Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol Δ8-isomerase, a cholesterogenic enzyme with multiple functions

Abstract

Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Recently, mutations of SI have been found to be associated with Conradi-Hünermann syndrome in humans. To investigate the in vitro and in vivo modes of molecular regulation of SI and its role in cholesterol biosynthesis in mammals, we isolated a full-length cDNA encoding rat SI. The deduced amino-acid sequence of rat SI predicts a 230-residue protein (26737 Da) with 87% and 80% amino-acid identity to mouse and human counterparts. The rat SI gene was mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FISH). The biological function of the cloned rat SI cDNA was verified by overexpressing recombinant Myc-SI in Saccharomyces cerevisiae. It showed a characteristic pattern of inhibition on exposure to trans-2-[4-(1,2-diphenylbuten-1-yl)phenoxy]-N,N-dimethylethylamine (tamoxifen; IC(50)=11.2 microM) and 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A; IC(50)=4.2 microM), 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 both age groups was restricted to liver, where a 70% reduction in mRNA levels was observed in 2-year-old rats. The FISH studies revealed ubiquitous expression of SI mRNA in rat hepatocytes. The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells. Treatment 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 enzymic 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. With this newly cloned rat SI cDNA, it becomes possible to gain molecular understanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic diseases.

Sterol Regulatory Element-binding Protein-2 Interacts with Hepatocyte Nuclear Factor-4 to Enhance Sterol Isomerase Gene Expression in Hepatocytes

“…Although SI is an important enzyme possessing unique features in the cholesterol biosynthesis pathway, to date little is known about its regulation. A lone previous paper has addressed the sterol-mediated regulation of SI mRNA levels (18). Here we report that the SI gene promoter, like other SREBPresponsive genes, contains a cluster of essential responsive elements for SREBPs, Sp1 and NF-Y.…”

Cholesterol-induced conformational change in SCAP enhanced by Insig proteins and mimicked by cationic amphiphiles

“…Examples of proteins whose activity is altered by cationic amphiphiles include G protein-coupled receptors, ion channels, membrane transporters, phospholipases, sphingomyelinase, sterol isomerases, and calmodulin (23)(24)(25)(26)(27)(28). Specificity is indicated by the finding that the activity of these proteins is altered by some cationic amphiphiles, but not by others.…”

“…SCAP appears to have a specific sensitivity to a subgroup of cationic amphiphiles, but this subgroup is not the same as the ones that affect other hydrophobic proteins. For example, the sterol C 8 -C 7 isomerase is inhibited by low concentrations of trifluoperazine, but it is also inhibited by similarly low concentrations of U18666A and ifenprodil, compounds that had minimal effect on SCAP conformation when added as high as 100 M (26,27). The sensitivity pattern of SCAP is perhaps closest to that of the soluble protein calmodulin, whose activity is inhibited by trifluoperazine ϭ chlorprothixene Ͼ chlorpromazine Ͼ clomipramine Ͼ haloperidol Ͼ promazine Ͼ imipramine Ͼ promethazine (24).…”

Gene expression profiling of androgen receptor antagonists in the rat fetal testis reveals few common gene targets

“…Both array analysis and real-time RT-PCR results indicated that vincolozin treatment significantly decreased the expression of Ebp and Idi, the protein products of both genes are involved in non-ratelimiting steps in cholesterol synthesis [44,45]. In human prostate cancer cells, androgen can induce the expression of lipogenic genes through the mediation of sterol regulatory element binding proteins (SREBPs) [46].…”