The Effects Of FXR On SR-BI And The Mechanism

Background: Plasma concentrations of high-density lipoprotein (HDL) cholesterol are well known to be inversely related to the incidence of atherosclerosis and coronary heart disease. Scavenger receptor type B class I as HDL receptor plays an important role in reverse cholesterol transport, protected against AS. The nuclear farnesoid X receptor plays an important role in in glucose metabolism, cholesterol metabolism, TG metabolism. And there is a closely link with the FXR lacking and AS. Whether depletion of FXR induce or inhibit AS, remains controversial. FXR and SR-BI both are highly expressed in liver, then whether FXR could regulate SR-BI to inhibit AS? The study on the effect of FXR on SR-BI will provide useful information with regarding to the physiological and pathological roles FXR and SR-BI play in cholesterol metabolism, and supply a potential drug target.Objective: the present study investigated the effect of FXR on SR-BI expression and the mechanism by which FXR enhances the expression of the SR-BI.Methods: endothelial cells, hepatocyte and hepatoma cell lines were employed for investigation. Three cell lines were stimulated with the given different concentrations of FXR agonists, respectively. Using RT-PCR, Real-time PCR and western blot detected the SR-BI expression levels. Bioinformatics analysis (http://www.nubiscan.unibas.ch/) indicated that there is a potential FXR binding site (DR8) in the SR-BI promoter region (-1200/-59bp). Luciferase reporter assay detected the SR-BI promoter activity with FXR. And EMSA was performed to detect FXR protein associated with SR-BI. RT-PCR and Real-time PCR was performed to detected PPARγexpression. The combination of GW9662 with GW4064 treat HepG2, RT-PCR, Real-time PCR and western blot were performed to detect the SR-BI expression. Luciferase reporter assay detected the SR-BI promoter activity with GW9662. With RT-PCR, Real-time PCR and western blot, we detected SR-BI expression treatment with PPARγagonist troglitazone. In vivo study, upon treatment of FXR agonsit CDCA, SR-BI mRNA and protein were analysed with RT-PCR, Real-time PCR and Western Blot.Results: (1) treatment with FXR agonists, FXR could up-regulate SR-BI expression in concentration-dependent manner;(2) in HepG2, FXR enhanced SR-BI promoter activity, and up-regulate SR-BI expression through FXR binding site(DR8) in SR-BI promoter region;(3) FXR induced PPARγat transcription level, PPARγantagonist GW9662 repressed the SR-BI promoter activity which was stimulated by FXR;(4) in HepG2, PPARγcould up-regulate SR-BI expression;(5) in C57BL/6 mice FXR up-regulated the expression of SR-BI.Conclusion: (1) we identified SR-BI was a target gene of FXR, which could be directly up-regulated by FXR. There is a putative FXR binding site (DR8) within the SR-BI promoter region;(2) we also investigated the indirect mechanism that FXR mediates SR-BI through PPARγ;(3) in vivo study, we demonstrated that FXR could up-regulate SR-BI in mice.The study on the effect of FXR on SR-BI will provide useful information with regarding to the physiological and pathological roles FXR plays in cholesterol metabolism, and suggests a new therapy.