These results indicate that bile acids may coordinately regulate biliary bile acids and cholesterol secretion. Induction of hepatic, but not intestinal cholesterol and bile acid transporters may result in increased biliary cholesterol and bile acid secretion with subsequent fecal elimination in Cyp7a1-tg mice. To test if increased hepatic Abcg5/g8 expression in Cyp7a1-tg mice could be due to bile acid activation of FXR, we treated mouse hepatocytes with bile acids or a specific FXR agonist GW4064 and analyzed Abcg5/g8 mRNA expression levels. As shown in Fig. 4B, CDCA, CA

and learn more GW4064 treatment all significantly induced Abcg5/g8 mRNA expression levels in mouse hepatocytes. CDCA induction of Abcg5/g8 was stronger than CA, which is consistent with CDCA being a more efficacious FXR ligand. Furthermore, treating primary human hepatocytes with CDCA, CA, and GW4064 also induced ABCG5/G8 mRNA (Fig. 4C) and protein expression (Fig. 4D), suggesting that FXR induction of ABCG5/G8 is conserved in human hepatocytes. To our surprise, an LXR agonist TO901317 or cholesterol did not induce ABCG5/G8 mRNA in human hepatocytes (Fig. 4C), in contrast to a previous report that LXR induce mouse Abcg5 and Abcg8 mRNA expression.10 These data suggest that LXR may differentially

regulate ABCG5 and ABCG8 expression Lapatinib mouse in mouse and human hepatocytes. To further elucidate the molecular mechanism of FXR regulation of Abcg5/g8 gene expression, we performed Abcg5 promoter/luciferase (luc) reporter assays in HepG2 cells. We found that the Abcg5 reporter activities of the reporter plasmids −2041-luc, −1420-luc, −1160-luc, and −918-luc were strongly induced by GW4064 treatment. Reporter activities of shorter constructs −679-luc and −431-luc were not affected. These assays defined a functional FXR response element (FXRE) located

between nucleotides −680 and −918 on the Abcg5 promoter (Fig. 5A). Analysis of nucleotide sequences in this region identified an inverted repeat with one-base spacing (IR1) located between Sitaxentan nucleotides −682 to −669 on the Abcg5 promoter (or +309 and +322 of abcg8 in intron 1), which is a typical FXRE (Fig. 5A). EMSA showed that FXR/RXRα heterodimer bound to this putative FXRE, and that binding was abolished by excess of unlabeled probes containing the known FXRE from small heterodimer partner (SHP), or fatty acid synthase (FAS) genes, or by antibody supershift assay using an antibody against FXR (Fig. 5B). We then performed ChIP assays using mouse liver and intestine nuclei. ChIP assays showed that FXR occupied the Abcg5/g8 promoter in the mouse liver (Fig. 5C), but not in mouse intestine (Fig. 5D). A positive control showed that FXR occupied the Shp gene promoter in both mouse liver and mouse intestine (Fig. 5C,D).