REGULATION OF CHOLESTEROL 7-ALPHA-HYDROXYLASE EXPRESSION BY STEROLS IN PRIMARY RAT HEPATOCYTE CULTURES

The importance of cholesterol and ''oxysterols'' in the regulation of cholesterol 7 alpha-hydroxylase is not clear. Previous in vivo studies suggest that cholesterol may up-regulate cholesterol 7 alpha-hydroxyl ase, the rate-limiting enzyme in bile acid biosynthesis, but these stu dies are open to question as they were carried out in whole animals. T herefore, we used primary rat hepatocytes, cultured in serum-free medi um, to determine the effects of cholesterol on the regulation of chole sterol 7 alpha-hydroxylase. Squalestatin, a specific squalene synthase inhibitor, was used to block sterol but not isoprenoid biosynthesis i n this system. Squalestatin (1 mu M) decreased cholesterol 7 alpha-hyd roxylase specific activity to undetectable levels and decreased steady -state mRNA and transcriptional activity to 13% and 47% of controls, r espectively. Mevalonolactone (2 mM) failed to restore cholesterol 7 al pha-hydroxylase specific activity or steady-stale mRNA levels in squal estatin-treated cells. Addition of cholesterol, delivered in beta-cycl odextrin, to squalestatin-treated cells restored cholesterol 7 alpha-h ydroxylase specific activity and steady-state mRNA to control levels i n a concentration (25 mu M to 200 mu M) -dependent manner. In contrast , the individual addition of selected ''oxysterols'' (5-cholesten-3 be ta,7 alpha-diol; 5 alpha-cholestan-3 beta,6 alpha-diol; cholestan-3 be ta, 5 alpha,6 beta-triol; 5-(25R)-cholesten-3 beta,26-diol, all al 50 mu M) failed to restore cholesterol 7 alpha-hydroxylase mRNA levels in squalestatin-treated cells. These experiments provide evidence that c holesterol rather than ''oxysterols'' regulate cholesterol 7 alpha-hyd roxylase gene expression. Squalestatin (1 mu M) treatment increased HM G-CoA reductase specific activity by 229% of controls. Addition of cho lesterol (200 mu M), but not mevalonolactone (2 mM), to squalestatin-t reated cells decreased HMG-CoA reductase specific activity to 19% of c ontrol. The primary rat hepatocyte culture system in conjunction with a specific squalene synthetase inhibitor should be a useful model for elucidating the mechanism of regulation of cholesterol 7 alpha-hydroxy lase gene expression by sterols.