Deglycosylated human follitropin: characterization and effects on adenosine cyclic 3',5'-phosphate production in porcine granulosa cells

Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin. We report here the chemical characterization and biological properties of deglycosylated human follitropin (degly hFSH). Results indicate that deglycosylation of hFSH by HF removes 89% of the total carbohydrate without disruption of the peptide chain or significant loss of amino acid residues. However, a change in the conformation of the molecule was observed by measurement of the far-ultraviolet circular dichroic spectrum. The degly hFSH showed a 44% reduction in binding when tested in a FSH radioimmunoassay utilizing a polyclonal antibody. Binding of the degly hFSH to FSH-responsive tissues showed that the altered hormone bound with equal or better avidity than the intact hormone while the association constants were approximately the same for both preparations. The degly hFSH alone did not stimulate the FSH-stimulatable adenylyl cyclase (AC) activity of cellular homogenates of small follicle porcine granulosa cells. Furthermore, degly hFSH was a potent antagonist of hFSH-stimulatable AC activity when coincubated with intact hFSH. In intact granulosa cells, both the hFSH and the degly hFSH stimulated cAMP production and release by these cells. However, the degly hFSH was one-tenth as effective as the intact hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT2 cells: sterol biosynthesis, phosphorylation, degradation, and statin inhibition

We have previously identified a CHO cell line (UT2 cells) that expresses only one 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein which is localized exclusively in peroxisomes [Engfelt, H.W., Shackelford, J.E., Aboushadi, N., Jessani, N., Masuda, K., Paton, V.G., Keller, G.A., and Krisans, S.K. (1997) J. Biol. Chem. 272, 24579-24587]. In this study, we utilized the UT2 cells to determine the properties of the peroxisomal reductase independent of the endoplasmic reticulum (ER) HMG-CoA reductase. We demonstrated major differences between the two proteins. The peroxisomal reductase is not the rate-limiting enzyme for cholesterol biosynthesis in UT2 cells. The peroxisomal reductase protein is not phosphorylated, and its activity is not altered in the presence of inhibitors of cellular phosphatases. Its rate of degradation is not accelerated in response to mevalonate. Finally, the degradation process is not blocked by N-acetyl-Leu-Leu-norleucinal (ALLN). Furthermore, the peroxisomal HMG-CoA reductase is significantly more resistant to inhibition by statins. Taken together, the data support the conclusion that the peroxisomal reductase is functionally and structurally different from the ER HMG-CoA reductase.     

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.     

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 synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives 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.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.     

Gonadotropins and cyclic adenosine 3',5'-monophosphate (cAMP) alter the morphology of cultured human granulosa cells

Morphological changes in human granulosa cells in culture were observed by phase, fluorescent, scanning electron and transmission electron microscopy following the addition of human chorionic gonadotropin (hCG), luteinizing hormone (LH), 8-bromocyclic adenosine 3',5'-monophosphate (cAMP) and cytochalasins B and D. In response to these agents, polygon-shaped granulosa cells with granular cytoplasm became rounded, leaving fingerlike processes attached to the substratum and adjacent cells. The changes in cell shape were accompanied by a centripetal movement of mitochondria and lysosomes to a perinuclear location. The morphological alterations appeared to be mediated by cyclic AMP and to be the result of a dismantling and reorganization of microfilament-containing stress fibers. Follicle-stimulating hormone (FSH), prolactin (PRL), growth hormone (GH), and human placental lactogen (hPL) did not provoke cell shape changes. We conclude that tropic hormones capable of stimulating progestin secretion by luteinized granulosa cells cause a change in cell structure in vitro which leads to a redistribution of organelles involved in steroid synthesis. The possible relationship of the cytoskeleton to steroidogenesis is considered.     

Inhibitors of sterol synthesis: effects of a 7α-alkyl analog of 3β-hydroxy-5α-cholest-8(14)-en-15-one on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells and on serum cholesterol levels and other parameters in rats

The 7α-methyl analog (II) of 3β-hydroxy-5α-cholest-8(14)-en-15-one (I) was prepared by chemical synthesis and evaluated with respect to its effects on HMG-CoA reductase activity in CHO-K1 cells and on serum cholesterol levels in rats. The 7α-methyl substitution had no detectable effect on the potency of I in lowering HMG-CoA reductase activity in the cultured cells. In contrast, the 7α-methyl substitution had a marked effect on the action of I in the suppression of food consumption in rats. Whereas II was less potent than I in lowering serum cholesterol levels in rats, it did so at dosage levels at which only slight or moderate effects on food consumption were observed. Full ¹H and ¹³C-NMR assignments for II and intermediates in its synthesis have been presented. Conformational analysis, based on ¹H-¹H coupling constants, NMR shieldings and force-field calculations, indicated that the 7α-methyl substitution had virtually no effect on the conformation of the 15-ketosterol apart from minor distortions of ring B.     

Inhibitors of sterol synthesis. Chemical synthesis and spectral properties of 3 beta-hydroxy-5 alpha-cholesta-8(14),24-dien-15-one, 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one, and 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

Side-chain functionalized delta 8(14)-15-ketosterols have been synthesized from 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (VI) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Oxidation of VI to the 24-aldehyde VII, followed by Wittig olefination with isopropyltriphenylphosphonium iodide gave 3 beta-acetoxy-5 alpha-cholesta-8(14),24-dien-15-one (VIII), which was hydrolyzed to the free sterol IX. Oxymercuration of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one (IV). Hydroboration-oxidation of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one (V) as a 5:4 mixture of the 24R and 24S epimers. 1H and 13C nuclear magnetic resonance (NMR) assignments and mass spectral fragmentation patterns, supported by high-resolution measurements, are presented for IV and its 3 beta-acetate, V, VII, VIII, and IX. Characterization of IV by NMR and of trimethylsilyl ethers of IV and V by gas chromatography-mass spectrometry was compatible with spectral data for samples of IV and V isolated previously after incubation of I with rat liver mitochondria in the presence of NADPH. Sterols IV, V, and IX were very potent in lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells; their potency was comparable to that of I.     

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)     

Regulation of low density lipoprotein receptor gene expression in cultured human granulosa cells: roles of human chorionic gonadotropin, 8-bromo-3',5'-cyclic adenosine monophosphate, and protein synthesis

Treatment of human granulosa cells with human CG (hCG) or an analog of its second messenger, cAMP, promotes a rapid increase in low density lipoprotein (LDL) receptor mRNA content. After 1 h of treatment with 8-bromo-cAMP, an appreciable increase in hybridizable LDL receptor mRNA was found which increased to apparently maximal levels within 4-6 h. Treatment of the granulosa cells with 25-hydroxycholesterol, in the presence of aminoglutethimide, resulted in a reduction in LDL receptor mRNA content within 6 h of treatment. However, hCG or 8-bromo-cAMP were able to stimulate an increase in LDL receptor mRNA content in the presence of this inhibitory signal. We further investigated the mechanism by which tropic agents increased mRNA content. While inhibition of RNA synthesis with actinomycin D blocked the hCG or cAMP-induced rise in LDL receptor mRNA content, inhibition of protein synthesis with cycloheximide augmented basal or hCG- or cAMP-stimulated LDL receptor mRNA levels. We conclude that human steroidogenic cells possess a cAMP-mediated mechanism for rapid upregulation of LDL receptor mRNA which is distinct from, and supercedes, cholesterol negative feedback of LDL receptor gene expression. The actions of hCG and 8-bromo-cAMP do not require ongoing protein synthesis. Indeed, a cycloheximide-sensitive mechanism modulates receptor mRNA levels in these cells such that the effects of hCG and 8-bromo-cAMP are enhanced when cells are pretreated with this drug.