Effects of a novel lanosterol 14 alpha-demethylase inhibitor on the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hep G2 cells.
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A 32-carboxylic acid derivative of lanosterol (SKF 104976) was found to be a potent inhibitor of lanosterol 14 alpha-demethylase (14 alpha DM). 14 alpha DM activity in a Hep G2 cell extract was inhibited 50% by 2 nM SKF 104976. Exposure of intact cells to similar concentrations of the compound resulted in the inhibition of incorporation of [14C]acetate into cholesterol with concomitant accumulation of lanosterol as well as a 40-70% decrease in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity. SKF 104976 did not effect low density lipoprotein uptake and degradation in Hep G2 cells, suggesting that HMGR and low density lipoprotein receptor activity were not coordinately regulated under these conditions. Reduction of the flux of carbon units in the sterol synthetic pathway by as much as 80% did not alter the suppressing effect of SKF 104976 on HMGR activity. However, under conditions where sterol synthesis was almost completely blocked by lovastatin, HMGR activity was not suppressed by SKF 104976. Mevalonate, at concentrations that did not decrease HMGR activity, was able to restore the inhibiting effect of SKF 104976 on HMGR activity. The rapid inhibition (2-3 h) of HMGR activity by SKF 104976 to 30-60% of the level in controls was not dependent on the initial amount of HMGR enzyme present. These findings suggest that upon inhibition of 14 alpha DM by SKF 104976, a mevalonate-derived precursor regulates HMGR activity, even when the sterol synthetic rate is considerably reduced and when HMGR protein levels are very high. In Hep G2 cells, formation of oxylanostenols from [3H]mevalonate reached a maximum between 1 and 10 nM SKF 104976 and was negligible at higher concentrations. This result suggests that oxylanostenols are not the key mediators of the modulation of HMGR in Hep G2 cells upon 14 alpha DM inhibition. (+info)
Comparative calorimetric and spectroscopic studies of the effects of lanosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes.
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We carried out comparative DSC and Fourier transform infrared spectroscopic studies of the effects of cholesterol and lanosterol on the thermotropic phase behavior and organization of DPPC bilayers. Lanosterol is the biosynthetic precursor of cholesterol and differs in having three rather than two axial methyl groups projecting from the beta-face of the planar steroid ring system and one axial methyl group projecting from the alpha-face, whereas cholesterol has none. Our DSC studies indicate that the incorporation of lanosterol is more effective than cholesterol is in reducing the enthalpy of the pretransition. Lanosterol is also initially more effective than cholesterol in reducing the enthalpies of both the sharp and broad components of the main phase transition. However, at sterol concentrations of 50 mol %, lanosterol does not abolish the cooperative hydrocarbon chain-melting phase transition as does cholesterol. Moreover, at higher lanosterol concentrations ( approximately 30-50 mol %), both sharp and broad low-temperature endotherms appear in the DSC heating scans, suggestive of the formation of lanosterol crystallites, and of the lateral phase separation of lanosterol-enriched phospholipid domains, respectively, at low temperatures, whereas such behavior is not observed with cholesterol at comparable concentrations. Our Fourier transform infrared spectroscopic studies demonstrate that lanosterol incorporation produces a less tightly packed bilayer than does cholesterol, which is characterized by increased hydration in the glycerol backbone region of the DPPC bilayer. These and other results indicate that lanosterol is less miscible in DPPC bilayers than is cholesterol, but perturbs their organization to a greater extent, probably due primarily to the rougher faces and larger cross-sectional area of the lanosterol molecule and perhaps secondarily to its decreased ability to form hydrogen bonds with adjacent DPPC molecules. Nevertheless, lanosterol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers, although this phase is not as tightly packed as comparable cholesterol/DPPC mixtures. (+info)
Cholesterol starvation induces differentiation of human leukemia HL-60 cells.
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Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G(2) phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-alpha demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D(3), which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal-regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Delta(7)-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy. (+info)
Lanosterol biosynthesis in the prokaryote Methylococcus capsulatus: insight into the evolution of sterol biosynthesis.
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A putative operon containing homologues of essential eukaryotic sterol biosynthetic enzymes, squalene monooxygenase and oxidosqualene cyclase, has been identified in the genome of the prokaryote Methylococcus capsulatus. Expression of the squalene monooxygenase yielded a protein associated with the membrane fraction, while expression of oxidosqualene cyclase yielded a soluble protein, contrasting with the eukaryotic enzyme forms. Activity studies with purified squalene monooxygenase revealed a catalytic activity in epoxidation of 0.35 nmol oxidosqualene produced/min/nmol squalene monooxygenase, while oxidosqualene cyclase catalytic activity revealed cyclization of oxidosqualene to lanosterol with 0.6 nmol lanosterol produced/min/nmol oxidosqualene cyclase and no other products observed. The presence of prokaryotic sterol biosynthesis is still regarded as rare, and these are the first representatives of such prokaryotic enzymes to be studied, providing new insight into the evolution of sterol biosynthesis in general. (+info)
4-Methyl sterols regulate fission yeast SREBP-Scap under low oxygen and cell stress.
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In fission yeast, orthologs of mammalian SREBP and Scap, called Sre1 and Scp1, monitor oxygen-dependent sterol synthesis as a measure of cellular oxygen supply. Under low oxygen conditions, sterol synthesis is inhibited, and Sre1 cleavage is activated. However, the sterol signal for Sre1 activation is unknown. In this study, we characterized the sterol signal for Sre1 activation using a combination of Sre1 cleavage assays and gas chromatography sterol analysis. We find that Sre1 activation is regulated by levels of the 4-methyl sterols 24-methylene lanosterol and 4,4-dimethylfecosterol under conditions of low oxygen and cell stress. Both increases and decreases in the level of these ergosterol pathway intermediates induce Sre1 proteolysis in a Scp1-dependent manner. The SREBP ortholog in the pathogenic fungus Cryptococcus neoformans is also activated by high levels of 4-methyl sterols, suggesting that this signal for SREBP activation is conserved among unicellular eukaryotes. Finally, we provide evidence that the sterol-sensing domain of Scp1 is important for regulating Sre1 proteolysis. The conserved mutations Y247C, L264F, and D392N in Scp1 that render Scap insensitive to sterols cause constitutive Sre1 activation. These findings indicate that unlike Scap, fission yeast Scp1 responds to 4-methyl sterols and thus shares properties with mammalian HMG-CoA reductase, a sterol-sensing domain protein whose degradation is regulated by the 4-methyl sterol lanosterol. (+info)
Hypoxia stimulates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase through accumulation of lanosterol and hypoxia-inducible factor-mediated induction of insigs.
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Endoplasmic reticulum-associated degradation of the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase represents one mechanism by which cholesterol synthesis is controlled in mammalian cells. The key reaction in this degradation is binding of reductase to Insig proteins in the endoplasmic reticulum, which is stimulated by the cholesterol precursor lanosterol. Conversion of lanosterol to cholesterol requires removal of three methyl groups, which consumes nine molecules of dioxygen. Here, we report that oxygen deprivation (hypoxia) slows demethylation of lanosterol and its metabolite 24,25-dihydrolanosterol, causing both sterols to accumulate in cells. In addition, hypoxia increases the amount of Insig-1 and Insig-2 in a response mediated by hypoxia-inducible factor (HIF)-1alpha. Accumulation of lanosterol together with increased Insigs accelerates degradation of reductase, which ultimately slows a rate-determining step in cholesterol synthesis. These results define a novel oxygen-sensing mechanism mediated by the combined actions of methylated intermediates in cholesterol synthesis and the hypoxia-activated transcription factor HIF-1alpha. (+info)
Histone deacetylase 3 down-regulates cholesterol synthesis through repression of lanosterol synthase gene expression.
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In vertebrates, a key step in the biosynthesis of cholesterol and steroid hormones is the conversion of (S)-2,3-oxidosqualene to lanosterol. The enzyme that catalyzes this complex cyclization/rearrangement step via the protosteryl cation intermediate is lanosterol synthase ((S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7). Because of the crucial role that lanosterol synthase plays in cholesterol biosynthesis, there is great interest in the identification of drugs that target this enzyme for anticholesteremic purposes. Although most studies on lanosterol synthase in the past have focused on the structural and biochemical functions of this enzyme, almost nothing is known concerning how the synthesis of lanosterol synthase is regulated. Here, we report that histone deacetylase 3 (HDAC3) represses transcription from the lanosterol synthase promoter. Overexpression of HDAC3 decreases, whereas knockdown of HDAC3 by small interfering RNA increases, endogenous lanosterol synthase mRNA in cells. Similarly, in transient transfection assays, overexpression of HDAC3 decreases, whereas depletion of HDAC3 increases, expression of a reporter gene under the control of the lanosterol synthase promoter. Stable cell lines that overexpress HDAC3 show a decrease in lanosterol synthase mRNA and have lower cholesterol concentrations compared with parental cells. Extensive promoter analyses coupled with chromatin immunoprecipitation assays reveal that the transcription factor YY1 binds to and recruits HDAC3 to the lanosterol synthase promoter. Together, our results demonstrate that HDAC3 represses the synthesis of a key regulatory enzyme and reveal a novel mechanism by which the cholesterol biosynthetic pathway can be regulated. (+info)
Effectors of rapid homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase.
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The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol. (+info)