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1.
Life Sci ; 254: 117791, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32416166

RESUMO

AIMS: Sepsis-induced acute kidney injury (SI-AKI) is the fifth most common cause of hospital-acquired acute kidney injury. Pannexin1 (Panx1) triggers inflammation and apoptosis which act as crucial factors in the pathogenesis of SI-AKI. We aimed to investigate the expression of Panx1 and its role on the inflammation and apoptosis in SI-AKI. MATERIALS AND METHODS: SI-AKI model was established by lipopolysaccharide (LPS) injection in mice and LPS-treated HK-2 cells in vitro. Panx1 was inhibited by pretreating with carbenoxolone (CBX) or small interfering RNA in vivo and vitro, respectively. The expression of Panx1 was determined by qPCR, western blot and immunohistochemistry (IHC). Kidney damage was evaluated by kidney function, histopathological examination and AKI biomarkers. Inflammatory cytokines were detected by qPCR and ELISA. Apoptosis was detected by TUNEL staining and the expression of apoptosis-related proteins. The activation of nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome was measured by western blot. KEY FINDINGS: Panx1 increased in LPS-induced SI-AKI mice and HK-2 cells, as well as in SI-AKI patients. CBX alleviated the renal function and pathological damage, as well as decreased the mRNA of kidney injury molecule (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Inhibiting Panx1 decreased the production of IL-1ß, IL-6 and TNF-α, as well as tubular cell apoptosis in SI-AKI. Inhibiting Panx1 suppressed inflammatory cytokines and apoptosis via inhibiting NLRP3 inflammasome activation and regulating apoptotic protein Bax and Bcl2 expression, respectively. SIGNIFICANCE: These observations suggest that pharmacological inhibition of Panx1 might be a potential approach in the clinical therapy of SI-AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Apoptose/efeitos dos fármacos , Carbenoxolona/farmacologia , Conexinas/antagonistas & inibidores , Citocinas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Lesão Renal Aguda/complicações , Lesão Renal Aguda/patologia , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Biomarcadores/metabolismo , Conexinas/biossíntese , Humanos , Inflamassomos/metabolismo , Inflamação/complicações , Inflamação/prevenção & controle , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Lipopolissacarídeos , Masculino , Camundongos , Proteínas do Tecido Nervoso/biossíntese , RNA Interferente Pequeno/farmacologia , Sepse/complicações
2.
Medicine (Baltimore) ; 99(14): e19054, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32243356

RESUMO

Lung adenocarcinoma (LUAD), a form of lung cancer, is reported to cause first and second-order cancer morbidity to men and women in China, respectively. We assessed the mRNA expression of GJB2 in LUAD patients in our study, based on data acquired from the cancer genome atlas (TCGA) and so as to increase further knowledge into the biological pathways involved in LUAD pathogenesis related to GJB2.Information on gene expression and comparing clinical data were recognized and downloaded from TCGA. Gene set enrichment analysis (GSEA) created an arranged list of all genes is indicated by their connection with GJB2 expression.Our study cohort included 265 (54.5%) female and 221 (36.0%) male patients. The scatter plot and paired plot showed the difference of GJB2 expression between normal and tumor samples (P < .01). Overall survival (OS) analysis demonstrated that LUAD with GJB2 -high had a more terrible prognosis than that with GJB2 -low (P < .01). Multivariate analysis with the cox proportional hazards model indicated that the expression of Cx26 (HR: 1.00; 95%CI: 1.00-1.01; P = .041) and stage (HR: 1.95; 95%CI: 1.23-3.09; P = .003) were independent prognostic factors for patients with LUAD. The GSEA results showed that cytosolic DNA sensing pathway, apoptosis, cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity, regulation of actin cytoskeleton, toll-like receptor signaling pathway, small cell lung cancer and pathways in cancer are differentially enriched in GJB2 high expression phenotype.Our study confirmed the significantly high levels of Cx26 expression in LUAD patients with several observed clinical features. GJB2 may be a potentially useful prognostic molecular biomarker of bad survival in LUAD, while further experimental ought to be performed to demonstrate the biologic effect of GJB2.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Conexinas/biossíntese , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Adenocarcinoma de Pulmão/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/fisiologia , Biomarcadores Tumorais , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/fisiologia , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/biossíntese , Transdução de Sinais/fisiologia , Carcinoma de Pequenas Células do Pulmão/patologia , Análise de Sobrevida
3.
Am J Physiol Renal Physiol ; 317(6): F1649-F1655, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31630543

RESUMO

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK.P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK.P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.


Assuntos
Cistos/patologia , Nefropatias/patologia , Rim Policístico Autossômico Recessivo/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/urina , Animais , Sistemas CRISPR-Cas , Conexinas/biossíntese , Conexinas/genética , Cistos/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Técnicas de Inativação de Genes , Nefropatias/genética , Mutagênese Insercional , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Rim Policístico Autossômico Recessivo/genética , Gravidez , Ratos , Receptores Purinérgicos P2X7/genética , Sódio/metabolismo
4.
Neurochem Int ; 128: 196-205, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31034913

RESUMO

Mutations in the GJB2 gene (which encodes Connexin26 (Cx26)) are the most common cause of non-syndromic deafness. Previous studies showed that an extensive knockout of the Gjb2 gene in cochlear epithelium can cause severe deafness, significant hair cell (HC) loss and failure of pillar cells (a type of supporting cell, PCs) to differentiate in mice. This study aimed to establish different mouse models with gradient reductions of cochlear Cx26 expression and to investigate the effect of different reduced levels of cochlear Cx26 expression on hearing and development of PCs. According to the reduction in the levels of cochlear Cx26, these models were named high knockdown (KD), middle KD and low KD group. In the low KD group, the mice showed normal hearing and well-developed PCs. In the high KD group, up to 90 percent of supporting cells (SCs) lost Cx26 expression. These mice exhibited severe deafness, rapid hair cell degeneration and juvenile PCs. In the middle KD group, nearly half of SCs lost Cx26 expression. However, these mice showed a moderate deafness and a late-onset hair cell loss. Moreover, nearly all the PCs in mice of this group were in a partially differentiated state. These results indicated that reduction of postnatal expression of cochlear Cx26 induces hearing loss in a dose-dependent manner. Null Cx26 in a few SCs affects the developmental status of PCs and the hair cell degeneration pattern. The abnormal developmental status of PCs may be a potential cause of Gjb2-related hearing loss.


Assuntos
Cóclea/metabolismo , Cóclea/ultraestrutura , Conexinas/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Perda Auditiva/metabolismo , Animais , Cóclea/patologia , Conexinas/genética , Perda Auditiva/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos
5.
J Invest Dermatol ; 139(4): 909-918, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30389492

RESUMO

Pannexin-3 (Panx3) is a gap junction protein that is required for regulating cell cycle exit and the differentiation of osteoblasts and chondrocytes during skeletal development. However, the role of Panx3 in skin tissue regeneration remains unclear. After dorsal skin punch biopsies, Panx3-knockout mice exhibited a significant delay in wound healing with insufficient re-epithelialization, decreased inflammatory reaction, and reduced collagen remodeling. Panx3 expression coincided with inflammatory reactions both in vivo and in vitro. By applying exogenous tumor necrosis factor-α to mimic inflammation in vitro, Panx3 expression was induced in HaCaT cells. In addition, Panx3 depletion reduced epithelial-mesenchymal transition during skin wound healing. A protein essential for signaling in epithelial-mesenchymal transition, transforming growth factor-ß interacted with Panx3 by modulating intracellular adenosine triphosphate levels and thereby enhanced HaCaT cell migration ability with Panx3 overexpression. In conclusion, Panx3 plays a key role in the skin wound healing process by controlling keratinocytes and keratinocyte-mesenchyme cross-talk via hemichannel and endoplasmic reticulum Ca2+ channel functions, which differs from another gap junction, connexin 43 (Cx43), during skin wound healing.


Assuntos
Conexinas/metabolismo , Regulação da Expressão Gênica , RNA/genética , Pele/metabolismo , Cicatrização , Animais , Diferenciação Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Colágeno/metabolismo , Conexinas/biossíntese , Conexinas/deficiência , Conexinas/genética , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Junções Comunicantes/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Pele/lesões , Pele/patologia
6.
Neurosci Lett ; 695: 19-24, 2019 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28711343

RESUMO

The many roles of innexins, the molecules that form gap junctions in invertebrates, have been explored in numerous species. Here, we present a summary of innexin expression and function in two small, central pattern generating circuits found in crustaceans: the stomatogastric ganglion and the cardiac ganglion. The two ganglia express multiple innexin genes, exhibit varying combinations of symmetrical and rectifying gap junctions, as well as gap junctions within and across different cell types. Past studies have revealed correlations in ion channel and innexin expression in coupled neurons, as well as intriguing functional relationships between ion channel conductances and electrical coupling. Together, these studies suggest a putative role for innexins in correlating activity between coupled neurons at the levels of gene expression and physiological activity during development and in the adult animal.


Assuntos
Conexinas/biossíntese , Sinapses Elétricas/metabolismo , Gânglios dos Invertebrados/metabolismo , Animais , Crustáceos/metabolismo , Junções Comunicantes/metabolismo , Atividade Motora/fisiologia , Neurônios/metabolismo
7.
Neurosci Lett ; 695: 91-99, 2019 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28545922

RESUMO

Connexins are a family of integral membrane proteins most of which form gap junctions and many of which form hemichannels as well. Mutations in at least 9 of the 21 genes encoding human connexin proteins cause human diseases. Mutations in GJB1 (Cx32), expressed in both Schwann cells and oligodendrocytes, cause both a form of inherited peripheral neuropathy and a variety of CNS symptoms. Mutations in GJC2 (Cx47), expressed in oligodendrocytes cause three disorders: a severe early onset dysmyelinating disorder, Pelizaeus-Merzbacher-Like disease (PMLD1 or HLD2); hereditary spastic paraplegia (SPG44), which has a milder phenotype and later onset; and a subclinical leukodystrophy. The clinical phenotypes and genetics associated with each disorder will be reviewed, focusing on features which may provide clues to pathogenesis. In vitro and animal model data which may shed light on these phenotypes will then be discussed along with recent work which may impact on therapeutic approaches for these disorders.


Assuntos
Conexinas/biossíntese , Doenças Desmielinizantes/metabolismo , Neuroglia/metabolismo , Animais , Conexinas/genética , Conexinas/metabolismo , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Humanos , Mutação , Neuroglia/patologia , Células de Schwann/metabolismo , Células de Schwann/patologia
8.
Acta Histochem ; 120(6): 558-565, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30100173

RESUMO

Previously we have demonstrated that the GJ protein connexin 30.2 (Cx30.2) is expressed in pancreatic beta cells and endothelial cells (ECs) of the islet. In the present study, we address whether Cx30.2 is expressed in the exocrine pancreas, including its vascular system. For this, adult mouse pancreatic sections were double labeled with specific antibodies against Cx30.2 and CD31, an endothelial cell marker, or with anti-α-actin smooth muscle, a smooth muscle cell (SMC) marker or anti-mucin-1, a marker of epithelial ductal cells, using immunofluorescence (IF) studies. Cx30.2-IF hot spots were found at junctional membranes of exocrine ECs and SMCs of blood vessels. Furthermore, Cx30.2 was localized in mucin-1 positive cells or epithelial ductal cells. Using immunohistochemistry (IHC) studies, it was found that in vessels and ducts of different diameters, Cx30.2 was also expressed in these cell types. In addition, it was found that Cx30.2 is already expressed in these cell types in pancreatic sections of 3, 14 and 21 days postpartum. Moreover, this cell specific pattern of expression was also found in the adult rat, hamster and guinea pig pancreas. Expression of Cx30.2 mRNA and protein in the pancreas of all these species was confirmed by RT-PCR and Western blot studies. Overall, our results suggest that intercellular coupling mediated by Cx30.2 intercellular channels may synchronize the functional activity of ECs and SMCs of vascular cells, as well as of epithelial ductal cells after birth.


Assuntos
Conexinas/biossíntese , Endotélio Vascular/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Ductos Pancreáticos/crescimento & desenvolvimento , Animais , Cricetinae , Endotélio Vascular/citologia , Células Epiteliais/citologia , Cobaias , Camundongos , Ductos Pancreáticos/citologia , Ratos
9.
J Tissue Eng Regen Med ; 12(4): e1936-e1949, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29222846

RESUMO

Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three-dimensional (3D) microenvironment provided by a polysaccharide-based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large-pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two-dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell-cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast-specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi-scid/IL-2Rγnull ) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.


Assuntos
Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células , Conexinas/biossíntese , Dextranos/química , Glucanos/química , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Tecidos Suporte/química , Animais , Células da Medula Óssea/citologia , Xenoenxertos , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Porosidade
10.
Interact Cardiovasc Thorac Surg ; 26(1): 139-145, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29049831

RESUMO

OBJECTIVES: The purpose of this study was to evaluate the effect of CX37 gene silence on myocardial fractional flow reserve (FFR). METHODS: A total of 90 male pigs were randomly divided into saline, mock and 3 different doses (5, 10 and 20 µl) of CX37 viral suspension groups that could induce coronary plaque formation with high-fat diet. After performing myocardial FFR by intravascular ultrasound, different doses of CX37 viral suspension, saline and mock small interfering RNA (siRNA) were transfected into the related coronary. The FFR, the myocardial enzymes and the cardiac structures and functions of the pigs were detected at baseline, 4th, 8th and 12th week after transfection, respectively. RESULTS: Repeated measures analysis of variance comparison showed that the difference in the FFR among the 5 groups was statistically significant (F = 27.0, P < 0.01). Post hoc analysis showed that FFR were highest in the siRNA CX37 group (20 µl), followed by the siRNA CX37 group (10 µl) and the siRNA CX37 group (5 µl), and lowest in the mock and saline groups. Left ventricular end-diastolic diameter was significantly smaller and ejection fraction was obviously higher in the 3 siRNA CX37 groups compared with the untreated groups. CONCLUSIONS: Our study showed that FFR levels increased along with decreased doses of siRNA CX37 lentivirus, indicating that siRNA CX37 lentivirus may reduce the risk of coronary atherosclerosis and provide a potential approach to treat coronary heart disease.


Assuntos
Conexinas/genética , Estenose Coronária/genética , Reserva Fracionada de Fluxo Miocárdico/fisiologia , Regulação da Expressão Gênica , Placa Aterosclerótica/genética , RNA/genética , Animais , Western Blotting , Conexinas/biossíntese , Estenose Coronária/metabolismo , Estenose Coronária/fisiopatologia , Modelos Animais de Doenças , Humanos , Masculino , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/fisiopatologia , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Elementos Silenciadores Transcricionais , Suínos
11.
Biochim Biophys Acta Biomembr ; 1860(1): 72-82, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28279657

RESUMO

Pannexins are a 3-membered family of proteins that form large pore ion and metabolite channels in vertebrates. The impact of pannexins on vertebrate biology is intricately tied to where and when they are expressed, and how they are modified, once produced. The purpose of this review is therefore to outline our current understanding of transcriptional and post-translational regulation of pannexins. First, we briefly summarize their discovery and characteristics. Next, we describe several aspects of transcriptional regulation, including cell and tissue-specific expression, dynamic expression over development and disease, as well as new insights into the underlying molecular machinery involved. Following this, we delve into the role of post-translational modifications in the regulation of trafficking and channel properties, highlighting important work on glycosylation, phosphorylation, S-nitrosylation and proteolytic cleavage. Embedded throughout, we also highlight important knowledge gaps and avenues of future research. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.


Assuntos
Conexinas/biossíntese , Regulação da Expressão Gênica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Transcrição Genética/fisiologia , Animais , Conexinas/genética , Humanos , Especificidade de Órgãos/fisiologia
12.
Invest Ophthalmol Vis Sci ; 58(13): 5654-5665, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29098296

RESUMO

Purpose: Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment. Methods: The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1α (IL1α) injection. Prior to EPCP transplantation, IL1α-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy. Results: Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1α injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration. Conclusions: Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation.


Assuntos
Conexinas/genética , Células Epiteliais/transplante , Regulação da Expressão Gênica , Aparelho Lacrimal/metabolismo , Proteínas do Tecido Nervoso/genética , Síndrome de Sjogren/genética , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Conexinas/biossíntese , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/terapia , Células-Tronco/metabolismo
13.
J Biol Regul Homeost Agents ; 31(3): 567-577, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28952288

RESUMO

In this study we investigated the expression of connexins Cx36, Cx37, Cx40, Cx43, and Cx45 mRNAs during real-time cellular proliferation in vitro. The oral mucosa cells were isolated from 80 pubertal crossbred Landrace gilts. The cells were transferred into primary in vitro culture (IVC) and cultured for 30 days. The cells were collected to RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using real-time cell analysis (RTCA). We found an increased expression of Cx43 mRNA after 30 days of IVC as compared to control (P<0.05). The expression level of Cx36 was significantly decreased after 30 days. The expression of Cx37, Cx40 and Cx45 mRNAs was not changed. The expression of Cx43 was statistically increased when compared to Cx40, Cx37, Cx45 and Cx36 (P<0.001, for all time periods, respectively). We confirmed the expression of selected connexins in porcine buccal mucosa cells during their long-term primary IVC, which suggests the existence of functional gap junction connections (GJCs) communication network between these cells. We also confirmed the observations of other authors that Cx43 plays a substantial role in GJC structure. However, the increased expression of Cx43 in buccal mucosa cells, accompanied with their proliferation during real-time primary culture, is presented, to our knowledge, for the first time.


Assuntos
Proliferação de Células , Conexinas/biossíntese , Regulação da Expressão Gênica , Mucosa Bucal/metabolismo , Animais , Células Cultivadas , Mucosa Bucal/citologia , Cultura Primária de Células , Suínos
14.
Am J Physiol Heart Circ Physiol ; 313(4): H810-H827, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28710068

RESUMO

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.


Assuntos
Potenciais de Ação/fisiologia , Fibroblastos/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Conexina 43/biossíntese , Conexinas/biossíntese , Junções Comunicantes/fisiologia , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/ultraestrutura , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
15.
J Clin Invest ; 127(7): 2647-2661, 2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-28604388

RESUMO

The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma.


Assuntos
Potenciais Evocados Visuais , Junções Comunicantes/metabolismo , Glaucoma/metabolismo , Neurônios Retinianos/metabolismo , Animais , Conexinas/biossíntese , Conexinas/genética , Junções Comunicantes/genética , Junções Comunicantes/patologia , Regulação da Expressão Gênica , Glaucoma/genética , Glaucoma/patologia , Glaucoma/fisiopatologia , Camundongos , Camundongos Knockout , Neurônios Retinianos/patologia
16.
Cell Death Dis ; 8(5): e2773, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28492539

RESUMO

The role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx), is unclear. Using cervix specimens (313 CaCx, 78 controls) and CaCx cell lines, we explored relationships among Cx expression, prognostic variables and mechanisms that may link them. In CaCx specimens, Cx32 was upregulated and cytoplasmically localized, and three other Cx downregulated, relative to controls. Cx32 expression correlated with advanced FIGO staging, differentiation and increased tumor size. In CaCx cell lines, Cx32 expression suppressed streptonigrin/cisplatin-induced apoptosis in the absence of functional GJ. In CaCx specimens and cell lines, expression of Cx32 upregulated epidermal growth factor receptor (EGFR) expression. Inhibition of EGFR signaling abrogated the anti-apoptotic effect of Cx32 expression. In conclusion, upregulated Cx32 in CaCx cells produces anti-apoptotic, pro-tumorigenic effects in vivo and vitro. Abnormal Cx32 expression/localization in CaCx appears to be both a mechanism and biomarker of chemotherapeutic resistance.


Assuntos
Apoptose , Biomarcadores Tumorais/biossíntese , Conexinas/biossíntese , Receptores ErbB/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias do Colo do Útero/metabolismo , Conexinas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Feminino , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
17.
Mol Cell Biochem ; 433(1-2): 27-40, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28337705

RESUMO

Cardiac surgery with extracorporeal circulation is characterized by different degrees of myocardial ischemia/reperfusion, which is often associated with postoperative atrial fibrillation (POAF). We have previously shown that a novel preventive therapy based on the reinforcement of the antioxidant system using omega-3 fatty acids plus antioxidant vitamin supplementation applied to patients undergoing cardiac surgery reduces POAF occurrence. We hypothesized that oxidative stress and nitrosative stress are involved in the development of an arrhythmogenic substrate by their effect on connexins (Cx40, Cx43 and Cx45) abundance and distribution pattern. Therefore, we have assessed the effect of redox status on atrial tissue in patients undergoing cardiac surgery. Placebo/POAF and supplemented/POAF patients showed 276 and 170% higher reactive oxygen species (ROS) levels and 223 and 96% higher nitrotyrosine residues levels, respectively, compared to sinus rhythm (SR). In POAF tissue, antioxidant supplementation prevented Cx40 and Cx43 lateralization on cardiomyocyte sarcolemma, keeping them at the intercalated disks. POAF samples showed Cx40 heterogeneous distribution pattern, presenting tissue areas lacking this protein (49 and 55% lower levels in placebo/POAF and supplemented/POAF groups, respectively, compared to SR). Of note, Cx45 overexpression occurred in POAF, being 211 and 167% higher in placebo/POAF and supplemented/POAF groups, respectively, compared to SR. It is concluded that treatment with omega-3 fatty acids and antioxidant vitamins reduces oxidative and nitrosative stress and prevents Cx40/Cx43 lateralization in atrial tissue likely contributing to POAF prevention. However, it failed to fully prevent POAF occurrence because these compounds have no effects on the normalization of Cx40 down-regulation and Cx45 up-regulation, which may promote POAF.


Assuntos
Antioxidantes/administração & dosagem , Procedimentos Cirúrgicos Cardíacos , Conexinas/biossíntese , Circulação Extracorpórea , Ácidos Graxos Insaturados/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Tirosina/análogos & derivados , Vitaminas/administração & dosagem , Feminino , Átrios do Coração/metabolismo , Átrios do Coração/cirurgia , Humanos , Masculino , Tirosina/metabolismo
18.
Oncol Rep ; 37(4): 2121-2128, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28260043

RESUMO

Recently, epithelial-mesenchymal transition (EMT) has been reported to be an important mechanism of drug resistance in numerous types of cancer cells, including hepatocellular carcinoma (HCC). However, the underlying mechanisms remain to be fully elucidated. Connexin (Cx)32 plays a crucial role in hepatocarcinogenesis. The present study investigated the role of Cx32 in the regulation of chemotherapy-induced EMT in HCC. We found that the expression levels of Cx32 and E-cadherin were clearly decreased in HCC tissues compared with the corresponding paracancerous tissues, while the expression level of vimentin was significantly enhanced in HCC tissues. The expression of Cx32 had a strong correlation with the expression of E-cadherin and vimentin. In an in vitro study, a doxorubicin (DOX)-resistant liver cell line HepG2/DOX was established from parental HepG2 cells. The results showed that HepG2/DOX cells acquired EMT characteristics, with a decreased expression level of E-cadherin and an enhanced expression level of vimentin, and possessed high migratory abilities and invasiveness. Meanwhile, Cx32 was significantly decreased in the HepG2/DOX cells. Knockdown of Cx32 by shRNA in HepG2 cells induced EMT, while overexpression of Cx32 converted EMT to mesenchymal-epithelial transition (MET) in the HepG2/DOX cells. These results suggest that Cx32 is an important regulator of DOX-induced EMT in HCC. Cx32 could be considered as a novel target to reverse DOX resistance in HCC.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Conexinas/biossíntese , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas/genética , Caderinas/biossíntese , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Proliferação de Células/genética , Conexinas/genética , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Invasividade Neoplásica/genética , Vimentina/biossíntese
19.
Toxicology ; 375: 1-9, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27894940

RESUMO

Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3ß-hydroxysteroid dehydrogenase (3ßHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows.


Assuntos
DDT/toxicidade , Diclorodifenil Dicloroetileno/toxicidade , Placenta/efeitos dos fármacos , Placenta/metabolismo , Hormônios Placentários/biossíntese , RNA Mensageiro/biossíntese , Animais , Bovinos , Conexinas/biossíntese , Conexinas/genética , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inseticidas/toxicidade , Hormônios Placentários/genética , Hormônios Placentários/metabolismo , Gravidez , Proteínas da Gravidez/biossíntese , Proteínas da Gravidez/genética , RNA Mensageiro/genética
20.
Sci Rep ; 6: 38266, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27910899

RESUMO

Drug studies in animal models have implicated pannexin1 (Panx1) in various types of pain, including trigeminal hypersensitivity, neuropathic pain and migraine. However, the tested drugs have limited specificity and efficacy so that direct evidence for Panx1 contribution to pain has been lacking. We here show that tactile hypersensitivity is markedly attenuated by deletion of Panx1 in a mouse model of chronic orofacial pain; in this model, trigeminal ganglion Panx1 expression and function are markedly enhanced. Targeted deletion of Panx1 in GFAP-positive glia or in neurons revealed distinct effects. Panx1 deletion in GFAP-positive glia cells prevented hypersensitivity completely, whereas deletion of neuronal Panx1 reduced baseline sensitivity and the duration of hypersensitivity. In trigeminal ganglia with genetically encoded Ca2+ indicator in GFAP-positive glia or in neurons, both cell populations were found to be hyperactive and hyper-responsive to ATP. These novel findings reveal unique roles for GFAP-positive glial and neuronal Panx1 and describe new chronic pain targets for cell-type specific intervention in this often intractable disease.


Assuntos
Dor Crônica/metabolismo , Conexinas/biossíntese , Regulação da Expressão Gênica , Hiperestesia/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neuroglia/metabolismo , Neurônios/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Dor Crônica/genética , Dor Crônica/patologia , Conexinas/genética , Hiperestesia/genética , Hiperestesia/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neuroglia/patologia , Neurônios/patologia , Gânglio Trigeminal/patologia
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