Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol Delta(8)-isomerase, a cholesterogenic enzyme with multiple functions

Sterol Delta (8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil bindin g protein or sigma receptor, catalyses the conversion of the 8-ene isomer i nto the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Re cently, mutations of SI have been found to be associated with Conradi-Huner mann syndrome in humans. To investigate the in vitro and in vivo modes of m olecular regulation of SI and its role in cholesterol biosythesis in mammal s, we isolated a full-length cDNA encoding rat SI. The deduced amino-acid s equence of rat SI predicts a 230-residue protein (26 737 Dal with 87 % and 80 % amino-acid identity to mouse and human counterparts, The rat SI gene w as mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FI SH). The biological function of the cloned rat SI cDNA was verified by over expressing recombinant Myc-SI in Saccharomyces cerevisiae. It showed a char acteristic pattern of inhibition on exposure to trans-2-[4-(1,2-diphenylbut en-1-yl)phenoxy]-N,N-dimethylethylamine (tamoxifen; IC50 = 11.2 muM) and 3 beta-[2-(diethylamino)ethoxy]androst-5-en- 17-one (U18666A; IC50 = 4.2 muM) , two well known potent inhibitors of SI. Northern-blot analysis of 3-week- old rats compared with 2-year-old rats showed that SI mRNA expression in bo th age groups was restricted to liver, where a 70 % reduction in mRNA level s was observed in 2-year-old rats. The FISH studies revealed ubiquitous exp ression of SI mRNA in rat hepatocytes. The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells. Tre atment of H4IIE cells grown in medium supplemented with fetal bovine serum with tamoxifen for 24 h resulted in a dose-dependent induction of SI mRNA, with a concomitant suppression of sterol regulatory element binding protein -1 mRNA. Interestingly, this effect was not seen in emopamil-treated cells. The in vivo experiments also indicate that both mRNA expression and enzymi c activity of SI in liver were induced approx. 3-fold in rats fed 5 %, (w/w ) cholestyramine plus 0.1 %, (w/w) lovastatin in normal chow for 2 weeks. W ith this newly cloned rat SI cDNA, it becomes possible to gain molecular un derstanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic disea ses.