Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K(i) of 320 microM, over 10(4) higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.     

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.     

Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in melon (Cucumis melo L. reticulatus)

We have isolated a cDNA for Cm-HMGR, encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in melon (Cucumis melo L. reticulatus; Genbank Accession No. AB021862). Cm-HMGR encodes a polypeptide of 588 amino acids that contains two transmembrane domains and a catalytic domain. Database searches revealed that Cm-HMGR shows homology to HMG1 (63.7%) and HMG2 (70.3%) of tomato, to HMG1 (77.2%) and HMG2 (69.4%) of Arabidopsis thaliana, and to HMGR of tobacco (72.6%). Functional expression in a HMG-CoA reductase-deficient mutant yeast showed that Cm-HMGR products mediate the synthesis of mevalonate. Northern analysis revealed that the level of Cm-HMGR mRNA in the fruit increased after pollination and markedly decreased at the end of fruit enlargement. During ripening, Cm-HMGR mRNA levels increased markedly in the fruit. In parallel with mRNA expression, Cm-HMGR activity increased after pollination, whereas no Cm-HMGR activity was detectable during fruit ripening. Our results suggest that Cm-HMGR is important during early post-pollination development of the fruit in melon.     

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the rate of sterol synthesis diminish in cultures with high cell density

The specific activity of HMG-CoA reductase, the major rate-limiting enzyme in the sterol biosynthetic pathway, declined linearly with increasing cell density in four different lines of mammalian cell cultures. As expected, this caused the rates of sterol synthesis from [14C]acetate to decline in a parallel manner. The decrease in reductase activity in the dense cultures was also correlated with decreased incorporation of [14C]acetate into fatty acids and [3H]thymidine into DNA. In contrast, the activities of two enzymes, NADH dehydrogenase and 5'-nucleotidase, which are not involved in lipid synthesis, were independent of changes in cell density. The simplest explanation for these data is tht HMG-CoA reductase and the synthesis of sterol and fatty acids are regulated in concordance with the rate of cell growth and proliferation.     

Differential regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, synthase, and low density lipoprotein receptor genes.

The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.     

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: A comparison and immunological study of purified solubilized preparations,and alteration of enzyme levels by cholestyramine feeding

Some properties of various preparations of solubilized 3-hydroxy-3-methylglutaryl CoA reductase from rat liver are described. One, prepared by solubilization with deoxycholate, has been brought to a level of purity such that only a sińgle component is detected by polyacrylamide gel electrophoresis. A second preparation, solubilized by high salt concentration and heat treatment, has also been purified to a high level of purity so that only minor contaminants are detected. The deoxycholate-solubilized 3-hydroxy-3-methylglutaryl CoA reductase has a molecular weight of 197,000–202,000. Electrophoresis of both preparations treated with mercaptoethanol on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed one band with a molecular weight of 65,000. The data are consistent with the trimeric structure consisting of three polypeptide chains of apparently identical molecular weight. An antiserum to the deoxycholate-solubilized preparation has been prepared. Despite major differences among these preparations in specific activity, in stability to cold, and in the requirement of high salt concentration for preservation, both samples react in the same manner to the antibody and are immunologically indistinguishable. A preparation solubilized by freeze-thawing also reacts with the antiserum. Possible reasons for the variations in specific activity are considered, and it is concluded that specific activity changes cannot be reliably related to protein concentration unless the protein is isolated.Application of the immunological assay to an analysis of the effect of feeding cholestyramine to rats shows that compared to normals the diurnal cycle is unchanged but the rate of enzyme protein synthesis in the cholestyramine-fed rats is greatly accelerated. However, the first-order rate constant for degradatation of enzyme protein remains essentially unchanged throughout the falling phases of the cycle. The specific activity relationships of the enzyme protein of cholestyramine-fed rats appear to be altered when compared to that of normally fed controls.     

Inhibitors of sterol synthesis. Chemical synthesis of 5 beta-cholest-8-ene-3 beta,15 alpha-diol and its effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells

5 beta-Cholest-8-ene-3 beta,15 alpha-diol, prepared by hydroboration of 5 beta-cholesta-8,14-dien-3 beta-ol, was determined to have the 14 alpha-H,15 alpha-OH configuration by comparisons of observed and calculated lanthanide-induced shifts for the 3-tertbutyldimethylsilyl derivative. The 3 beta,15 alpha-diol was found to exist partially in a conformation in which ring B is a 5 beta, 6 alpha-half chair and the axial-equatorial orientation of ring A substituents is reversed. This conformation has been observed previously for 3 beta-(p-bromobenzoyloxy)-5 beta-cholesta-8,14-diene and for some cis-decalin derivatives. 5 beta-Cholest-8-ene-3 beta,15 alpha-diol was found to be highly active in the lowering of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells and only slightly less active than the corresponding sterol (5 alpha-cholest-8-ene-3 beta,15 alpha-diol) with the trans A-B ring junction.     

Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway)

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of beta-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.     

Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis.

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.     

Effects of compactin,a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor,on the growth of alfalfa (Medicago sativa) seedlings and the rhizogenesis of pepper (Capsicum annuum) explants

The effects of compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the growth of alfalfa seedlings in vivo and the rhizogenesis of pepper explants in vitro were investigated. Compactin added to the agar medium inhibited the elongation of roots and hypocotyls of etiolated alfalfa seedlings. The growth inhibition was accompanied by strict inhibition of sterol synthesis. Addition of mevalonic acid, the direct product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, together with compactin relieved the growth inhibition. The sterol level in the seedlings was also protected against the lowering effect of compactin. Similarly, the rhizogenetic process of cultured explants of pepper was inhibited by compactin and relieved by mevalonic acid. Several isoprenoid end products were tested in combination with compactin to determine which compounds, if any, might be limiting for growth. Exogenously supplied isoprenoids failed to relieve the growth inhibition of seedlings. In contrast, they partly relieved the growth inhibition of explants, suggesting their important role in plant growth. During the course of these experiments, it was also found that brassinolide caused remarkable growth inhibition and twisting of alfalfa seedlings.     

CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species

CS-514 is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol synthesis. For the microsomal enzyme from rat liver, the mode of inhibition is competitive with respect to hydroxymethylglutaryl-CoA, and the Ki value is 2.3 X 10(-9) M. CS-514 also strongly inhibited the sterol synthesis from [14C]acetate in cell-free enzyme systems from rat liver and in freshly isolated rat hepatocytes; the concentrations required for 50% inhibition were 0.8 ng/ml and 2.2 ng/ml, respectively. On the other hand, the inhibition by CS-514 was much less in the cells from nonhepatic tissues such as freshly isolated rat spleen cells, and cultured mouse L cells and human skin fibroblasts. In addition, the cellular uptake of 14C-labeled CS-514 by isolated rat spleen cells or mouse L cells was less than one-tenth of that by isolated hepatocytes. These differences between hepatic and nonhepatic cells were further confirmed by the fact that CS-514 orally administered to rats inhibited sterol synthesis selectively in liver and intestine, the major sites of cholesterogenesis. CS-514 markedly reduced serum cholesterol levels in dogs, monkeys and rabbits, including Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia in man, but did not reduce those in rats and mice. In the former case, preferential lowering of atherogenic lipoproteins was observed in all of the animals tested. The biliary neutral sterols significantly decreased, whereas the amount of biliary bile acids was not affected by administration of the drug to dogs.     

Stimulation of the proliferation of the Madin-Darby canine kidney (MDCK) epithelial cell line by high-density lipoproteins and their induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

MDCK Cells seeded on extracellular matrix- (ECM) coated dishes and exposed to medium supplemented with high-density lipoproteins (HDLs, 750 micrograms protein/ml) and transferrin (10 micrograms/ml) have a proliferative rate, final cell density, and morphological appearance similar to those of cells grown in serum-supplemented medium. The mitogenic stimulus provided by HDLs is not limited by the initial cell density at which cultures are seeded, nor is it limited in time, since cells grown in medium supplemented with transferrin and HDLs grew to at least 50 generations. The presence of HDLs in the medium is required in order for cells to survive, since cells actively proliferating in the presence of medium supplemented with HDLs and transferrin begin to die within 2 days after being transferred to medium supplemented only with transferrin. Low-density lipoprotein (LDL) is mitogenic for MDCK cells when present at low concentrations (from 2.5 to 100 micrograms protein/ml). Above 100 micrograms protein/ml, LDL is cytotoxic and therefore cannot support cell proliferation at an optimal rate. The mitogenic effect of HDLs is also observed when cells are maintained on fibronectin-coated dishes. However, the proliferative rate of the cells is suboptimal and cultures cannot be passaged on this substrate indefinitely, as they can be on ECM-coated dishes. A close association between the ability of HDLs to support cell proliferation and their ability to induce the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is observed. HMG CoA reductase activity is 18 times higher (70 pmoles/min/10(6) cells) in proliferating cells than in confluent, nondividing cells (4 pmoles/min/10(6) cells). The HMG Coa reductase activity of sparse cells is more sensitive to induction by HDLs (eight-fold higher than control cells) than is the enzyme activity of confluent cells (two-fold higher than control levels). The dose-response relationship between the abilities of HDLs to support proliferation and to induce HMG CoA reductase activity are similar. The time course of the stimulation of proliferation and the increase in enzyme activity of sparse, quiescent cells after exposure to HDLs are parallel. The HMG CoA reductase activity of sparse MDCK cells is induced six-fold by exposure to compactin, a competitive inhibitor of HMG CoA reductase. This induction of HMG CoA reductase is prevented by mevalonic acid, not affected by LDL, and synergistically enhanced by simultaneous exposure to HDLs. HDLs effect a rescue from the cytotoxic effect of compactin, whereas LDL does not.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitors of sterol synthesis. Characterization of beta,gamma-unsaturated analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells

Treatment of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism, with perchloric acid in methanol resulted in its partial isomerization to the beta,gamma-unsaturated 15-ketosterols, 3 beta-hydroxy-5 alpha,14 beta-cholest-8-en-15-one (2) and 3 beta-hydroxy-5 alpha,14 beta-cholest-7-en-15-one (3), which were easily separated from 1 by chromatography. Isomers 1, 2, and 3 could be distinguished by their chromatographic retention times as well as by their physical and spectral properties. Reduction of 2 with sodium borohydride gave 5 alpha,14 beta-cholest-8-ene-3 beta,15 beta-diol (4), for which the C-15 configuration was established from the lanthanide-induced shifts of its 3 beta-tert-butyldimethylsilyl ether. 1H and 13C NMR chemical shift differences between 2, 3, and 4 indicated the involvement of variable populations of conformers that differ in the flexible C-D ring system and in the side chain. Compounds 2, 3, and 4 lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.     

The inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by sterol regulatory element binding protein cleavage-activating protein requires four phenylalanine residues in span 6 of HMG-CoA reductase transmembrane domain

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway. This endoplasmic reticulum membrane protein contains a cytosolic catalytic domain and a transmembrane domain with eight membrane spans that are necessary for sterol-accelerated degradation. Competition experiments showed that wild-type transmembrane domains of HMGR and sterol regulatory element binding protein cleavage-activating protein (SCAP) blocked sterol-accelerated degradation of intact HMGR and HMGal, a model protein containing the membrane domain of HMGR linked to Escherichia coli beta-galactosidase. However, mutant transmembrane domains of HMGR and SCAP whose sterol-sensing functions were abolished did not inhibit sterol-accelerated degradation of HMGR and HMGal. In addition, our mutagenesis studies on HMGal indicated that four Phe residues conserved in span 6 of HMGR and the sterol-sensing domains of other sterol-related proteins are required for the regulated degradation of HMGR. These results suggest that HMGR and SCAP compete for binding to a sterol-regulated regulator protein, and this binding may need the four Phe residues.     

3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product.

The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.     

Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells

The effects of ketoconazole, a lanosterol demethylase and cytochrome P450 inhibitor, on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity and sterol biosynthesis were studied in rat intestinal epithelial cell cultures (IEC-6). Incubation of cells with 0.15-2 microM ketoconazole resulted in a concentration-dependent inhibition of reductase activity. As the drug concentration approached 15 microM, the reductase activity returned to control values, and at 30 microM ketoconazole, a stimulation of enzyme activity was observed. The drug had no effect on reductase activity in homogenates of IEC-6 cells. Ketoconazole (0.15-30 microM) caused a concentration-dependent inhibition of the incorporation of [3H] mevalonolactone into cholesterol with a concomitant accumulation of radioactivity in methyl sterols; e.g. lanosterol and 24,25-epoxylanosterol. Interestingly, the incorporation of radioactivity into polar sterols showed a biphasic response which was inversely proportional to the biphasic response of reductase activity. Thus, incorporation of [3H]mevalonolactone into polar sterols increased at low concentrations of ketoconazole (0.15-2 microM) and decreased to control values at high concentrations of the drug. Treatment of cells with ketoconazole (30 microM) and [3H]mevalonolactone followed by removal of the drug and radiolabel resulted in an inhibition of reductase activity and a redistribution of radioactivity from lanosterol and 24,25-epoxylanosterol to cholesterol and polar sterols. These results suggested that the inhibition of reductase activity at low concentrations of ketoconazole (less than 2 microM) was due to a formation of regulatory polar sterols generated from the methyl sterols. At high concentrations of ketoconazole (30 microM) where no suppression in reductase activity was observed, the conversion of exogenously added [3H]24(S),25-epoxylanosterol to polar sterols was prevented. Exogenously added 24,25-epoxylanosterol inhibited reductase activity in a dose-dependent fashion, and ketoconazole (30 microM) prevented the inhibition caused by low concentrations of epoxylanosterol. The drug, however, was unable to prevent the dose-dependent suppression of reductase activity by 25-hydroxylanosterol, a reduced form of 24,25-epoxylanosterol. These results indicated that 24,25-epoxylanosterol per se was not an inhibitor of reductase activity but could be metabolized to regulatory polar sterols through a cytochrome P-450 dependent reaction which was sensitive to ketoconazole. Treatment of cells with ketoconazole totally abolished the inhibition of reductase activity by low density lipoprotein (LDL).(ABSTRACT TRUNCATED AT 400 WORDS)