Regulation of Lipoprotein Lipase by the Oxysterol Receptors, LXRα and LXRβ
2001; Elsevier BV; Volume: 276; Issue: 46 Linguagem: Inglês
10.1074/jbc.m107823200
ISSN1083-351X
AutoresYuan Zhang, Joyce J. Repa, Karine Gauthier, David J. Mangelsdorf,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoLipoprotein lipase (LPL) is a key enzyme for lipoprotein metabolism and is responsible for hydrolysis of triglycerides in circulating lipoproteins, releasing free fatty acids to peripheral tissues. In liver, LPL is also believed to promote uptake of high density lipoprotein (HDL)-cholesterol and thereby facilitate reverse cholesterol transport. In this study we show that the Lpl gene is a direct target of the oxysterol liver X receptor, LXRα. Mice fed diets containing high cholesterol or an LXR-selective agonist exhibited a significant increase in LPL expression in the liver and macrophages, but not in other tissues (e.g. adipose and muscle). Studies inLxr-deficient mice confirmed that this response was dependent more on the presence of LXRα than LXRβ. Analysis of theLpl gene revealed the presence of a functional DR4 LXR response element in the intronic region between exons 1 and 2. This response element directly binds rexinoid receptor (RXR)/LXR heterodimers and is sufficient for rexinoid- and LXR agonist-induced transcription of the Lpl gene. Together, these studies further distinguish the roles of LXRα and β and support a growing body of evidence that LXRs function as key regulators of lipid metabolism and are anti-atherogenic. Lipoprotein lipase (LPL) is a key enzyme for lipoprotein metabolism and is responsible for hydrolysis of triglycerides in circulating lipoproteins, releasing free fatty acids to peripheral tissues. In liver, LPL is also believed to promote uptake of high density lipoprotein (HDL)-cholesterol and thereby facilitate reverse cholesterol transport. In this study we show that the Lpl gene is a direct target of the oxysterol liver X receptor, LXRα. Mice fed diets containing high cholesterol or an LXR-selective agonist exhibited a significant increase in LPL expression in the liver and macrophages, but not in other tissues (e.g. adipose and muscle). Studies inLxr-deficient mice confirmed that this response was dependent more on the presence of LXRα than LXRβ. Analysis of theLpl gene revealed the presence of a functional DR4 LXR response element in the intronic region between exons 1 and 2. This response element directly binds rexinoid receptor (RXR)/LXR heterodimers and is sufficient for rexinoid- and LXR agonist-induced transcription of the Lpl gene. Together, these studies further distinguish the roles of LXRα and β and support a growing body of evidence that LXRs function as key regulators of lipid metabolism and are anti-atherogenic. lipoprotein lipase high density lipoprotein sterol regulatory element binding protein-1 peroxisome proliferator-activated receptor liver X receptor rexinoid receptor LXR response element direct repeat spaced by four bases ATP-binding cassette tumor necrosis factor Dulbecco's modified Eagle's medium Chylomicrons and very low density lipoprotein particles are the main carriers of triglycerides in plasma. LPL1 is synthesized in parenchymal cells and secreted and transported to the endothelial surface, where it is activated to hydrolyze triglycerides present in circulating large lipoproteins (1Eckel R.H. N. Engl. J. Med. 1989; 320: 1060-1068Crossref PubMed Scopus (846) Google Scholar). Free fatty acids and monoglycerides are released and taken up by tissues to be either re-esterified for storage, used for fuel or lipid synthesis. LPL activity is also essential for the processing of triglyceride-rich lipoproteins into HDL (2Strauss J.G. Frank S. Kratky D. Hammerle G. Hrzenjak A. Knipping G. von Eckardstein A. Kostner G.M. Zechner R. J. Biol. Chem. 2001; 276: 36083-36090Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In addition to its enzymatic activity, LPL has been proposed to be a bridging factor that facilitates the uptake of HDL-associated cholesterol esters into the liver (3Rinninger F. Kaiser T. Mann W.A. Meyer N. Greten H. Beisiegel U. J. Lipid Res. 1998; 39: 1335-1348Abstract Full Text Full Text PDF PubMed Google Scholar). LPL is a member of a family of lipase enzymes that also includes hepatic lipase and pancreatic lipase (4Kirchgessner T.G. Svenson K.L. Lusis A.J. Schotz M.C. J. Biol. Chem. 1987; 262: 8463-8466Abstract Full Text PDF PubMed Google Scholar, 5Kirchgessner T.G. Chuat J.C. Heinzmann C. Etienne J. Guilhot S. Svenson K. Ameis D. Pilon C. d'Auriol L. Andalibi A. Schotz M.C. Galibert F. Lusis A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9647-9651Crossref PubMed Scopus (184) Google Scholar). Although hepatic lipase and pancreatic lipase expression are limited to liver and pancreas, respectively, LPL is expressed at high levels in heart, adipose tissue, skeletal muscle, kidney, and mammary gland, and at lower levels in liver, adrenal, and brain (4Kirchgessner T.G. Svenson K.L. Lusis A.J. Schotz M.C. J. Biol. Chem. 1987; 262: 8463-8466Abstract Full Text PDF PubMed Google Scholar, 6Komaromy M.C. Schotz M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1526-1530Crossref PubMed Scopus (123) Google Scholar, 7Lowe M.E. Rosenblum J.L. Strauss A.W. J. Biol. Chem. 1989; 264: 20042-20048Abstract Full Text PDF PubMed Google Scholar, 8Kirchgessner T.G. LeBoeuf R.C. Langner C.A. Zollman S. Chang C.H. Taylor B.A. Schotz M.C. Gordon J.I. Lusis A.J. J. Biol. Chem. 1989; 264: 1473-1482Abstract Full Text PDF PubMed Google Scholar, 9Semenkovich C.F. Chen S.H. Wims M. Luo C.C. Li W.H. Chan L. J. Lipid Res. 1989; 30: 423-431Abstract Full Text PDF PubMed Google Scholar). LPL activity is regulated by a variety of nutritional factors, as well as insulin, glucocorticoids, catecholamines, pro-inflammatory cytokines, and thiazolidinediones (10Enerback S. Gimble J.M. Biochim. Biophys. Acta. 1993; 1169: 107-125Crossref PubMed Scopus (176) Google Scholar, 11Schoonjans K. Peinado-Onsurbe J. Lefebvre A.M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. EMBO J. 1996; 15: 5336-5348Crossref PubMed Scopus (1007) Google Scholar). Depending on the stimulus, LPL activity can also vary greatly between tissues. For example, in response to cytokines LPL expression is decreased in adipose tissue and increased in liver (12Enerback S. Semb H. Tavernier J. Bjursell G. Olivecrona T. Gene. 1988; 64: 97-106Crossref PubMed Scopus (66) Google Scholar, 13Chajek-Shaul T. Friedman G. Stein O. Shiloni E. Etienne J. Stein Y. Biochim. Biophys. Acta. 1989; 1001: 316-324Crossref PubMed Scopus (82) Google Scholar, 14Hardardottir I. Grunfeld C. Feingold K.R. Curr. Opin. Lipidol. 1994; 5: 207-215Crossref PubMed Scopus (266) Google Scholar). LPL gene expression is also regulated by a number of transcription factors, including SP1, SREBP-1, and the peroxisome proliferator-activated receptors, PPARα and PPARγ (11Schoonjans K. Peinado-Onsurbe J. Lefebvre A.M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. EMBO J. 1996; 15: 5336-5348Crossref PubMed Scopus (1007) Google Scholar, 15Yang W.S. Deeb S.S. J. Lipid Res. 1998; 39: 2054-2064Abstract Full Text Full Text PDF PubMed Google Scholar, 16Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (824) Google Scholar, 17Schoonjans K. Gelman L. Haby C. Briggs M. Auwerx J. J. Mol. Biol. 2000; 304: 323-334Crossref PubMed Scopus (65) Google Scholar, 18Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (693) Google Scholar, 19Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (671) Google Scholar, 20Sartippour M.R. Renier G. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 104-110Crossref PubMed Scopus (49) Google Scholar). LXRα and LXRβ are members of the nuclear hormone receptor superfamily that form heterodimers with RXRs and can be activated by both RXR and LXR ligands (21Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). RXR/LXR heterodimers activate their target genes by binding to specific response elements (termed LXREs) that contain a hexameric nucleotide direct repeat spaced by four bases (DR4). LXRs are activated by naturally occurring oxysterol ligands (22Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1428) Google Scholar, 23Janowski B.A. Grogan M.J. Jones S.A. Wisely G.B. Kliewer S.A. Corey E.J. Mangelsdorf D.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 266-271Crossref PubMed Scopus (776) Google Scholar, 24Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar) and regulate the expression of a number of genes involved in cholesterol metabolism (24Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar, 25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar, 26Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (304) Google Scholar, 27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar, 28Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 29Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar, 30Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (822) Google Scholar, 31Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 32Berge K.E. Tian H. Graf G.A., Yu, L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1328) Google Scholar, 33Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (561) Google Scholar). Studies in Lxr-deficient mice have helped elucidate the role of LXRs as one of the body's main sensors for regulating sterol homeostasis (25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar, 27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar, 34Alberti S. Schuster G. Parini P. Feltkamp D. Diczfalusy U. Rudling M. Angelin B. Bjorkhem I. Pettersson S. Gustafsson J.A. J. Clin. Invest. 2001; 107: 565-573Crossref PubMed Scopus (316) Google Scholar,35Claudel T. Leibowitz M.D. Fievet C. Tailleux A. Wagner B. Repa J.J. Torpier G. Lobaccaro J.M. Paterniti J.R. Mangelsdorf D.J. Heyman R.A. Auwerx J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2610-2615Crossref PubMed Scopus (256) Google Scholar). Mice harboring Lxr-null mutations lack the ability to sense dietary cholesterol and as a consequence fail to regulate a number of lipid metabolic pathways, including cholesterol absorption and transport, and bile acid synthesis. Several lines of evidence suggest that, in addition to sterol metabolism, LXRs reciprocally regulate fatty acid metabolism. For example, in wild-type mice dietary cholesterol induces a marked increase in fatty acid synthesis and accumulation of hepatic triglycerides, whereas cholesterol levels are maintained at normal levels due to increased catabolism. This phenotype is reversed in Lxrα knockout animals, which accumulate high levels of cholesterol but show no increase in hepatic fatty acid synthesis and have normal triglyceride levels (25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar). Recently, we have shown that the cholesterol-induced elevation in fatty acid synthesis is due to the direct activation by LXRs of the gene encoding SREBP-1c (36Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1394) Google Scholar,37Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1373) Google Scholar). SREBP-1c is the primary transcription factor responsible for regulating fatty acid synthesis in the liver and peripheral tissues (16Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (824) Google Scholar, 38Horton J.D. Shimomura I. Curr. Opin. Lipidol. 1999; 10: 143-150Crossref PubMed Scopus (267) Google Scholar). We have suggested that the coordinate regulation of fatty acid and cholesterol metabolism by LXRs provides a means by which the body may protect itself from elevated levels of cholesterol. These findings prompted us to investigate other lipid-regulating genes as potential targets of LXRα and LXRβ action. In this work we show that cholesterol-induced LPL gene expression in the liver is directly regulated by RXR/LXR heterodimers in a tissue-specific manner and that in vivo this regulation is mediated predominantly by LXRα. These studies define a new mechanism for governing tissue-specific LPL expression and further expand the role of the LXRs as key regulators of lipid metabolism. LG268, 22(R)-hydroxycholesterol, and T0901317 were acquired from Ligand Pharmaceuticals, Steraloids Inc., and Tularik, Inc, respectively. Lipoprotein-deficient serum was obtained from Intracel Corp. (Rockville, MD). Mouse Lpl gene sequences were obtained from GenBank™. The promoter region was polymerase chain reaction-amplified from mouse genomic DNA using primers 5′-CCTTAGAAAACGGATCGTAGACTACTCAAC-3′ and 5′-CCGCTCGAGCACTCTTCTCGCTTCTAGAGGCGTCTG-3′. A fragment spanning nucleotide −289 to +752 of the promoter was cloned intoSmaI-XhoI sites of pGL2-basic vector (Promega). Mutations at the DR4.2 site (+635) were created by SOE (Splicing by Overlap Extention) polymerase chain reaction (39Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene. 1989; 77: 61-68Crossref PubMed Scopus (2614) Google Scholar) using primers 5′-CTGTAGTGAGGGGTGGTGAGGTCCCTATAGGGAA-3′ and 5′-CTATAGGGACCTCACCACCCCTCACTACAGCTTTG-3′ (mutated nucleotides are underlined). To create DR4.1-TK-LUC and DR4.2-TK-LUC, oligonucleotides (sequences shown in Fig. 4) withBamHI overhang sequences were ligated into theBamHI site of the TK-LUC vector. All constructs were verified by sequencing. Animal experiments were approved by the Institution Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center and were conducted as described previously (27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar). Mice were fed ad libitum Teklad 7001 rodent diet supplemented with cholesterol and receptor agonists or vehicle. The rexinoid LG268 was solubilized at 4.5 mg/ml in a vehicle containing 0.9% carboxymethylcellulose, 9% polyethylene glycol 400, and 0.05% Tween 80 and provided in the diet to give a final concentration of 30 mg/kg body weight. The LXR agonist T0901317 was solubilized in a vehicle containing 1% methylcellulose and 1% Tween 80 and administered by oral gavage or provided in the diet at a dose of 50 mg/kg body weight. RNA was extracted and used in Northern analysis as described previously (25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar). For these experiments poly(A)+ RNA (5 μg/lane) or total RNA (10 μg/lane) was separated on 1% formaldehyde agarose gels, transferred to nylon membranes, and probed with 32P-labeled human LPL or mouse actin or cyclophilin cDNAs. The human LPL cDNA probe has been described previously (40Auwerx J.H. Deeb S. Brunzell J.D. Peng R. Chait A. Biochemistry. 1988; 27: 2651-2655Crossref PubMed Scopus (98) Google Scholar). Peritoneal macrophages were obtained from thioglycolate-injected male mice of wild-type orLxrα/β−/− genotype as described in a previous study (31Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Cells were pooled from four wild-type or fiveLxrα/β−/− mice and distributed on two plates, one for each treatment condition. Cells were allowed to adhere for 7 h in DMEM containing10% fetal bovine serum and penicillin/streptomycin. The medium was then replaced with DMEM supplemented with 10% lipoprotein-deficient serum, penicillin/streptomycin, and either vehicle (Me2SO) or 10 μm LXR agonist (T0901317) and incubated for 42 h. The human embryonic kidney cell line, HEK 293, was maintained at 37 °C, 5% CO2 in DMEM containing 10% fetal bovine serum. Transfections were performed in 96-well plates in media containing 10% dextran-charcoal-stripped fetal bovine serum using the calcium phosphate coprecipitation technique (41Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar). Ligands were added at final concentrations of 0.1 μm LG268 and 5 μm22(R)-hydroxycholesterol. All transfection data were normalized using an internal β-galactosidase marker and represent the mean of triplicate assays. Electrophoretic mobility shift assay was performed as described previously (42Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (898) Google Scholar). Sequences of the Lpl DR4.1, DR4.2, and DR4.2m elements are shown in Figs. 4 and 5. Competitor DR4 containing the perfect tandem repeat of AGGTCA was described previously (42Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (898) Google Scholar). After electrophoresis, the gel was dried at 80 °C for 1.5 h and autoradiographed with intensifying screens at −80 °C overnight. Previous work has shown that the hepatic accumulation of triglycerides in cholesterol-fed mice is dependent on the expression of LXRα (25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar). To explore the possibility that changes in hepatic LPL expression are associated with this response, wild-type,Lxrα−/−, Lxrβ−/−, andLxrα/β−/− mice were treated with various dietary regimens for 12 h or 7–10 days and then sacrificed, and hepatic LPL expression was examined by Northern analysis. On a low cholesterol diet in the absence of added agonists, LPL expression in the liver was barely detectable regardless of genotype (Vehicle lanes in Fig. 1). After feeding a diet rich in cholesterol for 7 days, which is known to generate endogenous oxysterol LXR ligands (43Zhang Z. Li D. Blanchard D.E. Lear S.R. Erickson S.K. Spencer T.A. J. Lipid Res. 2001; 42: 649-658Abstract Full Text Full Text PDF PubMed Google Scholar), LPL mRNA expression was increased 2.5- to 5-fold in wild-type mice (Fig. 1, A and B). In parallel experiments, treatment with a potent synthetic LXR agonist (27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar) resulted in a 12- to 17-fold increase in LPL mRNA in wild-type mice (Fig. 1, A and B). Elevation of LPL mRNA was also evident after just one 12-h treatment with LXR agonist (Fig.1 C), suggesting that Lpl is a direct LXR target gene. Treatment of wild-type mice for 10 days (Fig. 1 C) or 12 h (Fig. 1 D) with the RXR agonist LG268 (27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar) also resulted in activation of hepatic LPL gene expression, consistent with the notion that RXR works as a permissive heterodimer with LXR (42Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (898) Google Scholar). Significantly, hepatic LPL expression induced by either LXR or RXR agonists was virtually absent in mice lacking both LXRs (Fig. 1,C and D). The RXR/LXR-induced expression of LPL was still observed in the Lxrβ knockout mice (Fig.1 B) but was not present in the Lxrα knockout mice (Fig. 1 A). These data indicate that LPL expression in the liver is regulated by the RXR/LXR heterodimer. In addition, because the liver expresses both LXRα and β subtypes, these data suggest that Lpl is predominantly an LXRα target gene. It is of interest that in these experiments high cholesterol diets did not induce LPL expression in either the Lxrα or βknockout animals (Fig. 1, A and B), even though the potent synthetic agonist did. At present, the significance of this finding is unknown but could reflect differences in the pharmacology of the synthetic agonists versus endogenous ligands, or differences in the strain background of the Lxrα knockout (which is in an A129 background) versus Lxrβknockout (which is in a C57BL/6 and A129 mixed background) animals. In any case, these experiments show unequivocally that the induction of LPL expression requires the expression of at least one LXR subtype. We next looked at regulation of LPL expression in other known LXR target tissues. Macrophages and adipose tissue express high levels of LXR proteins and are known to regulate several LXR target genes (27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar, 31Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 36Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1394) Google Scholar). LPL expression in peritoneal macrophages was stimulated 2-fold after a 42-h treatment with LXR agonist (Fig.2 A). Although the induction was not as high as that seen in liver (Fig. 1), the effect in macrophages was consistently reproducible (in four independent experiments). It is also of significance to note that the basal expression in macrophages was higher, which affected the fold induction but not the maximal level of expression induced by the LXR agonist. This induction was absent in macrophages isolated fromLxrα/β double-knockout mice. In contrast, in adipose tissue, which expresses higher basal levels of LPL, the LXR agonist exhibited no significant effect on LPL expression in either mouse genotype after a 12-h or 10-day oral administration of the drug (Fig.2, B and C, respectively). We also looked at expression of LPL in heart, muscle, kidney, intestine, and adrenals, tissues that are known to express LPL. Similar to the results found in adipose tissue, none of these other tissues exhibited LXR-dependent regulation of LPL (Fig. 2 D). These data support the conclusion that LPL expression is differentially regulated by LXR in a tissue-specific manner. The mouse Lpl gene promoter and partial coding sequences were obtained from GenBank™. A computer-assisted search for potential LXR response elements was performed. The search revealed two DR4-like sequences (Fig.3 A), one at −274 to −259 (DR4.1, 5′-TAAATCagtgTAAACC-3′) and another at +635 to +650 (DR4.2, 5′-TGACCGgtggTGACCT-3′). Electrophoresis mobility gel shift assays were performed to investigate the direct binding of receptors to each sequence. An oligonucleotide containing the DR4.2 sequence was radiolabeled and used in the experiments shown in Fig. 3 (Band C). The DR4.2 oligo produced a significant band shift when incubated with in vitro translated RXR/LXRα protein (Fig. 3 B, lane 4) but not when incubated with either receptor alone (lanes 2 and 3). Binding of the RXR/LXRα heterodimer was completely inhibited by a 10- and 50-fold molar excess of either unlabeled DR4.2 (Fig. 3 B,lanes 5 and 6) or the canonical DR4 LXRE (lanes 11 and 12) oligonucleotide identified previously (42Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (898) Google Scholar). This inhibition was specific, because a mutated version of the DR4.2 element (see Fig. 5 A for sequence) was unable to compete for binding, even at a 50-fold molar excess (Fig.3 B, lanes 7 and 8). In contrast to the DR4.2 element, the DR4.1 element was unable to compete for binding to the RXR/LXRα heterodimer (Fig. 3 B, lanes 9 and10), suggesting that this site does not function as an LXRE. We also looked at the ability of LXRβ to bind to the two sites. LXRβ protein was radiolabeled by in vitro translation to a specific activity that was similar to LXRα and tested in the band-shift assay. In contrast to LXRα (Figs. 3 B and3 C, lane 6), the RXR/LXRβ heterodimer bound only weakly (∼10-fold less) to the DR4.2 oligonucleotide (Fig.3 C, lane 4). As expected, LXRβ exhibited no binding to the DR4.1 site (data not shown). Similar results were seen using either human or mouse receptor proteins. To test the ability of the DR4-like elements to function as LXR response elements, one copy of either the DR4.1 or DR4.2 elements was cloned into the luciferase reporter gene TK-Luc and cotransfected into HEK 293 cells with or without expression plasmids for mouse LXRα, LXRβ, and RXRα receptors. After treatment with ethanol vehicle, rexinoid LG268, LXR ligand 22(R)-hydroxycholesterol, or both ligands, the cells were harvested and assayed for luciferase activity (Fig. 4, A and B). Consistent with the band-shift results in Fig. 3, only the DR4.2 element was able to mediate RXR/LXR-dependent transactivation of the reporter gene. In addition, LXRα-mediated transcription was significantly higher than that of LXRβ, further supporting the notion that the Lpl gene is more selectively activated by LXRα than LXRβ. To demonstrate that the DR4.2 element identified above functions as an LXRE in the context of the Lpl gene promoter, the mouse Lpl gene from −289 to +752 was cloned into the luciferase vector pGL2 and the resultant reporter gene (pLPLwt-Luc) was tested for LXR-dependent transactivation. This portion of the Lpl gene contains both the DR4.1 and DR4.2 sequences (Fig.5 A). A similar construct in which the DR4.2 site was mutated to destroy LXR binding (see Fig.3 B, lanes 7 and 8) was also tested. As shown in Fig. 5 B, the wild-type promoter conferred significant RXR/LXRα-dependent transactivation to theLpl promoter, whereas the mutated version in which the DR4.2 site was altered (pLPLmut-Luc) did not. We conclude from these data that the Lpl promoter contains a functional LXRE located within the first intron of the gene. In a similar experiment LXRβ was also able to mediate activation of the wild-type promoter, albeit at a significantly reduced level (data not shown). Taken together, these results support the conclusion that the Lplgene is a direct target of LXR-mediated regulation by oxysterols. Furthermore, these data suggest that this regulation is governed primarily by LXRα. In this study we show that the mouse Lpl gene is a direct target of the oxysterol receptor, LXR. The activation ofLpl gene transcription by LXR is mediated via an LXRE in the first intron of the mouse gene. This LXRE is conserved in the humanLpl gene as well, indicating that LXR responsiveness is also conserved in humans. Furthermore, we show that LXR regulation of LPL expression is tissue-specific. LPL is markedly up-regulated by RXR/LXR agonists in liver and to a lesser extent in macrophages but not in adipose, muscle, kidney, adrenal, intestine, or heart. One of the questions raised by these findings is what is the physiological basis of the tissue-specific regulation of LPL by LXRs. LPL is the rate-limiting enzyme that catalyzes the hydrolysis of lipoprotein triglycerides for uptake of fatty acids into adjacent tissues. Thus, in tissues such as adipose and muscle, LPL activity is required to meet the high demand these tissues have for free fatty acids. Despite the fact that LXRs are expressed abundantly in adipose and are known to regulate other adipose target genes, such as SREBP-1c, it is of significance that Lpl is not regulated by LXRs in this tissue. One explanation for this lack of regulation may be that subtle changes in the expression of LPL are masked by the already high basal level of LPL expression in adipose. However, it is worth noting that other transcription factors, in particular PPARγ (see below), are able to regulate Lpl in this tissue. A more likely explanation is that tissue-specific regulation of Lpl by LXRs in liver and macrophages is directly linked to the role LXRs play in maintaining whole body cholesterol balance. One attractive hypothesis is that LXR up-regulates LPL to help the body clear the serum of cholesterol-rich lipoproteins (via macrophages) and transport this cholesterol (via HDL) back to the liver for catabolism and elimination. At present the function of LPL in macrophages and liver is controversial and just beginning to be elucidated. LPL has been suggested to have both pro- and anti-atherogenic properties in these tissues (44Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar). For example, in macrophages LPL has been shown to facilitate uptake of cholesterol esters, presumably by remodeling triglyceride-rich chylomicrons and very low density lipoprotein into chylomicron remnants and low density lipoprotein that are then taken up by macrophages (45Lindqvist P. Ostlund-Lindqvist A.M. Witztum J.L. Steinberg D. Little J.A. J. Biol. Chem. 1983; 258: 9086-9092Abstract Full Text PDF PubMed Google Scholar). Under pathogenic conditions, the excess cholesterol build-up in macrophages would lead to the development of foam cells. However, under non-pathogenic conditions, this cholesterol ester may be converted to oxysterols and free cholesterol that are effluxed out of the macrophage into HDL and transported to the liver. In the liver, LPL expression is normally undetectable in adult animals (9Semenkovich C.F. Chen S.H. Wims M. Luo C.C. Li W.H. Chan L. J. Lipid Res. 1989; 30: 423-431Abstract Full Text PDF PubMed Google Scholar, 46Vilaro S. Llobera M. Bengtsson-Olivecrona G. Olivecrona T. Biochem. J. 1988; 249: 549-556Crossref PubMed Scopus (34) Google Scholar), but as shown by this study hepatic LPL expression is up-regulated markedly by high cholesterol diets and LXR agonists (Fig.1). Several recent studies implicate the role of hepatic LPL expression as being anti-atherogenic by helping to facilitate reverse cholesterol transport through the HDL pathway. Strauss et al. (2Strauss J.G. Frank S. Kratky D. Hammerle G. Hrzenjak A. Knipping G. von Eckardstein A. Kostner G.M. Zechner R. J. Biol. Chem. 2001; 276: 36083-36090Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) showed that adenoviral-mediated expression of LPL in Lpl-deficient mice is necessary and sufficient to promote maturation of HDL. It has also been shown that LPL can mediate the selective uptake of HDL-cholesterol into liver cells by a mechanism that evidently does not involve the enzymatic activity of the lipase (3Rinninger F. Kaiser T. Mann W.A. Meyer N. Greten H. Beisiegel U. J. Lipid Res. 1998; 39: 1335-1348Abstract Full Text Full Text PDF PubMed Google Scholar). Our observation that LPL is induced by LXR and its agonists and that cholesterol induction of LPL is LXR-dependent is consistent with the recent identification of other LXR target genes in macrophages and liver that are in the reverse cholesterol transport pathway. These genes include the ABC sterol transport proteins ABCA1, ABCG1, ABCG5, and ABCG8; apolipoprotein E; cholesterol ester transfer protein; and cholesterol 7α-hydroxylase (24Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar, 25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar, 26Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (304) Google Scholar, 27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar, 28Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 29Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar, 30Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (822) Google Scholar, 31Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 32Berge K.E. Tian H. Graf G.A., Yu, L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1328) Google Scholar, 33Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (561) Google Scholar). 2J. J. Repa and D. J. Mangelsdorf, manuscript in preparation. Together these findings support a growing body of evidence that demonstrates LXRs are key sensors of sterol metabolism and maintain normal cholesterol balance by promoting sterol efflux from peripheral cells, increasing circulating HDL-cholesterol, increasing hepatic sterol catabolism and excretion, and inhibiting further sterol absorption (25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar, 27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar, 34Alberti S. Schuster G. Parini P. Feltkamp D. Diczfalusy U. Rudling M. Angelin B. Bjorkhem I. Pettersson S. Gustafsson J.A. J. Clin. Invest. 2001; 107: 565-573Crossref PubMed Scopus (316) Google Scholar, 35Claudel T. Leibowitz M.D. Fievet C. Tailleux A. Wagner B. Repa J.J. Torpier G. Lobaccaro J.M. Paterniti J.R. Mangelsdorf D.J. Heyman R.A. Auwerx J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2610-2615Crossref PubMed Scopus (256) Google Scholar). Another conclusion that can be extrapolated from this and other recent studies is that LPL expression is coordinately regulated by multiple dietary factors via nuclear receptors. Here we have shown that sterol-mediated regulation of Lpl requires the LXRs. Previous work has shown that, in macrophages, high glucose induces LPL expression, in part through enhanced expression of PPARα (20Sartippour M.R. Renier G. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 104-110Crossref PubMed Scopus (49) Google Scholar). Fatty acids and other PPARα agonists also induce LPL expression in the liver through a pathway that may involve LXR activation (11Schoonjans K. Peinado-Onsurbe J. Lefebvre A.M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. EMBO J. 1996; 15: 5336-5348Crossref PubMed Scopus (1007) Google Scholar, 47Tobin K.A. Steineger H.H. Alberti S. Spydevold O. Auwerx J. Gustafsson J.A. Nebb H.I. Mol. Endocrinol. 2000; 14: 741-752Crossref PubMed Scopus (185) Google Scholar). TNF has also been shown to enhance both fatty acid synthesis and LPL activity in liver (12Enerback S. Semb H. Tavernier J. Bjursell G. Olivecrona T. Gene. 1988; 64: 97-106Crossref PubMed Scopus (66) Google Scholar, 14Hardardottir I. Grunfeld C. Feingold K.R. Curr. Opin. Lipidol. 1994; 5: 207-215Crossref PubMed Scopus (266) Google Scholar). The mechanism of TNF stimulation in these cases is also believed to be indirect, suggesting that either PPAR or LXR may be modulating TNF induction of LPL. Finally, in adipocytes, LPL expression is specifically induced by PPARγ agonists (11Schoonjans K. Peinado-Onsurbe J. Lefebvre A.M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. EMBO J. 1996; 15: 5336-5348Crossref PubMed Scopus (1007) Google Scholar), but not by LXR agonists (Fig. 2), even though LXRα is abundantly expressed in adipose tissue and can potently up-regulate other adipose target genes such as SREBP-1c (36Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1394) Google Scholar). These data support the notion that the LXRs (oxysterol receptors) and PPARs (fatty acid receptors) define two distinct, but overlapping metabolic pathways that govern fatty acid metabolism in response to different dietary lipids (i.e.cholesterol and fatty acids). A final intriguing observation from this work is the finding that LXRα is a more selective regulator of Lpl than LXRβ. In liver, LPL synthesis has been reported to be confined to Kupffer cells (48Camps L. Reina M. Llobera M. Bengtsson-Olivecrona G. Olivecrona T. Vilaro S. J. Lipid Res. 1991; 32: 1877-1888Abstract Full Text PDF PubMed Google Scholar), although there is evidence that in newborn rats LPL is expressed in hepatocytes (49Burgaya F. Peinado J. Vilaro S. Llobera M. Ramirez I. Biochem. J. 1989; 259: 159-166Crossref PubMed Scopus (39) Google Scholar). Although it is possible that part of the LXRα selectivity for LPL expression is attributable to differential cell type expression of the two LXR genes, we note that the in vitro data on Lpl promoter binding and activation by the LXRs (Figs. 3 and 4) support the idea that LXRα has a higher affinity than LXRβ for the Lpl LXRE. In either case, the identification of Lpl as an LXRα-selective target gene is in keeping with the previous notion that LXRα and LXRβ have distinct targets in vivo (25Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar, 34Alberti S. Schuster G. Parini P. Feltkamp D. Diczfalusy U. Rudling M. Angelin B. Bjorkhem I. Pettersson S. Gustafsson J.A. J. Clin. Invest. 2001; 107: 565-573Crossref PubMed Scopus (316) Google Scholar). Because LXR agonists have pharmacologic effects that are both desirable (e.g.increased reverse cholesterol transport) (27Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar, 35Claudel T. Leibowitz M.D. Fievet C. Tailleux A. Wagner B. Repa J.J. Torpier G. Lobaccaro J.M. Paterniti J.R. Mangelsdorf D.J. Heyman R.A. Auwerx J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2610-2615Crossref PubMed Scopus (256) Google Scholar) and undesirable (e.g. hypertriglyceridemia) (37Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1373) Google Scholar), the identification of specific LXRα or LXRβ agonists or antagonists may have considerable therapeutic potential. We thank Dr. Richard Heyman at X-Ceptor Therapeutics and Dr. Bei Shan at Tularik for RXR and LXR agonists, respectively; Dr. Peter Tontonoz for reagents for macrophage experiments; and Dr. Johan Auwerx for human LPL cDNA. We thank members of the Mango laboratory for critically reading the manuscript.
Referência(s)