Contribution of Accelerated Degradation to Feedback Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and Cholesterol Metabolism in the Liver

Abstract

Accumulation of sterols in endoplasmic reticulum membranes stimulates the ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which catalyzes a rate-limiting step in synthesis of cholesterol. This ubiquitination marks HMGCR for proteasome-mediated degradation and constitutes one of several mechanisms for feedback control of cholesterol synthesis. Mechanisms for sterol-accelerated ubiquitination and degradation of HMGCR have been elucidated through the study of cultured mammalian cells. However, the extent to which these reactions modulate HMGCR and contribute to control of cholesterol metabolism in whole animals is unknown. Here, we examine transgenic mice expressing in the liver the membrane domain of HMGCR (HMGCR (TM1-8)), a region necessary and sufficient for sterol-accelerated degradation, and knock-in mice in which endogenous HMGCR harbors mutations that prevent sterol-induced ubiquitination. Characterization of transgenic mice revealed that HMGCR (TM1-8) is appropriately regulated in the liver of mice fed a high cholesterol diet or chow diet supplemented with the HMGCR inhibitor lovastatin. Ubiquitination-resistant HMGCR protein accumulates in the liver and other tissues disproportionately to its mRNA, indicating that sterol-accelerated degradation significantly contributes to feedback regulation of HMGCR in vivo Results of these studies demonstrate that HMGCR is subjected to sterol-accelerated degradation in the liver through mechanisms similar to those established in cultured cells. Moreover, these studies designate sterol-accelerated degradation of HMGCR as a potential therapeutic target for prevention of atherosclerosis and associated cardiovascular disease.

Keywords: cholesterol metabolism; endoplasmic reticulum (ER); endoplasmic-reticulum-associated protein degradation (ERAD); isoprenoid; lipid metabolism.

FIGURE 1.

Generation of Tg-HMGCR (TM1–8) mice expressing HMGCR (TM1–8) in the liver and hmgcr knock-in mice expressing ubiquitination-resistant HMGCR. A, schematic of transgenic construct used to generate Tg-HMGCR (TM1–8) mice. The transgenic construct contains a cDNA fragment encoding transmembrane domains 1–8 (corresponding to amino acids 1–348) of hamster HMG-CoA reductase followed by three copies of the T7 epitope under control of the human apoE promoter and its hepatic control region. B, total RNA extracted from the indicated tissues of four male Tg-HMGCR (TM1–8) mice (12–14 weeks of age) fed ad libitum a chow diet was pooled and subjected to quantitative real-time RT-PCR using transgene-specific primers as described under “Experimental Procedures.” The relative amount of transgene mRNA was calculated using the comparative threshold cycle (C_T) method and the mouse glyceraldehyde 3-phosphate dehydrogenase mRNA as an invariant control. C, detergent lysates of the indicated tissue from the same animals used in B were prepared and pooled as described under “Experimental Procedures.” Aliquots of pooled lysates (45 μg of protein/lane) were subjected to SDS-PAGE, and immunoblot analysis was carried out with anti-T7 IgG (against HMGCR (TM1–8)) and anti-gp78 IgG. D, targeting strategy for constructing the hmgcr knock-in allele harboring mutations of lysines 89 (K89R) and 248 (K248R) to arginine. Flippase recognition target (FRT) sites and FRT3 sites are indicated by small black and green triangles, respectively. Neo, neomycin resistance gene; Hygro, hygromycin resistance gene. The location of two primer sets used for genotyping is denoted by arrows. E, genomic DNA isolated from the tails of mice of the indicated genotype were amplified by PCR using primer set A and primer set B and fractionated on 2% agarose gels. Bands corresponding to the K89R and K248R alleles were visualized by staining of gels with ethidium bromide.

FIGURE 2.

Analysis of cholesteryl esters, cholesterol biosynthetic intermediates, and sterol synthesis in WT and hmgcr knock-in mice. Male mice (6–8 weeks of age, 5–6 mice/group) were fed ad libitum a chow diet before study. Livers (A–C) or brains and testes (E) were collected, and the amounts of free cholesterol, cholesteryl esters, and cholesterol biosynthetic intermediates were determined by a colorimetric assay (A and E) or by LC-MS/MS (C) as described under “Experimental Procedures.” Error bars, S.E. D, male mice (6–8 weeks of age, 5–6 mice/group) were fed a chow diet ad libitum and injected intraperitoneally with ³H-labeled water (50 mCi in 0.2 ml of isotonic saline). One hour later, tissues were removed for measurement of ³H-labeled fatty acids and digitonin-precipitable sterols. Each bar represents the mean ± S.E. of the values from 5 or 6 mice. 24,25-DHL, 24,25-dihydrolanosterol; t-MAS, testis-specific meiosis-activating sterol; 7-Dehydrochol., 7-dehydrocholesterol. *, p < 0.05; **, p < 0.01; N.S., not significant.

FIGURE 3.

Levels of endogenous HMGCR in livers of WT and hmgcr knock-in mice. Thirteen-week-old male WT, hmgcr^WT/Ki, and hmgcr^Ki/Ki littermates (4 mice/group) were fed an ad libitum chow diet before study. Livers of mice were subjected to subcellular fractionation as described under “Experimental Procedures.” Aliquots of resulting membrane (Memb., 30 μg of protein/lane) and nuclear extract (N.E., 20–50 μg of protein/lane) fractions for each group were pooled and subjected to immunoblot analysis using antibodies against endogenous HMGCR, SREBP-1, SREBP-2, Insig-1, Insig-2, Scap, calnexin, and LSD-1. Although shown in separate panels, Scap and calnexin serve as loading controls for the HMGCR immunoblot. For mRNA analysis (A), equal amounts of RNA from individual mice were subjected to quantitative real-time RT-PCR using primers against the HMGCR mRNA and apoB mRNA as an invariant control. The relative amount of HMGCR protein in hmgcr knock-in mice was determined by quantifying the band corresponding to HMGCR using Image J software and normalizing it to the amount of HMGCR mRNA. Error bars, S.E.

FIGURE 4.

Hepatic HMGCR accumulates in tissues of hmgcr knock-in mice, due to resistance to ubiquitination. A, membrane extract fractions were obtained from the liver, kidney, spleen, brain, and testis of 6–7-week-old male WT and hmgcr^Ki/Ki mice fed an ad libitum chow diet (5 mice/group). Aliquots of membrane extract fractions for each group were pooled and subjected to immunoblotting (30 μg/lane) for HMGCR and calnexin (top). Total RNA from each tissue was reverse-transcribed, and aliquots of cDNA were pooled for each group. cDNA was subjected to quantitative real-time PCR (middle) as indicated under “Experimental Procedures.” The relative amount of HMGCR protein was obtained as described in the legend to Fig. 3 (bottom). Each bar represents the mean ± S.E. (error bars) of triplicate samples. B, 8–10-week-old male WT, hmgcr^WT/Ki, and hmgcr^Ki/Ki littermates (4 mice/group) were fed an ad libitum chow diet before study. Aliquots of liver lysates for each group were pooled and immunoprecipitated with anti-HMGCR polyclonal antibodies and immunoblotted for ubiquitin or HMGCR (left). To adjust the amount of HMGCR protein subjected to immunoprecipitation, liver lysates were diluted as indicated. Ten percent of the lysates were subjected to immunoblotting for HMGCR, Scap, and UBXD8 (right). C, total RNA from livers of mice used in Fig. 3 was separately isolated. Equal amounts of RNA from individual mice were subjected to quantitative real-time PCR using apoB mRNA as an invariant control. Each value represents the amount of mRNA relative to that in WT mice, which was arbitrarily set as 1. Each bar represents the mean ± S.E. of data from five mice. FPPS, farnesyl pyrophosphate synthase; HMGCS, HMG coenzyme A synthase; LDL-R, LDL-receptor; FAS, fatty acid synthase; SCD-1, stearoyl coenzyme A desaturase-1; GGPS, geranylgeranyl pyrophosphate synthase; GPAT, glycerol-3-phosphate acyltransferase; ACS, acetyl coenzyme A synthetase; ACC, acetyl coenzyme A carboxylase; ABCG5 and ABCG8, ATP-binding cassette subfamily G member 5 and 8, respectively.

FIGURE 5.

Dietary cholesterol suppresses expression of HMGCR (TM1–8) in Tg-HMGCR (TM1–8) mouse livers and endogenous HMGCR in hmgcr knock-in mouse livers. A–C, male mice (6–8 weeks of age, 4 mice/group) were fed an ad libitum chow diet supplemented with the indicated amount of cholesterol for 5 days. A and C, aliquots of membrane (Memb.) and nuclear extract (N.E.) fractions from homogenized livers (10–30 μg of total protein/lane) were analyzed by immunoblot analysis with anti-T7 IgG (against HMGCR (TM1–8)) and antibodies against the indicated proteins. B, equal amounts of RNA from the individual mice used in A and C were subjected to quantitative real-time RT-PCR using primers against the HMGCR (TM1–8) mRNA; apoB mRNA was used as an invariant control. Values represent the amount of HMGCR (TM1–8) mRNA relative to that in transgenic mice fed a chow diet, which is arbitrarily defined as 1. Bars, mean ± S.E. (error bars) of data from four mice. *, a nonspecific cross-reactive band. D and E, male WT, hmgcr^WT/Ki, and hmgcr^Ki/Ki littermates (6–8 weeks of age, 4 mice/group) were fed an ad libitum chow diet supplemented with 2% cholesterol as indicated for 5 days. Aliquots of membrane (30 μg of protein/lane) and nuclear extract (20–50 μg of protein/lane) fractions from homogenized livers were analyzed by immunoblot as described in the legend to Fig. 3. Although shown in separate panels, Scap and calnexin serve as loading controls for the HMGCR immunoblot. For mRNA analysis (D), equal amounts of RNA from individual mice were subjected to quantitative real-time RT-PCR as described in the legend to Fig. 3A. Values represent the amount of mRNA relative to that in WT mice, which was arbitrarily set as 1. Bars, mean ± S.E. of data from four mice. The relative amount of HMGCR protein in hmgcr knock-in mice was determined as described in the legend to Fig. 3.

FIGURE 6.

Effect of dietary cholesterol on expression of mRNAs encoding components of the Scap-SREBP pathway in livers of Tg-HMGCR (TM1–8) and hmgcr knock-in mice. Total RNA from livers of mice used in Fig. 5, A and D (4 mice/group) was separately isolated. Equal amounts of RNA from the individual mice were subjected to quantitative real-time RT-PCR using primers against the indicated gene; apoB mRNA was used as an invariant control. Each value represents the amount of mRNA relative to that in WT and transgenic mice (A) or WT mice (B) fed a chow diet, which is arbitrarily defined as 1. Bars, mean ± S.E. (error bars) of data from four mice. Squal. Syn., squalene synthase.

FIGURE 7.

Cholesterol deprivation enhances expression of HMGCR (TM1–8) in livers of transgenic mice and endogenous HMGCR in livers of hmgcr knock-in mice. A–C, male mice (6–8 weeks of age, 5 mice/group in A and B or 3 mice/group in C) were fed an ad libitum chow diet in the absence or presence of the indicated concentration of lovastatin for 5 days. A and C, aliquots of membrane and nuclear extract fractions (10–30 μg protein/lane) from homogenized livers were analyzed by immunoblot as described in the legend to Fig. 5, A and C. Asterisks denote nonspecific cross-reactive bands. Equal amounts of RNA from individual mice used in A were subjected to quantitative real-time RT-PCR as described in the legend to Fig. 5B. Values represent the amount of HMGCR (TM1–8) mRNA relative to that in chow-fed transgenic mice, which is arbitrarily defined as 1. Bars, mean ± S.E. (error bars) of data from five mice. D and E, male WT and hmgcr^Ki/Ki littermates (6–8 weeks of age, 4 mice/group) were fed an ad libitum chow diet in the absence or presence of 0.2% lovastatin for 5 days. Aliquots of membrane (Memb., 30 μg protein/lane) and nuclear extract (N.E., 20–50 μg of protein/lane) fractions from homogenized livers were analyzed by immunoblot as described in the legend to Fig. 3. Although shown in separate panels, Scap and calnexin serve as loading controls for the HMGCR immunoblot. For mRNA analysis (D), equal amounts of RNA from individual mice were subjected to quantitative real-time RT-PCR as described in the legend to Fig. 3A. Values represent the amount of mRNA relative to that in WT mice, which was arbitrarily set as 1. Bars, mean ± S.E. of data from four mice. The relative amount of HMGCR protein in hmgcr knock-in mice was determined as described in the legend to Fig. 3.

FIGURE 8.

Effect of cholesterol deprivation on expression of mRNAs encoding components of the Scap-SREBP pathway in livers of Tg-HMGCR (TM1–8) and hmgcr knock-in mice. Total RNA from livers of mice used in Fig. 7, A and D (4 or 5 mice/group) was separately isolated. Equal amounts of RNA from the individual mice were subjected to quantitative real-time RT-PCR using primers against the indicated gene; apoB mRNA was used as an invariant control. Each value represents the amount of mRNA relative to that in WT and transgenic mice (A) or WT mice (B) fed a chow diet, which is arbitrarily defined as 1. Bars, mean ± S.E. (error bars) of data from four or five mice.

FIGURE 9.

Effect of fasting and refeeding on expression of HMGCR (TM1–8) in livers of transgenic mice and endogenous HMGCR in livers of hmgcr knock-in mice. A–C, male WT and Tg-HMGCR (TM1–8) mice (6–8 weeks of age, 4 mice/group) were subjected to fasting and refeeding as described under “Experimental Procedures.” A and B, aliquots of membrane and nuclear extract fractions from homogenized livers (10–30 μg of protein/lane) were analyzed by immunoblot as described in the legend to Fig. 5. C, equal amounts of RNA from individual mice used in A and B were subjected to quantitative real-time RT-PCR as described in the legend to Fig. 5B. Values represent the amount of HMGCR (TM1–8) mRNA relative to that in chow-fed transgenic mice, which is arbitrarily defined as 1. Bars, mean ± S.E. (error bars) of data from four mice. Metabolic parameters of WT and Tg-HMGCR (TM1–8) mice subjected to fasting and refeeding are provided in Table 4. D and E, male WT and hmgcr^Ki/Ki littermates (6–8 weeks of age, 4 mice/group) were subjected to fasting and refeeding as described in A. Aliquots of membrane (Memb., 30 μg of protein/lane) and nuclear extract (N.E., 20–50 μg of protein/lane) fractions from homogenized livers were subjected to immunoblot analysis as described in the legend to Fig. 3. Although shown in separate panels, Scap and calnexin serve as loading controls for the HMGCR immunoblot. Equal amounts of RNA from individual mice were subjected to quantitative real-time RT-PCR as described in the legend to Fig. 3A. Values represent the amount of mRNA relative to that in WT nonfasted mice, which was arbitrarily set as 1. Bars, mean ± S.E. of data from four mice. The relative amount of HMGCR protein in hmgcr knock-in mice was determined as described in the legend to Fig. 3.

FIGURE 10.

Effect of fasting and refeeding on expression of mRNAs encoding components of the Scap-SREBP pathway in livers of Tg-HMGCR (TM1–8) and hmgcr knock-in mice. Total RNA from livers of mice used in Fig. 9, A and D (4 mice/group) was separately isolated. Equal amounts of RNA from individual mice were subjected to quantitative real-time RT-PCR using primers against the indicated gene; apoB mRNA was used as an invariant control. Each value represents the amount of mRNA relative to that in control, nonfasted mice, which was arbitrarily set as 1. Bars, mean ± S.E. (error bars) of data from four mice. PEPCK, phosphoenolpyruvate carboxykinase.