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Molecular and Cellular Biology, April 2008, p. 2626-2636, Vol. 28, No. 8
0270-7306/08/$08.00+0     doi:10.1128/MCB.01575-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Phosphorylation of Liver X Receptor {alpha} Selectively Regulates Target Gene Expression in Macrophages{triangledown} ,{dagger}

Inés Pineda Torra,1 Naima Ismaili,1 Jonathan E. Feig,2 Chong-Feng Xu,4 Claudio Cavasotto,3,{ddagger} Raluca Pancratov,4 Inez Rogatsky,5 Thomas A. Neubert,4 Edward A. Fisher,2 and Michael J. Garabedian1*

Departments of Microbiology and Urology,1 Medicine and Cell Biology,2 Pharmacology and the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016,4 MolSoft LLC, La Jolla, California 92037,3 Hospital for Special Surgery, Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 100215

Received 27 August 2007/ Returned for modification 26 September 2007/ Accepted 28 January 2008


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ABSTRACT
 
Dysregulation of liver X receptor {alpha} (LXR{alpha}) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXR{alpha} target gene selectivity is achieved by modulation of LXR{alpha} phosphorylation. Under basal conditions, LXR{alpha} is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXR{alpha} S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXR{alpha} S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXR{alpha}-responsive genes.


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INTRODUCTION
 
The liver X receptors (LXRs) are nuclear receptors that are activated by oxysterol cholesterol metabolites (67) and by a number of synthetic nonsteroidal agonists (26, 46, 48). Two different genes have been described, LXR{alpha} (NR1H3) and LXRβ (NR1H2). LXR{alpha} expression is restricted to macrophages and tissues involved in lipid metabolism, whereas LXRβ is more ubiquitous (67). LXRs heterodimerize with the 9-cis retinoid X receptor (RXR) and bind to a DNA motif termed the LXR response element (LXRE) (62).

LXRs regulate cholesterol homeostasis by modulating the transcription of genes involved in its catabolism, storage, absorption, and transport. LXR expression and activation with ligand have also been shown to modulate atherogenesis. LXR{alpha}/β-deficient mice show enhanced lipid-loaded foam cell accumulation (47). Macrophage-specific ablation of LXR increases, whereas its activation decreases, atherosclerotic lesions (28, 52). By regulating the expression of the membrane ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, LXR controls the efflux of free cholesterol from lipid-laden cells (6, 67). Additionally, synthetic LXR agonists reduce atherosclerosis progression in mouse models (26), which correlates with the induction of genes involved in cholesterol transport (67).

LXR ligands may also reduce atherosclerosis by limiting the production of inflammatory mediators in the artery wall. Activated LXR inhibits the expression of macrophage inflammatory genes and reduces inflammation in vivo (24). LXR signaling in macrophages is also crucial for antimicrobial responses, and LXR{alpha}-regulated expression of the apoptosis inhibitor expressed in macrophages (AIM) appears to mediate macrophage survival upon bacterial infection (25, 53).

Moreover, LXR activation leads to increased hepatic lipogenesis and plasma triglyceride levels via the induction of the sterol regulatory element-binding protein 1c (SREBP-1c) (46). Elevated triglyceride concentrations are considered an independent risk factor for atherosclerosis (3), and this represents an important obstacle in the pharmacological development of LXR agonists as therapeutic agents. Ideally, clinically relevant LXR activators would be tissue- and gene-specific modulators with favorable coronary antiatherogenic and anti-inflammatory properties void of the less favorable hepatic lipogenic effects. Because both LXR isotypes are able to modulate the expression of genes involved in the cholesterol efflux pathway in macrophages and LXR{alpha} is considered the predominant isotype controlling hepatic lipogenesis, a popular approach has been to develop LXRβ-selective ligands (30, 39). We hypothesized as an alternative approach that modulation of posttranslational modifications of the receptor, such as phosphorylation, may finely tune LXR{alpha} actions in a gene-specific manner, as has been suggested for other nuclear receptors (8).

Nuclear receptor activity can be regulated by phosphorylation (41, 61). Previous studies suggested that different signaling pathways may contribute to LXR{alpha} phosphorylation (22, 32, 50). Here, we provide evidence of selective regulation of gene expression by modulating LXR{alpha} phosphorylation at serine 198 (S198) in macrophages. LXR{alpha} is phosphorylated in cultured macrophages, as well as in macrophages of atherosclerotic lesions. We also show that LXR{alpha} phosphorylation at S198 in macrophages modulates its transcriptional activity and restricts the repertoire of LXR{alpha}-responsive genes, revealing a novel role for phosphorylation in LXR{alpha} function. This could be exploited for the development of therapeutic agents against a number of metabolic diseases.


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MATERIALS AND METHODS
 
Plasmids and site-directed mutagenesis. A FLAG tag was introduced N-terminal to the human LXR{alpha} (hLXR{alpha}) cDNA on the pcDNA3-hLXR{alpha} template by PCR using primers listed in Table S1 in the supplemental material and subcloned into pcDNA3 to yield pcDNA3-FLAG-hLXR{alpha}. A mutagenesis kit (Stratagene) was used to introduce a serine-to-alanine mutation into S198 of LXR{alpha}. LZRSpBMN-GFP retroviral vectors carrying the FLAG-tagged wild-type (WT) or S198A mutant LXR{alpha} cDNA were generated. DNA was sequenced to confirm all constructs.

Mass spectrometry. 293T cells were transfected with pcDNA3-FLAG-hLXR{alpha} and treated with T0901317 (1 µM), vehicle (dimethyl sulfoxide), or 20% fetal bovine serum (FBS) for 18 h. The cells were lysed, and LXR{alpha} was immunoprecipitated with anti-FLAG antibody M2 agarose (Sigma), eluted with a FLAG3 peptide (Sigma), and resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The stained LXR{alpha} band was excised, digested with trypsin, and subjected to mass spectrometry (see the supplemental material). Negative charges on acidic groups were neutralized by methyl esterification (31, 63) before mass spectrometry in positive- and negative-ion modes using a Waters matrix-assisted laser desorption ionization quadrupole-time of flight Ultima mass spectrometer. Tandem mass spectra of the LXR{alpha} phosphopeptide in positive-ion mode were acquired to confirm the identity of the phosphopeptide and to determine the phosphorylated amino acids.

Cell culture, retrovirus production, and infection. 293T and RAW 264.7 cells were obtained from the ATCC and maintained in Dulbecco's modified Eagle's medium with 10% FBS and 20 µg/ml gentamicin. Recombinant retroviruses were produced by transfecting LZRSpBMN-GFP, LZRSpBMN-GFP/LXR{alpha}, or LZRSpBMN-GFP/S198A into 293GP cells as described previously (66). Virus-containing supernatants were overlaid on RAW cells as described previously (15). Cells infected with either the retroviral vector devoid of an LXR{alpha} sequence (RAW-VO [vector only]), the FLAG-tagged WT LXR{alpha} (RAW-LXR{alpha}), or mutant S198A (RAW-S198A) were sorted for green fluorescent protein expression by fluorescence-activated cell sorting. These cell lines therefore represented a pool of multiple LXR{alpha}-expressing clones. For phosphorylation experiments, cells were serum starved (1% FBS) overnight and incubated with either vehicle (dimethyl sulfoxide) or T0901317 for 2 h prior to cell lysis. For cholesterol loading, cells were incubated with soluble cholesterol-cyclodextrin complexes as described previously (43). Cholesterol accumulation was verified by measuring total and free cholesterol (43). The cholesteryl ester content was 0.5 µg/mg protein in control cells versus 7.1 µg/mg protein in cholesterol-treated cells. For mRNA analysis, cells were cultured in medium with 10% lipoprotein-deficient serum (Sigma).

Antibody production. An hLXR{alpha} S198 phosphopeptide corresponding to amino acids (aa) 189 to 202 (189-ATSLPPRASS[PO4]PPQI-202) was synthesized (Anaspec) and used to immunize rabbits (Covance). Serum that displayed high phosphospecificity was selected and affinity purified. For dephosphorylation of LXR{alpha}, proteins were separated and transferred, and membranes were incubated in phosphatase buffer without or with 400 units of lambda phosphatase (Upstate) at 4°C for 16 h. The membranes were probed with the phospho-S198 antibody, stripped, and reprobed with a FLAG polyclonal antibody. Antibody recognizing total LXR{alpha} (phosphorylated and nonphosphorylated) was generated as described above using an hLXR{alpha} peptide, 198-SPPQILPQLSPEQL-211. High-titer antibodies were affinity purified and tested for LXR{alpha} immunoreactivity (see Fig. S2C in the supplemental material).

Cell extracts, immunoprecipitation, and Western blot analysis. Whole-cell extracts were prepared as described previously (58). Nuclear and cytoplasmic extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce). FLAG-LXR{alpha} proteins were immunoprecipitated with a FLAG M2 monoclonal antibody agarose. Protein extracts were analyzed by immunoblotting as described previously (58). Phospho-Ser198, monoclonal M2, or polyclonal FLAG (Sigma); CK{alpha} polyclonal (Cell Signaling; catalog no. 2656) and CKβ monoclonal (Affinity Bioreagents; catalog no. MA1-5004); {alpha}-tubulin monoclonal (Covance; catalog no. MMS-489P); and hsp90 monoclonal (BD Transduction Laboratories; catalog no. 610418) antibodies were used at 1: 2,000 dilutions. For nuclear receptor corepressor (NCoR) coimmunoprecipitation assays, RAW-LXR{alpha} or RAW-S198A cells treated with vehicle or T0901317 for 2 h were cross-linked for 20 min in phosphate-buffered saline with 1.6 mM DSP [dithiobis(succinimidyl)propionate] (Pierce) prior to whole-cell extract preparation. FLAG-LXR{alpha} proteins were immunoprecipitated with a FLAG M2 monoclonal antibody agarose, and protein extracts were analyzed by immunoblotting them with a specific antibody to NCoR (Affinity Bioreagents; catalog no. PA1-844A).

Animals and diets. Animal procedures were approved by the IACUC. apoE–/– mice in a C57BL6 background were weaned at 4 weeks of age onto a "Western-type diet" (21% fat, 0.15% cholesterol; Dyets, Inc.) for 4 months to develop advanced aortic lesions. The mice were euthanized, and the aortic roots were excised.

Immunohistochemistry. The aortic roots were fixed in buffered formalin and embedded in paraffin blocks. Serial 4-µm-thick tissue sections were mounted and stained as described previously (51). Affinity-purified anti-phospho-Ser198 (1:100) or anti-LXR{alpha} (1:250) antibodies were employed, and samples were visualized with 3,3'-diaminobenzidine. For assessment of the macrophage content, sections were incubated with the macrophage-specific antibody CD68 (1:200). Slides were counterstained with hematoxylin, dehydrated, and mounted with Permount (Dako Corp.).

Molecular modeling. Modeling of peptides was performed at MolSoft as described previously (29). Global energy minimization simulations were performed with two peptides, one with S198 phosphorylated and one without: 187-AHATSLPPARSSPPQILPQLSPEQLGMIEKLVAA-220. For each peptide, 40 parallel independent biased-probability Monte Carlo (1) global energy optimizations were performed, starting from randomized conformations. The simulations of each peptide were terminated after 200 million energy evaluations. During the simulations, low-energy conformations were merged and sorted by energy, and redundant conformations were eliminated.

Real-time PCR. Total RNA from RAW 264.7 cells was extracted, and cDNA was synthesized and amplified on a LightCycler (Roche) as described previously (38). The primers are listed in Table S1 in the supplemental material.

Mouse primary macrophage isolation and culture. Bone marrow-derived or peritoneal macrophages were prepared from SV129 x C57BL/6 mice as described previously (4). Tibias and femurs were isolated, and the bone marrow was flushed out with Dulbecco's modified Eagle's medium. The cells were seeded in RPMI-10% FBS and supplemented with 10 ng/ml macrophage colony-stimulating factor (Peprotech). After 7 days in culture, the bone marrow-derived macrophages were treated with T0901317 (5 µM) for 4 h and harvested for LXR{alpha} protein analysis.

Cloning, expression, and purification of glutathione S-transferase (GST)-LXR{alpha} (161 to 448). Residues 161 to 448 of the hLXR{alpha} protein were amplified by PCR using primers listed in Table S1 in the supplemental material. The PCR fragment was cloned into the BamHI-XhoI sites of the pGEX-4T-1 vector. The fidelity of the cloning was checked by sequencing and restriction enzyme analysis. Proteins were expressed in transformed Escherichia coli BL21(DE3)pLysS grown in LB and purified and eluted in 10 mM reduced glutathione buffer as described previously (65). The proteins were quantitated by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining using bovine serum albumin as a standard.

In vitro kinase assays. Recombinant CK2{alpha} subunit purified from Sf9 cells (Cell Signaling) was used to phosphorylate GST-LXR{alpha} (161 to 448) as described by the manufacturer. GST-LXR{alpha} (161 to 448) (2 µg as a 50% GST bead slurry) was incubated at 30°C in kinase buffer containing 60 mM HEPES-NaOH, pH 7.5, 200 µM ATP, 3 mM MgCl2, 3 mM MnCl2, and 1.2 mM dithiothreitol for 10 to 20 min. The reaction was terminated by adding SDS buffer and boiling the mixture for 5 min. Phosphorylated LXR{alpha} was detected with the phospho-S198 antibody, stripped, and reprobed with a polyclonal LXR{alpha} antibody.

ChIP. Assays were performed as described previously (34) with the following modifications. After treatments (2 h), the cells were washed twice in phosphate-buffered saline and first cross-linked for 20 min at room temperature with 1.6 mM DSP (Pierce) before formaldehyde cross-linking. The sonication and immunoprecipitation buffer consisted of 10 mM Tris, 1 mM EDTA, 140 mM NaCl, 5% glycerol, 1% Triton X-100, and 1x freshly added protease inhibitor cocktail (Sigma). Washes were performed as described in the Upstate chromatin immunoprecipitation (ChIP) protocol, except that the buffers did not contain any SDS detergent. DNA (2 µl) was amplified by real-time PCR using the Sybr green JumpStart Taq Ready Mix (Sigma). Primers used to amplify the mouse ABCA1 or SREBP1c LXRE or the mouse CCL24 LXR binding region are listed in Table S2 in the supplemental material. Immunoprecipitations were performed with the following antibodies: anti-NCoR polyclonal (ABR), anti-FLAG M2 agarose (Sigma), anti-acetylated H3 (Lys9 and Lys14) (Milllipore), anti-p300 (C-20) (Santa Cruz), anti-Pol II clone 8WG16 (Covance), and nonimmune immunoglobulin G (Sigma).

Small interfering RNA (siRNA) transfection. RAW-LXR{alpha} (WT) cells (4 x 106) were transfected with 4 µg of either the On-Targetplus siControl nontargeting pool or the On-Targetplus Smartpool for mouse NCoR (Dharmacon) with the Amaxa Nucleofector and solution V, as described by Reily et al. (40). The cells were then plated, and after 36 h, the cells were incubated with vehicle or agonists for another 24 h before being harvested.


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RESULTS
 
LXR{alpha} is phosphorylated in cholesterol-loaded macrophages in vitro and in atherosclerotic lesions in vivo. To identify sites of LXR{alpha} subjected to phosphorylation in vivo, we immunoprecipitated ectopically expressed N-terminally FLAG-tagged hLXR{alpha} proteins from HEK293T cells for tandem mass spectrometry analysis. We identified a phosphorylation site on LXR{alpha} at S198 located in the hinge region of the receptor (Fig. 1A; see Fig. S1A and B in the supplemental material). This residue is conserved between human, rat, and mouse LXR{alpha} proteins, suggesting possible conservation of function (see Fig. S1C in the supplemental material). In contrast, LXRβ lacks a corresponding phosphorylation motif, indicating that different signaling pathways may modulate the two LXRs (see Fig. S1D in the supplemental material).


Figure 1
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  		FIG. 1. LXR{alpha} is phosphorylated in cholesterol-loaded macrophages in vitro and in vivo. (A) Domain structure of hLXR{alpha} with the S198 phosphorylation site depicted in red. (B) RAW 264.7 cells infected with a retroviral vector only (VO) or stably expressing WT or S198A FLAG-hLXR{alpha} were treated with 40 µg/ml cholesterol (Chol) in the form of soluble cholesterol-cyclodextrin complexes for 48 h. Nuclear extracts were prepared, and LXR{alpha} was immunoprecipitated with a FLAG antibody. Phosphorylated (P-LXR{alpha}) and total LXR{alpha} were detected by immunoblotting with a P-S198 and FLAG antibody, respectively. Experiments were performed three times with similar results. A representative blot is shown. (C) Aortic-root sections of apoE–/– mouse lesions were immunostained with antibodies against total (LXR{alpha}) and phospho-S198 (P-LXR{alpha}) LXR{alpha} as described in Materials and Methods. Total and P-S198-LXR{alpha} are prominent in macrophage foam cell-rich areas of the atherosclerotic lesion (solid arrows). Staining of LXR{alpha} was also observed in the SMCs of the medium (open arrows). Sections were examined for macrophage content with a CD68 antibody. No staining was evident when the primary antibody was omitted (Neg. CTRL). Objective magnification, x200 (x400 for the insets).

To further analyze LXR{alpha} phosphorylation in vitro and in vivo, we generated antisera specifically recognizing the S198 phosphorylation site within LXR{alpha}. Specific immunoreactivity of the phospho-S198 antibody was demonstrated by its ability to recognize WT phosphorylated LXR{alpha}, but not phosphorylation-deficient S198A LXR{alpha} (see Fig. S2A in the supplemental material). In contrast, the phospho-S198 antibody efficiently recognized LXR{alpha} carrying the serine- or threonine-to-alanine mutations S207A and T236A (see Fig. S2A in the supplemental material), indicating specificity for the S198 site. Additionally, when LXR{alpha} was dephosphorylated with lambda phosphatase, the phospho-S198 antibody failed to recognize the LXR{alpha} protein (see Fig. S2B in the supplemental material). Thus, we had developed a novel antibody that specifically recognized the phospho-S198 site within LXR{alpha}.

Given the major role LXR{alpha} plays in modulating cholesterol metabolism and efflux in lipid-engorged macrophages, we next investigated LXR{alpha} phosphorylation in cholesterol-loaded macrophages. Pools of cell clones stably expressing either WT or S198A-mutated LXR{alpha} were generated by retroviral infection of RAW 264.7 cells, which lack endogenous LXR{alpha} but express LXRβ. Nuclear extracts from LXR{alpha}-expressing RAW 264.7 cells loaded with cholesterol showed enhanced WT (and not S198A) LXR{alpha} phosphorylation compared to untreated cultures (Fig. 1B). We then assessed the expression of total LXR{alpha} protein in the atherosclerotic plaque of the apoE-deficient-mouse model of atherosclerosis by immunohistochemistry. Total LXR{alpha} antibody (see Fig. S2C in the supplemental material) markedly stained macrophage-rich regions (Fig. 1C) within the plaques present in the aortic roots from apoE–/– mice fed a cholesterol-rich diet. Some staining was also observed in smooth muscle cells (SMCs) (Fig. 1C), which is in agreement with reports showing the expression of LXR{alpha} in this cell type (7, 11). Phospho-S198 staining was detectable in macrophages of the atherosclerotic lesion and, albeit to a lower extent, in SMCs (Fig. 1C). Thus, both LXR{alpha} ectopically expressed in cultured cholesterol-loaded macrophages and endogenous LXR{alpha} within loaded foam cells of the atherosclerotic lesion were phosphorylated. These findings suggest that phosphorylation of the receptor may modulate LXR{alpha} transcriptional actions in the atherosclerotic lesion, which in turn may affect receptor target gene expression.

LXR{alpha} phosphorylation is induced by ligand. Given that nuclear receptor phosphorylation is frequently ligand regulated (61), we investigated whether that is the case for LXR{alpha}. As shown in Fig. 2A, phosphorylation of S198 occurs in the absence of ligand and is enhanced by the LXR agonist T0901317 only in RAW 264.7 cells expressing the WT receptor. Induction of S198 LXR{alpha} phosphorylation was detectable at 0.1 µM T0901317 (peaking at 1 µM; data not shown) and within 1 h of treatment and was sustained for at least 8 h (Fig. 2B). Phosphorylation was also induced by the LXR oxysterol ligand 24S,25-epoxycholesterol (EC), but not by the RXR ligand 9-cis-retinoic acid (9cRA) (Fig. 2C). Additionally, basal and ligand-induced phosphorylation of endogenous LXR{alpha} at S198 was observed in primary bone marrow-derived macrophages (see Fig. S2D in the supplemental material). These data indicate that LXR phosphorylation at S198 is present under basal conditions and is enhanced by agonists in a dose- and time-dependent manner, suggesting that phosphorylation at this site may modulate LXR{alpha} transcriptional activities.


Figure 2
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  		FIG. 2. LXR ligands induce phosphorylation of LXR{alpha} at S198. (A) RAW cells stably expressing FLAG-hLXR{alpha} (WT or S198A) were incubated with or without 5 µM T0901317 ligand for 2 h. Extracts were prepared, and LXR{alpha} was detected as described in the legend to Fig. 1B using a phospho-S198 (P-LXR{alpha}) or FLAG antibody as a measure of total LXR{alpha}. (B) The T0901317 ligand induces LXR{alpha} phosphorylation in a time-dependent manner. RAW-LXR{alpha} cells were incubated with 5 µM T0901317 (T) ligand for the times shown, and phosphorylation was analyzed by immunoblotting as described above. (C) LXR{alpha} phosphorylation is induced by oxysterol ligands. RAW-LXR{alpha} cells were treated with either T0901317 (T1317) (1 µM), 9cisRA (1 µM), or 24S EC (10 µM). LXR{alpha} was immunoprecipitated with FLAG antibody, and phosphorylation was analyzed by immunoblotting as described for panel A.

Endogenous LXR{alpha} target genes are sensitive to S198-LXR{alpha} phosphorylation. To determine whether phosphorylation at S198 influences LXR{alpha}-mediated gene regulation, we examined the expression of well-characterized LXR target genes in RAW 264.7 cells stably transduced with either the WT LXR{alpha}-expressing or the empty retroviral vector (VO). Consistent with previous reports (25, 27, 55), expression of LXR{alpha} in RAW 264.7 cells enhanced the induction of the LXR ABCA1, AIM, and SREBP1c target genes in response to LXR ligands (see Fig. S3 in the supplemental material). It should be noted that endogenous LXRβ in these cells may be sufficient to drive the ligand-dependent expression of genes such as the ABCA1 gene (55).

Next, to determine the impact of S198 phosphorylation on LXR{alpha} function, we investigated LXR target gene expression in RAW 264.7 cells stably expressing the phosphorylation mutant S198A. RAW-S198A macrophages showed a dramatically enhanced ligand-dependent expression of LPL and AIM compared to the WT LXR{alpha}-transduced cells (Fig. 3A), indicating that S198 phosphorylation exerts an inhibitory effect on the LXR{alpha}-regulated transcription of these genes. In contrast, transcript levels of ABCA1, SREBP1c, ABCG1, and PLTP (phospholipid transfer protein) were similar in both WT and S198A LXR{alpha}-expressing cells (Fig. 3A and data not shown), indicating that the effect of S198 phosphorylation on LXR{alpha} transcriptional activity is gene specific. The differential regulation of target genes by WT versus S198A LXR{alpha} was also observed when LXR{alpha} was activated by the oxysterol ligand EC (see Fig. S3 in the supplemental material). Consistent with the gene expression data, the activities of WT and S198A receptors were similar when tested in transient-transfection experiments on an ABCA1 promoter fragment containing the LXRE (reference 10 and data not shown). Furthermore, the S198A mutant displayed no major difference in expression (Fig. 3B) or subcellular localization (see Fig. S3C in the supplemental material) compared to the WT and thus cannot account for the observed differential gene expression. Together, these results indicate that LXR{alpha} phosphorylation can selectively affect the transcription of LXR-responsive genes.


Figure 3
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  		FIG. 3. LXR{alpha} S198 phosphorylation affects the expression of specific target genes. (A) RAW-LXR{alpha} (WT or S198A) cells were incubated with vehicle or T0901317 ligand (T) at the indicated concentrations for 24 h. Transcripts were analyzed by real-time reverse transcription-PCR. The values indicate expression of target genes normalized to cyclophilin (CYCLO) mRNA levels and are presented relative to the expression in WT vehicle-treated cells, which was set as 1. Experiments were repeated three times with similar results. A representative experiment is shown. (B) Protein expression levels of LXR{alpha} in RAW 264.7 cells infected with a retroviral vector only (VO) or FLAG-hLXR{alpha} (WT or S198A) incubated in the presence or absence of T0901317, as in panel A. Whole-cell extracts were analyzed by immunoblotting them with FLAG antibody (LXR{alpha}) and with an antibody against hsp90 to monitor protein loading.

Molecular modeling of LXR{alpha} phosphorylation at S198. We next characterized the possible effect of S198 phosphorylation on the structure of LXR{alpha}. To date, only the LXR{alpha} ligand binding domain (aa 207 to 447) crystal structure in a heterodimer with RXRβ has been solved (49). However, S198 is located in the hinge region of LXR{alpha}, close to the reported LXR{alpha} corepressor interaction surface (aa 262 to 291) (21), and phosphorylation in the hinge region of at least one other nuclear receptor, SF-1, has been shown to modulate the interaction with coactivators and corepressors (12, 20). Using a computational structure prediction approach, extensive global energy minimization simulations were performed with the aa 187-to-220 peptide with and without a phosphate group at S198. Due to computational constraints, we can model only 25-residue peptides, which do not account for potential tertiary contacts from other parts of the receptor. This approach, however, can provide insight into local structural changes that occur upon S198 phosphorylation and has been successfully employed to determine local tertiary structures of WT and mutated androgen receptor peptides (29). The predicted ensemble of equilibrium conformations of the nonphosphorylated peptide exhibits a mainly disordered structure, in agreement with previous reports (59), with a slightly structured conformation toward the N terminus (Fig. 4, left). Interestingly, phosphorylation of S198 appears to induce a marked trend toward a more structured conformation at the C terminus of the peptide, with the peptide adopting a helical structure (Fig. 4, right). Hence, phosphorylation of S198 is predicted to induce a structural change in the LXR{alpha} hinge domain, which we hypothesize could influence cofactor recruitment, ultimately modulating LXR{alpha} activity.


Figure 4
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  		FIG. 4. LXR{alpha} S198 phosphorylation is predicted to result in structural changes in LXR{alpha}. Shown are representative low-energy conformations of the nonphosphorylated (WT) and phosphorylated (P-S198) peptides encompassing the S198 site. Peptides are colored blue (N-term) to red (C-term), and S198 is depicted. Note that the phosphorylated peptide displays a more structured C terminus.

The phosphorylation status of LXR{alpha} determines expression of CCL24. Our molecular-modeling experiments suggest that phosphorylation causes structural changes in LXR{alpha}. We therefore hypothesized that there may exist genes whose expression is regulated exclusively by the nonphosphorylated receptor and investigated the expression of genes that were previously proposed to be LXR{alpha} targets but were subsequently found to be unaffected by LXR ligands. One such gene is that for the chemokine CCL24, whose expression in the liver is LXR{alpha} dependent upon Listeria infection but is not regulated by ligand exposure (25), suggesting that the signaling pathways triggered by infection versus LXR ligands modulate the receptor in a different manner or that CCL24 is not a traditional LXRE-bearing target gene. Consistent with previous reports that differentiated macrophages produce little CCL24 (60), its expression was nearly undetectable in either RAW-VO or RAW-LXR{alpha} WT cells (cycle number above 40) and was largely unaffected by T0901317 (Fig. 5A). Remarkably, in RAW-S198A macrophages, CCL24 basal expression was markedly induced compared to WT LXR{alpha}-expressing cells (Fig. 5A) and was further dramatically increased by T0901317 in a dose-dependent manner (Fig. 5B). These results demonstrate that in RAW264.7 macrophages, CCL24 regulation by LXR{alpha} is determined by the receptor phosphorylation status and that lack of S198 phosphorylation allows specific gene activation.


Figure 5
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  		FIG. 5. LXR{alpha} phosphorylation status determines expression of CCL24. RAW-VO (VO) and RAW-LXR{alpha} (WT or S198A) cells were incubated for 24 h in medium with vehicle or 1 µM (A) or the indicated concentrations (B) of T0901317 LXR ligand. CCL24 transcripts were analyzed by real-time reverse transcription-PCR. The values indicate expression levels of CCL24 normalized to cyclophilin (CYCLO) levels and are presented relative to the CCL24 expression in WT vehicle-treated cells, which was set as 1.

Inhibition of CK2-mediated LXR{alpha} S198 phosphorylation induces CCL24 expression upon ligand activation. To identify the LXR{alpha}-targeting kinase(s), T0901317 ligand-dependent S198 phosphorylation was examined in the presence or absence of a panel of kinase inhibitors. Pretreatment with staurosporine (a broad-spectrum kinase inhibitor) or 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT) (a specific inhibitor of CK2) (35) reduced both basal and ligand-induced phosphorylation of LXR{alpha} at S198 (Fig. 6A and not shown). In contrast, mitogen-activated protein kinase (U1026), protein kinase A (PKA) (H89), and phosphatidylinositol 3-kinase (LY294002) inhibitors did not affect phosphorylation of LXR{alpha} irrespective of ligand presence (Fig. 6A and not shown). CK2{alpha} also phosphorylated LXR{alpha} at S198 in vitro (see Fig. S4 in the supplemental material), suggesting that CK2 may be a bona fide S198 kinase. Consistent with the idea that reduced phosphorylation of LXR{alpha} modulates the expression of certain phosphorylation status-dependent target genes, such as CCL24, inhibition of WT LXR{alpha} phosphorylation by the CK2 inhibitor DMAT potently increased T0901317-dependent expression of CCL24 (Fig. 6B). The induction of CCL24 expression by DMAT was not observed in RAW-S198A cells, indicating that this effect is due to LXR{alpha} dephosphorylation by the CK2 inhibitor. These results further demonstrate that CCL24 expression in RAW cells is dependent on the phosphorylation status of LXR{alpha}.


Figure 6
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  		FIG. 6. CK2 inhibitors reduce LXR{alpha} phosphorylation at S198 and trigger regulation of CCL24 expression by LXR ligands. (A) Basal and ligand-induced phosphorylation of LXR{alpha} is reduced by the CK2 inhibitor DMAT. RAW-LXR{alpha} cells were incubated for 1 h with kinase inhibitors (10 µM LY294002 [phosphatidylinositol 3-kinase], 10 µM H89 [PKA], staurosporine [Staurosp.] [a broad-spectrum inhibitor], and 10 µM DMAT [CK2]) and then treated with 5 µM T0901317 for 2 h (CTRL, control non-kinase inhibitor-treated cells). LXR{alpha} was immunoprecipitated with a FLAG antibody. Phosphorylated and total LXR{alpha} were detected by immunoblotting them with a P-S198 and a FLAG antibody, respectively. (B) CCL24 is regulated by the T0901317 LXR ligand upon inhibition of LXR{alpha} phosphorylation. RAW-LXR{alpha} cells were treated for 1 h with the CK2-specific inhibitor DMAT (10 µM) prior to incubation with T0901317 (T) (1 µM) for 16 h. CCL24 transcripts were analyzed and presented as in Fig. 4. CYCLO, cyclophilin.

The RXR ligand 9cRA inhibits ligand-induced phosphorylation of LXR{alpha} and confers transcriptional regulation of CCL24. RXR is thought to be a permissive heterodimeric partner of LXRs (62), and RXR ligands enhance the effect of LXR agonists on LXR target genes. Recently, in the case of the vitamin D receptor (VDR)/RXR heterodimer, the RXR ligand 9cRA was shown to supplement the agonistic activities of a weak VDR ligand and to restore transcriptional activity to a VDR ligand binding domain mutant (44). Thus, we hypothesized that 9cRA can confer LXR ligand responsiveness to CCL24. Indeed, in the presence of the RXR ligand 9cRA, CCL24 induction by T0901317 in the RAW LXR{alpha} cells was markedly enhanced (Fig. 7A), reaching expression levels similar to those observed in untreated RAW-S198A cells (see Fig. S5 in the supplemental material). Expression of other LXR target genes, such as ABCA1, was not influenced in a similar manner by the addition of 9cRA (see Fig. S5B in the supplemental material). Concomitantly with enhanced CCL24 expression, 9cRA reduced the level of T0901317-dependent S198 phosphorylation (Fig. 7B). This synergistic activation of CCl24 expression in the presence of 9cRA and the concomitant inhibition of LXR{alpha} phosphorylation were also observed when the RXR selective ligand bexarotene was employed (see Fig. S6 in the supplemental material), indicating that these effects are mediated by RXR. Importantly, the synergistic action of T0901317 and 9cRA on CCL24 expression was lost in RAW-S198A cells (16-fold versus 2.7-fold over T0901317 alone in WT and S198A cells, respectively), suggesting that it is S198 phosphorylation that mediates this effect (see Fig. S5A in the supplemental material). Thus, RXR agonists reduce LXR{alpha} S198 phosphorylation, which is translated into changes in LXR{alpha}/RXR-regulated gene expression of phosphorylation-sensitive genes, such as the CCL24 gene.


Figure 7
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  		FIG. 7. RXR ligands induce CCL24 expression by inhibiting LXR{alpha} phosphorylation in RAW cells. (A) RXR ligands, in combination with LXR ligands, synergistically induce CCL24 expression in RAW cells. RAW-LXR{alpha} (WT) cells were treated with vehicle or 1 µM T0901317 in the absence or presence of 1 µM 9cRA. CCL24 expression was analyzed by real-time reverse transcription-PCR. The values indicate expression levels of CCL24 normalized to cyclophilin (CYCLO) mRNA levels and are presented relative to the expression in vehicle-treated cells, which was set as 1. The error bar indicates the standard deviation. (B) RXR ligands inhibit LXR{alpha} phosphorylation in RAW cells. RAW-LXR{alpha} cells were cultured for 3 h in the absence or presence of either T0901317 (T) (5 µM), 9cRA (1 µM), or a combination of T0901317 (5 µM) and increasing concentrations of 9cRA (0.1, 0.5, and 1 µM). Nuclear extracts were prepared, and phosphorylation (P-LXR) and the total level (LXR) of LXR{alpha} were detected by immunoblotting, as described in the legend to Fig. 1. hsp90 antibody was used to visualize protein loading. (C and D) LXR and RXR ligands modulate CK2{alpha} and CK2β localization in RAW cells. RAW-LXR{alpha} cells were cultured as described in the legend to Fig. 6B. Nuclear and cytoplasmic extracts were prepared, and CK2{alpha} (C) and CK2β (D) levels were detected by immunoblotting. hsp90 antibody was used to ensure equal protein loading (data not shown), and quantification by densitometry of nuclear versus cytoplasmic CK2{alpha} (C) and nuclear CK2β (D) normalized to hsp90 levels is presented. One representative experiment is shown. The experiment was repeated twice with similar results.

Regulation of CK2{alpha} and CK2β expression by LXR/RXR ligands. To gain more insight into the mechanism of the modulation of LXR{alpha} phosphorylation by RXR ligands, we investigated the possible effects of T0901317 and 9cRA on CK2. CK2 consists of two catalytic subunits ({alpha} and {alpha}') and a regulatory subunit (β) existing as an {alpha}2β2, {alpha}{alpha}'β2, or {alpha}'2β2 configuration (14). CK2 activity is proportional to the expression of its subunits (18). In addition, it has recently been shown that nucleocytoplasmic shuttling is a major mode of regulating CK2 activity and specificity (57). Therefore, we first explored the subcellular distribution of the CK2 {alpha} and β subunits. CK2{alpha} was present in the nucleus and cytoplasm of RAW-LXR{alpha} cells (Fig. 7C), and incubation with T0901317 ligand did not change the distribution of the subunit. However, addition of increasing concentrations of 9cRA- to T0901317 ligand-treated cells led to a slight shift of CK2{alpha} from the nucleus to the cytoplasm. As CK2{alpha} catalytic subunit phosphorylates LXR{alpha} in vitro (see Fig. S4 in the supplemental material), these findings suggest that its subunit redistribution could in part account for the reduction in T0901317 ligand-dependent LXR{alpha} phosphorylation in response to 9cRA. The β regulatory subunit was found only in the nucleus (Fig. 7D), consistent with the reported asymmetrical distribution of different subunits within the same cell (14, 45). Remarkably, the amount of the β subunit in the nucleus was increased by the T0901317 ligand and reduced by 9cRA in a dose-dependent manner. Thus, the nuclear concentration of the β subunit correlates with LXR{alpha} phosphorylation levels. Given that formation of the CK2 tetrameric structure imparts maximal kinase activity, it is tempting to speculate that the β subunit is involved in the ligand-modulated phosphorylation of LXR{alpha}. Collectively, our findings show that LXR/RXR ligands differentially affect the levels of active CK2 in the nucleus, which are associated with the modulation of LXR{alpha} phosphorylation and function.

Identification of an LXR/RXR-responsive region in the CCL24 gene. Regulatory regions in the CCL24 gene have not been described. Therefore, we next aimed to identify an LXR/RXR ligand-responsive region in the CCL24 gene that could mediate the observed changes in its transcript levels. Acetylation of histones, and histone 3 (H3) in particular, are known epigenetic marks that define active transcriptional-regulatory regions, such as promoters and enhancers (42). Therefore, we analyzed H3 acetylation of the mouse CCL24 gene by ChIP throughout 10 kb of genomic sequence from 7.0 kb upstream of the transcription start site to 2 kb downstream of the stop codon. This ChIP scanning approach identified a region located 1.6 kb downstream of the stop codon (d1.6) of the CCL24 gene that displayed the highest H3 acetylation in RAW-LXR{alpha} cells upon LXR/RXR ligand activation (Fig. 8A and data not shown). This region showed no increase in H3 acetylation in response to T0901317 alone (data not shown), suggesting that activation of both LXR and RXR is required for its H3 hyperacetylation.


Figure 8
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  		FIG. 8. Identification of an LXR/RXR-responsive region in the CCL24 gene. ChIP assays were employed to examine Pol II and p300 recruitment, H3 acetylation (AcH3), and LXR{alpha} binding to the CCL24 gene in a region located 1.6 kb downstream from the stop codon (d1.6). (A) RAW-LXR{alpha} (WT) cells were incubated with vehicle or 5 µM T0901317 in combination with 1 µM 9cRA for 2 h, and chromatin was prepared and processed as described in Materials and Methods. Precipitated DNA was amplified by real-time PCR using primers located between +1597 and +1694 (d1.6) and –6684 and –6597 (–7.0), normalized to input chromatin levels, and reported as induction (n-fold) relative to vehicle-treated samples (set as 1). Amplification of background immunoprecipitated DNA with preimmune immunoglobulin G is also shown. Samples were measured in triplicate, and the results are means ± standard deviations for a representative experiment. (B) LXR{alpha} binds to a sequence downstream of the CCL24 gene. LXR{alpha} binding to the 3' sequence of CCL24 was examined by ChIP in RAW-VO (VO) and RAW-LXR{alpha} (WT) cells treated as for Fig. 7A using antibodies against FLAG. For each treatment, LXR{alpha} binding in WT cells was normalized for input chromatin levels and reported as induction (n-fold) over signal in VO cells (which represents the background for FLAG antibody). As an additional negative control, an unrelated region 7 kb upstream of the start site (–7.0) was also amplified by PCR. DNA samples were measured in triplicate, and the results are means ± standard deviations for a representative experiment.

Enhancer elements act through the recruitment of multiprotein complexes, which include cofactors and RNA polymerase II (Pol II) (13, 33). To test whether the d1.6 region could represent an LXR/RXR-regulated region in the CCL24 gene, we first analyzed occupancy of the cofactor p300 and Pol II by ChIP. Similar to the LXRE-containing regions in the ABCA1 and SREBP1c LXR{alpha} target genes (see Fig. S7C and D in the supplemental material), both Pol II and p300 were recruited to the d1.6 region upon simultaneous LXR/RXR ligand treatment of RAW-LXR{alpha} (WT) cells (Fig. 8A). To determine whether LXR{alpha} binds to this sequence downstream of the CCL24 gene, we performed ChIP assays in RAW-VO (VO) and RAW-LXR{alpha} (WT) cells using antibodies against the FLAG tag on LXR{alpha} under conditions in which LXR{alpha} recruitment to the ABCA1 and SREBP1c LXREs was observed (see Fig. S7A and B in the supplemental material). At the d1.6 sequence of CCL24, LXR{alpha} was significantly enriched in WT LXR{alpha} cells, and this binding was enhanced upon treatment with LXR/RXR ligands compared to VO cells (Fig. 8B). These findings indicate that the combination of T0901317 plus 9cRA enhances CCL24 gene expression in RAW-LXR{alpha} cells, likely by inducing recruitment of the p300 coactivator and Pol II to the d1.6 region downstream of the CCL24 gene.

NCoR participates in the regulation of CCL24 gene expression by dephosphorylated LXR{alpha}. It has been shown that in the absence of ligand, LXR/RXR heterodimers bound to LXREs complex with corepressors, such as NCoR, thereby inhibiting target gene transcription (21). Recently, Ghisletti et al. defined a novel pathway by which ligand-induced sumoylated LXR is specifically targeted to promoters of proinflammatory genes, where it prevents the removal of NCoR complexes upon the addition of proinflammatory stimuli (16). This suggests that posttranslational modifications of LXR{alpha} may specially influence the binding and recruitment of NCoR to target gene promoters. We thus explored the possibility that activation of CCL24 gene expression by S198A is mediated by NCoR. As expected from previous reports, coimmunoprecipitation studies performed in RAW cells revealed that LXR{alpha} was bound to NCoR in the absence, but not in the presence, of T0901317 ligand (not shown). However, no detectable binding to the phosphorylation mutant S198A was observed, despite the fact that similar amounts of LXR{alpha} protein were immunoprecipitated and NCoR expression levels were similar in WT and S198A cells (not shown). Next, we investigated whether NCoR was differentially recruited by phosphorylated and nonphosphorylated LXR{alpha} to the endogenous CCL24 in the LXR{alpha} regulatory region. In RAW-LXR{alpha} WT cells, NCoR was recruited to the d1.6 region compared to an upstream region (–7.0 kb) that does not bind LXR{alpha}. NCoR binding was only slightly reduced upon addition of an LXR ligand and was more significantly reduced by combination with 9cRA (Fig. 9A), which induces LXR{alpha} dephosphorylation (Fig. 7). This suggests that NCoR recruitment to the CCL24 regulatory region is affected by changes in LXR{alpha} phosphorylation. In agreement with this observation, NCoR was barely present at the d1.6 region in RAW-S198A cells compared to RAW-LXR{alpha} WT cells under similar conditions. These data also indicate that in the absence of ligand, this transcriptional corepressor is present at higher levels on this regulatory sequence in the CCL24 gene in WT-expressing cells than in the S198A cells, which could explain, at least in part, higher CCL24 levels in cells expressing the phosphorylation-deficient mutant. This prompted us to speculate that inhibition of NCoR expression in RAW-LXR{alpha} cells would result in enhanced CCL24 expression. Reduction of NCoR levels in RAW-LXR{alpha} cells by NCoR-specific siRNA (Fig. 9D) led to an increase in the basal and T0901317 ligand-induced expression of CCL24 compared to cells transfected with control siRNA (Fig. 9C), indicating that NCoR is important for maintaining CCL24 expression repressed in RAW-LXR{alpha} cells. Consistent with our previous results, CCL24 levels were highest in cells with reduced expression of NCoR in the presence of T0901317 and 9cRA, when LXR{alpha} phosphorylation is also lower (Fig. 7). Reduction of NCoR expression significantly affected only basal ABCA1 levels (not shown), which is in agreement with previous observations that demonstrated that NCoR contributes to the derepression of ABCA1 basal expression (56). Similarly, mRNA levels of the AIM gene, another LXR{alpha} target gene whose expression is influenced by LXR{alpha} phosphorylation (Fig. 4A), were largely unaffected by NCoR siRNA transfection (not shown). This suggests that NCoR is an important component in the regulation of target gene expression by LXR{alpha} phosphorylation status in a promoter-dependent manner and that other factors are likely to play a role in the regulation of the different LXR{alpha} phosphorylation-sensitive target genes.


Figure 9
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  		FIG. 9. Impaired NCoR recruitment by dephosphorylated LXR{alpha} induces CCL24 expression. ChIP assays were performed using chromatin isolated from RAW-LXR{alpha} (WT) (A) or WT and RAW-S198A (S198A) cells (B) treated as described in the legend to Fig. 7. NCoR-specific antibody precipitates were isolated and subjected to PCR using primers against an unrelated region 7 kb upstream of the start site (–7.0) or the LXR binding region (d1.6). PCR results from immunoprecipitated samples were normalized to the PCR amplification of input chromatin. For each condition, samples were measured in triplicate, and the results are means ± standard deviations as percentage recruitment of NCoR at the d1.6 region compared to the non-LXR{alpha} binding –7-kb region, which was set as 100%. Shown is a representative experiment. DMSO, dimethyl sulfoxide. (C) RAW-LXR{alpha} cells (WT) were transfected with either a nonspecific pool of siRNA duplexes (iCTRL) as a control or a pool of siRNA duplexes specific for mouse NCoR (iNCoR). After 36 h, the cells were treated with the indicated agonists for 24 h. CCL24 mRNA levels were measured by real-time reverse transcription (RT)-PCR. The values indicate expression levels of target genes normalized to cyclophilin (CYCLO) transcripts and are plotted relative to the expression in vehicle-treated cells transfected with control duplexes, which was set as 1. (D) RAW cells were transfected as indicated in panel C. NCoR mRNA levels were measured by real-time RT-PCR and represented as in panel C. Protein levels were evaluated by immunoblotting using an anti-NCoR antibody.


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DISCUSSION
 
The functional significance of phosphorylation for LXR{alpha} actions has not been elucidated in detail. Our results show that macrophage LXR{alpha} phosphorylation at S198 affects the transcriptional activity of the receptor in a gene-specific manner (Fig. 3A) and restricts the repertoire of genes regulated by LXR{alpha} (Fig. 5).

A recent study showed that in liver, PKA-induced phosphorylation of LXR{alpha} results in a reduction of SREBP expression (64). This was shown to be mediated by decreased binding of the phosphorylated LXR{alpha}/RXR heterodimer to the SREBP LXRE and impaired interaction between phospho-LXR{alpha} and RXR by PKA activators (64). In RAW-LXR{alpha} macrophage cells treated with PKA inhibitors, we did not detect changes in basal or ligand-induced LXR{alpha} phosphorylation at S198 using our phosphospecific antibodies (not shown). Thus, regulation of LXR{alpha} phosphorylation by PKA appears to be tissue specific. Additionally, it is plausible that other residues, in addition to S198, are phosphorylated by PKA. Indeed, using in vitro kinase assays, Yamamoto et al. showed that PKA induces phosphorylation of multiple domains of mouse LXR{alpha}, including the N-terminal part of the receptor (1 to 163), the hinge region (162 to 326), and the C-terminal domain (325 to 445) (64). The authors also present two forms of LXR{alpha} bearing double mutations (S195A and S196A, the latter being equivalent to S198A in the human sequence, and T290 and S291) that show resistance to PKA inhibition of SREBP-1c promoter activity. Although we did not study phosphorylation of LXR{alpha} by PKA, our mass spectrometry assays detected S198A as the only phosphorylated residue in LXR{alpha}. These data strongly suggest that other residues are phosphorylated upon PKA signaling. Concerning the effects of LXR{alpha} phosphorylation on DNA binding and its heterodimerization with RXR, we did not observe reduced binding of the S198A mutant to different LXREs or impaired interaction between RXR and LXR{alpha} upon changes in LXR{alpha} phosphorylation by gel shift assays (not shown). This further highlights important tissue- and cell-specific differences in the signaling pathways regulating LXR{alpha} actions and their functional consequences. Interestingly, Yamamoto et al. showed that PKA phosphorylation of LXR{alpha} reduced the recruitment of the SRC-1 coactivator and diminished binding to NCoR in mammalian two-hybrid assays, which is consistent with our findings.

We identified LXR target genes whose regulation is largely insensitive to S198 phosphorylation (ABCA1, ABCG1, SREBP1c, or PLTP), in agreement with a recent report by Chen et al. (9), and genes whose expression is modulated by the phosphorylation status of the receptor (AIM and LPL). In this group of genes, a lack of LXR{alpha} S198 phosphorylation correlates with higher ligand-dependent transcriptional efficiency, suggesting that phosphorylation may represent a negative feedback mechanism to limit the response of certain target genes to the LXR ligand.

Although kinase inhibitor studies indicate that LXR{alpha} S198 phosphorylation is DMAT sensitive (Fig. 6) and that DMAT is a highly specific CK2 inhibitor, siRNA knockdown of the CK2{alpha} catalytic subunit did not efficiently reduce LXR{alpha} S198 phosphorylation (data not shown). This may reflect residual CK2{alpha} expression or show that the other CK2{alpha}' subunit is sufficient to phosphorylate LXR{alpha} at S198. Alternatively, another DMAT-sensitive kinase may be involved.

Our studies with the S198A mutant suggest that no major effects of phosphorylation are observed on LXR{alpha} expression, subcellular localization, or LXR{alpha} heterodimerization with RXR to bind to an LXRE in vitro (Fig. 2 and data not shown). One plausible hypothesis is that LXR{alpha} adopts different conformations depending on its phosphorylation state, which in turn may facilitate the recruitment of corepressors and/or release of coactivators in a promoter-selective fashion, as shown for PPAR{gamma} (17, 36). Consistent with this idea, phosphorylation of LXR{alpha} at S198 appears to promote a conformational change in the receptor (Fig. 4), although at present it is unclear how this change would affect tertiary contacts with other parts of the receptor.

An attractive candidate for such preferential binding to the S198-phosphorylated LXR{alpha} is the corepressor NCoR. This corepressor has been implicated in the promoter-dependent transrepression of proinflammatory genes, with ligand-dependent LXR sumoylation preventing the release of NCoR-containing complexes upon inflammatory stimuli (16). Moreover, NCoR dissociation leads to a promoter-specific derepression of LXR target genes in the absence of LXR (56). Indeed, we observed reduced recruitment of NCoR by the nonphosphorylated LXR{alpha}-S198A (not shown). However, reducing NCoR expression did not proportionally affect the expression of other phosphorylation-sensitive target genes, such as the AIM gene (not shown). This points to additional mechanisms involving other negative coregulators and/or additional factors binding to the LXRE-adjacent sequences, as shown by Wagner et al. (56), likely mediating the regulation of AIM expression in an LXR{alpha} phosphorylation-sensitive manner.

This study also provides the first evidence that endogenous LXR{alpha} S198 phosphorylation occurs in cholesterol-loaded cells in atherosclerotic lesions of apoE-deficient mice. Interestingly, both LXR target genes that are selectively overexpressed in macrophages bearing nonphosphorylated LXR{alpha} S198A (AIM and LPL) have been implicated in atherogenesis. AIM has been shown to play roles in the protection of macrophages against apoptosis (25, 53) and in the development of atherosclerosis (2), with AIM-deficient mice displaying less progression of the plaque in early stages of the disease. In addition, macrophage LPL is considered proatherogenic (4, 5, 23, 54), presumably by inducing foam cell accumulation in the atherosclerotic lesion. This may point to a specific role for LXR{alpha} phosphorylation in the pathogenesis of atherosclerosis, although that is beyond the scope of this study and remains to be tested.

Importantly, our studies reveal that changes in the phosphorylation of LXR{alpha} expand the range of LXR{alpha}-responsive genes. We report the chemokine CCL24 gene as a phosphorylation status-specific LXR{alpha}-responsive gene in macrophages (Fig. 5). To understand the mechanistic basis of CCL24 induction, we identified a genomic site downstream of the CCL24 coding region to which LXR{alpha} is recruited (Fig. 8). LXR{alpha} binds to this region, albeit weakly, even in the absence of ligand, which suggests that LXR{alpha} binding per se is not sufficient to enhance CCL24 expression. Additionally, in the context of RXR activation by 9cRA, a condition under which LXR{alpha} phosphorylation is reduced and the WT LXR{alpha} activates CCL24, hyperacetylation of H3 occurs, along with recruitment of the p300 coactivator (Fig. 8B) and release of NCoR (Fig. 9A and B). Also noteworthy is the fact that nonphosphorylated S198A-LXR{alpha} exhibits diminished recruitment of NCoR to the CCL24 gene, and indeed, CCL24 expression in macrophages appears to be sensitive to NCoR, since a partial reduction of NCoR expression resulted in a significant increase in CCL24 levels (Fig. 9C).

CCL24, also called eotaxin 2, belongs to a family of chemokines that coordinate the recruitment of inflammatory cells to sites of allergic inflammation (37). CCL24 binds the chemokine receptor CCR3, which is expressed on the surfaces of eosinophils, Th2 lymphocytes, basophils, and mast cells and, as recently shown, in atherogenic plaques (19). We predict that in cell types where LXR{alpha} is phosphorylated in response to LXR ligands, such as oxysterol-loaded macrophages, CCL24 will not be expressed. In contrast, when LXR{alpha} is not phosphorylated (e.g., in macrophages exposed to 9cRA or CK2 inhibitor or in macrophages expressing S198A), CCL24 expression will be enhanced by LXR ligands. Although the role of CCL24 in atherosclerosis has not been established, it is tempting to speculate that LXR{alpha}-dependent expression of CCL24 could recruit cells to control inflammation and plaque formation or help in lesion repair.

LXR{alpha} phosphorylation is reduced by 9cRA, which appears to recapitulate selective gene activation by LXR{alpha}-S198A. This finding suggests that RXR is not a passive binding partner but rather an active participant in gene regulation by LXR{alpha}, imparting new characteristics to the LXR/RXR heterodimers. This may generate a "composite" LXR/RXR surface that recruits a distinct cofactor or allows new contacts with DNA and/or neighboring transcription factors to activate genes selectively. Alternatively, activated RXR molecules associated with other heterodimeric partners may be responsible for the effects observed on LXR{alpha} phosphorylation.

Indeed, close to the downstream CCL24 regulatory region located between +1063 and +1078 from the stop codon, a degenerate direct-repeat 4 element (AGGTCAgagaAGGGCG), rather than the canonical direct-repeat 4 site found in established LXR target genes, is present. Thus, nonconsensus sequences may serve as bona fide LXREs in the context of the hypophosphorylated LXR{alpha} to regulate novel target genes. Such gene-specific modulation of LXR{alpha} activities by phosphorylation could be exploited to screen for compounds that would confine LXR{alpha} to its phosphorylated or nonphosphorylated conformation to allow selective gene expression in pathological settings.


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ACKNOWLEDGMENTS
 
We thank D. Mangelsdorf, M. Pagano, B. Staels, and A. Tall for plasmids and reagents; P. Gonzalez Santamaria and T. Bashir for advice on the retroviral infections; N. Swenson, R. Ruoff, and S. Logan for help with the immunohistochemistry; T. Macatee for tissue sections; and M. Kennedy for preparing the primary macrophages. We are also grateful to J. Nwachukwu, S. Logan, N. Tanese, and T. Claudel for critically reading the manuscript.

This work was supported by an AHA postdoctoral grant (I.P.T.); NIH grants 2P30CA016087-239025, 1S10RR017990 (T.A.N.), and HL084312 (E.A.F.); and Philip Morris USA Inc. (M.J.G.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, NYU School of Medicine, New York, NY 10016. Phone: (212) 263-7662. Fax: (212) 263-8276. E-mail: garabm01{at}med.nyu.edu Back

{triangledown} Published ahead of print on 4 February 2008. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back

{ddagger} Present address: School of Health Information Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030. Back


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Molecular and Cellular Biology, April 2008, p. 2626-2636, Vol. 28, No. 8
0270-7306/08/$08.00+0     doi:10.1128/MCB.01575-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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