Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis by a non-sterol mevalonate-derived product in Mev-1 cells. Apparent translational control

A somatic cell mutant (Mev-1) auxotrophic for mevalonate by virtue of a complete lack of detectable 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activity has been shown to demonstrate a requirement for a non-sterol mevalonate-derived product for regulation of synthesis of HMG-CoA reductase. A comparison of the effects of 25-hydroxycholesterol and the combination of 25-hydroxycholesterol and mevalonate on HMG-CoA reductase activity, synthesis, and mRNA levels in Mev-1 is presented in this report. The results show a close correlation between activity, rate of synthesis, and mRNA levels for Mev-1 cells treated with 25-hydroxycholesterol alone. Under the conditions of these experiments these effects are relatively small (approximately a 4-fold decrease). A much larger inhibition of HMG-CoA reductase activity and rate of synthesis (approximately 50-fold) is observed upon treatment of Mev-1 cells with a combination of 25-hydroxycholesterol and mevalonate. Yet, under these conditions mRNA levels are still reduced by only a factor of 4. These results are interpreted to suggest that the non-sterol mevalonate-derived regulatory product of HMG-CoA reductase acts by a translational control mechanism.     

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.     

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.     

Defective regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant.

A somatic cell mutant of the CHO-K1 cell selected to be resistant to the killing effects of 25-hydroxycholesterol in the absence of cholesterol is shown to be defective in the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity by 25-hydroxycholesterol, cholesterol, and lipoproteins, thus maintaining the enzyme activity found in cells in the absence of exogenous sterol constitutively. The mutants phenotype is shown to be dominant with respect to the wild type. Actinomycin D and cycloheximide prevent the increase of HMG-CoA reductase activity that occurs in the CHO-K1 cell when cholesterol is removed from medium. Degradation of the enzyme, measured during inhibition of protein synthesis by cycloheximide, occurs at the same rate in the mutant as in the wild type. Kinetic studies indicate that the Km for two substrates, the activation energy, and a break in the Arrhenius plot are the same for HMG-CoA reductase determined in wild type and mutant cells. From these studies it is concluded that the mutant is defective in the regulation of synthesis of HMG-CoA reductase. Of the four processes which determine cellular cholesterol levels: biosynthesis, esterification, efflux, and uptake, only biosynthesis is altered, demonstrating that these processes are not co-ordinately controlled as has been suggested previously.     

Post-translational isoprenylation of cellular proteins is altered in response to mevalonate availability

Cells incorporate isoprenoid products derived from mevalonate (MVA) into several unique proteins. The aim of this study was to delineate the effects of blocking MVA synthesis on the covalent isoprenylation of these proteins in murine erythroleukemia cells. Inhibition of protein synthesis with cycloheximide prevented the incorporation of [3H]MVA into proteins, suggesting that isoprenylation normally occurs immediately after synthesis of the polypeptides. However, incubation of cells with lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, for as little as 1 h prior to addition of cycloheximide rendered the isoprenylation step insensitive to cycloheximide. Lovastatin had no apparent effect on the stability of the isoprenylated proteins, but the development of cycloheximide insensitivity during the lovastatin preincubation was dependent on synthesis of new protein during that period. Addition of 50-200 microM MVA to the culture medium eliminated the effects of preincubation with lovastatin. Preincubation of cells with 25-hydroxycholesterol, which suppresses the synthesis and enhances the degradation of HMG-CoA reductase but is not a competitive enzyme inhibitor, did not induce cycloheximide-insensitivity of the isoprenylation reaction. The results suggest that blocking MVA synthesis with lovastatin causes a rapid depletion of isoprenoid groups available for protein modification. Consequently, there is an accumulation of non-isoprenylated substrate proteins. Shifts in the ratio of modified vs. unmodified proteins in response to MVA availability may have implications for the changes in cell morphology, cell proliferation and HMG-CoA reductase gene expression that occur when cells are subjected to MVA deprivation.     

Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells

Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.     

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.     

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.     

Treatment of CHO-K1 cells with 25-hydroxycholesterol produces a more rapid loss of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity than can be accounted for by enzyme turnover

A key enzyme in the regulation of mammalian cellular cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). It is well established that treatment with the compound 25-hydroxycholesterol lowers HMG-CoA reductase activity in cultured Chinese hamster ovary (CHO-K1) cells. After brief incubation (0-4 h) with 25-hydroxycholesterol (0.5 microgram/ml), cellular HMG-CoA reductase activity is decreased to 40% of its original level. This also occurs in the presence of exogenous mevinolin, a competitive inhibitor of HMG-CoA reductase which has previously been shown to inhibit its degradation. The inhibition of HMG-CoA reductase activity by 25-hydroxycholesterol is complete after 2 h. Radio-immune precipitation analysis of the native enzyme under these conditions shows a degradation half-life which is considerably longer than that of the observed inhibition. Studies with sodium fluoride, phosphatase 2A, bacterial alkaline phosphatase and calf alkaline phosphatase indicate that the observed loss of activity is not due to phosphorylation. These data are not consistent with described mechanisms of HMG-CoA reductase activity regulation by phosphorylation or degradation but are consistent with a novel mechanism that regulates the catalytic efficiency of this enzyme.     

Regulation of cholesterol biosynthesis and esterification by 25-hydroxycholesterol in a macrophage-like cell line: uncoupling by progesterone

The coordinated control of cholesterol biosynthesis and esterification by 25-hydroxycholesterol was studied in the macrophage-like cell line P388D1. Since 25-hydroxycholesterol rapidly stimulated incorporation of [3H]oleate into the cholesteryl ester fraction of these cells, we have tested the possibility that the well-known inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) by 25-hydroxycholesterol might be the indirect consequence of an increased cholesterol esterification rather than a direct effect on HMG-CoA reductase. The experimental results show that progesterone, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), when added together with 25-hydroxycholesterol, abolished the increased cholesterol esterification without affecting the inhibition of HMG-CoA reductase by 25-hydroxycholesterol. Thus, uncoupling cholesterol esterification had no effect on 25-hydroxycholesterol's ability to inhibit HMG-CoA reductase. Unexpectedly, pretreatment of P388D1 cells with 25-hydroxycholesterol resulted in no elevation of ACAT activity as measured in broken cell preparations. Therefore, the possibility that 25-hydroxycholesterol stimulated cholesteryl ester formation by increasing the amount of cholesterol available for esterification, rather than by acting directly on ACAT activity, was considered. Labeling experiments using [14C]-cholesterol have provided evidence for this assumption.     

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.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by reversible phosphorylation: modulation of enzymatic activity by low density lipoprotein, sterols, and mevalonolactone

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase exists in interconvertible active and inactive forms in cultured fibroblasts from normal and familial hypercholesterolemic subjects. The inactive form can be activated by endogenous or added phosphoprotein phosphatase. Active or partially active HMG-CoA reductase in cell extracts was inactivated by a ATP-Mg-dependent reductase kinase. Incubation of phosphorylated (inactive) HMG-CoA reductase with purified phosphoprotein phosphatase was associated with dephosphorylation (reactivation) and complete restoration of HMG-CoA reductase activity. Low density lipoprotein, 25-hydroxycholesterol, 7-ketocholesterol, and mevalonolactone suppressed HMG-CoA reductase activity by a short-term mechanism involving reversible phosphorylation. 25-Hydroxycholesterol, which enters cells without the requirement of low density lipoprotein-receptor binding, inhibited the HMG-CoA reductase activity in familial hypercholesterolemic cells by reversible phosphorylation. Measurement of the short-term effects of inhibitors on the rate of cholesterol synthesis from radiolabeled acetate revealed that HMG-CoA reductase phosphorylation was responsible for rapid suppression of sterol synthesis. Reductase kinase activity of cultured fibroblasts was also affected by reversible phosphorylation. The active (phosphorylated) reductase kinase can be inactivated by dephosphorylation with phosphatase. Inactive reductase kinase can be reactivated by phosphorylation with ATP-Mg and a second protein kinase from rat liver, designated reductase kinase kinase. Reductase kinase kinase activity has been shown to be present in the extracts of cultured fibroblasts. The combined results represent the initial demonstration of a short-term regulation of HMG-CoA reductase activity and cholesterol synthesis in normal and receptor-negative cultured fibroblasts involving reversible phosphorylation of both HMG-CoA reductase and reductase kinase.     

Differential translational effects of myristic acid and eicosapentaenoic acid on 3-hydroxy-3-methylglutaryl-CoA reductase from Reuber H35 hepatoma cells

The mechanisms by which saturated and polyunsaturated fatty acids may exert their effects on levels of blood cholesterol and human atherosclerosis have not been fully established. In this work, we studied the translational effects of myristic (14:0) and eicosapentaenoic (20:5) acids on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Reuber H35 hepatoma cells. This enzyme is an intrinsic membrane, 96-kDa protein whose proteolysis releases an enzymatically active, 52- to 56-kDa, soluble fragment. We optimized an immunoblot procedure for quantifying small amounts of both the native and the soluble forms of HMG-CoA reductase from Reuber H35 hepatoma cells. We demonstrated that the upregulation of HMG-CoA reductase by a acid is due to an increase of the HMG-CoA reductase protein; therefore, protein synthesis would be required for the increase of HMG-CoA reductase activity caused by this fatty acid. In contrast, the downregulation of HMG-CoA reductase caused by eicosapentaenoic acid is not due to decreased protein synthesis, since similar levels of protein were found in the presence and absence of this fatty acid. Results obtained with cycloheximide as a protein-synthesis inhibitor confirm these findings.     

Studies on the relationships between 7α-hydroxylation and the ability of 25- and 27-hydroxycholesterol to suppress the activity of HMG-CoA reductase

The metabolism of 25-hydroxycholesterol in different cell types was studied and the role of 7α-hydroxylation for the effect of 25-hydroxycholesterol on the activity of HMG-CoA reductase was determined. Human diploid fibroblasts (HDF) and the human melanoma cell line SK-MEL-2 converted 25-hydroxycholesterol into 7α,25-dihydroxycholesterol and 7α,25-dihydroxy-4-cholesten-3-one while the virus-transformed fibroblast line 90VA-VI, the colon carcinoma cell line WiDr and the breast cancer cell line MDA-231 did not express 7α-hydroxylase activity. The 7α-hydroxylation of 25-hydroxycholesterol in HDF could be stimulated by dexamethasone and cortisol and inhibited by metyrapone. An unidentified, possibly 4-hydroxylated, metabolite was formed by 90VA-VI cells and a polar, probably conjugated, metabolite was formed by WiDr cells. The 7α-hydroxylated metabolites of 25-hydroxycholesterol suppressed the activity of HMG-CoA reductase to a similar extent as 25-hydroxycholesterol in HDF but not in 90VA-VI cells, while the 7α-hydroxylated metabolites of 27-hydroxycholesterol suppressed the activity of HMG-CoA reductase also in 90VA-VI cells. The suppression of HMG-CoA reductase activity by 25- and 27-hydroxycholesterol was decreased or abolished by dehydroepiandrosterone or pregnenolone which have little or no effect on the 7α-hydroxylation. The results indicate that 7α-hydroxylation is not directly involved, positively or negatively, in the action of 25- or 27-hydroxycholesterol as suppressors of HMG-CoA reductase activity.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.