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Previous Article | Next Article 
Molecular and Cellular Biology, November 2003, p. 7756-7766, Vol. 23, No. 21
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.21.7756-7766.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Florent Lalanne,1* Marie C. Gerbod-Giannone,1 Jennifer R. Molina,1 Xuan Fu,2 Erik G. Lund,2 Lorraine J. Gudas,3 and Alan R. Tall1
Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032,1 Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, Rahway, New Jersey 07065,2 Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 100213
Received 5 February 2003/ Accepted 25 July 2003
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ABSTRACT |
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/RXR activated the human ABCA1 promoter, via the same direct repeat 4 (DR4) promoter element as LXR/RXR. Chromatin immunoprecipitation analysis in macrophages confirmed the binding of RAR/RXR to the ABCA1 promoter DR4 element in the presence of ATRA, with weaker binding of RAR
/RXR, and no binding of RARß/RXR. However, in macrophages from RAR-/- mice, TTNPB still induced ABCA1, in association with marked upregulation of RAR, suggesting that high levels of RAR can compensate for the absence of RAR. Dose-response experiments with ATRA in mouse primary macrophages showed that other LXR target genes were weakly induced (ABCG1 and SREBP-1c) or not induced (apoE and LXR). The more specific RAR activator TTNPB did not induce SREBP-1c in mouse primary macrophages or liver. These studies indicate a direct role of RAR/RXR in induction of macrophage ABCA1. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ABCA1 is upregulated in cholesterol-loaded cells, as a result of increased transcription mediated by the oxysterol-activated nuclear receptors liver X receptor (LXR)/retinoid X receptor (RXR) acting on a direct repeat nuclear receptor binding site spaced by 4 nucleotides (DR4) in the proximal promoter of the ABCA1 gene (6, 31, 33). Treatment of animals with LXR activators reduces atherosclerosis, and bone marrow transplantation experiments indicate a specific antiatherogenic function of LXRs and ABCA1 in macrophages (16, 38). LXRs target a battery of genes mediating cholesterol efflux, transport and excretion and have emerged as major drug targets (5). However, LXRs also act at the promoter of sterol regulatory element-binding protein-1c (SREBP-1c), a master transcriptional regulator of genes of fatty acid and triglyceride synthesis, resulting in fatty liver and hypertriglyceridemia (3, 12, 30).
Vitamin A and its derivatives, the retinoids, exert many biological activities at different stages of development. They are crucial for the normal development of the embryo and are later essential for cell proliferation, differentiation, and apoptosis (7, 17). Two classes of nuclear receptors mediate these biological effects: RXRs and retinoic acid receptors (RARs). Each of these classes consists of three isoforms (, ß, and ) (11, 24, 25, 29, 49). RXR is activated by 9-cis-retinoic acid (9-cRA), whereas RAR is activated by all-trans-retinoic acid (ATRA) and 9-cRA (1). In vivo, dimeric RXR/RAR typically binds to promoter elements consisting of direct repeats spaced by five nucleotides (DR5) (14). 13-cRA and ATRA are in clinical use, and retinoids are under active investigation for several different conditions.
In the present study we examined a possible role of retinoids in the regulation of macrophage cholesterol efflux and ABCA1 gene expression. We found that RAR ligands, ATRA and TTNPB (4-[E-2-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl] benzoic acid), upregulate the ABCA1 gene, unexpectedly acting at the noncanonical DR4 element of the ABCA1 promoter. These studies suggest a broader role of retinoids acting through a specific RAR isoform (RAR) in the regulation of macrophage functions, including cholesterol efflux and transport.
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MATERIALS AND METHODS |
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Animals.
Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were housed in a temperature- and light-controlled facility. RAR-/- mice and their control littermates RAR+/+ were maintained on a C57BL/6J genetic background and genotyped by Southern blot. Mice were aged matched for each experiment. All animal procedures were approved by the Institutional Animal Care and Research Advisory Committee at Columbia University.
Cell cultures and transfection experiments. Human HEK293 cells were purchased from the American Type Culture Collection (Manassas, Va.) and maintained in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum, 100 U of penicillin/ml, and 100 mg of streptomycin/ml. Primary peritoneal macrophages were isolated from C57BL/6J male mice 6 to 8 weeks old intraperitoneally injected with 1 ml of 30% thioglycolate. After 72 h, macrophages were collected by washing the peritoneal cavity with phosphate-buffered saline (PBS) and cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, and 100 mg of streptomycin/ml for 48 h before the experiment. Human monocyte-derived macrophages were prepared and cultured as described previously (10).
Transfection experiments were performed in 24-well plates with Lipofectamine Plus reagent according to the manufacturer's instructions (Invitrogen). Cells were transfected with 12.5 ng of phRLTK (Renilla; Promega, Madison, Wis.)/well, 0.15 µg of reporter DNA (containing hABCA1 proximal promoter)/well, and 0.15 µg of each receptor (pCMX-hRXR, pCMX-hRAR, pCMX-hRARß, and pCMX-hRAR)/well and pcDNA3.1 (to a final total of 0.45 µg/well) if necessary. The transfected cells were cultured in DMEM with 10% lipoprotein-deficient serum, 100 U of penicillin/ml, and 100 mg of streptomycin/ml in the presence of 0.1 µM TTNPB or its vehicle for 36 h. Luciferase activity was then measured by using the Dual Luciferase assay system (Promega) and normalized with Renilla.
Cholesterol efflux assays. Macrophages were cholesterol loaded and radiolabeled overnight in DMEM 0.2% bovine serum albumin (BSA; Sigma) containing 50 µg of acetylated low-density lipoprotein and 1 µCi of [3H]cholesterol (51.2 Ci/mmol; NEN/Life Science Products, Boston, Mass.)/ml in the presence or absence of ATRA or TO-901317. Cells were washed with PBS, equilibrated for 30 min in DMEM-0.2% BSA, and then incubated for 4 h in the efflux media containing DMEM-0.2% BSA and 10 µg of purified apoA-I/ml in the presence or absence of the different ligands. The media was then collected, and cells were lysed with a 0.1% NaOH-0.1% sodium dodecyl sulfate (SDS) solution. After determination of the radioactivity recovered in the medium and cell lysate by liquid scintillation counting, cholesterol efflux was calculated as the percentage of the radioactivity recovered in the media over the total radioactivity (cells plus media) after subtraction of the nonspecific apoA-I-free media. Cholesterol efflux assays were performed in triplicates or quadruplicates.
Western blot analysis. Protein extracts from macrophages were prepared by lysing the cells in modified radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing a protease inhibitor cocktail (Complete EDTA Free; Roche). Protein content in the extracts was determined by using the DC protein assay (Bio-Rad, Hercules, Calif.). Equal amounts of protein (40 to 50 µg) were separated by electrophoresis with 4 to 15% acrylamide gradient gels (Bio-Rad) and then transferred to nitrocellulose membrane (Trans-Blot Transfer Medium; Bio-Rad). Membranes were probed with anti-ABCA1 antibody (Novus Biologicals, Littleton, Colo.) and anti-ß-actin antibody (Sigma) according to the manufacturer's recommendations. Immunoblots were developed by using a chemiluminescent detection system (Super Signal West Pico chemiluminescent substrate; Pierce, Rockford, Ill.).
Nuclear protein extracts from tissues and macrophages were isolated according to the following method. Tissue samples and cells were first homogenized on ice and then lysed in lysis buffer (10 mM Tris [pH 7.5], 3 mM MgCl2, 10 mM NaCl, 0.5% NP-40) containing protease inhibitor cocktail. The nucleus was then pelleted by centrifugation for 10 min at 6,000 x g, immediately resuspended in a nucleus suspension buffer (250 mM Tris [pH 8.0], 60 mM KCl, 1 mM dithiothreitol), and then rotated for 1 h. After centrifugation for 5 min at 6,000 x g, the solubilized nuclear proteins were separated by electrophoresis with a 4 to 15% gradient gel (Bio-Rad), transferred to nitrocellulose membrane (Trans-Blot transfer medium; Bio-Rad), and probed with anti-RAR and anti-RAR antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.) and their respective horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's recommendations. The intensities of the bands were quantified by using ImageQuant and normalized to ß-actin.
EMSA.
RARs and LXR and RXR proteins were in vitro translated by using the TNT-coupled wheat germ extract systems (Promega). Double-strand oligonucleotides corresponding to the wild-type ABCA1 DR4 element 5'-ACTGGGCTTTGACCGATAGTAACCTCTGCGCTCG-3' and the mutated sequence 5'-ACTGGGCTTTGTGTGATAGTACTATCTGCGCTCG-3' were used, and electrophoresis mobility shift assays (EMSAs) were done with 32P-labeled probes as described previously (6). For competition experiments, a 30-fold molar excess of cold unlabeled competitor DNA relative to labeled DNA was used. In antibody experiments, the mixture was first incubated for 10 min at room temperature with 0.4 to 1 µg of anti-RAR or anti-RXR rabbit polyclonal antibody (Santa Cruz Biotechnology). The oligonucleotides 5'-ACTGCAGTGACCGCCAGTAACCCCAGC-3' and 5'-ACTGGGACGCCCGCTAGTAACCCCGGC-3' were, respectively, used in EMSA analysis of DR4 elements a and b of the murine SREBP-1c promoter.
Plasma and hepatic lipid analysis. Mice were fasted for 3 h before blood collection. Plasma was separated by centrifugation and kept at -80°C until lipid analysis. Liver tissue samples (50 to 75 mg) were homogenized in PBS. Lipids were then extracted with chloroform-methanol (2/1 [vol/vol]) and redissolved in isopropanol. Triglyceride and cholesterol were measured in plasma and in the liver lipid extracts by using commercial kits (Wako Chemicals, Neuss, Germany).
RNA analysis.
Total RNA was isolated from mouse peritoneal macrophages or 
50 mg of mouse liver tissue by using the RNeasy Mini kit (Qiagen, Valencia, Calif.) or RNA-Bee reagent (Tel-Test, Inc., Friendswood, Tex.), respectively, according to the manufacturer's protocol. Real-time quantitative PCR assays were performed by using the Mx4000 Quantitative PCR System (Stratagene, La Jolla, Calif.). Briefly, 5 µg of total RNA was treated with RNase-free DNase I (Ambion, Austin, Tex.), and first-strand cDNA was synthesized with oligo(dT)12-18 by using a Superscript II RNase H- reverse transcriptase reagent kit (Invitrogen) according to the manufacturer's protocol. For quantification of mouse ABCA1, ABCG1, SREBP-1c, LXR, ApoE, and fatty acid synthase (FAS) mRNA levels, each amplification mixture contained 62.5 ng of cDNA, appropriate concentrations of forward and reverse primers and of dual-labeled fluorogenic probe (Biosearch Technologies, Novato, Calif.), and 12.5 µl of TaqMan Universal PCR master mix (Applied Biosystems, Foster City, Calif.). PCR thermocycling parameters were 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 60°C for 1 min. All samples were analyzed for ß-actin expression in the same run. Quantitative expression values were extrapolated from standard curves for the gene of interest with 10-fold dilutions of cDNA (in triplicate). Each sample was normalized to ß-actin, triplicates were averaged, and relative mRNA levels were determined. The following mouse primers and probes were used: mouse ABCA1 (mABCA1) forward (F) (5'-GGTTTGGAGATGGTTATACAATAGTTGT-3'), mABCA1 reverse (R) (5'-CCCGGAAACGCAAGTCC-3'), and mABCA1 TaqMan probe (5'-FAM-CGAATAGCAGGCTCCAACCCTGACC-BHQ-3'); mABCG1 F (5'-CCATGAATGCCAGCAGCTACT-3'), mABCG1 R (5'-CACTGACACGCACACGGACT-3'), and mABCG1 TaqMan probe (5'-FAM-TGCCGCAATGACGGAGCCC-BHQ-3'); mSREBP-1c F (5'-GGAGCCATGGATTGCACATT-3'), mSREBP-1c R (5'-CCTGTCTCACCCCCAGCATA-3'), and mSREBP-1c TaqMan probe (5'-FAM-CAGCTCATCAACAACCAAGACAGTGACTTCC-BHQ-3'); mApoE F (5'-CCTGAACCGCTTCTGGGATT-3'), mApoE R (5'-GCTCTTCCTGGACCTGGTCA-3'), and mApoE TaqMan probe (5'-FAM-AAAGCGTCTGCACCCAGCGCAGG-BHQ-3'); mLXR F (5'-GCTCTGCTCATTGCCATCAG-3'), mLXR R (5'-TGTTGCAGCCTCTCTACTTTGGA-3'), and mLXR TaqMan probe (5'-FAM-TCTGCAGACCGGCCCAACGTG-BHQ-3'); mFAS F (5'-GGCATCATTGGGCACTCCTT-3'), mFAS R (5'-GCTGCAAGCACAGCCTCTCT-3'), and mFAS TaqMan probe (5'-FAM-CCATCTGCATAGCCACAGGCAACCTC-BHQ-3'); and mß-actin F (5'-AGAGGGAAATCGTGCGTGAC-3'), mß-actin R (5'-CAATAGTGATGACCTGGCCGT-3'), and mß-actin TaqMan probe (5'-JOE-CACTGCCGCATCCTCTTCCTCCC-BHQ-3').
For quantification of mouse Cyp26 mRNA levels, each amplification mixture (25 µl) contained 62.5 ng of cDNA, 100 nM concentrations of reverse and forward primers, and 2.5 µl of 10x SYBR Green PCR master mix (Perkin-Elmer Life Sciences, Boston, Mass.). Quantitative expression values were extrapolated from standard curves for Cyp26 expression with 10-fold dilutions of cDNA (in triplicate). Each sample was normalized to ß-actin that was deduced from TaqMan assays, triplicate results were averaged, and relative Cyp26 mRNA levels were determined. The primers mCyp26 F (5'-GCCGCGAGGCACTCCAGTGCT-3') and mCyp26 R (5'-CCCAGCAGGATGCGCATGGCGAT-3') were used. RNA measurements from human monocyte-derived macrophages were performed as described previously (10, 44).
Chromatin immunoprecipitation (ChIP) assays.
Mouse peritoneal macrophages were treated or not with 1 µM ATRA for 24 h and then incubated with 1% formaldehyde in cell culture media for 20 min. Thereafter, cells were washed twice with ice-cold PBS and lysed in buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), and protease inhibitor cocktail (Roche). Samples were sonicated three times with 10-s pulses at 4°C. After centrifugation the samples were diluted 1:10 in buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), and 167 mM NaCl and precleared with 80 µl of salmon sperm DNA-protein A-agarose for 2 h at 4°C. Samples were then centrifuged to pellet the agarose beads, and immunoprecipitation was performed on the supernatant by using anti-RAR, anti-RARß, anti-RAR polyclonal antibodies or an equal amount of normal rabbit immunoglobulin G (Santa Cruz Biotehnology) overnight at 4°C. The antibody-protein-DNA complexes were then precipitated by 60 µl of salmon sperm DNA-protein A-agarose for 1 h at 4°C. The precipitates were washed sequentially in low-salt immune complex buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl), hight-salt immune complex buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl), LiCl immune complex buffer (0.25 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]) prior to two final washes in Tris-EDTA buffer. The protein-DNA complexes were eluted by using a 1% SDS-0.1 M NaHCO3 solution. Cross-linked DNA was reversed by incubation at 65°C for 6 h in presence of 5 M NaCl, and proteins were digested at 45°C for 1 h with proteinase K. Immunoprecipitated DNA fragments were purified by using a QIAquick PCR purification kit (Qiagen). Samples were analyzed by PCR with the primers 5'-CCACGTGCTTTCTGCTGAGT-3' and 5'-TGCCGCGACTAGTTCCTTTT-3' (nucleotides 1306 to 1325 and 1426 to 1445 of the ABCA1 promoter; GenBank accession number AF275948).
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RESULTS |
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2-fold. At doses of 1 and 5 µM ATRA, the increase in cholesterol efflux was comparable to that occurring in macrophages treated with the LXR activator TO-901317 (Fig. 1B). ABCA1 protein levels were dose dependently increased after ATRA treatment, paralleling effects on cholesterol efflux (Fig. 1C). ABCA1 protein was also increased in human primary monocytes/macrophages treated with ATRA (Fig. 1C). To determine whether the increase in ABCA1 protein and cholesterol efflux might be caused by induction of ABCA1 gene expression, we measured ABCA1 mRNA by quantitative real-time PCR. This revealed an increase in the mRNA that was parallel to the dose response of protein and cholesterol efflux (Fig. 2A), a finding consistent with increased gene expression as the underlying mechanism.
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, and a weaker and somewhat inconsistent induction of ABCG1 and SREBP-1c (Fig. 2A). Human primary monocytes/macrophages showed a more general induction of LXR target genes by ATRA. There was a strong induction of both ABCA1 and ABCG1 and weaker but significant increases in LXR and SREBP-1c. However, compared to these other genes, the fold induction was more pronounced for ABCA1 at lower doses of ATRA (0.5 and 1 µM), a difference that was observed in repeated experiments. ApoE, another macrophage LXR/RXR target (19, 23), was not induced by ATRA either in human or mouse primary macrophages even at the higher dose of ATRA. TTNPB induces macrophage ABCA1 but not SREBP-1c. ATRA is a ligand for RARs but not RXRs (15). However, there may be a small amount of spontaneous conversion of ATRA to other retinoids, such as 9-cRA, that are ligands for RXRs (15), raising the possibility that some of the alterations in gene expression in response to ATRA might reflect activation of RXRs. Thus, we also treated mouse macrophages with TTNPB, a synthetic RAR pan-agonist that does not activate RXRs (24). This resulted in accumulation of ABCA1 protein, which is similar to the effects of ATRA (Fig. 1D). There was also an induction of macrophage ABCA1 mRNA by TTNPB, but no effect on SREBP-1c mRNA (Fig. 3A). Thus, TTNPB causes a specific induction of mouse macrophage ABCA1, strongly suggesting a response mediated via RARs.
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6-fold versus 30-fold in murine macrophages), suggesting the metabolism of TTNPB in human macrophages.
RAR/RXR activates the ABCA1 promoter via a DR4 element.
To further assess the possibility of a direct activation of the ABCA1 promoter by RARs, we used human ABCA1 promoter-reporter constructs in transfection studies and treated the cells with TTNPB. HEK293 cells were transfected with the human ABCA1 promoter (bp -928 to bp +101) linked to luciferase, as well as each of the three different isoforms of RAR (, ß, and ) and RXR (Fig. 4A). Although RAR and RARß failed to activate the ABCA1 promoter, either in presence or the absence of TTNPB, RXR/RAR increased luciferase activity by 2.8-fold in the basal state and by 4.0-fold in the presence of TTNPB. Transactivation of the ABCA1 promoter by RAR was not dependent on cotransfection with RXR (Fig. 4B), indicating that RAR activates the ABCA1 promoter, either by acting as a homodimer or as a heterodimer with endogenous RXR.
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and RXR, in the presence of TTNPB. We also included a construct containing the full-length promoter containing point mutations in the DR4 element that are known to abolish the interaction with RXR/LXR (6). Deleting the region from bp -928 to bp -101 had no effect on the hABCA1 promoter response to retinoids. Surprisingly, the mutation in the DR4 element abolished the RAR-mediated activation of the hABCA1 promoter. This suggests that the DR4 element mediates the response of the ABCA1 promoter to RAR and retinoids.
To further assess the possibility of a direct interaction between RAR and the human ABCA1 promoter, we carried out gel shift assay by using oligonucleotides consisting of the DR4 element. RAR/RXR formed a specific complex on the human ABCA1 promoter (Fig. 4D, lane 2) that was specifically competed by an unlabeled oligonucleotide containing the DR4 element (lane 3) but not by a mutant DR4 element (lane 7). Antibodies raised against RXR or RAR abolished the complex and gave rise to supershifted complexes (lanes 4 and 5, asterisks), indicating that the complex consists of RAR/RXR. An antibody raised against ROR, an irrelevant nuclear receptor, had no effect (lane 6). We also determined whether RAR and RARß could bind the ABCA1 DR4 element. Each of the three RAR isoforms formed a specific complex on the ABCA1 DR4 element but only in the presence of RXR (data not shown), confirming that complexes are heterodimers of RAR/RXR. These results show direct binding of RAR/RXR to the ABCA1 promoter, involving the same DR4 promoter element that binds LXR/RXR. In contrast to the transactivation assay in transfected 293 cells (Fig. 4A), the binding was not specific for a particular RAR isoform. This could indicate that specificity in the transactivation assay depends on RAR isoform-specific sets of coactivators or corepressors present in HEK 293 cells.
We also sought to determine whether there might be a comparable binding of RAR/RXR to the promoters of other LXR target genes. The SREBP-1c promoter contains two LXR/RXR binding sites (Fig. 4E). However, neither element bound to RAR/RXR or competed with the RAR/RXR complex formed on the ABCA1 DR4 element (Fig. 4F shows data for the DR4b element) or bound any other RAR isoform (not shown). This finding suggests that some of the genes more weakly induced by ATRA (Fig. 2) may be indirect targets.
ChIP of RAR in mouse macrophages.
We carried out further experiments to verify a direct effect of RARs on the mouse macrophage ABCA1 DR4 promoter element. First, to evaluate the expression of RAR protein in different mouse tissues, Western blots were performed on nuclear extracts. This revealed a high level of expression of RAR in spleen, adipose and lung, with much lower levels in liver, intestine and kidney (Fig. 5A). RAR protein was also well expressed in primary macrophage cultures from mice and humans. RAR was found to be highly expressed in adipose tissue, lung, and spleen, whereas RARß was widely expressed in different tissues (data not shown), similar to the distribution of their cognate mRNAs (49). Whereas RARß protein was not detected in murine macrophages (not shown), RAR protein was detected at low levels in wild-type macrophages (Fig. 5D).
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to the DR4 element (Fig. 5B, lane 5). There was a much weaker signal for RAR (lane 3) and no signal for RARß (lane 4). Similar specific binding of RAR was observed for three different macrophage preparations. Interestingly, the signal for of RAR was much stronger when cells were treated with ATRA (Fig. 5B, lane 1 versus lane 5). In other experiments, we observed that incubation of macrophages with ATRA weakly induced RAR protein (<2-fold) (data not shown), suggesting a predominant effect of ATRA on RAR binding to the DR4 element rather than an induction of RAR itself.
Response of ABCA1 in RAR-deficient macrophages.
To further evaluate a possible specific role of RAR in the upregulation of ABCA1, we examined macrophages from RAR-/- mice (21). Recovery of thioglycolate-elicited macrophages was considerably lower in RAR-/- mice than in controls (ca. 1/3 the number of cells), but cellular morphology appeared similar to that seen with controls, perhaps indicating a role of RAR in the migration of macrophages into tissues. To evaluate the upregulation of ABCA1, macrophages were treated with 5 µM TTNPB. This experiment showed that ABCA1 was still induced in RAR-/- macrophages, a finding similar to the results in macrophages from RAR+/+ controls (Fig. 5C). Similar results were obtained in two separate experiments performed on macrophages pooled from four mice per group. Interestingly, nuclear RAR was substantially increased (20-fold) in RAR-deficient macrophages (Fig. 5D) and decreased in response to TTNPB treatment (2-fold). These findings suggest autoregulation of RAR expression, both between and within RAR isoforms. Most likely the upregulation of RAR compensates for the deficiency of RAR, leading to induction of ABCA1 expression by TTNPB. Even though RAR did not increase ABCA1 promoter activity in HEK293 cells, it is possible that the cell-specific context in macrophages allows a response to increased RAR, albeit weaker than the RAR response, a finding consistent with the ChIP assay results.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/RXR on the ABCA1 promoter. Even though the effect is mediated via the LXR-binding site in the ABCA1 promoter, other LXR target genes, including SREBP-1c, were modestly induced by ATRA, possibly by indirect mechanisms. RAR is highly expressed in macrophages but not highly expressed in liver (Fig. 5A). These findings suggest a role of RAR in the regulation of macrophage cholesterol efflux via ABCA1.
Surprisingly, the effects of RAR/RXR on the promoter of ABCA1 were found to be mediated via a noncanonical DR4 element, previously implicated in the activity of LXR/RXR (6). This initially raised the possibility that effects of ATRA on ABCA1 gene expression could reflect conversion to other retinoids such as 9-cRA with subsequent activation of RXR in LXR/RXR complexes. However, multiple lines of evidence accrued to indicate a direct action of RAR/RXR on the DR4 element, including transactivation and gel shift assays and, most compellingly, direct demonstration of binding in ChIP analysis of the ABCA1 promoter in macrophages. Moreover, macrophage ABCA1 was induced by TTNPB, a synthetic agonist that is specific for RAR and not RXR. A number of other LXR target genes were evaluated and showed weak or no induction in mouse macrophages but stronger induction in human macrophages. Thus, it is possible that in part the effect of ATRA in human macrophages reflects induction of LXR, as suggested in a report that appeared while the present study was under review (43), or conversion to other retinoids that act on RXR. However, even in human macrophages induction of ABCA1 was more prominent than that of other LXR target genes at lower doses, a finding consistent with a direct effect of RAR on the human ABCA1 promoter, as shown in the transactivation and gel shift assays (Fig. 4A). Noncanonical binding of RAR/RXR to a DR4 element has been described (39, 40), although the functional implications of such binding have not been previously shown.
Transactivation assays in HEK293 cells and ChIP analysis in macrophages indicated a selective effect of RAR/RXR complexes on the ABCA1 promoter, a finding consistent with the high expression of RAR in macrophages. However, there was a weak signal above background for RAR in the ChIP analysis of the ABCA1 promoter, and induction of ABCA1 by TTNPB in macrophages from RAR-/- mice indicated that the effect on ABCA1 was not completely specific for the RAR isoforms (Fig. 5C). This likely reflected a 20-fold upregulation of RAR in RAR-deficient macrophages. These findings indicate partial compensation between the different RAR isoforms in relation to macrophage functions consistent with the evidence of such compensation in embryos from mice with knockouts of the various RAR isoforms (20, 21, 28). This functional redundancy between RAR and RAR has also been shown in RAR-null F9 cells, where basal expression of RAR did not induce RAR responsive genes, but responsiveness of RAR target genes was observed when RAR was overexpressed (37). The ability of ABCA1 to respond to RAR in macrophages but not HEK293 cells could potentially reflect the presence of different sets of coregulators in the different cell types and may explain the modest effects of RARs on the ABCA1 promoter in HEK293 cells. Although our studies have focused on the role of RAR activators in ABCA1 gene expression, RAR may have an essential role in macrophage differentiation and function, as suggested by the low recovery of macrophages in RAR-deficient mice. Interestingly, RAR is also highly expressed in adipocytes and forced expression in preadipocytes blocks the program of adipocyte differentiation (46). Further studies are indicated to define the more general roles of RAR in macrophage differentiation and functions.
Our findings suggest convergent signaling of retinoids and oxysterols on the macrophage ABCA1 promoter. A convergence of RAR and LXR signaling pathways might be related to events occurring in the embryo, involving first RAR and later LXR. Mice lacking the RAR gene, together with one or both copies of RARß display severe interdigital webbing associated with a low number of apoptotic cells and an increase of cell proliferation in the interdigital necrotic zone (8). Mouse embryos deficient in ABCA1 also exhibit delayed clearance of interdigital webbing accompanied by the accumulation of apoptotic corpses (13). Interestingly, ABCA1 appears to be the human homolog of Caenorhabditis elegans ced-7, which functions in engulfment of cell corpses during apoptosis (45). Chimini and coworkers have suggested that ABCA1 promotes engulfment of apoptotic cells (13). Therefore, it is possible that ABCA1 levels in macrophages might be controlled by retinoids at the initiation of the tissue remodeling process and later by cellular sterol content, reflecting ongoing phagocytosis of cholesterol-rich corpses.
Current clinical use of retinoids is related to their properties as cellular differentiating agents. 13-cRA is used to treat severe cystic acne (9, 34). ATRA induces remissions in ca. 80% of patients with acute promyelocytic leukemia. Retinoids are promising chemopreventive agents, and clinical studies have also demonstrated their effectiveness in reversing premalignant lesions, such as leukoplakia, and in preventing second primary tumors of the head and neck and also liver and breast cancer (35). ATRA is also in clinical trials for the treatment of emphysema (26).
A major concern in the use of retinoids has been the induction of hypertriglyceridemia, often accompanied by reduced HDL levels, as well as elevations of transaminase in plasma, probably resulting from the development of fatty liver. The molecular mechanisms of these side effects are not well understood and might be due to the low specificity of the retinoids used. For some retinoids, they may be related to activation of RXR, induction of SREBP-1c, and genes of fatty acid synthesis. Other mechanisms of dyslipidemia may be related to induction of apoCIII. Indeed, 13-cRA (isotretinoin) increases hepatic apoCIII expression at a transcriptional level, providing an explanation for hypertriglyceridemia (42). apoCIII delays the catabolism of triglyceride-rich particles and increases atherosclerosis (27). Vu-dac et al. showed that this regulation is mediated by RXR and not by RAR (42). Our studies indicate a modest induction of SREBP-1c by ATRA and suggest that this might be a mechanism underlying the fatty liver and hypertriglyceridemia associated with clinical use of this agent.
Since RAR is not highly expressed in the liver and RAR activators are able to stimulate macrophage cholesterol efflux via ABCA1, it is conceivable that RAR selective activators might have an interesting spectrum of properties that includes increased macrophage cholesterol efflux while tending to spare fatty liver and some of the proatherogenic effects of other retinoids. However, it seems unlikely that RAR activators will prove useful in the treatment of atherosclerosis in view of their wide spectrum of adverse side effects.
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ACKNOWLEDGMENTS |
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We thank R. M. Evans for providing the pCMX expression plasmids for RAR, RARß, and RAR, and Pierre Chambon for providing RAR gamma deficient mice. We also thank C. Mendelsohn for helpful discussions.
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FOOTNOTES |
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Present address: INSERM U539, CHU Hotel Dieu, 44000 Nantes, France.
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REFERENCES |
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