Regulation of chol rol 7~hydroxyl expression by sterols in primary rat he cultures

The importance of cholesterol and "oxysterols" in the regulation of cholesterol 7a-hydroxylase is not clear. Previous in vivo studies suggest that cholesterol may up-regulate cholesterol 7a-hydroxylase, the rate-limiting enzyme in bile acid biosynthe- sis, but these studies are open to question as they were carried out in whole animals. Therefore, we used primary rat hepato- cytes, cultured in serum-free medium, to determine the effects of cholesterol on the regulation of cholesterol 7a-hydroxylase. Squalestatin, a specific squalene synthase inhibitor, was used to block sterol but not isoprenoid biosynthesis in this system. Squalestatin (1 pM) decreased cholesterol 7a-hydroxylase specific activity to undetectable levels and decreased steady-state mRNA and transcriptional activity to 13% and 47% of controls, respectively. Mevalonolactone (2 mM) failed to restore cholesterol 7a-hydroxylase specific activity or steady-state mRNA levels in squalestatin-treated cells. Addition of cholesterol, delivered in p- cyclodextrin, to squalestatin-treated cells restored cholesterol 7a-hydroxylase specific activity and steady-state mRNA to con- trol levels in a concentration (25 pM to 200 pM) -dependent manner. In contrast, the individual addition of selected "oxysterols" (5-cholesten-3fl,7a-diol; 5a-cholestan-3fl,6a-diol; cholestan-3b, 5a,Gfl-triol; 5-(25R)-cholesten-3fl,26-diol, all at 50 pM) failed to restore cholesterol 7a-hydroxylase mRNA levels in squalestatin-treated cells. a These experiments provide evi- dence that cholesterol rather than "oxysterols" regulate cholesterol 7a-hydroxylase gene expression. Squalestatin (1 pM) treatment increased HMG-CoA reductase specific activity by 229% of controls. Addition of cholesterol (200 pM), but not mevalonolactone (2 mM), to squalestatin-treated cells decreased HMG-CoA reductase specific activity to 19% of control. 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 7a- hydroxylase gene expression by sterols.-Doerner, K. C., E. C. Gurley, Z. R Vlahcevic, and P. B. Hylemon. Regulation of cholesterol 7a-hydroxylase expression by sterols in primary rat hepatocyte cultures. J. Lipid Res. 1995. 36: 1168-1177.