4-4-10-trimethyl-trans-decal-3-ol and 25-hydroxycholesterol

Previously, we found that mevalonate-derived products together with an oxysterol regulated reductase synthesis at a posttranscriptional level. To determine which products were responsible for this regulation, either the squalene synthase inhibitor zaragozic acid A or the squalene cyclase inhibitor 4,4,10-beta-trimethyl-trans-decal-3beta-ol (TMD) was added to lovastatin-treated Syrian hamster cells in conjunction with mevalonate. Mevalonate alone decreased reductase synthesis 50% compared with lovastatin-treated cells. In contrast, when both zaragozic acid A and mevalonate were added to lovastatin-treated cells, there was no change in reductase synthesis. With either treatment, reductase mRNA levels did not change compared with lovastatin-treated cells. When both 25-hydroxycholesterol and mevalonate were added to lovastatin-treated cells, reductase synthesis and mRNA levels were decreased 95 and 50%, respectively. The 10-fold difference between changes in reductase synthesis and mRNA levels under these conditions reflects a specific effect of mevalonate-derived isoprenoids on reductase synthesis at the translational level. In contrast, coincubation of cells with mevalonate plus 25-hydroxycholesterol in the presence of zaragozic acid decreased reductase synthesis and mRNA levels 60 and 50%, respectively, compared with lovastatin-treated cells. Moreover, degradation of reductase was increased approximately 7-fold in cells treated with mevalonate alone but only 3-fold in cells treated with mevalonate and zaragozic acid A. These results indicate that isoprenoid products between mevalonate and squalene affect reductase at a posttranslational level by increasing degradation but do not regulate reductase synthesis at a posttranscriptional level. In contrast, when both TMD and mevalonate were added to lovastatin-treated cells, reductase synthesis was decreased approximately 50% with no corresponding decrease in reductase mRNA levels, similar to mevalonate only. Reductase degradation was increased approximately 7-fold under these conditions. Cellular incubation in TMD, mevalonate, and 25-hydroxycholesterol decreased reductase synthesis and mRNA levels 95 and 50%, respectively. From these results we concluded that mevalonate-derived nonsterols synthesized between squalene and lanosterol decrease reductase synthesis at a translational level-either alone or in combination with 25-hydroxycholesterol-and also increase reductase degradation.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cricetinae; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Lovastatin; Lyases; Mesocricetus; Mevalonic Acid; Naphthols; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic; Tricarboxylic Acids

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.

Topics: Acetyl-CoA C-Acetyltransferase; Animals; Cell Line; Chromatography, High Pressure Liquid; Cricetinae; Dose-Response Relationship, Drug; Hydroxycholesterols; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hydroxymethylglutaryl-CoA Synthase; Ketoconazole; Lanosterol; Mevalonic Acid; Naphthols; Squalene; Sterols