Cholesterol 7~hydroxylase: evidence for transcriptional regulation by cholesterol or metabolic products of cholesterol in the rat

Cholesterol 7a-hydroxylase, the rate-determining enzyme in the bile acid biosynthesis pathway, is regulated in a negative feedback manner by hydrophobic bile salts returning to the liver via the portal circulation. The role of cholesterol in the regulation of cholesterol 7a-hydroxylase and the interrelationship between the cholesterol and bile acid biosynthesis pathways remain controversial. The objective of the present study was to define the role of cholesterol in the regulation of cholesterol 7ahydroxylase and determine the molecular level of its control. In order to avoid intestinal or intravenous administration of cholesterol, we manipulated the flow of cholesterol within the hepatocytes by decreasing cholesterol synthesis with lovastatin in bile fistula rats (bile acid synthesis is up-regulated), or by increasing cholesterol supply by administering mevalonate, a precursor of cholesterol, to rats with intact enterohepatic circulation (bile acid synthesis is normal). In the first series of studies, lovastatin was administered as a single intravenous bolus (10 mg/kg) to rats with chronic bile fistula and to rats with intact enterohepatic circulation (cholesterol and bile acid synthesis is normal). Three hours after lovastatin administration, cholesterol 7a-hydroxylase specific activity, enzyme mass, mRNA, and gene transcriptional activity were decreased by 35%, 32%, 56%, and 34%, respectively, in rats with chronic bile fistula. In rats with intact enterohepatic circulation, lovastatin administration resulted in a similar decrease (34%) of cholesterol 7a-hydroxylase specific activity. In the second group of experiments, rats with intact enterohepatic circulation were administered a 180 pM bolus of mevalonate followed by a continuous infusion of I80 @bVh for 1.5, 3, 4.5, and 24 h prior to being killed. CoKtinuoUs infusion of mevalonate increased cholesterol 7a hydroxylase specific activity, mRNA levels, and transcriptional activity by an average of 2to %fold at all time intervals. I1p We conclude that under circumstances in which cholesterol is present in excess, cholesterol 7a-hydroxylase transcriptional activity is up-regulated and removal of cholesterol from the hepatocytes is facilitated by an increase of bile acid synthesis. When cholesterol availability is decreased, cholesterol 7ahydroxylase transcriptional activity is down-regulated leading to a decreased elimination of cholesterol via bile acid synthesis. In both instances, hepatic cholesterol homeostasis is effectively maintained.-Jones, M. P., W. M. Pandak, D. M. Heuman, J. Y. L. Chiang, P. B. Hylemon, and Z. R. Vlahcevic. Cholesterol 7a-hydroxylase: evidence for transcriptional regulation by cholesterol or metabolic products of cholesterol in the rat. J. Lipid Res. 1993. 34: 885-892. Supplementary key words bile acid synthesis mevalonate lovastatin enterohepatic circulation bile fistula The conversion of cholesterol into bile acids is initiated by the 7a-hydroxylation of cholesterol. This reaction is catalyzed by cholesterol 7cu-hydroxylase, the ratedetermining enzyme in the bile acid biosynthesis pathway (1, 2). It is generally agreed that the activity of this enzyme is principally regulated in a negative feedback manner by hydrophobic bile salts undergoing enterohepatic circulation (3, 4). Recently, several laboratories have purified and characterized cholesterol 7a-hydroxylase enzyme protein, and cloned its cDNA (5-9). These accomplishments cleared the way for studies of regulation of this important enzyme. It has been shown that cholesterol 7ahydroxylase mass and steady-state mRNA levels increase in rats following treatment with cholestyramine or biliary diversion (7, lo). In contrast, intraduodenal infusion of taurocholate to bile fistula rats significantly decreased cholesterol 7a-hydroxylase specific activity, enzyme mass, mRNA, and transcriptional activity (10). These latter findings provided evidence that taurocholate, ither directly or indirectly, regulates cholesterol 7a-hydroxylase at the level of gene transcription. Bile acid synthesis is the major pathway of cholesterol degradation in mammals and cholesterol availability appears to play an important role in the regulation of cholesterol 7a-hydroxylase. It has been shown that acute inhibition of cholesterol synthesis with the HMG-CoA Abbreviation: HMG, 3-hydroxy-3-methylglutaryl. 'To whom correspondence should be addressed at: Division of Gastroenterology, Medical College of Virginia, Box 711, Richmond, VA 23298. Journal of Lipid b e a r c h Volume 34, 1993 885 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom reductase inhibitor, lovastatin, or AY9944, an inhibitor of the last step in cholesterol synthesis, resulted in a prompt decrease of cholesterol 7a-hydroxylase activity and bile acid synthesis (11, 12). Effects of lovastatin, but not AY-9944, were preventable by a simultaneous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase (11). The molecular basis of downregulation of cholesterol 7a-hydroxylase by inhibition of cholesterol synthesis has not been established in these studies. In in vivo experiments, exogenous cholesterol was found to increase cholesterol 7a-hydroxylase activity at the level of gene transcription (10). However, these latter findings were questioned by Bjorkhem, Eggertsen, and Anderson (13), who reported that a diet high in cholesterol causes bile salt malabsorption in rats, implying that previously reported increase in cholesterol 7a-hydroxylase transcriptional activity (7, 10) may be a secondary phenomenon, Le., due to the effect of high dietary cholesterol on the intestinal bile salt absorption. Moreover, the same investigators were unable to demonstrate stimulatory effects of intravenously administered cholesterol on cholesterol 7a-hydroxylase activity (14). In order to determine whether cholesterol is a physiologic regulator of cholesterol 7a-hydroxylase, we administered an inhibitor of cholesterol synthesis (lovastatin) to rats with chronic bile fistula, and mevalonate, a precursor of cholesterol, to rats with intact enterohepatic circulation. Using these approaches, we avoided intestinal and intravenous routes of cholesterol administration and were able to study the effects of changes of cholesterol supply on cholesterol 7a-hydroxylase directly at the level of hepatocytes. The data obtained in this study provide evidence that alterations of cholesterol availability are coupled with parallel changes in cholesterol 7a-hydroxylase gene expression. Under circumstances of mevalonate overload, cholesterol 7a-hydroxylase is up-regulated, while in the presence of decreased cholesterol supply the enzyme is down-regulated. In either case, cholesterol homeostasis is effectively maintained. EXPERIMENTAL PROCEDURES