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• W2107255540 abstract "The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/–) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/– embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/– mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/– and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis. The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/–) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/– embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/– mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/– and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis. The mevalonate pathway produces isoprenoids that are essential for diverse cellular functions, ranging from cholesterol synthesis to growth control. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 1The abbreviations used are: HMG-CoA, 3-hydroxy-3-methyglutaryl-coenzyme A; ER, endoplasmic reticulum; E, embryonic day; MEF, mouse embryonic fibroblast; DMEM, Dulbecco's modified Eagle medium; FCS, fetal calf serum; LPDS, lipoprotein-deficient serum; LDL, low density lipoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.1The abbreviations used are: HMG-CoA, 3-hydroxy-3-methyglutaryl-coenzyme A; ER, endoplasmic reticulum; E, embryonic day; MEF, mouse embryonic fibroblast; DMEM, Dulbecco's modified Eagle medium; FCS, fetal calf serum; LPDS, lipoprotein-deficient serum; LDL, low density lipoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. reductase (EC 1.1.1.34), which catalyzes the conversion of HMG-CoA to mevalonate, is the rate-limiting enzyme in the mevalonate pathway (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4519) Google Scholar). Because of its major role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely studied. To ensure a steady mevalonate supply, the non-sterol and sterol end-products of mevalonate metabolism exert feedback regulation on the activity of this enzyme through multivalent mechanisms, including inhibition of transcription of the HMG-CoA reductase mRNA, blocking of translation, and acceleration of protein degradation, thus regulating the amount of reductase protein over a several hundred-fold range (reviewed in Refs. 1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4519) Google Scholar, 2Nakanishi M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1988; 263: 8929-8937Abstract Full Text PDF PubMed Google Scholar, 3Brown M.S. Goldstein J.L. J. Lipid Res. 1980; 21: 505-517Abstract Full Text PDF PubMed Google Scholar). Inhibitors of HMG-CoA reductase, statins, are potent hypocholesterolemic agents that exhibit some cholesterol-independent, or so-called pleiotropic, effects, that involve improving or restoring endothelial function, enhancing the stability of atherosclerotic plaque, and decreasing oxidative stress and vascular inflammation (4Takemoto M. Liao J.K. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1712-1719Crossref PubMed Scopus (1221) Google Scholar). Benefits of statins also extend beyond cardiovascular diseases, including a reduction in the risk of dementia (5Jick H. Zornberg G.L. Jick S.S. Seshadri S. Drachman D.A. Lancet. 2000; 356: 1627-1631Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar), Alzheimer's disease (6Wolozin B. Kellman W. Ruosseau P. Celesia G.G. Siegel G. Arch. Neurol. 2000; 57: 1439-1443Crossref PubMed Scopus (1336) Google Scholar), ischemic stroke (7Crouse 3rd, J.R. Byington R.P. Furberg C.D. Atherosclerosis. 1998; 138: 11-24Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), and osteoporosis (8Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1583) Google Scholar, 9Chan K.A. Andrade S.E. Boles M. Buist D.S. Chase G.A. Donahue J.G. Goodman M.J. Gurwitz J.H. LaCroix A.Z. Platt R. Lancet. 2000; 355: 2185-2188Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Most of these pleiotropic effects of statins are shown to be mediated by their ability to block the synthesis of non-sterol isoprenoid intermediates. However, it remains to be unraveled how the mevalonate pathway is affected in those disease processes, particularly in cells involved in atherogenesis. In addition, some of the collateral effects of statins have been found to be independent of HMG-CoA reductase (10Rao S. Porter D.C. Chen X. Herliczek T. Lowe M. Keyomarsi K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7797-7802Crossref PubMed Scopus (333) Google Scholar, 11Weitz-Schmidt G. Welzenbach K. Brinkmann V. Kamata T. Kallen J. Bruns C. Cottens S. Takada Y. Hommel U. Nat. Med. 2001; 7: 687-692Crossref PubMed Scopus (960) Google Scholar, 12Wagner A.H. Gebauer M. Guldenzoph B. Hecker M. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1784-1789Crossref PubMed Scopus (112) Google Scholar). In mammals, only one gene has been found to encode HMG-CoA reductase (13Reynolds G.A. Basu S.K. Osborne T.F. Chin D.J. Gil G. Brown M.S. Goldstein J.L. Luskey K.L. Cell. 1984; 38: 275-285Abstract Full Text PDF PubMed Scopus (266) Google Scholar). In yeast, fungi, and plants, on the other hand, more than one gene encode the enzyme. Yeast, for example, contains two functional genes for HMG-CoA reductase, HMG-CoA reductase 1 (HMG1) and HMG2. HMG1 and HMG2 are differently expressed, and, when HMG1 is deleted, HMG2 can replace the function of HMG1 (14Basson M.E. Thorsness M. Rine J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5563-5567Crossref PubMed Scopus (165) Google Scholar). Given the major role of HMG-CoA reductase in the mevalonate pathway, it is tempting to hypothesize that mammalian cells also have a second gene for the enzyme. In fact, although the classic form of the enzyme is a transmembrane protein anchored to the endoplasmic reticulum (ER), recent studies using a mutant cell line that lacks the ER isoform of the enzyme indicate the existence of a second isoform of the reductase exclusively localized in peroxisomes and that the peroxisomal activity might be due to a second gene (15Engfelt W.H. Shackelford J.E. Aboushadi N. Jessani N. Masuda K. Paton V.G. Keller G.A. Krisans S.K. J. Biol. Chem. 1997; 272: 24579-24587Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 16Engfelt W.H. Masuda K.R. Paton V.G. Krisans S.K. J. Lipid Res. 1998; 39: 2182-2191Abstract Full Text Full Text PDF PubMed Google Scholar, 17Aboushadi N. Shackelford J.E. Jessani N. Gentile A. Krisans S.K. Biochemistry. 2000; 39: 237-247Crossref PubMed Scopus (22) Google Scholar). We have previously shown that the targeted disruption of the gene for squalene synthase, the first committed enzyme of sterol synthesis, results in embryonic death at mid gestation with growth retardation and defective neural tube closure (18Tozawa R. Ishibashi S. Osuga J. Yagyu H. Oka T. Chen Z. Ohashi K. Perrey S. Shionoiri F. Yahagi N. Harada K. Gotoda T. Yazaki Y. Yamada N. J. Biol. Chem. 1999; 274: 30843-30848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In these mice, the non-sterol pathways are presumed to be retained, whereas de novo cholesterol synthesis is blocked. To further determine the physiological consequences of perturbation of the mevalonate pathway and gain some insights into a putative second gene for HMG-CoA reductase, we have generated and characterized mice defective in mevalonate synthesis by disrupting the gene for HMG-CoA reductase. Generation of HMG-CoA Reductase Knockout Mice—A replacement-type targeting vector was constructed; the 0.8-kb short arm spanning exons 14–15, and the 10-kb long arm fragment encompassing exons 2–12 were generated by PCR using genomic DNA from the 129/Sv mouse as template. Primers used were as follows: 5′-CCGCTCGAGAAAGGAGGCCTTTGATAGCACCAGCA-3′ (exon 14) and 5′-CCGCTCGAGCTTAGAGATCATGTTCATGCCCATGG-3′ (exon 15) for the short arm and 5′-GCGGCCGCTTTGTGGCCTCCCATCCCTGGGAAGTTATTGT-3′ (exon 2) and 5′-GCGGCCGCCTCTGCATCGCTAAGGAACTTTGCACCTTTCT-3′ (exon 12) for the long arm. Integrity of the amplified fragments was verified by Southern blot analysis and partial sequencing. After subcloning into pCR2.1 vector (Invitrogen, Carlsbad, CA), the fragments were cut out and inserted into the XhoI and NotI sites, respectively, of the pPolII-short-neo-bpA-HSVTK as described previously (19Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1265) Google Scholar). Thus, a 1.2-kb region spanning the exons 12–14 of the HMG-CoA reductase gene was replaced by a neomycin-resistant cassette, which was expected to abolish translation of the entire carboxyl half of the protein containing the catalytic activity (20Liscum L. Finer-Moore J. Stroud R.M. Luskey K.L. Brown M.S. Goldstein J.L. J. Biol. Chem. 1985; 260: 522-530Abstract Full Text PDF PubMed Google Scholar). After linearization by digestion with SalI, the vector was electroporated into JH1 embryonic stem cells (A gift from Dr. J. Herz at the University of Texas Southwestern Medical Center at Dallas). Targeted clones, which had been selected in the presence of G418 and 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-5-iodouracil, were identified by PCR using the following primers: primer 1, 5′-GATTGGGAAGACAATAGCAGGCATGC-3′ and primer 2, 5′-GCTGGGTACGGTAGCACAGTTATGGT-3′ (Fig. 1). Homologous recombination was verified by Southern blot analysis after EcoRI digestion using a 3′-flanking probe (Fig. 1). Targeted embryonic stem cell clones were injected into the C57BL/6J blastocysts yielding three lines of chimeric mice, which transmitted the disrupted allele through the germ line. All experiments reported here were performed with 129/Sv-C57BL/6J hybrid descendants (F1 and subsequent generations) of these animals consuming a normal chow diet (MF, Oriental Yeast, Tokyo, Japan). To avoid differences resulting from diurnal changes in the reductase activity (21Hwa J.J. Zollman S. Warden C.H. Taylor B.A. Edwards P.A. Fogelman A.M. Lusis A.J. J. Lipid Res. 1992; 33: 711-725Abstract Full Text PDF PubMed Google Scholar, 22Shapiro D.J. Rodwell V.W. Biochem. Biophys. Res. Commun. 1969; 37: 867-872Crossref PubMed Scopus (109) Google Scholar), mice were maintained on a strict 12-h light-dark cycle. One week prior to the measurement of reductase activities and sterol synthesis rates, mice were caged individually to minimize non-genetic variability (21Hwa J.J. Zollman S. Warden C.H. Taylor B.A. Edwards P.A. Fogelman A.M. Lusis A.J. J. Lipid Res. 1992; 33: 711-725Abstract Full Text PDF PubMed Google Scholar). Analysis of Embryos—Embryos were harvested from timed matings of heterozygous intercrosses. Genotyping of embryos was performed by Southern blotting of DNA isolated from fetal membranes or whole embryos. For genotyping of blastocysts, female heterozygote animals were superovulated with pregnant mare serum and human chorionic gonadotropin and mated to heterozygote males. Blastocysts were flushed from uteri at 3.5 days post coitum (E3.5), then lysed individually as described (23Feldman B. Poueymirou W. Papaioannou V.E. DeChiara T.M. Goldfarb M. Science. 1995; 267: 246-249Crossref PubMed Scopus (624) Google Scholar) and subjected to PCR analysis. Sense primers for mutant and wild-type allele were the primers 1 and 3, 5′-AGTCCCATAATCCTCTGCTTAGCTT-3′, respectively. Each allele was amplified separately with a common antisense primer, primer 4, 5′-CTACATTACCCTAAGCAGGCAATGT-3′ (Fig. 2A) for 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min. PCR products for each blastocyst were subjected to 2%-agarose gel electrophoresis followed by Southern blotting with an internal primer. Control PCR experiments with limiting dilutions of Hmgcr+/– tail genomic DNA template demonstrated that the sensitivities for detecting mutant or wild-type allele were comparable. Mevalonate Supplementation—To test whether supplementation of mevalonate to pregnant females could rescue the homozygous embryos, dl-mevalonic acid lactone (Sigma, St. Louis, MO) was infused via a miniosmotic pump (ALZET model 2004 osmotic pump, ALZA, Palo Alto, CA), which was implanted subcutaneously in female heterozygous mice. About 1 week after surgery, the mice were subjected to timed matings with heterozygous males. The pumps are designed to release the content constantly for 4 weeks, long enough to cover the entire pregnancy period. In a pilot experiment, plasma concentration of mevalonate rose from 25–35 ng/ml in non-treated females to 60,000–70,000 ng/ml in infused animals. Mouse Embryonic Fibroblasts—Primary mouse embryonic fibroblasts (MEFs) were prepared from 14.5-day-old embryos and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). MEFs were used within three passages. All experiments were done when the cells reached subconfluence. Each monolayer was washed three times with phosphate-buffered saline, after which fresh media containing either 10% (v/v) FCS or 5 mg/ml human lipoprotein-deficient serum (LPDS) with or without 100 nm simvastatin (Wako, Osaka, Japan) were added. Cells were incubated for 6 h and subjected to the following analyses. Simvastatin was activated by alkaline hydrolysis prior to use. HMG-CoA Reductase Activity Assay—For preparation of liver microsomes, animals were sacrificed during the early dark cycle, at a time when HMG-CoA reductase activity was at its peak of diurnal rhythm (21Hwa J.J. Zollman S. Warden C.H. Taylor B.A. Edwards P.A. Fogelman A.M. Lusis A.J. J. Lipid Res. 1992; 33: 711-725Abstract Full Text PDF PubMed Google Scholar). Livers were homogenized in a buffer containing 15 mm nicotinamide, 2 mm MgCl2, and 100 mm potassium phosphate, pH 7.4, and centrifuged at 10,000 × g for 20 min at 4 °C. The supernatants were centrifuged at 105,000 × g for 1 h at 4 °C, and the resultant pellets, comprising a microsome fraction, were washed, resuspended in the same buffer, and stored in aliquots at –80 °C. HMG-CoA reductase activities were measured essentially as described previously (24Kita T. Brown M.S. Goldstein J.L. J. Clin. Invest. 1980; 66: 1094-1100Crossref PubMed Scopus (264) Google Scholar). Briefly, the microsome fractions (∼50 μg) were incubated in 20 μl of a buffer containing 110 μm dl-[3-14C]HMG-CoA (4.5 μCi/μmol), 5 mm NADPH, 10 mm EDTA, 10 mm dithiothreitol, and 100 mm potassium phosphate, pH 7.4, at 37 °C for 30 min. Reaction was terminated by the addition of 10 μl of 2 n HCl and incubated for another 30 min at 37 °C to lactonize the mevalonate formed. The [14C]mevalonate was isolated by thin-layer chromatography and counted using [3H]mevalonate as an internal standard. For MEFs, cellular extracts were prepared as described (25Kaneko I. Hazama-Shimada Y. Endo A. Eur. J. Biochem. 1978; 87: 313-321Crossref PubMed Scopus (181) Google Scholar). Extracts (∼50 μg of protein) were incubated in 50 μl of a buffer containing 30 μm dl-[3-14C]HMG-CoA (20 μCi/μmol), 2.5 mm NADPH, 5 mm EDTA, 5 mm dithiothreitol, and 100 mm potassium phosphate, pH 7.4, at 37 °C for 120 min, then processed as in the assay of liver microsomes. HMG-CoA reductase activity is expressed as picomoles of [14C]mevalonate formed per minute per mg of protein. Measurement of Hepatic Cholesterol Synthesis—Cholesterol synthesis in the liver was estimated in littermate 12-week-old male mice (n = 6) during the mid light cycle as previously described (18Tozawa R. Ishibashi S. Osuga J. Yagyu H. Oka T. Chen Z. Ohashi K. Perrey S. Shionoiri F. Yahagi N. Harada K. Gotoda T. Yazaki Y. Yamada N. J. Biol. Chem. 1999; 274: 30843-30848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 26Eisele B. Budzinski R. Muller P. Maier R. Mark M. J. Lipid Res. 1997; 38: 564-575Abstract Full Text PDF PubMed Google Scholar). In brief, animals were given food and water ad libitum and injected intraperitoneally with [2-14C]acetate (37 kBq/kg body weight). After 1 h, animals were euthanized and the liver was removed. Two portions of the liver (200–300 mg/each) were saponified, and the digitonin-precipitable sterols were isolated for the measurement of radioactivities. The results were expressed as 14C dpm/100 mg of wet weight of liver/h. Northern Blot Analysis—Poly(A+) RNA was isolated and pooled from the livers of five animals. 1.2 μg were subjected to 1%-agarose gel electrophoresis in the presence of formalin. The fractionated RNA was transferred to Hybond N (Amersham Biosciences, Piscataway, NJ). The filters were hybridized to 32P-labeled cDNA probes: HMG-CoA reductase, HMG-CoA synthase, mevalonate kinase, farnesyl diphosphate synthase, squalene synthase, and LDL receptor. As a control for loading of mRNA, the same filter was hybridized with a GAPDH probe. Radioactivity was quantified with a PhosphorImager, and the results were corrected for loading with GAPDH, and the -fold increase in each mRNA was calculated. Other Assays—The content of cholesterol and triglycerides in plasma and liver was measured as described (18Tozawa R. Ishibashi S. Osuga J. Yagyu H. Oka T. Chen Z. Ohashi K. Perrey S. Shionoiri F. Yahagi N. Harada K. Gotoda T. Yazaki Y. Yamada N. J. Biol. Chem. 1999; 274: 30843-30848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 27Yokode M. Hammer R.E. Ishibashi S. Brown M.S. Goldstein J.L. Science. 1990; 250: 1273-1275Crossref PubMed Scopus (143) Google Scholar). Lipoproteins were fractionated by high-performance liquid chromatography as described (28Osuga J. Yonemoto M. Yamada N. Shimano H. Yagyu H. Ohashi K. Harada K. Kamei T. Yazaki Y. Ishibashi S. J. Clin. Invest. 1998; 102: 386-394Crossref PubMed Scopus (15) Google Scholar). Statistics—Data are represented as mean ± S.D. The Student's t test was used to compare the mean values between two groups. Embryonic Lethality of Hmgcr–/– Mice—A replacement-type vector, which allowed the deletion of exons 12–14 to abolish the translation of the entire carboxyl half of the enzyme, which is crucial for the catalytic activity (20Liscum L. Finer-Moore J. Stroud R.M. Luskey K.L. Brown M.S. Goldstein J.L. J. Biol. Chem. 1985; 260: 522-530Abstract Full Text PDF PubMed Google Scholar, 29Istvan E.S. Deisenhofer J. Biochim. Biophys. Acta. 2000; 1529: 9-18Crossref PubMed Scopus (117) Google Scholar), was constructed and used to generate heterozygous HMG-CoA reductase knockout mice (Hmgcr+/–). The heterozygous mutant mice appeared normal in their development and gross anatomy and were fertile. Intercrosses between heterozygotes yielded no viable offspring homozygous for the mutant Hmgcr allele (Hmgcr–/–) (+/+:+/–:–/– = 52:106:0, p < 0.0001, χ2 test) (Table I), indicating that Hmgcr expression is essential for the development and survival of embryos. To determine the developmental stage where the embryos were lethal, timed pregnancies of Hmgcr+/– matings were examined at E8.5 and 13.5 of gestation. No Hmgcr–/– embryos were identified at these time points (Table I). In addition, no resorbed embryos were detected, suggesting that death of Hmgcr null embryos occurs very early in the development, potentially prior to implantation. We therefore analyzed pre-implantation embryos. Superovulated Hmgcr+/– females were mated with Hmgcr+/– males, and blastocysts were isolated at E3.5. Blastocysts were genotyped directly by using PCR (Fig. 2). Among 74 phenotypically normal E3.5 blastocysts, 19 (26%) were found to be homozygotes (Table I and Fig. 2B). Therefore, at the blastocyst stage, Hmgcr–/– embryos were viable and identified at the expected Mendelian frequency. Hmgcr–/– blastocysts were morphologically indistinguishable from Hmgcr+/– or +/+ embryos.Table IGenotypes of offspring from intercrosses of Hmgcr+/– miceAgeGenotypeResorbedTotal+/++/--/-3 weeks521060NAaNA, not applicable.158E13.5370010E8.5-9.517260043E3.5134219NA74a NA, not applicable. Open table in a new tab Attempt to Rescue the Homozygotes by Mevalonate Supplementation—If the embryonic lethality of the homozygotes results from the deficiency of mevalonate and subsequent sterol and non-sterol products of the mevalonate pathway, it is conceivable that supplementation of mevalonate would reverse the phenotype. To test this hypothesis, we supplemented the pregnant Hmgcr+/– mice with mevalonate using an osmotic pump, which was implanted subcutaneously. Although this intervention yielded a marked increase in the maternal plasma mevalonate level, no viable Hmgcr–/– offspring was obtained among 31 mice from 5 litters (Table II).Table IIEffects of maternal mevalonate supplementation on the genotypes of offspring from heterozygous intercrossesAgeAppearanceGenotypeTotalNormalAbnormalaAbnormal embryos were with egg cylinder stage appearance at E9.5-10.5 and their precise genotyping was not successful.+/++/--/-3 weeks3101021031E9.5-10.539101623049a Abnormal embryos were with egg cylinder stage appearance at E9.5-10.5 and their precise genotyping was not successful. Open table in a new tab To determine whether mevalonate supplementation allows null embryos to develop beyond implantation, we studied embryos at E9.5–10.5. 10 out of 49 (20%) decidua were remarkably smaller than the others and contained embryos with appearance corresponding to those at E5.5–6.5 (the egg cylinder stage). The remaining 39 normal-sized decidua contained phenotypically normal embryos, none of which were genotyped as homozygote (Table II). Hmgcr+/+ female mice, which were supplemented with mevalonate, mated to the same Hmgcr+/– males and examined at E9.5, gave rise to 20 decidua with equivalent size, which contained properly developed concepti. These results suggest that mevalonate supplementation of the pregnant females enabled Hmgcr–/– embryos to implant but failed to support the development after egg cylinder stage. Analysis of Heterozygous Mice—The Hmgcr mRNA levels in Hmgcr+/– mice were slightly reduced in the liver (Fig. 3). Expression levels of four genes in the cholesterol biosynthetic pathway, both upstream and downstream of HMG-CoA reductase (encoding HMG-CoA synthase, mevalonate kinase, farnesyl diphosphate synthase, and squalene synthase), showed a 50–70% increase in the liver of heterozygotes, whereas the expression of the gene for LDL receptor remained unaffected. On the other hand, there was no significant change in the hepatic HMG-CoA reductase activities. Consistently, the amounts of [14C]acetate incorporated into digitonin-precipitable sterols in the liver were not different between the wild-type and heterozygous mice (Table III).Table IIIPhenotypic comparison of Hmgcr+/+ and Hmgcr+/– miceParameterNumber of miceGenotypeWild-typeHeterozygoteHMG-CoA reductase activity (pmol/mg protein/min)5129.6 ± 78.7117.5 ± 59.3[14C]Acetate incorporation into sterols (dpm/100 mg liver/h)61217.8 ± 793.41008.3 ± 436.3Liver cholesterol content (mg/g)53.5 ± 0.473.0 ± 0.44Liver triglyceride content (mg/g)515.2 ± 2.211.6 ± 6.3Plasma total cholesterol (mg/dl)1494.0 ± 19.4100.5 ± 30.9Plasma triglycerides (mg/dl)2082.3 ± 24.784.3 ± 35.3 Open table in a new tab Analysis of hepatic lipids demonstrated that both cholesterol and triglyceride content were unchanged. Plasma levels of total cholesterol and triglycerides did not differ in the two groups of mice. Plasma lipoprotein analysis by high-performance liquid chromatography revealed no discernible difference in the amount of each lipoprotein fraction (data not shown). Hmgcr+/– MEFs—To investigate the regulation of HMG-CoA reductase in Hmgcr+/– mice further, we isolated MEFs from Hmgcr+/– and Hmgcr+/+ embryos. Their growth in culture media was comparable. Fig. 4 summarizes HMG-CoA reductase activities of MEFs maintained for 6 h in the presence of either 10% FCS, or 5 mg/ml LPDS with or without 100 nm simvastatin, a competitive inhibitor of HMG-CoA reductase, which induces up-regulation of HMG-CoA reductase in vivo and in vitro. As expected, the enzyme activity in MEFs was up-regulated by the addition of LPDS and further induced by concomitant treatment with simvastatin. In each experimental condition, the HMG-CoA reductase activity was reduced by ∼50% in Hmgcr+/– MEFs. Increases in the enzymatic activities over the levels observed in the presence of FCS were approximately 2- and 12-fold in the presence of LPDS and in the presence of LPDS plus simvastatin, respectively, and did not differ significantly between heterozygous and wild-type MEFs. In the present study, we have demonstrated that HMG-CoA reductase is essential for the early development of the embryos. Null embryos were recovered at the blastocyst stage, but not at E8.5, indicating that loss of HMG-CoA reductase activity through targeted disruption of the gene in the germ line leads either to implantation failure or to embryonic death prior to implantation. Moreover, the lethal phenotypes of Hmgcr–/– embryos were not completely reversed by supplementation with mevalonate to the dams. Mammalian cells accelerate their growth rate dramatically upon implantation. Cholesterol synthesis in the embryos appears to begin at the peri-implantation stage (around E4–E5 in mice) (30Brewer L.M. Sheardown S.A. Brown N.A. Teratology. 1993; 47: 137-146Crossref PubMed Scopus (14) Google Scholar, 31Pratt H.P. Dev. Biol. 1982; 89: 101-110Crossref PubMed Scopus (29) Google Scholar). Cholesterol plays an essential role in mammalian embryonic development, including the covalent modification of the morphogenic sonic hedgehog signal pathway during early gestation (32Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-259Crossref PubMed Scopus (1086) Google Scholar). Nevertheless, defective cholesterol synthesis is unlikely to explain the peri-implantational lethality of Hmgcr–/– embryos, because mice lacking squalene synthase (Ss–/–), the first committed enzyme of sterol synthesis in the mevalonate pathway, are viable until around E9.5 even with gross growth retardation and defective neural tube closure (18Tozawa R. Ishibashi S. Osuga J. Yagyu H. Oka T. Chen Z. Ohashi K. Perrey S. Shionoiri F. Yahagi N. Harada K. Gotoda T. Yazaki Y. Yamada N. J. Biol. Chem. 1999; 274: 30843-30848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Observations in Ss–/– embryos suggest that, even in the complete absence of endogenous cholesterol biosynthesis, maternally supplied cholesterol could, even although incompletely, support the embryonic growth by E9.5. The phenotype of Hmgcr null mice is strikingly more severe than that of Ss–/– mice. In addition to loss of de novo cholesterol synthesis, disruption of mevalonate synthesis has many ramifications, including loss of non-sterol isoprenoids essential for protein isoprenylation modifications and potential perturbation on N-linked glycosylation through inhibition of dolichol synthesis (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4519) Google Scholar). Non-sterol isoprenoids serve as lipid attachment for a variety of intracellular signaling molecules, including small GTP-binding proteins, such as Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation (33Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2089) Google Scholar). Given the role that these proteins play in pathways regulating cell" @default.
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• W2107255540 title "Early Embryonic Lethality Caused by Targeted Disruption of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Gene" @default.
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