Liver X Receptor (LXR) Regulation of the LXRα Gene in Human Macrophages
2001; Elsevier BV; Volume: 276; Issue: 47 Linguagem: Inglês
10.1074/jbc.m106155200
ISSN1083-351X
AutoresKarl Whitney, Michael A. Watson, Bryan Goodwin, Cristin M. Galardi, Jodi M. Maglich, Joan G. Wilson, Timothy M. Willson, Jon L. Collins, Steven A. Kliewer,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoThe nuclear oxysterol receptors LXRα (NR1H3) and LXRβ (NR1H2) coordinately regulate the expression of genes involved in the transport and catabolism of cholesterol. In macrophages, LXR stimulates the transcription of genes encoding transporters involved in cholesterol efflux, which may limit the transformation of these cells into foam cells in response to lipid loading. Here, we report that natural and synthetic LXR ligands induce the expression of the LXRα gene in primary human macrophages and differentiated THP-1 macrophages. This regulation was not observed in primary human adipocytes or hepatocytes, a human intestinal cell line, or in any mouse tissue or cell line examined. The human LXRα gene was isolated, and the transcription initiation site delineated. Analysis of the LXRα promoter revealed a functional LXR/RXR binding site ∼2.9 kb upstream of the transcription initiation site. We conclude that LXRα regulates its own expression in human macrophages and that this response is likely to amplify the effects of oxysterols on reverse cholesterol transport. These findings underscore the importance of LXR as a potential therapeutic target for the treatment of atherosclerosis. The nuclear oxysterol receptors LXRα (NR1H3) and LXRβ (NR1H2) coordinately regulate the expression of genes involved in the transport and catabolism of cholesterol. In macrophages, LXR stimulates the transcription of genes encoding transporters involved in cholesterol efflux, which may limit the transformation of these cells into foam cells in response to lipid loading. Here, we report that natural and synthetic LXR ligands induce the expression of the LXRα gene in primary human macrophages and differentiated THP-1 macrophages. This regulation was not observed in primary human adipocytes or hepatocytes, a human intestinal cell line, or in any mouse tissue or cell line examined. The human LXRα gene was isolated, and the transcription initiation site delineated. Analysis of the LXRα promoter revealed a functional LXR/RXR binding site ∼2.9 kb upstream of the transcription initiation site. We conclude that LXRα regulates its own expression in human macrophages and that this response is likely to amplify the effects of oxysterols on reverse cholesterol transport. These findings underscore the importance of LXR as a potential therapeutic target for the treatment of atherosclerosis. liver X receptor LXR response elements 9-cis retinoic acid receptor high-density lipoprotein oxidized low-density lipoprotein adenosine triphosphate-binding cassette fetal bovine serum Dulbecco's modified Eagle's medium peripheral blood mononuclear cells rapid amplification of cDNA ends real-time quantitative polymerase chain reaction kilobase(s) base pair(s) The characterization of two closely related ligand-activated transcription factors, named liver X receptor (LXR)1 α (NR1H3) and LXRβ (NR1H2) (1Apfel R. Benbrook D. Lernhardt E. Ortiz M.A. Salbert G. Pfahl M. Mol. Cell. Biol. 1994; 14: 7025-7035Crossref PubMed Scopus (294) Google Scholar, 2Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (920) Google Scholar, 3Teboul M. Enmark E. Li Q. Wikstrom A.C. Pelto-Huikko M. Gustafsson J.A. Proc. 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LXR-dependent regulation of cholesterol efflux may prove to be an important antiatherogenic process amenable to pharmacological intervention with LXR agonists. In studies performed in macrophages and macrophage cell lines, we have discovered that one of the genes regulated by the LXRs is LXRα. Notably, this positive feedback loop is specific to human macrophages. We hypothesize that this regulatory pathway increases the magnitude of the biological response to rising cellular levels of cholesterol and other components of oxLDL and, thus, represents an important component of macrophage lipid physiology. THP-1 cells were maintained in suspension for passage and growth in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Irvine Scientific, Santa Ana, CA), 100 units/ml penicillin/100 μg/ml streptomycin (Irvine Scientific), 1 mm sodium pyruvate (Invitrogen), and 55 μmβ-mercaptoethanol (Sigma). Passaging was performed every 3–4 days at a 1:4 dilution. For experiments, 1 × 106cells/well were plated in 6-well plates in media supplemented with 100 ng/ml phorbol 12-myristate-13-acetate (Sigma) to induce differentiation. Cells were maintained in this media for 5 days prior to treatment with LXR agonists. RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS (Irvine Scientific), 2 mml-glutamine (Invitrogen) and 100 units/ml penicillin/100 μg/ml streptomycin (Invitrogen). Passaging was performed by scraping every 3–4 days at 1:3 dilutions. Cells were plated at 5 × 105 cells/well into 6-well plates or 5 × 106 cells into T75 flasks and dosing was begun when the cells had grown to ∼50% confluency. FHs74 cells were grown in Hybri-Care medium (ATCC, Manassas, VA) supplemented with 10% FBS and 30 ng/ml epidermal growth factor PBS (Sigma). Cells were passaged by trypsinization every 3–4 days at a 1:3 dilution. Cells were plated at 3 × 105 cells/well into 6-well plates or 3 × 106 cells into T75 flasks. Treatments began when cells were ∼50% confluent. Primary human adipocytes (Zen-Bio, Research Triangle Park, NC) were supplied in 6-well plates in a proprietary culture media. After cells arrived, the media was changed and the cells allowed to recover overnight, whereupon drug treatments commenced. Cells were ∼60–80% confluent at this time. Primary human hepatocytes pre-plated in 6-well plates were obtained from either Stephen Strom (University of Pittsburgh) or Clonetics (Walkersville, MD). Hepatocyte media consists of Williams' E (Invitrogen) supplemented with 2 mml-glutamine and ITS-G (insulin-transferrin-selenium-G, Invitrogen). Primary human macrophages were isolated and cultured as follows. Human blood was freshly drawn into heparin-treated tubes and diluted 1:3 with phosphate-buffered saline containing 2 mm EDTA. Peripheral blood mononuclear cells (PBMC) were collected by ficoll density separation by overlaying 25 ml of diluted blood on 25 ml of LymphoPrep reagent (Nycomed Pharma AS, Asker, Norway). After centrifugation according to the LymphoPrep protocol, the leukocyte layer was collected. Monocytes were isolated from PBMC by an indirect magnetic cell isolation technique. In this system, non-monocyte PBMC cell subsets are labeled with subset-specific antibody-coated magnetic beads (Monocyte Isolation Kit, Miltenyi Biotec, Auburn, CA) and then separated from unlabeled monocytes with a magnet (VarioMACS separator, Miltenyi Biotec). Unretained cells were found to be >95% monocytes by flow cytometry. Postseparation, monocytes were washed and maintained overnight at 1 × 106 cells/well in 6-well plates in DMEM containing 10% FBS, 2 mm glutamine, 100 units/ml penicillin/100 μg/ml streptomycin, and 0.1 ng/ml GM-CSF (BD PharMingen, Franklin Lakes, NJ). Cultured cells and cell lines were generally dosed with compounds dissolved in the same culture media in which they were grown. Treatments included the LXRα/β agonists GW3965 (32Oliver Jr., W.R. Shenk J.L. Snaith M.R. Russell C.S. Plunket K.D. Bodkin N.L. Lewis M.C. Winegar D.A. Sznaidman M.L. Lambert M.H. Xu H.E. Sternbach D.D. Kliewer S.A. Hansen B.C. Willson T.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5306-5311Crossref PubMed Scopus (966) Google Scholar), T0901317 (19Repa 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 (1150) Google Scholar), 22(R)-hydroxycholesterol (6Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1467) Google Scholar, 7Lehmann 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 (1043) Google Scholar), or 24(S),25-epoxycholesterol (7Lehmann 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 (1043) Google Scholar). Each cell line was treated according to an optimized schedule. Generally, the culture media was replaced with media containing vehicle (Me2SO or ethanol) or 1–10 μm drugs at 0 h. FHs74 cells were harvested for RNA isolation 48 h after the initial dosing; human macrophages and hepatocytes were treated a second time at 24 h and harvested 4 h later; THP-1 and RAW 264.7 cells were dosed a second time at 24 h and then harvested 24 h later; adipocytes were treated a second time at 48 h and harvested 4 h later. The transcriptional initiation site of the human LXRα gene was determined by rapid amplification of cDNA ends (5′-RACE) using a 5′/3′ RACE kit (Roche Molecular Biochemicals) and THP-1 total RNA prepared as described below. The primers used were based on the 5′-end of the published human LXRα cDNA (2Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (920) Google Scholar). The sequences are as follows: SP1, 5′-GGCCCCCAGCCACAAGGACAT-3′; SP2, 5′-CTCTTCCTGGAGCCCT-3′; SP3, 5′-CATTACCAAGGCACTG-3′. SP1–3 are nested primers with SP1 positioned the furthest downstream. Male C57-Bl/6 mice maintained on a regular chow diet were dosed twice daily by oral gavage with 0.5% methyl cellulose or 10 mg/kg GW3965 suspended in 0.5% methyl cellulose. After 2 days dosing, peritoneal macrophages, liver, and intestine were collected. Peritoneal macrophages were harvested by flushing the abdominal cavity with 10 ml ice-cold DMEM containing 10% FBS (Irvine Scientific). The media was withdrawn and spun at 3000 × g for 10 min in a tabletop centrifuge. Cell pellets were lysed in TRIzol reagent (Invitrogen) and RNA extracted according to the manufacturer's instructions. Intestine samples were removed and flushed with ice-cold saline. Liver and intestine samples were snap-frozen in liquid nitrogen and held at −80 °C prior to RNA isolation. Total RNA samples were diluted to 100 μg/ml and treated with 40 units/ml RNA-free DNase-I (Ambion, Austin, TX) for 30 min at 37 °C followed by inactivation at 75 °C for 5 min. Samples were quantitated by spectrophotometry or with the RiboGreen assay (Molecular Probes, Eugene, OR) and diluted to a concentration of 10 ng/μl. Samples were assayed in duplicate 25-μl reactions using 25 ng of RNA/reaction with PerkinElmer chemistry on an ABI Prism 7700 (Applied Biosystems, Foster City, CA). Gene-specific primers were used at 7.5 or 22.5 pmol/reaction and optimized for each gene examined, and the gene-specific fluorescently tagged probe was used at 5 pmol/reaction. In this system, the probe is degraded by Taqpolymerase during the amplification phase, releasing the fluorescent tag from its quenched state; amplification data is expressed as the number of PCR cycles required to elevate the fluorescence signal beyond a threshold intensity level. Fold induction values were calculated by subtracting the mean threshold cycle number for each treatment group from the mean threshold cycle number for the vehicle group and raising 2 to the power of this difference. Northern blot analysis was performed exactly as described elsewhere (33Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar). Briefly, 10 μg of total RNA was electrophoresed in a denaturing agarose gel and transferred to a nylon membrane (Hybond N+, Amersham Pharmacia Biotech) according to the manufacturer's instructions. Human LXRα and LXRβ cDNA corresponding to the ligand-binding domain was labeled with [α-32P]dCTP by random priming using a commercially available system (Megaprime, Amersham Pharmacia Biotech). Blots were sequentially probed with radiolabeled LXRα, LXRβ, and β-actin (CLONTECH Laboratories, Palo Alto, CA) using standard techniques. DNA-receptor protein interactions were examined by electrophoretic mobility shift assays as described elsewhere (33Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar). Competitor oligonucleotides were added at 5-, 15-, or 75-fold molar excess (mutant oligonucleotide added only at 75-fold molar excess). The binding reactions were resolved on a pre-electrophoresed 0.4 × TBE, 4% polyacrylamide gel at room temperature. Human LXRα, LXRβ, and RXRα proteins were synthesized from pSG5-hLXRα, LXRβ, and RXRα using the TNT T7-coupled reticulocyte system (Promega, Madison, WI). The oligonucleotides used in the experiments described here were as follows (sense strand only, with overhang and mutated nucleotides in lowercase and underlined, respectively: Rat CYP7A1; 5′-gatcCTTTGGTCACTCAAGTTCAAGT-3′, LXRE1; 5′-agctTGAATGACCAGCAGTAACCTCAGC-3′, mutLXRE1; 5′-agctTGAATG TTCAGCAGTA TTCTCAGC-3′. A 3.5-kb ApaI fragment of the human LXRα 5′-flanking domain was cloned directly upstream of a luciferase reporter gene in a modified pGL3-Basic vector (Promega) containing an ApaI site in the polylinker. This construct, named pGL3-hLXRα-3027/463, contains bases −3027 to +463 of the human LXRα gene and was obtained by ApaI digest of bacterial artificial chromosome clone RP1117G12 (GenBankTMAC018410). This construct was modified to generate deletion and point mutants as follows. pGL3-hLXRα-2677/463 was generated by digestion of the parent construct with SacI and lacks a fragment extending from the SacI site in the polylinker upstream of the insert to position −2677 within the insert; this construct lacks LXRE1. In pGL3-hLXRα-3027/463mut, four bases within LXRE1 from the parent construct were mutated using the Transformer site-directed mutagenesis system (CLONTECH Laboratories) and the following mutation primer (mutated nucleotides underlined): 5′-CAGGGGGTGAATGTTCAGCAGTATTCTCAGCAGCTTGC-3′. The pGL3-hLXRα-3027/463-Pst deletion mutant lacks LXRE2 and LXRE3 and was generated by digestion of the parent construct with PstI to exclude a fragment from bases −2895 to −217; pGL3-hLXRα-3027/463-Pst-mut was generated by PstI digestion of the pGL3-hLXRα-3027/463mut mutant. HepG2 cells were cultured and transfected exactly as described elsewhere (33Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar). All luciferase values were normalized to secreted placental alkaline phosphatase and are expressed as fold activation over the activity of the no receptor/vehicle condition for each construct. As part of a comprehensive search for LXR target genes in macrophages, we discovered that LXRα itself was up-regulated in differentiated THP-1 cells treated with the potent, synthetic LXR agonists GW3965 (32Oliver Jr., W.R. Shenk J.L. Snaith M.R. Russell C.S. Plunket K.D. Bodkin N.L. Lewis M.C. Winegar D.A. Sznaidman M.L. Lambert M.H. Xu H.E. Sternbach D.D. Kliewer S.A. Hansen B.C. Willson T.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5306-5311Crossref PubMed Scopus (966) Google Scholar) and T0901317 (19Repa 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 (1150) Google Scholar) as assessed by Northern blot analysis (Fig. 1). In multiple experiments, LXRα up-regulation in THP-1 cells averaged 4.2- and 6.2-fold with GW3965 and T0901317, respectively, as measured by RTQ-PCR (Table I). The natural LXR agonists 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol also robustly up-regulated LXRα (Table I), whereas the inactive enantiomer 22(S)-hydroxycholesterol (6Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1467) Google Scholar, 7Lehmann 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 (1043) Google Scholar) had no effect on gene expression in THP-1 cells or any other cells examined (data not shown). In sharp contrast, LXRβ expression was not regulated in THP-1 cells by treatment with the synthetic LXR agonists (Fig. 1).Table IRTQ-PCR analysis of LXRα, ABCA1, and ABCG1 expression in various cells and cell lines treated with various synthetic and natural LXR agonistsCell or cell lineDrugFold regulationLXRαABCA1ABCG1THP-1 human macrophage-like cell lineGW39654.29.260.1T09013176.212.210.324(S),25-epoxycholesterol3.94.065.422(R)-hydroxycholesterol4.94.44.6Human hepatocytesGW39651.15.139.7T09013170.73.132.4Human adipocytesGW39651.57.85.7T09013171.56.05.024(S),25-epoxycholesterol1.13.74.1FHs74 intestinal cell lineGW39651.17.383.1T09013170.98.591.824(S),25-epoxycholesterol0.99.2154.922(R)-hydroxycholesterol1.110.0114.6RAW 264.7 murine macrophage-like cell lineGW39651.18.94.224(S),25-epoxycholesterol0.84.03.622(R)-hydroxycholesterol1.34.22.8Mouse peritoneal macrophagesGW39651.66.210.7Human macrophagesGW39652.57.6113.8T09013173.616.2118.022(R)-hydroxycholesterol3.33.1NDCultured cells were treated with 1–10 μm of the indicated compounds, while peritoneal macrophages were obtained from mice dosed orally with 10 mg/kg GW3965. Gene expression levels were calculated from raw RTQ-PCR data as described under "Experimental Procedures" with the vehicle group expression set to 1.0 arbitrary unit. Data presented are averaged from multiple determinations on samples from one to six individual experiments. First entry, THP-1 cell data; middle entries, additional human and mouse cells and cell line data; last entry, primary human macrophage data. ND, not determined. Open table in a new tab Cultured cells were treated with 1–10 μm of the indicated compounds, while peritoneal macrophages were obtained from mice dosed orally with 10 mg/kg GW3965. Gene expression levels were calculated from raw RTQ-PCR data as described under "Experimental Procedures" with the vehicle group expression set to 1.0 arbitrary unit. Data presented are averaged from multiple determinations on samples from one to six individual experiments. First entry, THP-1 cell data; middle entries, additional human and mouse cells and cell line data; last entry, primary human macrophage data. ND, not determined. Additional experiments were performed to explore the tissue and species specificity of this effect. LXRα, ABCA1, and ABCG1 mRNA levels were assessed by RTQ-PCR in primary human hepatocytes, adipocytes, and the human intestinal cell line FHs74 treated with various synthetic and natural LXR agonists. Gene expression was also analyzed in drug-treated murine macrophage-like RAW 264.7 cells and primary peritoneal macrophages obtained from mice treated with GW3965. Whereas expression of the known LXR target genes ABCA1 and ABCG1 was substantially up-regulated by LXR agonists, little or no change was observed in LXRα expression levels (Table I). Similar results were obtained in additional tissues from drug-treated mice including liver and intestine (data not shown). In contrast, all three LXR target genes were up-regulated in primary human monocyte-derived macrophages (Table I), confirming the original results obtained with THP-1 cells. Overall, these results suggest the effect is specific to macrophages of human origin. LXRβ was not regulated by LXR agonists in any of the cells, cell lines, or tissues examined (data not shown). The genomic structure and promoter regions of the mouse LXRα and LXRβ genes have recently been examined in detail (34Alberti S. Steffensen K.R. Gustafsson J.A. Gene. 2000; 243: 93-103Crossref PubMed Scopus (79) Google Scholar). To begin dissecting the human LXRα 5′-flanking region, DDBJ/EMBL/GenBank high throughput genomic sequence data bases were queried with nucleotides corresponding to mouse Lxrα exon 1 (34Alberti S. Steffensen K.R. Gustafsson J.A. Gene. 2000; 243: 93-103Cros
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