Correlation of farnesoid X receptor coactivator recruitment and cholesterol 7 alpha-hydroxylase gene repression by bile acids

Cholesterol conversion to bile acids in the liver is regulated by the rate- limiting enzyme cholesterol 7 alpha -hydroxylase (CYP7A1). CYP7A1 activity is regulated by feedback repression by bile acids at the transcriptional le vel. The farnesoid X receptor (FXR), a member of the nuclear hormone recept or superfamily, was recently demonstrated to function as the bile acid rece ptor and its high level of expression in the liver implicates it in the tra nscriptional regulation of CYP7A1. This study compares the potencies of var ious bile acids in their ability to mediate recruitment of the transcriptio nal coactivator protein, steroid receptor coactivator-1 (SRC-1), to the FXR ligand binding domain with their ability to repress CYP7A1 expression in H epG2 cells. A mammalian two-hybrid assay was utilized to assess the ability of FXR to recruit SRC-1 in a ligand-dependent manner. Chenodeoxycholic aci d (CDCA) was the most potent and efficacious compound in the SRC-1 recruitm ent assay (EC50 = 11.7 muM) followed by deoxycholic acid (DCA; EC50 = 19.0 muM). Ursodeoxycholic acid (UDCA) displayed minimal activity while cholic a cid (CA) was inactive. In order to directly compare the potencies of the bi le acids in the coactivator recruitment assay to their ability to repress C YP7A1 expression, a branched DNA assay was developed to rapidly measure CYP 7A1 mRNA levels from HepG2 cells cultured in 96-well plates. The rank order and absolute potency was conserved (CDCA IC50 = 8.7 muM, DCA IC50 = 27.2 m uM, UDCA and CA inactive) consistent with bile acid repression of CYP7A1 be ing mediated by FXR. (C) 2000 Academic Press.