HMG-CoA Reductase Gene Expression Research Articles

Periodontal Infection and Dyslipidemia in Type 2 Diabetics: Association with Increased HMG-CoA Reductase Expression

Recent studies have suggested that the periodontal disease, chronic sub-clinical inflammation, is associated with atherosclerosis, although "cause or effect" relationship is still unclear. The aim of this study was to assess the association between the degree of periodontal infection and lipid profiles in diabetic subjects. Additionally, the association of such sub-clinical inflammation with HMG-CoA reductase gene expression was evaluated. One hundred and thirty-one non-obese relatively well-controlled Japanese type 2 diabetic patients were enrolled for the study. Although no significant association was observed between serum triglycerides, HLD-cholesterol and antibody titer to Porphyromonas gingivalis (Pg), the most predominant periodontal pathogen in adults, LDL-cholesterol was significantly associated with antibody titer to Pg. Concomitantly, the same works out to be true for total cholesterol. To understand the possible mechanisms underlying this association, we evaluated 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase gene expression in cultured HepG2 cells stimulated by either bacterial lipopolysaccharide (LPS) or inflammatory cytokines. Although Pg and E. coli LPS had no effect on HMG-CoA reductase gene expression, both tumor necrosis factor-alpha and interleukin-6 (IL-6), especially IL-6 at low concentration, markedly up-regulated HMG-CoA reductase gene expression. It can be concluded that Pg infection is associated with increased LDL-cholesterol in diabetic subjects, which may be accompanied by increased cholesterol synthesis by inflammatory cytokines.

Increased gene expression of liver SREBP-2 in experimental chronic renal failure

Sterol regulatory element-binding protein-2 (SREBP-2) is a transcription factor regarded as the main regulator of cholesterol homeostasis. Therefore, increased level of SREBP-2 could be responsible for hypercholesterolemia, which is observed in experimental chronic renal failure (CRF). This study was designed primary to evaluate the impact of experimental CRF (5/6 nephrectomy model) on rat liver SREBP-2 gene expression. In CRF rats, a twofold increase in SREBP-2 mRNA level, as well as in mature SREBP-2 protein abundance was found, when compared to control animals. It was associated with enhanced activity and mRNA abundance of liver HMG-CoA reductase, a rate-limiting enzyme for cholesterol biosynthesis. A twofold increase in liver cholesterologenesis rate was also noted. We conclude that experimental CRF is associated with increased liver SREBP-2 gene expression. This is probably the cause for enhanced HMG-CoA reductase gene expression and, consequently, for increase in liver cholesterol synthesis in CRF rats. Despite increased SREBP-2 gene expression we found LDL-receptor mRNA level to be lower than in controls, suggesting SREBP-2 independent mechanisms of LDL-receptor transcriptional regulation in CRF rats. Enhanced cholesterol synthesis and decreased LDL-receptor mRNA level are probably responsible for an almost fourfold increase in serum cholesterol concentration in CRF rats.

Effects of ezetimibe on LDL receptor- and HMG-CoA reductase-gene expression, on LDL receptor protein expression and on HMG-CoA reductase activity in mononuclear blood cells of healthy men – A randomized trial

Purpose of the study: Achieving target LDL-cholesterol (LDL-C) levels is of utmost importance, especially in patients with diabetes mellitus. The combination of simvastatin and the cholesterol absorption inhibitor ezetimibe is widely used to attain this goal. Purpose of the present study was to examine the effects of these agents and their combination on LDL-receptor- (LDLR) and HMG-CoA reductase-gene expression, on the LDLR protein expression and on the HMG-CoA reductase activity in peripheral blood mononuclear cells (PBMC).

Sex and Hormonal Status Modulate the Effects of Psyllium on Plasma Lipids and Monocyte Gene Expression in Humans

Psyllium (PSY) intake decreases plasma LDL cholesterol (LDL-C) in men and pre- and post-menopausal women while PSY effects on plasma triglycerides (TG) are sex related. A significant decrease in plasma TG was observed in men while postmenopausal women experienced an increase in plasma TG concentrations following PSY supplementation. To further explore the mechanisms by which sex and hormonal status influence the effects of PSY on plasma lipids, HMG-CoA reductase, LDL receptor and lipoprotein lipase (LPL) mRNA abundance were measured in mononuclear cells isolated from these subjects. The intervention followed a randomized crossover design in which participants were allocated to either 15 or 0 g (control) of PSY/d for 30 d. Compared to the control period, PSY intake induced a 20% increase in HMG-CoA reductase mRNA abundance (P < 0.05) while no significant changes in LDL receptor mRNA abundance were observed. In contrast, LPL mRNA abundance was 24% higher in men and 23% lower in postmenopausal women (P < 0.05) when comparing PSY with the control period. These results suggest that the LDL-C lowering induced by PSY was related to changes in HMG-CoA reductase gene expression in monocytes while the expression of LPL in this system was affected by sex and hormonal status.

PPARγ ligands, troglitazone and pioglitazone, up-regulate expression of HMG-CoA synthase and HMG-CoA reductase gene in THP-1 macrophages

Recently it has been reported that macrophages express a nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ). Using a ligand of PPARγ, troglitazone or pioglitazone, we have shown that the expression of two genes involved in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase, were increased by activation of PPARγ through a PPAR response element (PPRE) in THP-1 macrophages. In addition, treatment with troglitazone significantly increased the activity of HMG-CoA reductase and the amount of intracellular cholesterol. Thus, we conclude that PPARγ and its agonists increase the cholesterol content of macrophages by the increased expression of genes involved in cholesterol biosynthesis. These findings suggest that PPARγ may play a role in cholesterol metabolism in macrophages.

Increased rate of cholesterologenesis--a possible cause of hypercholesterolemia in experimental chronic renal failure in rats.

Hypercholesterolemia plays an important role in the lipid abnormalities in chronic renal failure (CRF). It is thought to contribute to both a progression of renal failure and atherosclerosis. Despite intensive research, the etiopathogenesis of hypercholesterolemia in CRF patients is still obscure. The present study was designed to evaluate the possible role of cholesterol overproduction in the development of hypercholesterolemia associated with experimental CRF. We found that plasma total cholesterol and cholesterol distributed in VLDL, LDL and HDL concentrations were significantly enhanced in CRF rats. Simultaneously, the rate of liver cholesterol biosynthesis in vivo (measured by determining the incorporation of tritium from tritiated water intraperitoneally injected into cholesterol ), liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and liver HMG-CoA reductase mRNA presence were elevated. Significant increases in activity of liver malic enzyme, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, NADPH-producing enzyme (required for cholesterol synthesis) have also been observed in CRF rats. In conclusion, the increased rate of liver cholesterol biosynthesis due to increase of HMG-CoA reductase and NADPH-producing enzyme gene expression could be one of the possible causes of hypercholesterolemia in CRF animals.

Effect of atorvastatin on serum lipid profile and HMG-CoA reductase gene expression in peripheral blood mononuclear cells in subjects with heterozygous familial hypercholesterolemia

Effect of atorvastatin on serum lipid profile and HMG-CoA reductase gene expression in peripheral blood mononuclear cells in subjects with heterozygous familial hypercholesterolemia

Angiotensin II atherogenicity in apolipoprotein E deficient mice is associated with increased cellular cholesterol biosynthesis

Angiotensin II (Ang II) was shown to be an important risk factor for accelerated atherosclerosis. Inhibition of Ang II action on the arterial wall by blocking its production with angiotensin converting enzyme (ACE) inhibitors, or by blocking binding to its receptors on cells with antagonists was shown to attenuate atherogenesis in animal model of atherosclerosis. We questioned whether Ang II atherogenicity is related to a stimulatory effect of Ang II on macrophage cholesterol biosynthesis. Angiotensin II injected intraperitoneally once a day (0.1 ml of 10 −7 M per mouse) for a period of 30 days, to the apolipoprotein E deficient mice increased the atherosclerotic lesion area by 95% ( P<0.01 vs. control), compared to placebo-injected mice, with no significant effect on blood pressure or on plasma cholesterol levels. On using mouse peritoneal macrophages (MPMs) that were harvested after intraperitoneally injection of Ang II, an increased rate of cellular cholesterol biosynthesis (measured as incorporation of [ 3H]acetate into cholesterol) by up to 90% ( P<0.01 vs. control) was observed. In mice treated with the ACE inhibitor, Fosinopril (25 mg/kg per day) a reduction in their MPM’s cholesterol synthesis by up to 70% ( P<0.01 vs. control) was obtained. In vitro studies in human monocyte-derived macrophages (HMDM), in MPMs from control BALB/c mice, and in J-774 A.1 macrophage-like cell line demonstrated up to 44, 34 and 30% stimulation of macrophage cholesterol biosynthesis, respectively, following cell incubation with 10 −7 M Ang II for 18 h at 37°C. The stimulatory effect of Ang II on macrophage cholesterol biosynthesis could be related to its interaction with the macrophage AT1 receptor, as Losartan (10 −5 M), an AT1 blocker, but not PD 123319 (10 −5 M), an AT2 blocker, prevented the stimulatory effect on macrophage cholesterol synthesis. Furthermore, in cells that lack the AT1 receptor (RAW macrophages), Ang II did not increase cellular cholesterol synthesis. Ang II increased macrophage 3-hydroxy-3-methyl glutaryl CoA (HMG CoA) reductase mRNA levels in a dose–dependent manner in J-774 A.1 macrophages and in MPM. Losartan, the AT1 receptor antagonist clearly attenuated this mRNA induction. We thus conclude that Ang II stimulation of macrophage cholesterol biosynthesis is related to its interaction with the AT1 receptor, followed by stimulation of macrophage HMG CoA reductase gene expression, which leads to increased cellular cholesterol biosynthesis, and can possibly result in macrophage cholesterol accumulation and foam cell formation.

Apoptosis of medulloblastoma cells in vitro follows inhibition of farnesylation using manumycin A.

Medulloblastoma is a malignant cerebellar tumor usually manifesting in childhood. We have previously shown that blocking the mevalonate pathway with lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits medulloblastoma proliferation and induces apoptosis in vitro. The underlying mechanism may involve blocking post-translational modification of important mitogenic signal-transduction proteins. We show that p21 ras processing is blocked by lovastatin, suggesting that inhibition of isoprenylation may be important in lovastatin-induced apoptosis. To test this hypothesis, manumycin A, an antibiotic which inhibits farnesyl protein transferase and thus farnesylation, was administered to 4 medulloblastoma cell lines in vitro. We found that blocking protein farnesylation with manumycin A was followed by apoptosis in a time- and dose-dependent manner. However, cell death induced by manumycin A was uniformly more rapid and efficient, requiring only 12 to 24 hr of treatment, than lovastatin-induced apoptosis, which required 36 to 96 hr (depending on the cell line tested). In addition, unlike lovastatin, which caused cell-cycle arrest in G1 phase and HMG-CoA reductase gene up-regulation, manumycin A had no effect on the cell cycle and resulted in down-regulation of HMG-CoA reductase gene expression. In both lovastatin- and manumycin A-treated cells, cellular cysteine protease precursor (CPP32) was activated, confirming the occurrence of apoptosis.

Compensatory Responses to Inhibition of Hepatic Squalene Synthase

The mechanism by which depletion of hepatic cholesterol levels, achieved by inhibition of squalene synthase, alters hepatic LDL receptor, HMG-CoA reductase, and cholesterol 7α-hydroxylase gene expression was investigated by measuring transcription rates, mRNA stability, rates of translation, translational efficiency, and levels of sterol response element binding proteins. It was found that the transcription of both hepatic LDL receptor and HMG-CoA reductase were increased about twofold. The increase in LDL receptor transcription occurred within 2 h after giving 2 mg/kg zaragozic acid A, a potent inhibitor of squalene synthase. This preceded the increase in transcription of HMG-CoA reductase that occurred at 4 h. Increases in the stability of both of these mRNAs were also observed. These changes account for the increases in LDL receptor and HMG-CoA reductase mRNA levels previously observed. The rate of transcription of hepatic cholesterol 7α-hydroxylase was decreased to about 25% of control within 3 h after administration of zaragozic acid A, which correlates with the decrease in this mRNA. The rates of translation, as determined by pulse labeling, of both hepatic HMG-CoA reductase and LDL receptor were increased two- to threefold. The translational efficiency of these two mRNAs was also increased as judged by polysome profile analysis. There was an increase in mRNA associated with the heaviest polysome fraction and a decrease in that associated with monosomes. No significant change was observed in the levels of sterol response element binding protein 2, the form that mediates induced transcription, in response to zaragozic acid A treatment, indicating that this protein might not be involved in mediating the observed transcriptional changes. An increase in sterol response element binding protein −1 was observed 30 min after giving zaragozic acid A. The results suggest that compensatory responses to depletion of squalene-derived products involve alterations in the rates of transcription, mRNA stability, and translational of key proteins involved in cholesterol homeostasis.

Gene expression of LDL receptor, HMG-CoA reductase, and cholesterol-7 alpha-hydroxylase in chronic renal failure.

Chronic renal failure (CRF) is associated with an atherogenic lipid profile and an increased risk of ischaemic cardiovascular disease. The associated hyperlipidaemia is reportedly ameliorated by erythropoietin (Epo) therapy. According to a recent report, rats studied 3 weeks after 5/6 nephrectomy and fed a high-protein diet exhibited increased activities of hepatic HMG-CoA reductase (HMG-CoAR) and cholesterol 7 alpha-hydroxylase (Ch-7 alpha-H), despite normal corresponding mRNA values. This study was designed to examine the effects of naturally progressing CRF of longer duration as well as those of Epo therapy on gene expressions of the key factors involved in hepatic cholesterol metabolism, i.e., LDL receptor (LDLR), HMG-CoAR, and Ch-7 alpha-H. Sprague-Dawley rats were randomized to the CRF group (5/6 nephrectomy), Epo-treated CRF group (given Epo 150 U/kg/twice weekly) and sham-operated, placebo-treated normal controls. They were allowed free access to regular rat chow and studied 6 weeks after surgery. Liver mRNAs and protein mass or activities of the above factors were studied. Plasma cholesterol concentration was significantly increased in the CRF group (P < 0.001) and was modestly lowered (P < 0.05) by Epo therapy. However, microsomal cholesterol concentration and LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA as well as HMG-CoAR activity, and Ch-7 alpha-H and LDLR protein mass measurements were virtually identical in the three groups. Thus, hepatic LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA and protein measurements in rats with CRF were similar to those of the normal control group representing an inappropriate response to the associated hypercholesterolemia. Epo therapy led to partial amelioration of CRF-associated hypercholesterolaemia with no discernible effect on hepatic tissue expression of the above factors.

Effect of micellar beta-sitosterol on cholesterol metabolism in CaCo-2 cells

CaCo-2 cells were used to address the effect of the plant sterol, beta-sitosterol, on cholesterol trafficking, cholesterol metabolism, and apoB secretion. Compared to cells incubated with micelles (5 mM taurocholate and 250 microM oleic acid) containing cholesterol, which caused an increase in the influx of plasma membrane cholesterol to the endoplasmic reticulum and increased the secretion of cholesteryl esters derived from the plasma membrane, beta-sitosterol did not alter cholesterol trafficking or cholesteryl ester secretion. Including beta-sitosterol in the micelle together with cholesterol attenuated the influx of plasma membrane cholesterol and prevented the secretion of cholesteryl esters derived from the plasma membrane. Stigmasterol and campesterol had effects similar to beta-sitosterol, although campesterol did not promote a modest influx of plasma membrane cholesterol. Including beta-sitosterol in the micelle with cholesterol decreased the uptake of cholesterol. Compared to cholesterol, 60% less beta-sitosterol was taken up by CaCo-2 cells. No observable esterification of beta-sitosterol was appreciated and the transport of the plant sterol to the basolateral medium was negligible. Cholesterol synthesis and HMG-CoA reductase activities were decreased in cells incubated with beta-sitosterol. This was associated with a decrease in reductase mass and mRNA levels. Cholesteryl ester synthesis and ACAT activities were unaltered by beta-sitosterol. Both stigmasterol and campesterol decreased reductase activity, but only campesterol increased ACAT activity. beta-sitosterol did not affect the secretion of apoB mass. The results suggest that beta-sitosterol does not promote cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. beta-sitosterol interferes with the uptake of micellar cholesterol causing less plasma membrane cholesterol to influx and less cholesteryl ester to be secreted. Despite its lack of effect on cholesterol trafficking, beta-sitosterol decreases cholesterol synthesis at the level of HMG-CoA reductase gene expression.

Hepatic HMG-CoA reductase gene expression during the course of puromycin-induced nephrosis

Increased production and depressed catabolism of lipoproteins play major roles in the pathogenesis of hypercholesterolemia of nephrotic syndrome (NS). However, the effect, if any, of NS on cholesterol biosynthetic capacity is uncertain. We examined the gene expression of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR, the rate limiting step in cholesterol biosynthesis) during the induction and chronic phase of puromycin (PAN)-induced NS in rats. The rats were randomized to NS (given i.p. puromycin aminonucleoside 130 mg/kg on day 1 and 60 mg/kg on day 14) and placebo-treated control groups. Subgroups of animals were sacrificed at days 5, 10, 20 and 30. The liver was harvested between 7 and 9 p.m. for measurements of HMG-CoAR and actin mRNAs, HMG-CoAR enzymatic activity and microsomal cholesterol concentration. In separate experiments, subgroups of animals with chronic NS (day 30) were studied in fed and 20-hour fasting states. A marked but transient rise in hepatic HMG-CoAR mRNA and HMG-CoAR enzymatic activity was observed following the onset and exacerbation of proteinuria within a few days after each puromycin injection. On each occasion, HMG-CoAR fell to the baseline level despite persistent severe hypercholesterolemia. In an attempt to examine the possible acute effect of PAN per se, experiments were repeated before and at short intervals (8 and 24 hr) after puromycin injection when proteinuria was absent and the drug exposure prominent. The HMG-CoAR mRNA and activity were virtually unchanged during this period, suggesting the lack of an acute effect of puromycin. Twenty-hour fasting led to a marked rise in HMG-CoAR mRNA and activity in animals with chronic NS but not in the controls. Microsomal cholesterol remained unchanged and comparable in the two groups at all points. Thus, the marked but transient rise in hepatic HMG-CoAR gene expression observed during the induction phase and with fasting during the chronic phase of PAN-induced NS may contribute to the generation and maintenance of hypercholesterolemia in this animal model.

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.

Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Gene Expression in FRTL-5 Cells: I. IDENTIFICATION AND CHARACTERIZATION OF A CYCLIC AMP-RESPONSIVE ELEMENT IN THE RAT REDUCTASE PROMOTER

Thyrotropin (TSH) increases 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene transcription in FRTL-5 rat thyroid cells, and the effect of TSH can be mimicked by cAMP. Sequence analysis of the rat reductase promoter has revealed a hitherto unnoticed cAMP-responsive element (CRE)-like octamer. This octamer is located between 53 and 60 nucleotides downstream of the sterol regulatory element 1; its first 6 nucleotides are identical to the consensus somatostatin CRE, and the entire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase CRE-like octamer (RED CRE) formed protein-DNA complexes with nuclear extracts from FRTL-5 cells, which could be prevented by unlabeled CRE-containing oligonucleotides whose flanking sequences were otherwise nonidentical. The complexes were specifically supershifted by anti-CREB antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the bacterial chloramphenicol acetyl transferase (CAT) under the control of the HMG-CoA reductase promoter displayed CAT activity, which was specifically stimulated by TSH. In contrast, CAT activity in FRTL-5 cells transfected with similar constructs carrying mutations in the reductase CRE was significantly lower and did not increase after TSH challenge. We suggest that the HMG-CoA reductase gene contains a functional CRE, important for TSH regulation of transcription. The data presented provide the molecular basis for a novel regulatory mechanism for HMG-CoA reductase gene expression in rat thyroid cells, which involves the direct effect of cAMP.

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.

Regulation of HMG-CoA reductase, apoprotein-B and LDL receptor gene expression by the hypocholesterolemic drugs simvastatin and ciprofibrate in Hep G2, human and rat hepatocytes

The comparative effects of simvastatin (a competitive inhibitor of HMG-CoA reductase) and ciprofibrate (another inhibitor of cholesterogenesis) on the incorporation of [ 14C]acetate and [ 3H]mevalonate into cholesterol HMG-CoA reductase activity, apo-B synthesis, LDL receptor, and their corresponding mRNAs, have been studied in the human hepatoma cell line Hep G2 and in human and rat hepatocytes in primary culture. Incubation of Hep G2 with simvastatin (0.01–1.5 μM) or ciprofibrate (25–100 μM) produced not only a marked inhibition of cholesterogenesis from [ 14C]acetate but also from [ 3H]mevalonate, an intermediate downstream of the HMG-CoA reductase reaction. However, in human and rat hepatocytes, cultured in similar conditions, simvastatin inhibited only the cholesterol synthesis from [ 14C]acetate, as expected. HMG-CoA reductase activity was greatly induced in Hep G2 and rat hepatocytes after incubation with simvastatin (up to 400% of controls), but not with ciprofibrate. Increased enzyme activity was accompanied by a higher cell content of reductase mRNA. Apo-B concentration in the medium of Hep G2 cells was 31% lower after 31 h incubation with simvastatin than in controls. However, neither simvastatin nor ciprofibrate modified the synthesis rate of apo-B or its mRNA level. Both LDL-receptor and its mRNA levels were raised by simvastatin at concentrations inhibiting cholesterol synthesis. Our data show that, in this human hepatoma cell line, HMG-CoA reductase competitive inhibition by simvastatin triggers a coordinate regulation of the expression of genes coding for reductase and LDL receptor but not for apo-B. Ciprofibrate, though efficient in inhibiting cholesterogenesis, did not induce the same regulatory reactions. The reason for this discrepancy is unknown.

Regulation of gene expression and synthesis and degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by micellar cholesterolin CaCo-2 cells.

To investigate whether, and by what mechanisms, luminal (dietary) cholesterol regulates cholesterol synthesis in human intestinal cells, HMG-CoA reductase activity, gene expression, synthesis, and degradation were investigated in CaCo-2 cells exposed to taurocholate micelles containing cholesterol. In cells incubated with cholesterol solubilized in 5 mM taurocholate and 30 microM monoolein, HMG-CoA reductase activity was decreased. 25-Hydroxycholesterol, delivered to the cells in the same manner as native cholesterol, was significantly more potent in inhibiting reductase activity and was used, therefore, to investigate mechanisms for sterol regulation. Cells incubated with taurocholate micelles without cholesterol lost cellular cholesterol into the medium causing an increase in HMG-CoA reductase activity and enzyme mass. Although steady-state levels of HMG-CoA reductase mRNA were increased under conditions of cholesterol efflux, synthesis rates of reductase protein were not increased. An increase in activity and enzyme mass in cells incubated with micelles alone, however, was accompanied by a significant decrease in the rate of degradation of reductase protein. In contrast, sterol influx from taurocholate micelles was associated with a marked decrease in HMG-CoA reductase activity and mass without altering mRNA levels except at high concentrations of the polar sterol which did decrease reductase mRNA levels by 50%. The absorption of apical sterol resulted in a significant decrease in the translational efficiency of reductase mRNA and a modest increase in the rate of degradation of the enzyme. Thus, although the primary function of the enterocyte is to transport luminal (dietary) cholesterol to other tissues of the body, apically derived cholesterol enters metabolic pools within the cell which regulates its own cholesterol synthesis. Dietary cholesterol, therefore, will regulate the contribution to the total body cholesterol pool of endogenously derived cholesterol from the intestine. The mechanism for this regulation of intestinal HMG-CoA reductase by luminal cholesterol occurs primarily at the post-transcriptional level.

Feedback regulation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol is not due to altered mRNA levels

Feeding rats diets containing 2% cholesterol markedly reduced hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity but had little effect on mRNA levels. Addition of mevalonolactone to the diet further decreased reductase activity independent of a change in mRNA levels. In contrast, farnesyl pyrophosphate synthetase mRNA levels and enzyme activity were decreased to similar degrees in response to dietary cholesterol. Addition of mevalonolactone to the diet did not further decrease farnesyl pyrophosphate synthetase activity. Dietary cholesterol and mevalonolactone had no effect on mRNA levels for "cellular nucleic acid-binding protein" which has been demonstrated to bind the sterol regulatory elements in the HMG-CoA reductase and farnesyl pyrophosphate synthetase promoters. Dietary cholesterol increased cholesterol 7 alpha-hydroxylase mRNA levels as expected. These results suggest that cholesterol-mediated feed-back regulation of hepatic HMG-CoA reductase gene expression does not occur at the level of transcription.