title: Ethanol-stimulated differentiated functions of human or mouse hepatic stellate cells are mediated by connective tissue growth factor.
abstract: OBJECTIVE
  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-β1), 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-β1- or CTGF-dependency of ethanol-induced CTGF, α-SMA, or collagen α1(I) was determined using small interfering RNA (siRNA) to TGF-β1 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-β1. 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-β1 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-β1- 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-β-dependent CTGF production, highlighting potential therapeutic benefits of targeting CTGF in alcoholic liver disease.
authors.affiliation: Center for Clinical and Translational Research, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, United States.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Alyssa L
authors.initials: AL
authors.lastname: Charrier
authors.forename: Andrew
authors.initials: A
authors.lastname: Leask
authors.forename: Samuel W
authors.initials: SW
authors.lastname: French
authors.forename: David R
authors.initials: DR
authors.lastname: Brigstock
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D055513:Connective Tissue Growth Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000633:toxicity*; D015870:Gene Expression / Q000187:drug effects; D055785:Gene Knockdown Techniques; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008108:Liver Diseases, Alcoholic / Q000378:metabolism / Q000473:pathology; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000494:pharmacology
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; C544013:alpha-smooth muscle actin, mouse; D055513:Connective Tissue Growth Factor; D000431:Ethanol