Recent data indicate that sterol carrier protein-2 (SCP-2) functions in the rapid movement of newly synthesized cholesterol to the plasma membrane (Puglielli, L., Rigotti, A., Greco, A. V., Santos, M. J., and Nervi, F. (1995) J. Biol. Chem. 270, 18723-18726). In order to further characterize the cellular function of SCP-2, we transfected McA-RH7777 rat hepatoma cells with a pre-SCP-2 cDNA expression construct. In stable transfectants, pre-SCP-2 processing resulted in an 8-fold increase in peroxisomal levels of SCP-2. SCP-2 overexpression increased the rates of newly synthesized cholesterol transfer to the plasma membrane and plasma membrane cholesterol internalization by 4-fold. There was no effect of SCP-2 overexpression on the microsomal levels of acyl-CoA:cholesterol acyltransferase and neutral cholesterol ester (CE) hydrolase; however, in the intact cell, CE synthesis and mass were reduced by 50%. SCP-2 overexpression also reduced high density lipoprotein-cholesterol secretion and apoA-I gene expression by 70% and doubled the rate of plasma membrane desmosterol conversion to cholesterol. We conclude that SCP-2 overexpression enhances the rate of cholesterol cycling, which reduces the availability of cholesterol for CE synthesis and alters the activity of a cellular cholesterol pool involved in regulating apoA-Imediated high density lipoprotein cholesterol secretion. The net result of these changes in cholesterol metabolism is a 46% increase in plasma membrane cholesterol content, the implications of which are discussed. Cellular free cholesterol is predominantly located in the plasma membrane (reviewed in Ref. 1). Cellular cholesterol content, however, is determined by the concerted action of intracellular enzymes and regulatory proteins as follows: ACAT 1 which catalyzes the synthesis of CE, sterol regulatory element binding proteins, which regulate the transcription of a number of genes involved in cholesterol metabolism (2, 3), and various cell type-specific metabolic reactions, e.g. lipoprotein secretion, steroidogenesis, and bile acid synthesis. Since cholesterol is highly insoluble in an aqueous environment, it has been postulated that sterol carrier protein-2 (SCP-2) regulates the movement and thus the availability of cholesterol for different cellular processes (4 -6). The evidence supporting this contention was initially derived in large part from studies demonstrating that the addition of purified SCP-2 stimulated the in vitro conversion of sterol intermediates to cholesterol (7) and cholesterol to 7␣-hydroxycholesterol (8) steroid hormones (9 -13) and cholesterol ester (14). More recent studies indicate that SCP-2 gene expression is regulated by changes in cellular cholesterol content (15-17); however, in these studies, a direct role in cholesterol trafficking and esterification was not demonstrated. The strongest support for a role in cellular cholesterol metabolism comes from studies on the role of SCP-2 in steroidogenesis. SCP-2 gene expression is coordinately regulated with steroid hormone syn...

The rate-limiting enzyme for mevalonate and cholesterol synthesis in mammalian cells is 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Control occurs through both transcriptional and post-transcriptional actions signaled by the end product, cholesterol, and by isoprenoid intermediates. End products of plant mevalonate metabolism, i.e., plant-derived isoprenoids, also suppress mammalian HMG-CoA reductase. Previous studies reported that isoprenoids suppress reductase synthesis at a post-transcriptional level. We tested the hypothesis that plant-derived isoprenoids also regulate mammalian HMG-CoA reductase synthesis at a post-transcriptional level by incubating lovastatin-treated C100 cells with mevalonate or a plant-derived isoprenoid (the monoterpenes, limonene, perillyl alcohol or geraniol) either alone or combined with the oxysterol, 25-hydroxycholesterol (25-OH C). Mevalonate decreased HMG-CoA reductase synthesis and mRNA levels by 65 and 66%, respectively (P < 0.05). The cyclic monoterpenes, limonene and perillyl alcohol, lowered HMG-CoA reductase synthesis by 70 and 89%, respectively (P < 0.05); although neither reduced HMG-CoA reductase mRNA levels (P = 0.88). Geraniol, an acyclic monoterpene, suppressed HMG-CoA reductase synthesis by 98% and lowered mRNA levels by 66% (P < 0.05). A combination of 25-OH C and either mevalonate or any three monoterpenes reduced HMG-CoA reductase mRNA levels (P < 0.05) compared with lovastatin-only treated cells. However, the dual combination of 25-OH C and either mevalonate or a monoterpene resulted in a greater decrease in HMG-CoA reductase synthesis than in mRNA levels. The difference between changes in HMG-CoA reductase synthesis and mRNA levels reflects a specific effect of isoprenoids on HMG-CoA reductase synthesis at the translational level. Mevalonate enhanced HMG-CoA reductase degradation, but no such effect was observed for the monoterpenes. These results indicate that the three plant-derived isoprenoids primarily suppress HMG-CoA reductase synthesis at a post-transcriptional level by attenuating HMG-CoA reductase mRNA translational efficiency.

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We reported previously that 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis is regulated at the translational level by mevalonate. To determine at what stage mevalonate affects reductase synthesis, we examined the distribution of reductase mRNA in polysomes from cells treated with lovastatin alone; lovastatin and 25-hydroxycholesterol; or lovastatin, 25-hydroxycholesterol, and mevalonate. In lovastatin-treated cells, reductase mRNA was primarily associated with heavy polysome fractions. When 25-hydroxycholesterol was added to lovastatin-treated cells, reductase mRNA levels were reduced approximately fourfold in all polysome fractions, with no accompanying redistribution of reductase mRNA into lighter polysome fractions. However, addition of both 25-hydroxycholesterol and mevalonate to lovastatin-treated cells shifted reductase mRNA from heavier to lighter polysome fractions. No change in the distribution of control beta-actin or ribosomal protein S17 mRNA occurred with any of the treatments. These results suggest that mevalonate suppresses reductase synthesis at the level of initiation. When the translation inhibitor cycloheximide was added to all three regimens, reductase mRNA shifted into heavy polysome fractions. Treatment with either lovastatin alone or lovastatin plus 25-hydroxycholesterol resulted in a 50% greater loss of reductase mRNA from the heavy polysome fractions compared to the same fractions from noncycloheximide-treated cells. No loss of reductase mRNA occurred when cycloheximide was added to cells treated with both 25-hydroxycholesterol and mevalonate. beta-Actin mRNA levels and polysome distribution were not significantly changed by cycloheximide under any of these conditions. Translationally mediated suppression of reductase mRNA did not occur when protein synthesis was inhibited with puromycin. Our results indicate that regulation of reductase mRNA levels is translation-dependent and is linked to the rate of elongation.

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