title: Differences in regulation of type I collagen synthesis in primary and passaged hepatic stellate cell cultures: the role of alpha5beta1-integrin.
abstract: Hepatic stellate cells (HSC) differ in their phenotype depending on the initiation and progression of their activation. Our hypothesis was that different mechanisms govern type I collagen synthesis depending on stage of HSC activation. We investigated the role of alpha(5)beta(1)-integrin as a regulator of type I collagen gene COL1A1 expression in primary and passaged HSC cultures using transgenic mouse containing type I collagen gene COL1A1 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The alpha(5)beta(1) protein levels increased during the activation and were highest in day 6 primary cultures but decreased in passaged HSC. CAT activity, reflecting COL1A1 expression, was upregulated by alpha(5)beta(1)-integrin. Inhibition of alpha(5)beta(1)-integrin by echistatin and blocking antibody resulted in reduced transgene activity only in early primary cultures (compared with the control, 53.3 +/- 12% echistatin and 58.8 +/- 7% blocking antibody, respectively, P < 0.05). Treatment of passaged HSC with either echistatin or blocking antibody had no effect. Fibronectin, an alpha(5)beta(1)-integrin ligand, increased transgene activity in primary (210 +/- 33%, P < 0.05) but not in passaged HSC cultures (119 +/- 8%). This alpha(5)beta(1)-integrin effect appears to be at least in part mediated by CCAAT enhancer binding protein-beta (C/EBPbeta), because fibronectin increased and alpha(5)-gene silencing by small interfering RNA decreased C/EBPbeta levels. In addition, C/EBPbeta knockout mice showed reduced type I collagen synthesis compared with wild-type littermates. Therefore alpha(5)beta(1)-integrin is an important regulator of type I collagen production in early primary HSC cultures but appears to have no direct role once the HSC are fully activated.
authors.affiliation: Department of Gastroenterology and Hepatology, Cleveland Clinic and Cleveland Clinic Lerner College of Medicine, 9500 Euclid Avenue, Cleveland, OH 44195, USA. dodigm@ccf.org
authors.forename: Milan
authors.initials: M
authors.lastname: Dodig
authors.forename: Ben
authors.initials: B
authors.lastname: Ogunwale
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Dasarathy
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Bingcheng
authors.initials: B
authors.lastname: Wang
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: McCullough
mesh_terms: D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000502:physiology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D024042:Collagen Type I / Q000096:biosynthesis*; D000090602:Collagen Type I, alpha 1 Chain; D020868:Gene Silencing; D039482:Integrin alpha5 / Q000235:genetics; D039081:Integrin alpha5beta1 / Q000502:physiology*; D008099:Liver / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D039482:Integrin alpha5; D039081:Integrin alpha5beta1; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase