RESUMO
BACKGROUND & AIMS: Connective tissue growth factor (CTGF) expression is intimately associated with hepatic fibrotic pathophysiology. In this study, CTGF production and action was investigated in ethanol-treated mouse primary hepatic stellate cells (HSC) or human LX-2 cells. METHODS: CTGF, transforming growth factor-beta1 (TGF-ß1), alpha-smooth muscle actin (α-SMA) or collagen α1(I) mRNA were quantified by real-time PCR after treatment of HSC with ethanol or acetaldehyde. CTGF protein production was assessed by immunoprecipitation or ELISA. Ethanol-stimulated CTGF transcription was investigated using CTGF promoter reporter constructs. The TGF-ß1- or CTGF-dependency of ethanol-induced CTGF, α-SMA, or collagen α1(I) was determined using small interfering RNA (siRNA) to TGF-ß1 or CTGF. RESULTS: In human steatohepatitis, CTGF was produced by presumptive activated HSC. In cultured human or mouse HSC, production of CTGF, α-SMA and/or collagen was increased by ethanol treatment, an effect mimicked by acetaldehyde and blocked by 4-methylpyrazole (4-MP) or N-acetylcysteine (NAC). CTGF promoter activity was stimulated in a sustained fashion by ethanol or TGF-ß1. Mutation of the Smad site or basal control element (BCE-1) in the CTGF promoter caused a 5-fold reduction in ethanol-stimulated CTGF promoter activity. Administration of TGF-ß1 siRNA or CTGF siRNA significantly decreased ethanol- or acetaldehyde-stimulated mRNA or protein levels of CTGF, α-SMA or collagen I in LX-2 cells. In mouse HSC, TGF-ß1- or ethanol-stimulated CTGF, α-SMA or collagen I were significantly attenuated by CTGF siRNA. CONCLUSIONS: Ethanol-induced α-SMA or collagen α1(I) in HSC are mediated via TGF-ß-dependent CTGF production, highlighting potential therapeutic benefits of targeting CTGF in alcoholic liver disease.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Etanol/toxicidade , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Actinas/genética , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Fator de Crescimento do Tecido Conjuntivo/genética , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células Estreladas do Fígado/patologia , Humanos , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Alcoholic chronic pancreatitis (ACP) is characterized by pancreatic necrosis, inflammation, and scarring, the latter of which is due to excessive collagen deposition by activated pancreatic stellate cells (PSC). The aim of this study was to establish a model of ACP in mice, a species that is usually resistant to the toxic effects of alcohol, and to identify the cell type(s) responsible for production of connective tissue growth factor (CTGF), a pro-fibrotic molecule. C57Bl/6 male mice received intraperitoneal ethanol injections for 3 weeks against a background of cerulein-induced acute pancreatitis. Peak blood alcohol levels remained consistently high in ethanol-treated mice as compared with control mice. In mice receiving ethanol plus cerulein, there was increased collagen deposition as compared with other treatment groups as well as increased frequency of alpha-smooth muscle actin and desmin-positive PSC, which also showed significantly enhanced CTGF protein production. Expression of mRNA for collagen alpha1(I), alpha-smooth muscle actin or CTGF were all increased and co-localized exclusively to activated PSC in ACP. Pancreatic expression of mRNA for key profibrotic markers were all increased in ACP. In conclusion, a mouse model of ACP has been developed that mimics key pathophysiological features of the disease in humans and which shows that activated PSC are the principal producers of collagen and CTGF. PSC-derived CTGF is thus a candidate therapeutic target in anti-fibrotic strategies for ACP.
