Induced collagen expression by hepatic stellate cells (HSC) is the hallmark of alcoholic liver fibrosis. Our laboratory recently demonstrated the potential link between depletion of PPARgamma in HSC and liver fibrogenesis and the reversal of HSC activation with PPARgamma ligands. The trainee will test the hypothesis that PPARgamma inhibits alpha1(I) collagen promoter in HSC line and liver fibrosis.
Specific aims i nclude determination of the specificity of PPARgamma-mediated inhibition of the promoter and elucidation of the mechanisms underlying the effect. The former will be achieved by transient transfection assays with the promoter-reporter gene and expression vectors for the members of nuclear receptors including PPARgamma. The latter will address PPARgamma-mediated negative cross-coupling with DNA-binding proteins for the promoter via DNase foot printing and gel shift assays and inhibition of co-activator recruitment by this nuclear protein which will be assessed by GST-fusion protein pull down assays and coimmununoprecipitation. PPARgamma-mediated suppression of the promoter will further be tested in vivo using the transgenic mice expressing GFP driven by the collagen promoter and adenoviral-mediated PPARgamma overexpression targeted to HSC by alpha-smooth muscle actin promoter. A similar approach will be taken to finally test the efficacy of PPARgamma overexpression for CCI[4] liver fibrosis in mice. These experiments will define the molecular basis for inhibition of collagen gene expression by PPARgamma and will test the potential of this mode if intervention as a novel therapy for alcoholic liver fibrosis.
