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J. Biol. Chem., Vol. 276, Issue 47, 43509-43515, November 23, 2001

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Liver X Receptor (LXR) Regulation of the LXR Gene in Human Macrophages^*

Karl D. Whitney, Michael A. Watson, Bryan Goodwin, Cristin M. Galardi, Jodi M. Maglich, Joan G. Wilson, Timothy M. Willson, Jon L. Collins, and Steven A. Kliewer

From GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27709

Received for publication, July 2, 2001, and in revised form, August 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The nuclear oxysterol receptors LXR (NR1H3) and LXR (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 LXR 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 LXR gene was isolated, and the transcription initiation site delineated. Analysis of the LXR promoter revealed a functional LXR/RXR binding site ~2.9 kb upstream of the transcription initiation site. We conclude that LXR 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The characterization of two closely related ligand-activated transcription factors, named liver X receptor (LXR)¹  (NR1H3) and LXR (NR1H2) (1-4), has provided important insights into the mechanisms underlying cellular and whole-body cholesterol homeostasis (5). The LXRs are activated by cholesterol derivatives and metabolites, including oxysterols such as 24(S),25-epoxycholesterol and 22(R)-hydroxycholesterol (6-10), and bind as heterodimers with the 9-cis retinoic acid receptor (RXR; NR2B1) to direct repeat four (DR-4) type sequence motifs, termed LXR response elements (LXREs), in the regulatory regions of target genes (1-4). Whereas LXR is expressed in most tissues, LXR is abundantly expressed in a more restricted set of tissues including the liver, kidney, spleen, intestine, and in macrophages (1-4). Over the past several years, the LXRs have been shown to regulate a number of genes involved in cholesterol absorption, transport, and excretion. For example, in the rodent liver, LXR stimulates the transcription of cholesterol 7 hydroxylase (CYP7A1) (11), the rate-limiting enzyme in the classical pathway for the conversion of cholesterol to bile acids (12, 13). When fed a cholesterol-rich diet, mice lacking functional LXR failed to up-regulate Cyp7a1 and accumulated copious amounts of cholesterol in their livers (11). Thus, LXR is critical for the elimination of excess cholesterol from the mouse liver.

More recently, the LXRs have been shown to have a central role in the regulation of reverse cholesterol transport, a process whereby excess cholesterol is transferred in high-density lipoprotein (HDL) particles from peripheral tissues to the liver for elimination from the body (14). The biology of the LXRs and their cholesterol transporter target genes is of particular disease relevance in macrophages since the accumulation of lipids in these cells is a critical step in their transformation into foam cells in fatty streak lesions in atherosclerotic vasculature subendothelium (15, 16). One of the principal vehicles for lipid accumulation is oxidized low density lipoprotein (oxLDL), which contains a number of oxysterol LXR ligands (17). In macrophages, the LXRs regulate the expression of the adenosine triphosphate-binding cassette (ABC) proteins A1 and G1 (18-22), which serve as free-cholesterol and phospholipid translocators enabling cholesterol efflux from the macrophage onto various acceptors, including nascent, cholesterol-poor HDL (18, 21, 23-26). Defective ABCA1 expression in humans is the basis for Tangier disease and is associated with cholesterol accumulation in peripheral tissues along with a near-total lack of cellular cholesterol efflux and plasma HDL (27-31). 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 LXR. 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- 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 µM -mercaptoethanol (Sigma). Passaging was performed every 3-4 days at a 1:4 dilution. For experiments, 1 × 10⁶ cells/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 mM L-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 mM L-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).

Drug Treatments-- Cultured cells and cell lines were generally dosed with compounds dissolved in the same culture media in which they were grown. Treatments included the LXR/ agonists GW3965 (32), T0901317 (19), 22(R)-hydroxycholesterol (6, 7), or 24(S),25-epoxycholesterol (7). 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.

Rapid Amplification of 5'-cDNA Ends (RACE)-- The transcriptional initiation site of the human LXR 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 LXR cDNA (2). 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.

Animal Treatments and Tissue Collection-- 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.

Real-time Quantitative Polymerase Chain Reaction (RTQ-PCR)-- 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 Taq polymerase 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 Protocol-- Northern blot analysis was performed exactly as described elsewhere (33). 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 LXR and LXR cDNA corresponding to the ligand-binding domain was labeled with [-³²P]dCTP by random priming using a commercially available system (Megaprime, Amersham Pharmacia Biotech). Blots were sequentially probed with radiolabeled LXR, LXR, and -actin (CLONTECH Laboratories, Palo Alto, CA) using standard techniques.

Electrophoretic Mobility Shift Assays-- DNA-receptor protein interactions were examined by electrophoretic mobility shift assays as described elsewhere (33). 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 LXR, LXR, and RXR proteins were synthesized from pSG5-hLXR, LXR, and RXR 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'-agctTGAATGTTCAGCAGTATTCTCAGC-3'.

Generation of Reporter Constructs-- A 3.5-kb ApaI fragment of the human LXR 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-hLXR-3027/463, contains bases 3027 to +463 of the human LXR gene and was obtained by ApaI digest of bacterial artificial chromosome clone RP1117G12 (GenBank^TM AC018410). This construct was modified to generate deletion and point mutants as follows. pGL3-hLXR-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-hLXR-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-hLXR-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-hLXR-3027/463-Pst-mut was generated by PstI digestion of the pGL3-hLXR-3027/463mut mutant.

Reporter Construct Transactivation Assays-- HepG2 cells were cultured and transfected exactly as described elsewhere (33). 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As part of a comprehensive search for LXR target genes in macrophages, we discovered that LXR itself was up-regulated in differentiated THP-1 cells treated with the potent, synthetic LXR agonists GW3965 (32) and T0901317 (19) as assessed by Northern blot analysis (Fig. 1). In multiple experiments, LXR 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 LXR (Table I), whereas the inactive enantiomer 22(S)-hydroxycholesterol (6, 7) had no effect on gene expression in THP-1 cells or any other cells examined (data not shown). In sharp contrast, LXR expression was not regulated in THP-1 cells by treatment with the synthetic LXR agonists (Fig. 1).

View larger version (70K): [in this window] [in a new window]   Fig. 1.   LXR is up-regulated in THP-1 cells in response to synthetic LXR agonists. Total RNA (10 µg) was prepared from differentiated THP-1 cells treated for a total of 48 h with vehicle or 1 µM of the indicated drugs. Northern blot analysis was performed with radiolabeled probes for human LXR (upper panel), LXR (middle panel), and glyceraldehyde-3-phosphate dehydrogenase.

View this table: [in this window] [in a new window]   Table I RTQ-PCR analysis of LXR, ABCA1, and ABCG1 expression in various cells and cell lines treated with various synthetic and natural LXR agonists 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. LXR, 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 LXR 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. LXR 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 LXR and LXR genes have recently been examined in detail (34). To begin dissecting the human LXR 5'-flanking region, DDBJ/EMBL/GenBank high throughput genomic sequence data bases were queried with nucleotides corresponding to mouse Lxr exon 1 (34). This search identified a BAC encompassing the 5'-end of the human LXR gene (GenBank^TM AC018410). The region surrounding the murine Lxr transcription initiation site (bases 248 to +521) was found to be highly homologous (~75%) to that in the human LXR gene. To determine the transcriptional initiation site for the human LXR gene, 5'-RACE was performed using total RNA prepared from THP-1 cells and primers located at the 5'-end of the published LXR cDNA (Fig. 2B). Despite the high degree of sequence homology between the mouse and human LXR genes, the positions of the major transcription initiation sites were not conserved. The vast majority (20 of 22 clones) of cDNA ends that were obtained by 5'-RACE started ~320 bp downstream from the mouse transcription initiation site (Fig. 2A). These clones delineated a novel exon (designated exon 1B) of the human LXR gene that is 134 bp in length and is not present in the previously described human LXR cDNA (2). A search of the DBEST data base of GenBank/EMBL/DDBJ sequences from EST Divisions demonstrated that nearly all entries containing sequence corresponding to the human LXR translational start site extend upstream to this same region and that none of them contains a sequence homologous to exon 1 of the mouse Lxr gene (data not shown). The 3'-donor site of exon 1B lies 1050 bp upstream of the 5'-acceptor site of exon 2, which contains the translational start site (Fig. 2, A and B). Two 5'-RACE clones mapped an additional exon (exon 1A, bases 290 to 261) in the region of the human LXR gene corresponding to exon 1 of the murine gene (Fig. 2A). Exon 1 of the murine Lxr gene contains two alternative splice donor sites (1a and 1b, positions +84 and +152, respectively). The splice donor site of the human exon 1A was identical to splice donor site 1a of the mouse Lxr gene (34). It is notable that the transcription initiation site in the murine Lxr gene was delineated using RNA prepared from liver (34), while the RNA used in this study was isolated from a macrophage-derived cell line. Hence, the observed differences may be the result of tissue-specific transcriptional events. Both the human and mouse LXR genes contain in-frame stop codons in exon 2 (34, 35) that prevent any contribution from exons 1A or 1B to the sequence of the LXR protein.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   The human LXR 5'-flanking region. A, alignment of the human LXR 5'-flank with the mouse gene indicating mouse exon 1 (34) (shaded box on lower strand), human exon 1A (dashed line on upper strand), and human exon 1B (shaded box on upper strand) as deduced from 5'-RACE and EST data base analysis. The number and position of cDNA ends as delineated by 5'-RACE are shown in boxes above the sequence. Sequence numbering is expressed relative to the transcriptional initiation site for each gene (indicated with an arrow). Exon 2 of the human LXR gene, which contains the translation start site, lies ~1 kb downstream of the sequence depicted here. B, schematic of the human LXR 5'-flanking region depicting the three potential LXREs (black boxes), exon 1A and exons 1B and 2 (open and shaded boxes, respectively). The indicated ApaI restriction sites were used to generate a reporter construct containing this portion of the 5'-flanking region. The positions of the primers used for 5'-RACE (SP1, SP2, and SP3) are shown.

Using a computer search algorithm, we identified three potential LXREs that conformed to the DR-4 consensus sequence in the 3 kb of sequence upstream from the human transcription initiation site. These sites were termed LXRE1, 2, and 3 (Fig. 2B) Interestingly, the LXRE2 and 3 sites are perfect duplicates, and moreover, the region extending ~200 bp upstream and ~100 bp downstream of the two LXREs is highly conserved (~75% homology). The functional significance of this observation is not clear. To explore the transcriptional activity of these LXREs in their genomic context, an ApaI fragment of the LXR gene containing exon 1 and upstream sequence covering all three putative response elements was inserted into a luciferase reporter gene vector (pGL3-Basic) directly upstream of the luciferase gene (named pGL3-hLXR-3027/463; Figs. 2B and 3). Deletion and point mutants of this construct were generated and are depicted in Fig. 3. Reporter construct activity was examined in transfection assays in a hepatocellular carcinoma cell line, HepG2. The transfections were performed in a liver cell line because of the difficulty of transfecting human macrophage cells. HepG2 cells were transfected with each reporter construct either without exogenous expression of receptors or in the presence of hRXR and either hLXR or hLXR expression vectors. Ligand treatments included vehicle or the synthetic LXR agonists GW3965 and T0901317. As shown in Fig. 3, pGL3-hLXR-3027/463 was activated in a receptor- and ligand-dependent fashion. Only weak induction of reporter activity in response to the LXR agonists was detected in HepG2 cells transfected with this reporter plasmid alone. Cotransfection of LXR and RXR expression plasmids resulted in an ~2-fold increase in reporter activity (Fig. 3). Addition of either LXR agonist to these cells increased reporter activity an additional 2-fold, for a total of 5- to 7-fold activation compared with the no receptor/vehicle condition. Virtually identical results were obtained when an LXR expression plasmid was substituted for LXR (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Activation of human LXR reporter constructs by LXR agonists. Luciferase reporter constructs containing a 3.5-kb ApaI fragment of the intact LXR upstream flanking region (bases 3027 to +463) or various mutations of this construct were transfected into HepG2 cells. Control cultures were transfected with the backbone pGL3-Basic vector. Cells were cotransfected either with an empty expression vector (pSG5) or expression vectors containing human RXR and either human LXR or LXR as indicated and then treated with vehicle (0.1% Me2SO) or 1 µM either GW3965 or T0901317 for 48 h. Data are expressed as fold induction over the normalized luciferase activity measured in the vehicle-treated/no receptor group. Error bars represent the mean ± S.D. from four separate transfection experiments.

To determine which of the three putative LXREs underlies LXR-mediated reporter activation, mutants were constructed that deleted or mutated LXRE1 or deleted both LXRE2 and LXRE3. The parent construct was compared side-by-side to each mutant construct both in the absence and presence of LXR/RXR and either vehicle, GW3965, or T0901317 (Fig. 3). Compared with the parent construct (pGL3-hLXR-3027/463), no basal or ligand-induced activity was observed with the mutant in which LXRE1 is deleted (pGL3-hLXR-2677/463) (Fig. 3). Very similar data were obtained with the pGL3-hLXR-3027/463mut, in which four nucleotides of LXRE1 are mutated Fig. 3). Deletion of LXRE1 also resulted in a loss of induction in response to LXR (data not shown). In contrast, deletion of nucleotides 2895 to 217 in the LXR promoter, which includes LXRE2 and LXRE3 (pGL3-hLXR-3027/463-Pst), did not reduce the activity of the LXR promoter. Instead, the basal and ligand-induced activities of this reporter construct were substantially increased over the parent construct (Fig. 3). We speculate that the deleted region contains a repressor element that may suppress LXR-dependent induction in cells other than macrophages. Site-directed mutagenesis of LXRE1 in this construct (pGL3-hLXR-3027/463-Pst-mut) eliminated the increases seen in basal and ligand-induced activities (Fig. 3). Overall, these results demonstrate that LXRE1 is the principal LXRE mediating LXR control of LXR expression.

We next sought to determine whether LXR/RXR heterodimers bind directly to LXRE1. Electrophoretic mobility-shift assays were performed with in vitro synthesized LXR, LXR, and RXR. As expected, both the LXR/RXR and LXR/RXR heterodimers bound efficiently to a radiolabeled LXRE derived from the rat CYP7A1 promoter (Fig. 4) (7). An oligonucleotide corresponding to LXRE1 competed efficiently with the rat CYP7A1 probe for binding to both the LXR/RXR and LXR/RXR heterodimers. Under the conditions employed, the LXRE1 oligonucleotide competed better than the unlabeled rat CYP7A1 oligonucleotide (Fig. 4). An oligonucleotide harboring a mutated LXRE1 failed to compete for binding (Fig. 4). Using radiolabeled LXRE1, we confirmed that both the LXR/RXR and LXR/RXR heterodimers bind directly to LXRE1 (Fig. 4). Together, these data provide evidence that the human LXR gene is regulated directly by the LXR/RXR heterodimers.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Electrophoretic mobility-shift analysis of LXRE1. Competition experiments were performed using a radiolabeled oligonucleotide probe corresponding to the DR4 LXRE from the rat CYP7A1 promoter (left panels) or LXRE1 from the human LXR gene (right panels). In addition to probe, binding reactions contained in vitro translated human RXR and/or LXR (top panels) or LXR protein (bottom panels). Some reactions also contained competitor oligonucleotides corresponding to either the rat CYP7A1 DR4 or LXRE1 from the human LXR promoter as indicated. Competitor rat CYP7A1 and LXRE1 oligonucleotides were added at 5-, 15-, or 75-fold molar excess, while the mutant LXRE oligonucleotide was added at 75-fold molar excess over the radiolabeled probe.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LXR is known to regulate a number of genes involved in cholesterol homeostasis (5, 36). The experiments described in this report reveal LXR as a novel LXR target gene in human macrophages. We hypothesize that this autoregulation increases the expression of LXR target genes such as ABCA1, ABCG1, and apolipoprotein (Apo) E in response to oxysterols derived from oxLDL (18-21, 37). The coordinate increase in the expression of these genes could then increase the efflux of excess cholesterol from macrophages onto acceptor proteins such as ApoA1 and ApoE for transport to the liver and elimination from the body (14, 36). Thus, the LXR autoregulatory loop provides a mechanism for efficiently amplifying the effects of oxysterols in macrophages and promoting reverse cholesterol transport. Interestingly, we did not observe stimulation of LXR gene expression by LXR agonists, indicating that autoregulation is restricted to the LXR gene. The physiological importance of this difference remains to be determined. The LXR gene was also recently shown to be directly regulated by peroxisome proliferator-activated receptor , which is activated by fatty acids and their metabolites (38). Thus, LXR gene expression is under the control of multiple receptors that sense dietary status. In cell-based reporter assays, both the LXR and LXR subtypes were capable of stimulating transcription of the LXR promoter. Since LXR and LXR are both expressed in macrophages, these data suggest that activation of either subtype can initiate this regulatory cascade.

Among the human cells examined, LXR was only up-regulated in primary macrophages. This macrophage-specific effect may be due to the restricted expression of positively acting transcription factors or coactivators in this cell type. Alternatively, tissue-specific repressors, perhaps interacting with a region of the LXR promoter bracketed by the PstI sites exploited in the deletion mutation experiments described above, may prevent induction in tissues where excessive activation of LXR target genes could be detrimental. Notably, we failed to detect a similar autoregulation of LXR gene expression by LXR agonists in murine macrophages or other mouse tissues or cell lines. The basis for these species differences is currently unknown. We speculate that the dynamic response to oxysterols as measured by cholesterol efflux may be greater in human macrophages than in rodent cells, perhaps reflecting an evolutionary response to the cholesterol-rich diet of humans as compared with rodents.

One possible implication of these findings is that polymorphisms in LXRE1 or other regulatory elements of the LXR promoter may impair the gene response to lipid loading. Thus, individuals harboring these mutations may have an increased risk of developing atherosclerosis. Interestingly, familial combined hyperlipidemia, a polygenic lipid disorder associated with the early onset of coronary artery disease, was recently found to have genetic linkage with a region of chromosome 11 that contains the LXR gene (39). It remains to be determined whether mutations in either the LXR gene or its regulatory regions contribute to this disease.

In conclusion, we have demonstrated that LXR is a direct target gene of LXR and LXR in human macrophages. Regulation is mediated by a single LXRE located 2.9 kb upstream of a previously unrecognized exon in the human LXR gene. LXR regulation is not seen in other human cell types or any murine tissue or cell examined. This macrophage-specific phenomenon may form an important component of the body's response to elevated cholesterol levels in general and may help prevent the transformation of macrophages into foam cells by amplifying the process of reverse cholesterol transport.

	Addendum

Laffitte et al. (40) have also recently described autoregulation of the LXR gene promoter.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: GlaxoSmithKline Research and Development, Five Moore Dr., Research Triangle Park, NC. Tel.: 919-483-5601; Fax: 919-315-6720; E-mail: sak15922@gsk.com.

Published, JBC Papers in Press, August 23, 2001, DOI 10.1074/jbc.M106155200

	ABBREVIATIONS

The abbreviations used are: LXR, liver X receptor; LXRE, LXR response elements; RXR, 9-cis retinoic acid receptor; HDL, high-density lipoprotein; oxLDL, oxidized low-density lipoprotein; ABC, adenosine triphosphate-binding cassette; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBMC, peripheral blood mononuclear cells; RACE, rapid amplification of cDNA ends; RTQ-PCR, real-time quantitative polymerase chain reaction; kb, kilobase(s); bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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