Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

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.     

Regulation of cholesterol synthesis in primary rat hepatocyte culture cells. Possible regulatory site at sterol demethylation.

Primary rat hepatocyte culture cells were used to study the acute regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in response to 25-hydroxycholesterol, 3 beta,5 alpha,6 beta-cholestantriol, and mevalonolactone. All three effectors caused a rapid suppression of HMG-CoA reductase activity. 25-Hydroxycholesterol also caused an increase in the ratio of newly synthesized methyl sterols to newly synthesized C27-sterols. Furthermore, in 25-hydroxycholesterol-treated cells, the relative contribution of delta 24-sterol precursors to the nonsaponifiable lipid fraction increased. Di- and trimethyl-diene sterols were the dominant methyl sterols synthesized in the presence of 25-hydroxycholesterol. 3 beta,5 alpha,6 beta-Cholestrantriol (50 microM) also caused a very strong (97%) suppression of sterol demethylation; 4,4-dimethylmonoene sterols were more prominent (23%) in cells treated with 3 beta,5 alpha,6 beta-cholestrantriol, than in cells treated with 25-hydroxycholesterol (2%). The rates of both unesterified and esterified sterol synthesis increased as a function of exogenous mevalonolactone concentration. C27-sterol synthesis was saturated at a concentration of (R)-mevalonolactone which produced only a 33% suppression of HMG-CoA reductase activity. However, there was a direct relationship between the accumulation of methyl sterols and the decrease in HMG-CoA reductase activity. With the aid of triparanol, it was demonstrated that the suppression of HMG-CoA reductase activity by mevalonolactone was linked with the ability of the cells to convert squalene-2,3-epoxide into sterols. The results described in the present article support an important and perhaps necessary relationship between the rate of methyl sterol conversion of C27-sterols and the suppression or inhibition of HMG-Coa reductase in primary hepatocyte culture cells.     

25-Hydroxycholesterol-induced elevations in 45Ca uptake: permeability changes in P815 cells

Certain oxysterols are capable of suppressing the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. We have previously demonstrated that treatment of P815 cells with 1 microgram 25-hydroxycholesterol/ml culture results in a rapid influx of 45Ca, and supplemental cholesterol prevents this from occurring. In this paper, we report on investigations into the means whereby this influx of calcium takes place. Through the use of respiratory inhibitors which prevent mitochondrial retention of calcium it was determined that the large increase in slow phase (intracellular) calcium uptake caused by 25-hydroxycholesterol treatment was related to mitochondrial uptake. The effects of various inhibitors of calcium uptake into cells, including verapamil, diltiazem, quinidine, ruthenium red, Co++, Mn++, were tested. Of these only Co++ and ruthenium red had any effect on 45Ca uptake. 25-Hydroxycholesterol has been shown to be capable of membrane insertion and this could result in plasma membrane permeability changes. To test this hypothesis P815 cells were treated with 1 microgram 25-hydroxycholesterol/ml or 5 micrograms mevinolin/ml culture. Mevinolin, being a water soluble competitive inhibitor of HMG-CoA reductase, should be unable to disrupt membrane architecture in a manner analogous to 25-hydroxycholesterol. While both inhibitors rapidly suppressed the synthesis of digitonin-precipitable sterols, only 25-hydroxycholesterol was able to increase 45Ca influx. The implications of these findings are discussed.     

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.     

8-bromoadenosine cyclic 3',5'-phosphate rapidly increases 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in human granulosa cells: role of cellular sterol balance in controlling the response to tropic stimulation

Exposure of cultured human granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP) resulted in a rapid increase in the content of the mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the de novo synthesis of cholesterol. HMG-CoA reductase mRNA levels increased within 2 h of stimulation and remained elevated for at least 6 h. Treatment of granulosa cells with 25-hydroxycholesterol, a soluble cholesterol analogue, in combination with aminoglutethimide to block conversion of cellular sterols to pregnenolone, resulted in suppression of HMG-CoA reductase mRNA. When cells were stimulated with 8-bromo-cAMP in the presence of 25-hydroxycholesterol and aminoglutethimide, the increase in HMG-CoA reductase mRNA provoked by the tropic agent was markedly attenuated. This indicates that 8-bromo-cAMP raises HMG-CoA reductase mRNA levels indirectly by accelerating steroidogenesis and depleting cellular sterol pools, thus relieving sterol-mediated negative feedback of HMG-CoA reductase gene expression. 25-Hydroxycholesterol in the presence of aminoglutethimide suppressed low-density lipoprotein (LDL) receptor mRNA, but 8-bromo-cAMP effected a significant stimulation of LDL receptor mRNA levels when added with hydroxysterol and aminoglutethimide. These findings reveal differential regulation of HMG-CoA reductase and LDL receptor mRNAs in the presence of sterol negative feedback.     

Inhibition of sterol biosynthesis in animal cells by 14 alpha-alkyl-substituted 15-oxygenated sterols

Reported herein are the results of investigations of the effects of a number of 14 alpha-alkyl-substituted 15-oxygenated sterols, prepared by chemical synthesis, on sterol biosynthesis and the levels of 3-hydroxy-3-methylglutaryl CoA reductase activity in L cells and in primary cultures of fetal mouse liver cells grown in serum-free media. Several of the compounds, most notably 14 alpha-ethyl-5 alpha-cholest-7-en-3 beta, 15 alpha-diol and 14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, were found to be extraordinarily potent inhibitors of sterol synthesis in these cells. For example, the latter compound caused a 50% inhibition of the incorporation of labeled acetate into digitonin-precipitable sterols in L cells in culture at a concentration of 6 X 10(-9) M.     

Cholesterol, 7-ketocholesterol and 25-hydroxycholesterol uptake studies and effect on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in human fibroblasts.

In human fibroblasts two oxidized derivatives of cholesterol, 7-ketocholesterol and 25-hydroxycholesterol, but not cholesterol itself, are potent inhibitors of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (mevalonate: NADP+ oxidoreductase (Co-enzyme A acylating), (EC 1.1.1.34), the rate-limiting enzyme in sterol biosynthesis. In addition, these derivatives of cholesterol are effective regulators in cells from homozygous familial hypercholesterolemic individuals. The differences in the inhibitory potencies of the sterols cannot be explained in terms of the amount of uptake into the cell.     

DNA synthesis and 3-hydroxy-3-methylglutaryl CoA reductase activity in PHA stimulated human lymphocytes: A comparative study of the inhibitory effects of some oxysterols with special reference to side chain hydroxylated derivatives

The effects of a series of oxygenated sterols on DNA synthesis and HMG-CoA reductase activity were tested in human lymphocytes. The cells were stimulated by PHA and cultured in cholesterol containing medium. The inhibitory effects of sterols on DNA synthesis were strictly related to the position and the configuration of the hydroxyl on the side chain, to the side chain conformation and integrity and to the structure of the sterol nucleus. The inhibition of HMG-CoA reductase activity was less dependent on these structural features since all the sterols tested were strong inhibitors. In our experimental conditions the inhibition of DNA synthesis was not related to the suppression of the HMG CoA reductase activity. The specificity of the structures required for DNA synthesis inhibition could be explained by the involvement of a specific hydroxysterol binding protein.     

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.     

Inhibitors of sterol biosynthesis. Synthesis and activities of ring C oxygenated sterols

The chemical syntheses of a number of C27 ring C oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in cultured mammalian cells. Thus, 5 alpha-cholest-7-ene-3 beta, 11 alpha-diol, 3 alpha-hydroxy-5 alpha-cholest-9(11)-en-12-one, and the previously unreported 11 alpha-hydroxy-5 alpha-cholest-7-en-3-one, 5 alpha-cholest-9(11)-ene-3,12-dione, and 3 beta-hydroxy-5 alpha-cholest-9 (11)-en-12-one have been synthesized. The effects of these compounds on the synthesis of digitonin-precipitable sterols from labeled acetate in mouse L cells and on the levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in the same cells have been investigated and compared with previously published data on other ring C oxygenated sterols. 5 alpha-Cholest-7-ene-3 beta, 11 alpha-diol was shown to be the most potent inhibitor of sterol synthesis.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.     

A possible translational control of 3-hydroxy-3-methylglutaryl coenzyme A reductase induction by ML-236B(Compactin) in isolated rat hepatocytes

ML-236B (“Compactin”), a competitive inhibitor of 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase, increased the cholesterol synthesis and the HMG-CoA reductase activity in isolated rat hepatocytes. These increases were prevented by 0.2 mM puromycin, but not by 10 μg/ml actinomycin D and 40 μg/ml α-amanitin. These results indicated that the increases in cholesterol synthesis and HMG-CoA reductase activity by ML-236B required the enzyme synthesis but not newly synthesized mRNA. The regulatory site of feed-back inhibition by cholesterol for the HMG-CoA reductase synthesis in liver may be at the translational level.     

Synthesis of sterols and 5-lipoxygenase products are required for the G1-S phase transition of interleukin-2-dependent lymphocyte proliferation

A murine killer T cell line, G-CTLL 1, whose proliferation depends on the presence of interleukin 2 (IL-2), was used to analyze the mechanism of IL-2 action with respect to sterol synthesis and arachidonate metabolism. De novo sterol synthesis was substantially enhanced much earlier than DNA synthesis, and the rate reached a maximum at 13 hr after the addition of IL-2. Compactin, which is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase, the enzyme in the rate-limiting step of the sterol synthesis), inhibited the IL-2-induced DNA synthesis. The addition of mevalonate, the product of HMG CoA reductase, prevented the inhibition of DNA synthesis by compactin, suggesting that the supply of a sufficient amount of sterol is an essential prerequisite for IL-2 action. The IL-2-induced DNA synthesis was also inhibited by AA861, a specific inhibitor of arachidonate 5-lipoxygenase, and by other lipoxygenase inhibitors such as nordihydroguaiaretic acid and esculetin. In contrast, indomethacin, an inhibitor of arachidonate cyclooxygenase, had no effect. These findings suggest that synthesis of 5-lipoxygenase products is also a prerequisite. The inhibition of DNA synthesis was effectively inhibited only when compactin or lipoxygenase inhibitors were added early enough to block the synthesis of sterols or 5-lipoxygenase products; addition of the reagents after 3 hr decreased the inhibition with time. Therefore, about 3 hr after the addition of IL-2, several drastic intracellular changes are assumed to begin and to lead to DNA synthesis.     

The effect of glucagon, norepinephrine, and dibutyryl cyclic AMP on cholesterol efflux and on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in rat hepatocytes.

Incubation of rat hepatocytes for 3 hours in a sterol-free medium containing 1.5% albumin resulted in efflux of cellular sterol into the medium and an increased activity of 3-hydroxy-3-methylglutaryl CoA reductase. The secretion of cholesterol was inhibited when cells were incubated with glucagon, norepinephrine, or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP also inhibited the induction of HMG-CoA reductase. Norepinephrine treatment resulted in a decrease in the synthesis and secretion of proteins but caused an increase in reductase activity. Insulin treatment had no effect either on reductase activity or on sterol efflux from rat hepatocytes.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in the human gastrointestinal tract

Activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) was measured in intestinal mucosa of the human gastrointestinal tract. Activity was highest in gastric mucosa (18.2 pmol per mg per min) and there was a constant low level in the small bowel and colon (approximately 10 pmol per mg per min). Phosphorylation/dephosphorylation modulation of intestinal reductase activity was demonstrated in normal mucosa. Expressed jejunal reductase activity was significantly higher in celiac sprue mucosa and mucosa from defunctionalized intestine of jejunoileal bypass patients. Enzyme activity also increased during 24-hr mucosal organ culture in the absence of exogenous cholesterol. Addition to the culture medium of pure cholesterol or 25-hydroxycholesterol dissolved in a small volume of ethanol suppressed the culture-induced increase to 86 +/- 3% and 69 +/- 5% of paired controls, respectively. This evidence suggests that a moderate degree of feedback regulation of intestinal cholesterol synthesis by luminal sterol occurs in man. Mucosal HMG-CoA reductase activity was also measured in patients with hyperlipoproteinemia. Patients with either predominant hypercholesterolemia or predominant hypertriglyceridemia lipid profiles had "normal" expressed reductase activity, but feedback regulation by free cholesterol could not be demonstrated in either group under these conditions.     

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells

The polytopic endoplasmic reticulum (ER)–localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myeloblasts. Mode of action of 25-hydroxycholesterol

25-Hydroxycholesterol inhibits cholesterol biosynthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Addition of 25-hydroxycholesterol to chicken myeloblasts caused a rapid inhibition of HMG-CoA reductase activity, producing approximately an 80% decrease in enzyme activity after 60 min. The mode of action of 25-hydroxycholesterol was determined by immunoprecipitating radiolabeled enzyme from 25-hydroxycholesterol-treated myeloblasts. The decline in enzyme activity due to addition of 25-hydroxycholesterol was not associated with increased levels of [32P]PO4 incorporation into the immunoprecipitated reductase polypeptide (Mr = 94,000). Hence, 25-hydroxycholesterol did not appear to regulate reductase activity by enzyme phosphorylation, as observed for other modulators of HMG-CoA reductase. However, 25-hydroxycholesterol was shown to inhibit reductase activity by causing a 350% increase in the relative rate of reductase degradation and a 72% decrease in the relative rate of reductase synthesis. These alterations in the rates of degradation and synthesis occurred rapidly (within 10-30 min after addition of 25-hydroxycholesterol) and can account completely for the 25-hydroxycholesterol-induced inhibition of enzyme activity. The rapid decline in the rate of synthesis of HMG-CoA reductase in 25-hydroxycholesterol-treated cells was not associated with concomitant changes in the levels of reductase mRNA; therefore, suggesting that 25-hydroxycholesterol must inhibit the rate of reductase synthesis by translational regulation. We also present evidence that mRNA purified from chicken myeloblasts codes for two reductase polypeptides of Mr = 94,000 and 102,000.     

Identification of regulatory oxysterols, 24(S),25-epoxycholesterol and 25-hydroxycholesterol, in cultured fibroblasts

Biosynthetically tritiated sterols from Chinese hamster lung (Dede) cells were fractionated by high performance liquid chromatography, and fractions were assayed for their ability to repress 3-hydroxy-3-methylglutaryl-CoA reductase in L cell cultures. Most of the activity found was associated with two oxysterols, 24(S),25-epoxycholesterol and 25-hydroxycholesterol. The identities of the two sterols were established by co-chromatography with authentic samples and by isotopic dilution and recrystallization. Only low levels of repressor activity were found in other fractions of the sterol extract. The endogenous concentrations of 24(S),25-epoxycholesterol (7.2 fg/cell) and 25-hydroxycholesterol (1.5 fg/cell) appear to be within the ranges required for the regulation of HMG-CoA reductase.