REGULATION OF BILE ACID SYNTHESIS--CHOLESTEROL 7ALPHA HYDROXYLASE

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In a classical feedback inhibitory loop, bile acids in the enterohepatic circulation repress the transcription of the rate-limiting enzyme of the bile acid biosynthetic pathway cholesterol 7 alpha-hydroxylase (C7alpha H). Two models have been proposed to account for this feedback inhibition. In a steroid hormone-type model, bile acids may bind to a cytosolic receptor, translocate to the nucleus, and interact directly with the C7alphaH promoter. Alternatively, bile acids may generate extranuclear signals, resulting in modification of a transcription factor required for C7alpha H gene expression. Data from our laboratories suggest that bile acids activate hepatocellular protein kinase C (PKC), and that PKC activation is required for the repression of C7alpha H transcription by taurocholate. Specific Aims: (1) To determine which PKC isoforms regulate C7alphaH transcription; (2) to characterize the mechanisms by which bile acids activate purified PKC isoforms; (3) to compare phorbol ester- and bile acid-induced events at the C7alphaH 5'-flanking region; and, (4) to explore the effects of the enterohepatic circulation of bile acids on hepatic and ileal mucosal PKC isoform activation as well as the possible implications of this activation on bile acid-regulated genes. Experimental design: In cultured rat hepatocytes (Obj. #1), Western immunoblotting and RNASE protection assays will be used to determine which PKC isoforms are translocated to hepatocellular membranes in response to bile acids, and which repress C7alpha H mRNA levels. In reconstituted assay systems with baculovirus- expressed PKC isoforms (Obj. #2), we will determine whether bile acids bind to the regulatory domain of PKC isoforms, or activate them indirectly by facilitating their association with membranes. In transfected rat hepatocytes (Obj. #4), RNase protection assays, immunohistochemistry, and in situ hybridization will be used to document the significance of bile acid-induced PKC isoform activation in regulating C7alphaH, the hepatic sinusoidal bile acid transporter, and the ileal bile acid transporter. Significance: We postulate that the flux of bile acids in the enterophepatic circulation may dynamically regulate the activity and turnover of hepatic and intestinal PKC isoforms, and that PKC isoforms in turn coordinate the activity of bile acid transporters and biosynthetic enzymes.