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 ␣51-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 ␣51 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 ␣51-integrin. Inhibition of ␣51-integrin by echistatin and blocking antibody resulted in reduced transgene activity only in early primary cultures (compared with the control, 53.3 Ϯ 12% echistatin and 58.8 Ϯ 7% blocking antibody, respectively, P Ͻ 0.05). Treatment of passaged HSC with either echistatin or blocking antibody had no effect. Fibronectin, an ␣51-integrin ligand, increased transgene activity in primary (210 Ϯ 33%, P Ͻ 0.05) but not in passaged HSC cultures (119 Ϯ 8%). This ␣51-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 ␣51-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.
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