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.     

Accumulation of regulatory oxysterols, 32-oxolanosterol and 32-hydroxylanosterol in mevalonate-treated cell cultures

In a previous publication (Saucier, S.E., A.A., Taylor, F.R., Spencer, T.A., Phirwa, S., and Gayen, A.K., J. Biol. Chem. (1985) 260, 14571-14579), we demonstrated that cultured Chinese hamster lung (Dede) cells contain 24(S),25-epoxycholesterol and 25-hydroxycholesterol in cellular concentrations within the range required to repress 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. In this paper, we show that the addition to the culture medium of a concentration of mevalonate high enough to repress the reductase by 90% resulted in the appearance of two new regulatory oxysterols. The two new sterols are believed to be 32-oxolanosterol and 32-hydroxylanosterol on the basis of high performance liquid chromatography (HPLC) retention times and mass spectrometric and nuclear magnetic resonance spectroscopic data and by NaBH4 reduction of the putative aldehyde to material which had the HPLC retention time of the putative alcohol. The cellular concentrations of 24(S),25-epoxycholesterol and 25-hydroxycholesterol were not significantly changed by the presence of mevalonate. However, there was approximately a 30% increase in total HMG-CoA reductase repressor units which can be ascribed to the 32-oxolanosterol and 32-hydroxylanosterol, where 1 unit equals the repressor activity of 1 ng of 25-hydroxycholesterol. These results support the idea that the level of HMG-CoA reductase activity in growing cell cultures is determined by intracellular oxysterol metabolites and that relatively small changes in their numbers or concentrations can alter the level of HMG-CoA reductase activity.     

Oxysterol binding protein

A binding protein is described for certain oxygenated derivatives of cholesterol which suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis in cultured mammalian cells. This protein is found in the cytosolic fraction of many cell types and is distinct from cytosolic proteins which bind cholesterol. The relative binding affinity of a wide variety of oxysterols correlates with their ability to suppress reductase and it is proposed that the binding protein functions as a receptor for endogenous regulatory oxysterols. The binding protein from cultured mouse fibroblasts (L cells) has been partially purified and characterized. Changes in its molecular form occur when a ligand is bound and further changes in form and binding kinetics occur at acid pH and in the presence of urea. Based on these changes a subunit model for the binding protein is presented.     

Characterization of oxysterol-binding protein in rat embryo fibroblasts and variations as a function of the cell cycle

A cell-free system assay involving cell freeze-thawing and protein fractionation by ammonium sulfate precipitation was developed to characterize a cytosol binding protein specific for oxysterols in rat embryo fibroblasts. This protein shared common characteristics with the oxysterol-binding protein described in L cells and in normal human lymphocytes: 8 S sedimentation coefficient, sterol-protein complex of Mr 160 600, saturability, high affinity (Kd in the range of 10(-9) M) and specificity for cholesterol derivatives oxidized on the side chain. These compounds were better inhibitors of DNA synthesis than the compounds oxidized on the nucleus, whereas no difference was found between sterols oxygenated either on the side chain or on the nucleus, as far as inhibition of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) was concerned. Macromolecular components capable of specifically binding 25-hydroxycholesterol were also detected in the fibroblast nucleus. The cytosol oxysterol-binding protein showed a sharp increase (5-fold) in the G2M phase of the cell cycle and in exponentially growing cells (maximal binding site number/cell: 43 500, versus 8850 in confluent cells). Neither the affinity nor the sedimentation coefficient of the protein changed in either situation. The quantitative (but not qualitative) variations of oxysterol-binding protein could be related to the inhibitory effect of 25-hydroxycholesterol on DNA synthesis, which becomes critical when this sterol is added in the G2M phase of the cell cycle.     

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.     

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts: I. Temporal relationships between HMG-CoA reductase activity,sterol biosynthesis and thymidine incorporation into DNA

Temporal relationships between hydroxymethylglutaryl-CoA reductase activity, biosynthesis of C₂₇ sterols, and [³H]thymidine incorporation into DNA were studied in a rat embryo fibroblast cell line synchronized by double thymidine block and cultured in cholesterol-containing medium. Cyclic variations of HMG-CoA reductase activity and C₂₇ sterols occurred, with two maxima in S and G₂M phases; the relative shortness of the G₁ phase (3 h) in these cells could be responsible for the shift of sterol synthesis in the S phase. No noticeable variation of the individual C₂₇ sterols was observed during the entire cell cycle. In each experiment, there was a good linear correlation between HMG-CoA reductase activity and C₂₇ sterol synthesis, but from one experiment to another, a given level of enzymatic activity led to varying levels of [2-¹⁴C]acetate incorporation into sterols. In our experimental conditions, total HMG-CoA reductase activity is measured, and the preceding observation could be explained by a varying degree of phosphorylation of the enzyme depending on the metabolic state of the cells at the start of the experiment. The cyclic variations of the enzyme activity seem to be due more to increased synthesis at given times of the cycle than to periodic dephosphorylation. We question the existence of a relationship between cell division and cyclic sterol synthesis occurring in cells cultured in cholesterol-containing medium.     

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.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant Chinese hamster ovary cell line

Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (CR-7), and revertant (RCR-7) cells. In contrast to WT and RCR-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of R-[2-14C]mevalonolactone revealed that CR-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but CR-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of 125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of 125I-LDL were also similar, suggesting that CR-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1-2 micrograms/ml) strongly and reversibly suppressed reductase activity in WT and RCR-7. CR-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of 3H-labeled mannose incorporation into cellular protein, but 3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.     

Effect of vitamin D3 derivatives on cholesterol synthesis and HMG-CoA reductase activity in cultured cells

Treatment of logarithmically growing rat intestinal epithelial cells (IEC-6) in culture with vitamin D3 (cholecalciferol), 25-hydroxy vitamin D3 (25-hydroxy cholecalciferol), 1,25-dihydroxy vitamin D3 (1,25-dihydroxycholecalciferol), and 24,25 dihydroxy vitamin D3 (24(R),25-dihydroxycholecalciferol), caused an inhibition of the cholesterol biosynthetic pathway at two separate sites. At concentrations greater than 2 micrograms/ml, the hydroxylated forms of vitamin D3 caused an accumulation of methyl sterols indicating an inhibition of lanosterol demethylation. Vitamin D3, however, had little effect on lanosterol demethylation. A second site of inhibition occurs at 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol biosynthesis at concentrations less than 2 micrograms/ml. All vitamin D3 compounds, except 1,25-dihydroxy vitamin D3, inhibited HMG-CoA reductase activity in a concentration-dependent manner. The lack of inhibition of HMG-CoA reductase activity by 1,25-dihydroxy vitamin D3 in IEC-6 cells was not due to impaired uptake, since 1,25-dihydroxy vitamin D3 caused an accumulation of methyl sterols under similar conditions. The inhibition of HMG-CoA reductase activity and cholesterol synthesis by vitamin D3 and 25-hydroxy vitamin D3 was also observed in other cell culture lines such as human skin fibroblasts (GM-43), transformed human liver cells (Hep G2), and mouse peritoneal macrophages (J-774). On the other hand, 1,25-hydroxy vitamin D3 showed effects on HMG-CoA reductase activity that varied with the cell line. In J-774 and human skin fibroblasts, 1,25-dihydroxy vitamin D3 showed a biphasic effect on reductase activity such that at low concentrations reductase activity was inhibited but was restored to control values at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 15-ketosterol is a liver X receptor ligand that suppresses sterol-responsive element binding protein-2 activity

Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-alpha and -beta (LXRalpha and LXRbeta). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.     

Oxygenation of desmosterol and cholesterol in cell cultures

In order to determine whether hydration of the delta 24 bond of desmosterol contributes to the formation of the regulatory oxysterol, 25-hydroxycholesterol, [3H]desmosterol was incubated with two cultured cell lines and the labeled products were analyzed. Small amounts of 25-hydroxycholesterol were formed with Chinese hamster lung (Dede) cell cultures, but not with mouse fibroblast (L) cell cultures. Apparently, desmosterol was converted into cholesterol, a process that does not occur in L cells, before 25-hydroxycholesterol takes place. No reliable evidence could be obtained for hydration of the delta 24 bond or for the reverse reaction upon incubation of [3H]25-hydroxycholesterol. Oxygenation of desmosterol occurred in both Dede and L cell cultures to give a mixture of 24(R)- and 24(S)-25-epoxy-cholesterol. This reaction, along with the production of 7-oxygenated sterols, may account for low levels of HMG-CoA reductase repressor activity previously found to be associated with delta 24 sterols.     

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites

Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) had the highest affinity for this microsomal binding site (Kd = 2.7 nM). Using 7-keto[3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol greater than 6-ketocholestanol greater than 7 beta-hydroxycholesterol = 7-ketocholesterol greater than cholesten-3 beta,5 alpha, 6 beta-triol = 7 alpha-hydroxycholesterol greater than 4-cholesten-3-one. All other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[3H]cholestanol for the same binding site. Second, the ability of different oxysterols and antiestrogens to inhibit 7-keto[3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[3H]cholestanol was similar to that of the antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.     

Oxysterol regulators of 3-hydroxy-3-methylglutaryl-CoA reductase in liver. Effect of dietary cholesterol

Hepatic regulatory oxysterols were analyzed to determine which oxysterols were present in livers of mice fed a cholesterol-free diet and whether repression of 3-hydroxy-3-methylglutaryl-CoA reductase following cholesterol feeding was accompanied by an increase in one or more oxysterols. Analysis of free and esterified sterols from mice fed a cholesterol-free diet resulted in the identification and quantitation of six regulatory oxysterols: 24-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, and 7-ketocholesterol. Following the addition of cholesterol to the diet for 1 or 2 nights, hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity declined and the levels of oxysterols, especially those of the side-chain-hydroxylated sterols, increased. Total 3-hydroxy-3-methylglutaryl-CoA reductase repressor units attributable to identified free oxysterols increased 2.5- and 6-fold after 1 and 2 nights, respectively, of cholesterol feeding. The amounts of esterified 24-, 25-, and 26-hydroxycholesterol also increased, with the increase in esterified 24-hydroxycholesterol being the greatest. The 24-hydroxycholesterol was predominantly the 24S epimer and the 26-hydroxycholesterol was predominantly the 25R epimer, indicating enzymatic catalysis of their formation. The observed correlation between increased levels of regulatory oxysterols and repression of 3-hydroxy-3-methylglutaryl-CoA reductase in cholesterol-fed mice is consistent with a hypothesis that intracellular oxysterol metabolites regulate the level of the reductase.     

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.