Bile acids are physiological agents required for the absorption, transport and disposal of lipid-soluble vitamins, steroids and xenobiotics. Bile acids are synthesized from cholesterol by two pathways. The major pathway starting with the rate-limiting enzyme, cholesterol 7a-hydroxylase, has been the focus of research. The gene CYP7A is regulated by a complex mechanism. A repressor and an activator region in the promoter have been mapped to contain elements responsive to bile acid, phorbol esters and retinoic acid. Yeast one-hybrid system will be used to isolate DNA-binding proteins isolated. Bile acid feedback is a physiological mechanism which not only inhibits CYP7A but also has a diverse effect on the expression of other lier genes. Imbalance in bile acid metabolism causes malabsorption of nutrients, proliferation of hepatocytes, cholestasis, liver cirrhosis and colon cancer in humans. Polymerase chain reaction based-differential display of mRNAs in hepatocytes will be used to identify genes differentially affected by bile acid. Bile acid synthesis pathway initiated with mitochondrial sterol 27- hydroxylase, and 27-hydorxycholeserol 7a-hydorxylase (oxysterol 7a- hydroxylase) has been uncovered recently. When cholesterol 7a- hydroxylase activity is expressed at a low level in neonatal animals, in liver disease, and in transgenic mice deficient the CYp7a gene, 27- hydroxycholestrerol synthesized in the extrahepatic tissues may be converted to bile acids in the liver as a compensatory mechanism. This pathway also regulate the levels of oxysterols which are potent repressors of cholesterol synthesis and transport. The CYP27 gene mutations have been identified in cerebrotendinous xanthomatosis but regulation of the CYP27 gene is unknown. The hypothesis that both sterol 27-hydorxylase and oxysterol-7a-hydroxylase may play important roles in regulation of bile acid and oxysterol syntheses will tested. Regulatory regions in the CYP27 upstream sequence will be mapped by transient transection assay of CYP27/luciferase reporter genes, DNase I foot printing and gel mobility shift assays. Oxysterol 7a-hydroxylase will be purified and cDNA will be cloned to study its role in the regulation of oxysterols and bile acid syntheses. The long term objective of this research project is to understand molecular mechanisms of regulation of bile acid synthesis and mechanisms of human diseases in bile acid metabolism and cholesterol homeostasis.
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