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Am J Physiol Gastrointest Liver Physiol 293: G154-G164, 2007. First published May 17, 2007; doi:10.1152/ajpgi.00432.2006
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LIVER AND BILIARY TRACT

Differences in regulation of type I collagen synthesis in primary and passaged hepatic stellate cell cultures: the role of {alpha}5beta1-integrin

Milan Dodig,1 Ben Ogunwale,2 Srinivasan Dasarathy,1 Min Li,1 Bingcheng Wang,2,3 and Arthur J. McCullough1

1Department of Gastroenterology and Hepatology, Cleveland Clinic and Cleveland Clinic Lerner College of Medicine; 2Division of Gastroenterology, MetroHealth Medical Center, School of Medicine at Case Western Reserve University; and 3Rammelkamp Research Center, MetroHealth Medical Center, Cleveland, Ohio

Submitted 20 September 2006 ; accepted in final form 10 May 2007

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}5beta1-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}5beta1 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}5beta1-integrin. Inhibition of {alpha}5beta1-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}5beta1-integrin ligand, increased transgene activity in primary (210 ± 33%, P < 0.05) but not in passaged HSC cultures (119 ± 8%). This {alpha}5beta1-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}5beta1-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.

integrins; C/EBPbeta; {alpha}5-integrin siRNA


Address for reprint requests and other correspondence: Address correspondence to: M. Dodig, Dept. of Gastroenterology and Hepatology, Cleveland Clinic and Cleveland Clinic Lerner College of Medicine, 9500 Euclid Ave., Cleveland, OH 44195 (e-mail: dodigm{at}ccf.org)






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