Regulation of Bile Acid Synthesis by Nuclear Receptors in Vivo

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Bile acids are required for intestinal absorption and biliary solubilization of cholesterol and lipids. In addition, bile acids play a crucial role in cholesterol homeostasis. There are two key enzymes in the bile acid biosynthetic pathways, cholesterol 7-hydroxylase/CYP7A1 (7-hydroxylase), which is the rate limiting and regulatory step of the "classic" pathway, and sterol 12-hydroxylase/CYP8B1 (12-hydroxylase), which is the specific enzyme for cholic acid synthesis. Expression of the 7- and 12-hydroxylase genes is highly regulated by feedback regulatory mechanisms, mainly at the transcriptional level. The molecular mechanisms involved in this regulation appear to be similar, and involve the coordinated activity of the farnesoid X receptor (FXR), the small heterodimer partner (SHP) and 1-fetoprotein transcription factor (FTF), also known as liver receptor homolog-1 (LRH-1), and hepatocyte nuclear factor-4 (HNF-4). All the studies done to date that have identified FTF as a factor involved in the feedback regulation of 7- and 12-hydroxylase transcription have been performed in tissue culture. Given the promiscuity of nuclear receptors in their DNA binding abilities, it is crucial to determine the role of FTF and HNF-4 in bile acid biosynthesis in vivo. The overall goal of this proposal is to understand the role and regulation of FTF and HNF-4 in the feedback regulation of bile acid biosynthesis in vivo. The P.I. hypothesizes that bile acids mediate the down regulation of the transcription of the 7- and 12-hydroxylases, by a two-step mechanism: first, bile acids suppress HNF-4 through a process mediated by the p38 MAP kinase pathway (a novel observation recently made in the P.I.'s lab), and increase FTF expression, which results in an increase in FTF binding to the overlapping FTF/HNF-4 recognition site found in the 7- and 12-hydroxylase promoters displacing HNF-4 from that site, and second, FTF becomes inactive through its heterodimerization with SHP. Preliminary studies suggest that some of the mechanisms involved in these processes are bile acid-specific and some of these mechanisms play a greater role in the regulation of 12-hydroxylase. The proposed specific studies are: 1) to characterize the role and mechanism of action of p38 kinase in the expression of the 7- and 12-hydroxylase genes; 2) to elucidate the role of the p38 pathway, FTF and HNF-4 in the bile acid-mediated regulation of bile acid synthesis in vivo, utilizing mice with suppressed p38 activity, and FTF+/- and SHP-/- mice; and 3) to characterize the displacement of HNF-4 by FTF from the 7- and 12- hydroxylase promoters, that is involved in the bile acid-mediated regulation of bile acid synthesis using the same mouse models. The successful completion of this study will provide us with new insights into the molecular mechanisms involved in the regulation of bile acid biosynthesis.