Sanger sequencing. Twenty-four patients were included in transcriptomic profiling with Ampliseq Transcriptome Human Gene Expression Core Panel. The entire patients group was included in qRT-PCR analysis of selected genes expression. Immunohistochemistry was used for visualization of selected protein. Results: We found USP8 mutation in 15 patients with CD and 4 SCAs. USP8 mutations determine molecular profile of the tumours as showed by hierarchical clustering and identification of 1648 genes differentially expressed in USP8-mutated and USP8-wild-type tumours. Mutations affect many molecular pathways as observed in Gene Set Enrichment analysis. USP8-mutated adenomas showed higher level of POMC, CDC25A, MAPK4 but lower level of CCND2, CDK6, CDKN1B than USP8-wt tumours. Eighty-seven genes differentially expressed between CD-related adenomas and SCAs were found, including those involved in cell signalling (GLI2, DLC1, TBX2, RASSF6), cell adhesion (GJA1, CDH6), ion transport (KCNN4, KCNJ5) and GABA signalling (GABBR2, GABRD). Conclusion: USP8 mutations occur in functioning and silent corticotrophinomas. They have pleiotropic effect, not limited to EGFR signalling, and affect expression levels of many genes involved in different pathways. Expression of GABA-related genes GABBR2, GNAL, GABARD and KCNJ5 correspond to functional status of the tumours.


Assuntos
Adenoma Hipofisário Secretor de ACT/genética , Adenoma Hipofisário Secretor de ACT/metabolismo , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Mutação/genética , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Ubiquitina Tiolesterase/genética , Adulto , Idoso , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo
13.
Chin Med J (Engl) ; 132(19): 2354-2361, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31567382

RESUMO

BACKGROUND: In our previous paper, we demonstrated that Connexin 43 (CX43) was highly expressed in bladder cancer (BC) tissues. But the molecular mechanism about microRNAs (miRNAs) regulation upstream of CX43 in BC has not been well elucidated and remains to be further studied. MicroRNA-139-5p (miR-139-5p) is a tumor suppressor in progression of multifarious cancers including BC. Nevertheless, the underlying mechanisms of CX43/miR-139-5p in tumorigenesis of BC are still not well illustrated. The specific objective of our study was to inquiry the effect of CX43/miR-139-5p on BC progression and its underlying mechanism. METHODS: The bioinformatics analysis softwares were applied to predict the miRNAs in the upstream of CX43. First, the expression levels of miR-139-5p in BC tissues (tumor) and paracancer tissues (normal) were investigated using the data from The Cancer Genome Atlas database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of miR-139-5p in three human BC cell lines 5637, T24, ECV-304 and a human bladder epithelial immortalized cell line SV-HUC-1 (normal control). Then si-CX43, si-control, miR-139-5p mimic, and its negative control (NC) were transfected into BC cell line ECV-304. The relationship of miR-139-5p and CX43 was analyzed by dual-luciferase reporter assay. The qRT-PCR and Western blotting were used to test the mRNA and protein expression level of CX43. The proliferation of ECV-304 and T24 cells were examined by cell counting kit-8. The migration and invasion of ECV-304 cells were tested by transwell assay. To determine whether miR-139-5p would affect cell proliferation, migration and invasion by targeting CX43, we executed the rescue assay. The comparison between two groups was analyzed by Student's t test, and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test. RESULTS: The expression of miR-139-5p was remarkably down-regulated in BC tissues (tumor vs. normal, 2.286 ±â€Š0.017 vs. 3.211 ±â€Š0.034, t = 11.540, P < 0.0001) and cell lines (P < 0.01 in all BC cell lines). Besides, we also indicated that over-expression of miR-139-5p reduced the proliferation of ECV-304 (P = 0.001) and T24 cells (P = 0.005). Moreover, miR-139-5p over-expression weakened the invasion (P = 0.001) and migration (P = 0.001) of ECV-304 cells. Furthermore, the relative luciferase activity of CX43-wild type construct was distinctly lessened by up-regulation of miR-139-5p (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.916 ±â€Š0.063 vs. 0.356 ±â€Š0.048, t = 7.085, P = 0.002), nevertheless the activity of CX43-mutant type construct was untouched (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.918 ±â€Š0.057 vs. 0.878 ±â€Š0.039, t = 0.577, P = 0.595). Finally, the rescue assay revealed that CX43 deletion enhanced the depressor effect of miR-139-5p on ECV-304 cell proliferation (P < 0.01), invasion (P = 0.028), and migration (P = 0.014). CONCLUSION: MiR-139-5p, as a tumor-suppressor, repressed cell proliferation, invasion, and migration in BC, which might be achieved by regulating CX43.


Assuntos
Conexina 43/genética , Genes Supressores de Tumor/fisiologia , MicroRNAs/fisiologia , Neoplasias da Bexiga Urinária/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Invasividade Neoplásica , Neoplasias da Bexiga Urinária/genética
14.
Int J Oncol ; 55(4): 845-859, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485592

RESUMO

Reports have highlighted an association between connexins (CXs) or gap junction proteins and non­small cell lung cancer (NSCLC). In the present study, it was aimed to elucidate the regulatory mechanism of CX26 and CX43 under hypoxic conditions in NSCLC. Clinical samples were collected for analysis of CX26 and CX43 expression and clinical cancerization followed by quantification of CX26 and CX43 expression. Following the establishment of an in vitro hypoxia model, P53/murine double minute­2 (MDM2) signaling pathway­, proliferation­ and epithelial­mesenchymal transition (EMT)­related genes were quantified to evaluate the influence of CX26 and CX43 on the biological functions of pulmonary epithelial cells in NSCLC. In addition, the proliferation and tumorigenicity of cancer cells were assessed by EdU staining and xenograft tumors, respectively. Decreased expression of CX26 and CX43 was found in cancer tissues compared with surrounding normal tissue. Hypoxia was shown to activate the P53/MDM2 axis and stimulate the downregulation, ubiquitination and degradation of CX26 and CX43, which were translocated from the membrane to the cytoplasm. Low levels of CX26 and CX43 were demonstrated to further promote EMT and the induction of the proliferation and tumorigenicity of cancer cells. These results were reflected by decreased E­cadherin expression and increased N­cadherin expression, along with increased cell migration, promoted cell proliferation ability and elevated relative protein expression of Oct4 and Nanog, and accelerated tumor growth, accompanied by a higher number of metastatic nodes. Taken together, the key observations of the present study demonstrate that the internalization of CX26 and CX43 promoted proliferation, EMT and migration and thus induced NSCLC via aberrant activation of the P53/MDM2 signaling pathway under hypoxic conditions.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Conexina 43/metabolismo , Conexinas/metabolismo , Células Epiteliais/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Hipóxia Celular , Proliferação de Células , Conexina 43/química , Conexina 43/genética , Conexinas/química , Conexinas/genética , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação
15.
Braz J Cardiovasc Surg ; 34(6): 711-722, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31545578

RESUMO

OBJECTIVE: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). METHODS: The DACT1 expression and its associations with the degree of fibrosis and ß-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of ß-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. RESULTS: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and ß-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in ß-catenin and subsequent partial colocalization of DACT1 and ß-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. CONCLUSION: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by ß-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibrilação Atrial/metabolismo , Conexina 43/metabolismo , Citoesqueleto/metabolismo , Miócitos Cardíacos/citologia , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Movimento Celular , Conexina 43/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Adulto Jovem
16.
Biomed Res Int ; 2019: 9103860, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275992

RESUMO

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare and potentially life-threatening disorder of the heart. The clinical spectrum of ARVC includes myocyte loss and fibro-fatty tissue replacement. With the progress of ARVC, the patient can present serious ventricular arrhythmias, heart failure, and even sudden cardiac death. Previous studies have demonstrated that desmosomes and intermediate junctions play a crucial role in the generation and development of ARVC. In this study, we enrolled a Chinese patient with suspicious ARVC. The patient suffered from right ventricular enlargement and less thickening of right ventricular wall. ECG record showed an epsilon wave. However, there was no obvious symptom in his parents. After whole-exome sequencing and data filtering, we identified a de novo mutation (c.1729C>T/p.R577C) of junction plakoglobin (JUP) in this patient. Bioinformatics programs predicted that this mutation was deleterious. Western blot revealed that, compared to cells transfected with WT plasmids, the expressions of desmoglein 2 (DSG2) and Connexin 43 were decreased overtly in cells transfected with the mutant plasmid. Previous studies have proven that the reduction of DSG2 and Connexin 43 may disturb the stability of desmosomes. In this research, we reported a novel de novo mutation (c.1729C>T/p.R577C) of JUP in a Chinese patient with suspicious ARVC. Functional research further confirmed the pathogenicity of this novel mutation. Our study expanded the spectrum of JUP mutations and may contribute to the genetic diagnosis and counseling of patients with ARVC.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Grupo com Ancestrais do Continente Asiático/genética , Mutação/genética , Sequenciamento Completo do Exoma , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Conexina 43/genética , Conexina 43/metabolismo , Desmogleína 2/genética , Desmogleína 2/metabolismo , Desmossomos/metabolismo , Humanos , Masculino , Adulto Jovem , gama Catenina/química , gama Catenina/genética
17.
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
18.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269652

RESUMO

Growing evidence suggests dietary antioxidants reduce the risk of several cancers. Grape seeds extracts (GSE) are a rich source of polyphenols known to have antioxidant, chemopreventive and anticancer properties. Herein, we investigated the in vitro effects and putative action mechanisms of a grape seed extract (GSE) on human breast cancer cells (MCF-7). The effects of GSE were evaluated on cell proliferation, apoptosis and gap-junction-mediated cell-cell communications (GJIC), as basal mechanism involved in the promotion stage of carcinogenesis. GSE (0.05-100 µg/mL) caused a significant dose- and time-dependent inhibition of MCF-7 viability and induced apoptotic cell death, as detected by Annexin-V/Propidium Iodide. Concurrently, GSE induced transient but significant enhancement of GJIC in non-communicating MCF-7 cells, as demonstrated by the scrape-loading/dye-transfer (SL/DT) assay and an early and dose-dependent re-localization of the connexin-43 (Cx43) proteins on plasma membranes, as assayed by immunocytochemistry. Finally, real-time-PCR has evidenced a significant increase in cx43 mRNA expression. The results support the hypothesis that the proliferation inhibition and pro-apoptotic effect of GSE against this breast cancer cell model are mediated by the GJIC improvement via re-localization of Cx43 proteins and up-regulation of cx43 gene, and provide further insight into the action mechanisms underlying the health-promoting action of dietary components.


Assuntos
Anticarcinógenos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/prevenção & controle , Extrato de Sementes de Uva/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Comunicação Celular/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Humanos , Células MCF-7 , Regulação para Cima/efeitos dos fármacos
19.
Neoplasma ; 66(6): 870-878, 2019 11.
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
20.
Biochim Biophys Acta Mol Basis Dis ; 1865(10): 2765-2773, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228617

RESUMO

Endotoxemia caused by bacterial lipopolysaccharides (LPSs) leads to severe skeletal muscular deterioration, starting with higher membrane permeability and decline in resting membrane potential (RMP). However, the molecular mechanism of such changes remains unclear. Here, we evaluated the possible involvement of connexin43- and connexin45-based hemichannels (Cx43 and Cx45 HCs, respectively) as putative mediators of sarcolemmal dysfunctions induced by LPS in control (Cx43fl/flCx45fl/fl) and Cx43/Cx45 expression-deficient (Cx43fl/flCx45fl/fl:Myo-Cre) skeletal mice myofibers. At 5 h of endotoxemia, control myofibers presented Cx43 and Cx45 proteins forming functional HCs. Additionally, myofibers from endotoxic control mice showed dye uptake in vivo, which was inhibited by carbenoxolone, a Cx HC blocker. A similar increase in membrane permeability was observed in myofibers freshly isolated from skeletal muscle of mice treated for 5 h with LPS, which was blocked by the Cx HC blocker and was absent in myofibers from mice simultaneously treated with LPS and boldine, which is a Cx HC blocker. The increase in sarcolemmal permeability was mimicked by isolated myofibers treated with pro-inflammatory cytokines (TNF-α and IL-1ß) and occurred at 5 h after treatment. Endotoxemia also induced a significant increase in basal intracellular Ca2+ signal and a drop in RMP in control myofibers. These two changes were not elicited by myofibers deficient in Cx43/Cx45 expression. Therefore, sarcolemmal dysfunction characterizing endotoxemia is largely explained by the expression of functional Cx43 and Cx45 HCs. Hence, current therapy options for individuals suffering from endotoxic shock could be greatly improved with selective Cx HC inhibitors avoiding the underlying skeletal muscle dysfunction.


Assuntos
Conexina 43/metabolismo , Conexinas/metabolismo , Endotoxemia/metabolismo , Músculo Esquelético/metabolismo , Animais , Cálcio , Membrana Celular/efeitos dos fármacos , Conexina 43/genética , Conexinas/genética , Citocinas , Modelos Animais de Doenças , Endotoxemia/complicações , Glucocorticoides , Lipopolissacarídeos/efeitos adversos , Masculino , Potenciais da Membrana , Camundongos , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Permeabilidade
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