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-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, but these studies are open to question as they were carried out in whole animals. Therefore, we used primary rat hepatocytes, cultured in serum-free medium, to determine the effects of cholesterol on the regulation of cholesterol 7 alpha-hydroxylase. Squalestatin, a specific squalene synthase inhibitor, was used to block sterol but not isoprenoid biosynthesis in this system. Squalestatin (1 microM) decreased cholesterol 7 alpha-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 7 alpha-hydroxylase specific activity or steady-state mRNA levels in squalestatin-treated cells. Addition of cholesterol, delivered in beta-cyclodextrin, to squalestatin-treated cells restored cholesterol 7 alpha-hydroxylase specific activity and steady-state mRNA to control levels in a concentration (25 microM to 200 microM) -dependent manner. In contrast, the individual addition of selected "oxysterols" (5-cholesten-3 beta, 7 alpha-diol; 5 alpha-cholestan-3 beta, 6 alpha-diol; cholestan-3 beta, 5 alpha,6 beta-triol; 5-(25R)-cholesten-3 beta,26-diol, all at 50 microM) failed to restore cholesterol 7 alpha-hydroxylase mRNA levels in squalestatin-treated cells. These experiments provide evidence that cholesterol rather than "oxysterols" regulate cholesterol 7 alpha-hydroxylase gene expression. Squalestatin (1 microM) treatment increased HMG-CoA reductase specific activity by 229% of controls. Addition of cholesterol (200 microM), 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 7 alpha-hydroxylase gene expression by sterols.
Doerner
K C
KC
Department of Medicine, Medical College of Virginia--Virginia Commonwealth, Richmond 23298, USA.
Gurley
E C
EC
Vlahcevic
Z R
ZR
Hylemon
P B
PB
D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002478:Cells, Cultured; D002784:Cholesterol / Q000008:administration & dosage / Q000494:pharmacology; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics*; D003505:Cyclodextrins; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors; D005786:Gene Expression Regulation / Q000187:drug effects*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013261:Sterols / Q000494:pharmacology*; D014233:Tricarboxylic Acids / Q000494:pharmacology; D047392:beta-Cyclodextrins
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003505:Cyclodextrins; D012333:RNA, Messenger; D013261:Sterols; D014233:Tricarboxylic Acids; D047392:beta-Cyclodextrins; C075117:squalestatin 1; C015367:mevalonolactone; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D002790:Cholesterol 7-alpha-Hydroxylase; D013186:Farnesyl-Diphosphate Farnesyltransferase; C031215:betadex; D008798:Mevalonic Acid