Hepatic cholesterol and bile acid synthesis are coordinated, since under most physiologic and experimental conditions the activities of HMG-C0A reductase (HMG-CoA-R) and cholesterol 7alpha-hydroxylase (C7alphaH), change in tandem. Both enzymes are subject to end product feedback regulation; HMG -CoA-R by cholesterol or oxidation products of cholesterol, and C7alphaH activity by bile salts. Recent data also show that hydrophobic bile salts down-regulate HMG-CoA-R, and cholesterol feeding, up-regulates C7alphaH activities. The molecular mechanism by which HMG-CoA-R and C7alphaH are regulated by bile salts have not been fully elucidated, nor is it known how the levels of microsomal cholesterol regulate C7alphaH. Recently, we obtained specific antibodies and cDNA probes for HMG-CoA-R and C7alphaH which will enable us to study the regulation of both enzymes simultaneously. Specific questions to be addressed in this grant proposal are: 1) What is the mechanism by which bile salts and cholesterol regulate C7alphaH? 2) What is the time course and concentration dependency of repression of C7alphaH and HMG-CoA-R by naturally occurring bile salts? 3) Is down regulation of C7alphaH by cholic acid secondary to its intestinal biotransformation to deoxycholic acid? 4) Is the mechanism of regulation of HMG-CoA-R by bile salts due to direct (post- transcriptional) or indirect effects of bile salts i.e. by facilitation of intestinal cholesterol absorption? 5) What is the role of steroid hormones (estrogen, dexamethasone) in the regulation of C7alphaH? 6) Can cultured hepatocytes, prepared by new and modified methods, be used to study regulation of C7alphaH and HMG-CoA-R? In addition, in collaboration with Drs. Chiang and Hylemon, we will study the regulation of 3beta-hydroxy-delta5-C27-steroid dehydrogenase/isomerase (3beta-HSD), and 12alpha-hydroxylase (12alphaH), two key enzymes in the bile acid biosynthetic pathway. The information obtained from these studies will be relevant to our understanding of factors responsible for the regulation of C7alphaH, the key enzyme in cholesterol degradation pathway and the mechanism by which cholesterol homeostasis is maintained in the liver.
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