The most important pathway for the catabolism and excretion of cholesterol in mammals is the formation of bile acids. Improper regulation of this pathway has widespread implications because the rate of elimination of cholesterol is a very important factor in diseases such as atherosclerosis, gallstone disease, and some lipid storage diseases. Two enzymes play major regulatory roles in bile acid synthesis, cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in the classic pathway, and sterol 12alpha-hydroxylase, the specific enzyme for cholic acid synthesis. Bile acids exert negative feedback regulation of their own synthesis by regulating the activity of these two enzymes. This regulation occurs at the level of the transcription of the genes. The applicant's laboratory has recently shown that alpha1-fetoprotein transcription factor (FTF) is crucial for the expression and the bile acid-mediated regulation of both 7alpha-and 12alpha-hydroxylase. This regulation functions through the interaction of FTF with the small heterodimer partner 1 (SHP). The Principal Investigators laboratory has also unveiled molecular details of how SHP and FTF interact, and has characterized the role of HNF-4 in the activation and regulation of 12alpha-hydroxylase expression. Additionally, they have discovered that sterol regulatory element binding protein 1 and 2 (SREBP-1 and-2) exert opposite effects on the 12alpha-hydroxylase promoter, which was highly unexpected. Understanding the molecular mechanisms regulating bile acid biosynthesis and its control will require knowledge of how SHP, a nuclear receptor co-factor that suppresses not only FTF activity but also many other nuclear receptors, specifically suppresses bile acid-regulated genes but not other genes that are activated by HNF-4, ER, RXR, TR, etc., and that are not regulated by bile acids. It will also require an understanding of the molecular mechanisms and function of the SREBP-2 mediated suppression of 12alpha-hydroxylase. To achieve this goal, the following specific aims will be pursued: 1) Characterization of the FTF/HNF-4 site within the 12alpha-hydroxylase promoter involved in the bile acid-mediated regulation of its transcription. 2) Elucidation of the molecular mechanisms y which the FTF/SHP pathway specifically regulates 12alpha-hydroxylase transcription. 3) Characterization of the molecular mechanisms involved in the SREBP-2- mediated suppression of the 12alpha-hydroxylase promoter. 4) Characterization of the significance and physiological role of the SREBP-2-mediated suppression of 2alpha-hydroxylase expression. These studies should provide an understanding of the molecular mechanisms involved in the regulation of bile acid synthesis.
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