Bile acid synthesis is regulated by cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme of the pathway, and sterol 12alpha-hydroxylase (CYP8B1) that catalyzes cholic acid (CA) synthesis and regulates the ratio of CA to chenodeoxycholic acid (CDCA) in bile. Transcription of the CYP7A1 and CYP8B1 is feedback inhibited by bile acids. We hypothesize that a nuclear receptor-mediated mechanism may be involved in regulation of genes in bile acid synthesis. CDCA has been shown to activate a nuclear hormone receptor, farnesoid X receptor (FXR) and induce a negative nuclear receptor, short heterodimer partner (SHP). SHP interacts with either alpha-fetoprotein transcription factor (FTF) and/or hepatocyte nuclear factor4 (HNF4) and represses CYP7A1 transcription. We hypothesize that similar mechanism may regulate CYP8B1 transcription but FTF and HNF4 differentially mediate FXR-dependent bile acid inhibition. Cholesterol stimulates CYP7A1 transcription by activating oxysterol receptor LXR only in the mouse model. In contrast, cholesterol inhibits human CYP7A1 and CYP8B1 transcription. We hypothesize that sterol response element binding proteins (SREBPs) that are known to stimulate sterol-suppressed genes also regulate the CYP7A1 and CYP8B1.
The specific aims of this research proposal are to use site-directed mutagenesis, promoter/reporter assays, and electrophoretic mobility shift assays to study bile acid regulation of CYP7A1 and CYP8B1 transcription by FTF and HNF4, and sterol regulation of these genes by SREBP-1c, and to identify bile acid and sterol response elements in the promoters. Our long-term objective is to study the mechanisms of bile acid synthesis and regulation and cholesterol homeostasis by nuclear receptors and SREBPs. Information obtained from this project is important for elucidation the molecular mechanism of human diseases such as atherosclerosis, cholestatic liver diseases and gallstone disease, and for developing new drug therapies targeted to nuclear receptors for lowering cholesterol by stimulating bile acid synthesis, secretion and transport.
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