HMGR mRNA Levels Research Articles

Three Structurally Different Polychlorinated Biphenyl Congeners (Pcb 118, 153, and 126) Affect Hormone Production and Gene Expression in the Human H295R In Vitro Model

Polychlorinated biphenyls (PCB) are ubiquitous environmental pollutants that have been linked to adverse health effects including endocrine disruption. This study compared the mono-ortho-substituted PCB 118 and di-ortho-substituted PCB 153 with the non-ortho-substituted PCB 126, for possible effects on steroid hormone production and on the expression of 10 genes encoding proteins involved in steroidogenesis. The H295R human adenocarcinoma cell line was used as an in vitro model. Cells were exposed for 48 h to solvent control (dimethyl sulfoxide, DMSO) or 6 different concentrations ranging from 40 pM to 4 μM of one of the three test compounds. All three congeners significantly increased the production of estradiol-17β. PCB 118 produced a rise in progesterone and cortisol in a concentration-dependent manner, similar to PCB 126. Testosterone was significantly reduced in response to PCB 153 but not PCB 118 or PCB 126. All three congeners elevated aldosterone at the highest concentration tested. A significant increase was observed in CYP11B2 mRNA levels in cells exposed to the three congeners. In addition, PCB 126 upregulated CYP19, 3β-HSD2, StAR, and HMGR mRNA levels at the highest concentration tested, and downregulated CYP21 at 40 nM. In conclusion, all three PCB congeners are capable of modulating steroidogenesis in H295R in a concentration-dependent manner, whereby the hormone profile following PCB 118 exposure resembles that of PCB 126. Where changes in gene expression profile are concerned, exposure to PCB 126 showed the greatest effects.

Site of pheromone biosynthesis and isolation of HMG‐CoA reductase cDNA in the cotton boll weevil, Anthonomus grandis

Isolated gut tissue from male cotton boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), incorporated radiolabeled acetate into components that co-eluted with monoterpenoid pheromone components on HPLC. This demonstrates that pheromone components of male A. grandis are produced de novo and strongly suggests that pheromone biosynthesis occurs in gut tissue. A central enzyme in isoprenoid biosynthesis is 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), and a full-length HMG-R cDNA was isolated from A. grandis. The predicted translation product was 54 and 45% identical to HMG-R from Ips paraconfusus and Drosophila melanogaster, respectively. HMG-R gene expression gradually increased with age in male A. grandis, which correlates with pheromone production. However, topical application of JH III did not significantly increase HMG-R mRNA levels.

Effects of atorvastatin on lipid metabolism in normolipidemic and hereditary hyperlipidemic, non-laying hens

As a result of a hereditable point mutation in the oocyte very low density lipoprotein (VLDL) receptor, sexually mature restricted ovulator (RO) female chickens ( Gallus gallus), first described as a non-laying strain, exhibit endogenous hyperlipidemia and develop atherosclerotic lesions. In a 20-day study, RO hens and their normolipidemic (NL) siblings were fed either a control diet, or the control diet supplemented with 0.06% atorvastatin (AT), a potent 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) inhibitor. Compared to NL hens, RO birds exhibited greatly elevated baseline plasma total cholesterol (CHOL) and triglyceride (TG) concentrations (1.56 vs. 4.55 g/l and 30.7 vs. 138.4 g/l, respectively). AT attenuated plasma CHOL and TG concentrations by 60.3% and 70.1%, respectively, in NL hens and by 45.1% and 34.3%, respectively, in RO hens. Messenger RNA levels of several key genes involved in hepatic VLDL assembly were suppressed in RO vs. NL hens, but were unaffected by AT. In contrast, AT elevated liver HMGR mRNA levels in NL and RO birds, but only NL hens exhibited an AT-associated increase in hepatic HMGR immunoreactive protein levels. Down-regulation of HMGR gene expression due to higher baseline levels of circulating CHOL may explain why RO birds responded less robustly than NL hens to AT administration.

The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase.

In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147-155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3'-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.

Changes in mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in chickens

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and cholesterol 7 alpha-hydroxylase (CYP7A1), essential enzymes of cholesterol synthesis and excretion, respectively, were isolated from a chicken liver cDNA library. When their recombinant proteins were overexpressed in HNK293 cells, corresponding enzyme activities were observed. The complete open reading frames of MHGR and CYP7A1 contained (i) 2625 base pairs (bp), predicting a protein of 875 amino acids, and (ii) 1539 bp, predicting a protein of 513 amino acids, respectively. By Northern blot analysis, chicken HMGR mRNA expression was detected in most tissues examined, however, the highest levels were found in liver, brain and ileum. CYP7A1 mRNA was detected only in the liver. Changes in chicken HMGR and CYP7A1 mRNA expression with nutritional state were examined and were shown to respond to certain nutritional treatments, i.e. fast refeeding and cholesterol supplementation. HMGR and CYP7A1 mRNA levels were significantly increased with maturation (i.e. egg producing), when compared to immature chickens. However, these stimulations were not associated with estrogen, although this does enhance triacylglycerol and very low density lipoprotein secretion by the chicken liver. The present study is the first to report the molecular characterization of HMGR and CYP7A1, key enzymes of cholesterol metabolism in avian species.

Physiological and Pharmacological Regulation of Hepatic 3-Hydroxy-3- Methylglutaryl Coenzyme A Reductase

The conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate, catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), is considered to be the ratelimiting step in the overall pathway of cholesterol biosynthesis. Although expressed in virtually every tissue, HMGR is normally expressed at the highest level in liver. Also the magnitude of regulation of HMGR in liver exceeds that in any other tissue. Thus, this review focuses on regulation of hepatic HMGR. Feedback regulation by the end product of the pathway, cholesterol, has been, perhaps, the most extensively studied of the physiological and pharmacological factors that act to regulate HMGR expression. This constitutes an important homeostatic mechanism to maintain cholesterol levels within rather narrow limits. A series of recent studies have now demonstrated that this regulation occurs at the level of mRNA translation - not at the level of transcription as has been widely accepted. The rapid stimulation of hepatic HMGR activity by insulin is due to increased transcription rather than a change in phosphorylation status. Glucagon opposes this effect. Insulins effect is mediated through phosphorylation of cAMP Response Element Binding Protein. Thyroid hormone acts, after a lag period of about 36 hrs, to markedly increase hepatic HMGR expression as a result of an increased rate of transcription as well as stabilization of the mRNA. Glucocorticoids oppose the mRNA stabilization. Statins act to increase hepatic HMGR protein levels as much as 700-fold through a combination of increased transcription, increased translation and stabilization of HMGR protein. Thyromimetics act to increase HMGR mRNA levels and also hepatic LDL receptor mRNA and protein levels. Oxylanosterols act to profoundly lower hepatic HMGR protein levels by acting to decrease the rate of translation. The physiological and pharmacological factors that modulate hepatic HMGR expression appear to employ a plethora of regulatory strategies. The molecular details of these regulatory mechanisms are the subjects of several current investigations. Keywords: hepatic 3-hydroxy-3-methylglutaryl, coenzyme a reductase, cholesterol biosynthesis, hmgr expression, hmgr protein levels

Regulation of hepatic low-density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol 7alpha-hydroxylase mRNAs in human liver.

To characterize the coordinate regulation of cholesterol metabolism in human liver, we simultaneously quantified mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and low- density lipoprotein receptors (LDLRs) in liver biopsies from 76 patients undergoing cholecystectomy. The three transcript levels were not different between untreated gallstone and gallstone-free patients and not significantly altered by 10-d exclusion of dietary cholesterol. Treatment with chenodeoxycholic acid suppressed CYP7A1 and to a lesser extent HMGR mRNA levels. Cholestyramine treatment increased CYP7A1, but also HMGR and LDLR mRNA, and statins only increased HMGR mRNA. Resin + statin treatment increased all mRNA species. In untreated patients, the mRNA levels of HMGR and LDLR were more strongly correlated (r = +0.60) than those of CYP7A1 and HMGR (r = +0.49) or CYP7A1 and LDLR (r = +0.21). In the treated patients, in whom bile acid synthesis was suppressed or stimulated, mRNA levels of CYP7A1 and HMGR (r = +0.84) as well as CYP7A1 and LDLR (r = +0.62) were more strongly correlated than those of HMGR and LDLR (r = +0.59). The coordinate control of HMGR and LDLR mRNA levels reflects their common regulation by shared transcriptional activation. In contrast, following changes in bile acid flux through the liver, CYP7A1 gene expression becomes a strong modulator of hepatic cholesterol metabolism.

Dietary Psyllium Increases Expression of Ileal Apical Sodium-Dependent Bile Acid Transporter mRNA Coordinately with Dose-Responsive Changes in Bile Acid Metabolism in Rats

Psyllium (PSY), a type of dietary fiber containing mainly soluble components, has been shown to decrease serum cholesterol concentrations in several species; however, mechanisms involved are not clearly defined. Four groups of 10 rats were fed semipurified diets containing 10% dietary fiber from cellulose and/or PSY for 21 d. Increasing levels of PSY were fed (0,3.33, 6.67 and 10% PSY) with the remaining 10% made up with cellulose. Liver cholesterol, cholesterol 7alpha-hydroxylase (CYP7A) activity and mRNA, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) mRNA, ileal apical sodium-dependent bile acid transporter (ASBT) mRNA, fecal bile acids and total steroids, and intestinal bile acid content were measured. All variables responded in a dose-dependent manner to PSY in the diet. Total liver cholesterol content was significantly reduced in all groups fed PSY compared to cellulose-fed controls [138(a), 105(b), 105(b) and 93(c) micromol (SEM = 4.2) for 0, 3.33, 6.67 and 10% PSY, respectively]. Activity of CYP7A was significantly greater in all groups fed PSY compared to the cellulose-fed controls [6.36(c), 16.92(b), 15.28(b) and 20.37(a) pmol x min(-1) x mg protein(-1) (SEM = 3.19) for 0, 3.33, 6.67 and 10% PSY, respectively]. These differences in CYP7A activity were similar to differences in CYP7A, HMGR and ASBT mRNA levels. Fecal bile acid and total steroid excretion as well as total intestinal bile acids were significantly greater in rats fed PSY-containing diets compared to 0% PSY-fed rats. These results suggest that the reduction in liver cholesterol involves modulating the size and composition of the bile acid pool via regulation of ileal ASBT, CYP7A and HMGR mRNA levels.

Select 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Inhibitors Vary in Their Ability to Reduce Egg Yolk Cholesterol Levels in Laying Hens through Alteration of Hepatic Cholesterol Biosynthesis and Plasma VLDL Composition

The inability to markedly attenuate cholesterol levels in chicken eggs has led to speculation that cholesterol is essential for yolk formation and that egg production would cease when yolk cholesterol deposition was inadequate for embryonic survival. However, this critical level hypothesis remains unproven. Here, we determine the relative responsiveness of laying hens to three select inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthesis. A control diet, either alone or supplemented with one of two dietary levels (0.03 or 0.06%) of atorvastatin, lovastatin, or simvastatin, was fed to White Leghorn hens for 5 wk. Liver cholesterol concentrations (mg/g tissue) were decreased (P ≤ 0.05) by each HMGR inhibitor; however, total liver cholesterol (mg) did not differ among treatments. Microsomal hepatic HMGR activities were increased one- to twofold in all HMGR inhibitor-treated groups, while HMGR mRNA levels were unaffected. Diameters of plasma VLDL particles, the main cholesterol-carrying yolk precursor macromolecules, were reduced (P ≤ 0.05) only in hens fed 0.06% atorvastatin, and the particles contained 38% less total cholesterol (P ≤ 0.05) than controls. Plasma total cholesterol concentrations were lowered (P ≤ 0.05) by both doses of atorvastatin (−56, −63%) and simvastatin (−36,−45%). Egg cholesterol contents were maximally reduced by 46% (P ≤ 0.05), 7% (P > 0.05), and 22% (P ≤ 0.05) in hens fed the 0.06% level of atorvastatin, lovastatin, and simvastatin, respectively, while overall egg production [−19% (P ≤ 0.05), +4% (P > 0.05), and -3% (P > 0.05)], was much less affected. We concluded that cholesterol per se may not be an obligatory component for yolk formation in chickens and, as such, may be amenable to further pharmacological manipulation

Down-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels and synthesis in Syrian hamster C100 cells by the oxidosqualene cyclase inhibitor [4′-(6-allyl-ethyl-amino-hexyloxy)-2′-fluoro-phenyl]-(4-bromophenyl)-methanone (Ro 48–8071): comparison to simvastatin

In vivo inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC, E.C. 5.4.99.7)—the enzyme which catalyzes the cyclization of monooxidosqualene to lanosterol—does not result in elevated 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) activity. This trait is attributed to increased levels of oxysterols, produced upon partial inhibition of OSC, that suppress HMGR and other sterol-responsive genes. The OSC inhibitor [4′-(6-allyl-ethyl-amino-hexyloxy)-2′-fluoro-phenyl]-(4-bromophenyl)-methanone (Ro 48–8071) was shown earlier to lower low-density lipoprotein (LDL) cholesterol in hamsters with no increase in hepatic HMGR, in contrast to simvastatin. To delineate the regulatory mechanism(s) by which Ro 48–8071 reduces cholesterol synthesis without raising HMGR levels, Syrian hamster C100 cells were incubated with either Ro 48–8071 or simvastatin, and their effects on cholesterol synthesis and LDL uptake, as well as on HMGR mRNA levels and rates of synthesis, were determined. Using RNase protection and radioimmunoprecipitation assays, we found that, in the absence of LDL in the culture medium, both HMGR mRNA levels and synthesis were reduced with concentrations of Ro 48–8071 inhibiting cholesterol synthesis by 50–75%, whereas LDL uptake was either reduced or unchanged. In contrast, simvastatin, at concentrations inhibiting cholesterol synthesis by the same 50–75%, increased both HMGR mRNA levels and synthesis, as well as LDL uptake. In the presence of LDL, HMGR mRNA levels and synthesis along with LDL uptake were little affected after incubation with Ro 48–8071. Still, simvastatin markedly increased both HMGR mRNA levels and synthesis in cells incubated in the presence of LDL, leaving LDL uptake unaffected. These data suggest that inhibition of OSC by Ro 48–8071 results in an indirect down-regulation of HMGR mRNA levels and synthesis.

Posttranscriptional Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Lens Epithelial Cells by Mevalonate-Derived Nonsterols

The ocular lens must continuously synthesize the cholesterol required to support membrane formation for its life-long growth. The roles of transcriptional and posttranscriptional mechanisms in controlling 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) protein levels in cultured lens epithelial cells were examined by measuring the effect of restricting exogenous cholesterol, endogenous cholesterol synthesis and mevalonate derived nonsterols upon HMGR protein and mRNA levels and upon the synthesis and degradation of HMGR protein. Sterols were restricted by culturing in lipoprotein deficient media and blocking 2,3-oxidosqualene cyclase with U18666A. Mevalonate derived nonsterols were additionally restricted by inhibition of HMGR activity with lovastatin. A 4-fold increase in HMGR protein levels due to restricting sterols with U18666A could be explained by comparably increased mRNA levels and enzyme protein synthesis. The very rapid turnover of HMGR protein (T12∼45 min) was unaffected. The additional restriction of mevalonate derived nonsterols increased HMGR protein levels to about 400-fold. A 10-fold slowing in the rate of enzyme degradation coupled with at least a 5-fold increase in mRNA levels likely accounted for this accumulated protein mass. The capacity of the nonsterol regulators to promote enzyme degradation appeared independent of sterols, since mevalonate restored rapid degradation of HMGR protein when 2,3-oxidosqualene cyclase activity was simultaneously blocked. Thus, in cultured lens epithelial cells, sterols appear to exert a modest influence on HMGR protein levels solely by suppressing transcription; whereas, mevalonate derived nonsterols exert major influence mainly by accelerating enzyme protein degradation. We speculate that nonsterol isoprenes might be important for preventing overexpression of cholesterol biosynthesis in the intact lens.

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that gluc...

Reduced hepatic LDL-receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase and sterol carrier protein-2 expression is associated with pregnancy loss in the diabetic rat

Hepatic low density lipoprotein receptor (LDLR), 3-hydroxy-3-methylglutaryl coenyzme A reductase (HMGR), cholesterol 7α-hydroxylase, and sterol carrier protein-2 are important proteins associated with the uptake, synthesis, degradation and transport of cellular cholesterol. Since cholesterol is critically important for steroid hormone synthesis and is an essential component in membrane biosynthesis, this study investigated whether these proteins are altered in the normal pregnant and streptozotocin (STZ)-induced diabetic pregnant rat. The goal of these experiments was to determine whether diabetic reproductive dysfunction is associated with a significant change in maternal cholesterol homeostasis. Diabetic animals were grouped based on their ability or inability to maintain pregnancy up to day 15 post-conception. LDLR and HMGR mRNA levels were significantly reduced in animals which did not maintain pregnancy whereas diabetic animals with fetuses had normal LDLR and HMGR mRNA levels. Hepatic LDLR, HMGR, and SCP2 protein levels were examined in normal pregnant and diabetic pregnant animals by Western blot analysis. SCP2 levels were reduced in all diabetic animals, particularly in the diabetic animals which lost their fetuses. The decline in SCP2 was associated with an increase in sterol carrier protein-X (SCPx), a protein related to SCP2. SCPx has been shown to have thiolytic activity and is thought to have a role in β-oxidation of fatty acids. HMGR was also significantly reduced in diabetic animals which lost their fetuses. Cholesterol 7α-hydroxylase mRNA was slightly, but not significantly, reduced in all diabetic animals. Serum very low density lipoprotein (VLDL) +LDL cholesterol increased significantly in the STZ-treated diabetic rats while the HDL cholesterol levels declined in these animals. Reduced hepatic LDLR and HMGR mRNA levels were consistently associated with reduced serum progesterone and an inability to maintain pregnancy. The results of this study suggest that the maintenance of maternal cholesterol metabolism is a critical factor directly associated with successful pregnancy outcome in the diabetic rat.

Diurnal Rhythms of Rat Liver Serine Dehydratase, D-Site Binding Protein, and 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase mRNA Levels Are Altered by Destruction of the Suprachiasmatic Nucleus of the Hypothalamus

It is generally accepted that a clock generating biological rhythms exists in the suprachiasmatic nuclei (SCN) of the hypothalamus in rodents. We have examined if diurnal variations of rat liver serine dehydratase (SDH), D-site binding protein (DBP), and 3-hydrogy-3-methylglutaryl coenzyme A reductase (HMGR) mRNA levels, each of which exhibits low values during the daytime (06:00-18:00) and high values during the dark period, are controlled by the SCN, The SCN were destructed electrolytically, and, 2 weeks later, rats were sacrificed at 06:00, 12:00, 18:00, and 24:00. Completeness of SCN lesions was assessed by the alteration of spontaneous locomotor activity and histological examination of the brains. The SDH and DBP mRNA levels in SCN-lesioned rats were very low at 06:00 and almost eliminated at 18:00, whereas the HMGR mRNA level at 06:00 was low at 06:00 and slightly increased at 18:00 compared to the control values. When determined at 12:00 and 24:00, the SDH, DBP, and HMGR mRNA levels in sham-operated rats were higher at 24:00 than at 12:00, whereas those in SCN-lesioned rats were higher at 12:00 than at 24:00. The hepatic β-actin and glycine N-methyltransferase mRNA levels showed no significant diurnal variation and were not affected by SCN lesions. These results indicate that the SCN also play a pivotal role in determining rhythmic gene expressions in liver.

Design and synthesis of seco-oxysterol analogs as potential inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene transcription.

The synthesis and biological activity of a series of seco-oxysterol analogs designed to be inhibitors of transcription of the gene for 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR) are described. The compound possessing the most significant activity, [1 alpha (E),4 beta]-3-[2-(4- hydroxy-1-methylcyclohexyl)ethenyl]-alpha,alpha-dimethylbenzenepentan ol (4, U-88156), inhibited (IC50 = 10 microM) the expression of beta-galactosidase (beta-gal) in a transfected human HepG2 cell line wherein the beta-gal gene was driven by a 5 kB segment of the promoter for hamster HMGR. Furthermore, using wild-type HepG2 cells, it was shown that 10 microM 4 reduced HMGR mRNA levels by 73% while stimulating LDL-receptor activity by 47%. In the same system, the related oxysterol, 25-hydroxycholesterol (1), at 10 microM lowered both HMGR mRNA levels and LDL-receptor activity by 58% and 64%, respectively. Overall HMGR activity in wild-type HepG2 cells was inhibited 30% by 4 at 10 microM. These findings collectively demonstrate that a seco-oxysterol analog is capable of regulating HMGR gene expression and that this regulation can occur without a concomitant attenuation of the level of LDL-receptor activity.

Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge.

Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge.

Tomato hydroxymethylglutaryl-CoA reductase is required early in fruit development but not during ripening.

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and the level of its mRNA have been determined at various stages of tomato fruit development. The HMGR reaction makes mevalonate, a necessary component in the synthesis of all isoprene containing compounds, such as sterols and carotenoids. A cDNA clone encoding the active site region of HMGR has been isolated from a tomato library derived from young-fruit mRNA. The clone hybridizes to a one- or two-copy fragment in high-stringency DNA gel blot analyses and detects an mRNA of approximately 3.0 kb. Both HMGR activity and mRNA levels are high in early stages of tomato fruit development, when rapid cell division occurs, as well as in the subsequent early stages of cellular expansion. In contrast, ripening fruit have very low levels of reductase activity and mRNA, even though large amounts of the carotenoid lycopene are synthesized during this period. Furthermore, in vivo inhibition of HMGR during early fruit stages disrupts subsequent development, whereas inhibition during later stages of fruit expansion has no apparent effect on ripening. We conclude that the pool of mevalonate responsible for the synthesis of phytosterols is synthesized primarily during the first half of tomato fruit development. In addition, the final period of fruit expansion and ripening is not dependent upon HMGR activity, but instead utilizes a preexisting pool of pathway intermediates or requires the use of salvage pathways in the cell.