Differences in regulation of type I collagen synthesis in primary and passaged hepatic stellate cell cultures: the role of 5 1-integrin

Dodig M, Ogunwale B, Dasarathy S, Li M, Wang B, McCullough AJ. Differences in regulation of type I collagen synthesis in primary and passaged hepatic stellate cell cultures: the role of 5 1integrin. Am J Physiol Gastrointest Liver Physiol 293: G154–G164, 2007. First published May 17, 2007; doi:10.1152/ajpgi.00432.2006.— 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 5 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 5 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 5 1-integrin. Inhibition of 5 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 5 1integrin ligand, increased transgene activity in primary (210 33%, P 0.05) but not in passaged HSC cultures (119 8%). This 5 1-integrin effect appears to be at least in part mediated by CCAAT enhancer binding protein(C/EBP ), because fibronectin increased and 5-gene silencing by small interfering RNA decreased C/EBP levels. In addition, C/EBP knockout mice showed reduced type I collagen synthesis compared with wild-type littermates. Therefore 5 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.