RESUMO
Adiponectin, an adipocyte-derived hormone, exerts pleiotropic biological effects on metabolism, inflammation, vascular homeostasis, apoptosis and immunity. Recently, adiponectin has been suggested to attenuate the progression of human dermal fibrosis. Connective tissue growth factor (CTGF) is induced in keloids and is thought to be participated in the formation of keloid fibrosis. However, the roles played by adiponectin in keloids remain unclear. In this study, we explored the effects of adiponectin on CTGF-induced cell proliferation, migration and the deposition of extracellular matrix (ECM) and their associated intracellular signalling pathways in keloid fibroblasts (KFs). We also explored possible mechanisms of keloid pathogenesis. Primary fibroblast cultures were established from foreskin biopsies and skin biopsies from patients with keloids. The expression of adiponectin and adiponectin receptors (adipoRs) was evaluated by reverse transcription-PCR (RT-PCR), quantitative real-time RT-PCR, immunofluorescence staining, and immunohistochemical analysis. Next, KFs and normal dermal fibroblasts (NFs) were treated with CTGF in the presence or absence of adiponectin. A cell counting kit-8 (CCK-8) and the Transwell assay were used to examine cell proliferation and migration. The level of the collagen I, fibronectin (FN) and α-smooth muscle actin (α-SMA) mRNAs and proteins were determined by quantitative real-time RT-PCR and western blotting. The effects of RNA interference (RNAi) targeting the adipoR genes were detected. Phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase-protein kinase (PI3K-Akt) were examined by western blotting to further investigate the signalling pathways. Furthermore, inhibitors of signal transduction pathways were investigated. The expression levels of adiponectin and adipoRs were significantly decreased in keloids compared with those in normal skin tissue. Adiponectin suppressed the CTGF-induced KFs, but not NFs, proliferation, migration and ECM production. Moreover, adiponectin inhibited the phosphorylation of AMPK, p38 and extracellular-regulated kinase (ERK), but not that of Jun N-terminal kinase (JNK) or Akt, in CTGF-treated KFs. The activity of adiponectin-mediated signalling pathways was attenuated by small interfering RNAs (siRNAs) targeting adipoR1 (but not siRNAs targeting adipoR2, T-cadherin or calreticulin), AMPK (Compound C), p38 (SB203580) inhibitors, and mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059). Based on our results, adiponectin suppresses CTGF-induced KFs proliferation, migration and ECM overproduction. One of the underlying mechanisms is the activation of the adipoR1, AMPK, p38, and ERK signalling pathways. Therefore, adiponectin may play an important role in the progression of keloids, suggesting a potential novel target for keloid treatment.
Assuntos
Adiponectina/metabolismo , Movimento Celular , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Queloide/metabolismo , Adiponectina/genética , Adulto , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Matriz Extracelular/genética , Feminino , Humanos , Queloide/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismoAssuntos
Neoplasias Esofágicas/genética , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ceratodermia Palmar e Plantar/genética , Povo Asiático/genética , China , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patologia , Humanos , Ceratodermia Palmar e Plantar/diagnóstico , Ceratodermia Palmar e Plantar/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Doenças Raras , Sequenciamento do ExomaRESUMO
Keloids are benign fibrous overgrowths that occur as a result of abnormal wound healing following cutaneous injury. MicroRNAs (miRNAs/miRs) are short noncoding RNAs that serve critical roles in numerous important biological processes, such as cell proliferation, differentiation and apoptosis. However, their role in keloid development remains largely unknown. In the present study, the role of miR30a5p, a miRNA regulated by Trichostatin A (TSA), in apoptosis within cultured keloid fibroblasts was investigated. An MTT assay was used to detect the proliferation of cultured keloid fibroblasts treated with TSA. Cell apoptosis and cell cycle phases were analyzed using flow cytometry. In addition, an miRNA microarray was performed to compare expression profiles between cultured keloid fibroblasts treated with or without 1,000 nM TSA. Reverse transcriptionquantitative polymerase chain reaction analysis was conducted to estimate miRNA expression levels. The direct target of miR30a5p was identified using a dualluciferase reporter assay. Western blotting was employed to assess protein expression levels in keloid fibroblasts. The results demonstrated that TSA inhibited the proliferation of keloid fibroblasts in a time and dosedependent manner. The miRNA microarray revealed alterations in the expression of numerous miRNA sequences in response to TSA when compared with controls. Notably, the expression of miR30a5p was downregulated in keloid tissues. In addition, overexpression of miR30a5p induced apoptosis by targeting Bcell lymphoma 2, which was similar to that observed in response to TSA. These results provide important information regarding a novel miR30a5pmediated signaling pathway induced by TSA treatment, and suggest a potential use for TSA and miR30a5p as effective therapeutic strategies for keloids.