Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 44–50

Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages Yuichi Ninomiya a,d,∗ , Toshimichi Yasuda b , Masashi Kawamoto b , Osafumi Yuge b , Yasushi Okazaki c,d a

c

Department of Immunology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan b Department of Anesthesiology and Critical Care, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan d Division of Translational Research, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan Received 9 March 2006; accepted 25 July 2006

Abstract Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H2 (PGH2 ) into prostaglandin E2 (PGE2 ). The mPGES-1-derived PGE2 is thought to help regulate inflammatory responses. On the other hand, excess PGE2 derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE2 in macrophages. These results suggest that LXR ligands diminish PGE2 production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages. © 2006 Elsevier Ltd. All rights reserved. Keywords: LXR; mPGES-1; PGE2 ; LPS; Macrophages

1. Introduction Prostaglandin E2 (PGE2 ) is a lipid metabolite of arachidonic acid (AA) that plays an important role in multiple physiological processes, including immune function [1,2], ∗ Corresponding author. Present address: Division of Translational Research, Research Center for Genomic Medicine, Saitama Medical School, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan. Tel.: +81 42 985 7319; fax: +81 42 985 7329. E-mail address: [email protected] (Y. Ninomiya).

0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2006.07.009

pain [3,4], fever [5], bone metabolism [6,7], and atherosclerosis [8]. PGE2 is released from cells and acts on the four types of PGE receptor (EP1, EP2, EP3, and EP4), which are linked to trimeric G protein signaling [9,10]. Various enzymes are involved in PGE2 biosynthesis, including phospholipase A2 (PLA2 ) isozymes, cyclooxygenase (COX) isozymes, and terminal PGE2 synthase (PGES) isozymes [11,12]. Several PLA2 isozymes help supply AA to COX isozymes. COX isomers catalyze the reaction of AA to prostaglandin H2 (PGH2 ) and terminal PGESs catalyze the reaction of PGH2 to PGE2 . As both COX-1 and

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cytosolic PGES (cPGES) are constitutively expressed in a wide variety of cells and tissues, this pathway is thought to be critical for the PGE2 production required to maintain tissue homeostasis [13,14]. Microsomal PGES-2 (mPGES-2) is also ubiquitously expressed, but its role has remained unclear [15,16]. On the other hand, the PGE2 production that is strongly induced by inflammatory stimuli such as lipopolysaccharide (LPS) and cytokines is exclusively induced by two key enzymes, cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES)-1 [17]. This concept is supported by experiments with mPGES-1 knockout mice. For example, macrophages derived from wild-type mice initiate PGE2 production after LPS treatment, but macrophages derived from mPGES-1−/− mice do not produce PGE2 [3,4,18–20]. Furthermore, macrophages transfected with mPGES-1 small interfering RNA (siRNA) exhibit reduced LPS-activated PGE2 production [4]. These results suggest that mPGES-1 acts as the primary enzyme involved in PGE2 induction by various inflammatory stimuli. In vivo and in vitro studies have shown that transcriptional activation of the mPGES-1 gene is dependent at least upon Toll-like receptor 4, MyD88, NF-IL6/C/EBP␤, and Egr-1 [18,22,23]. In contrast, the inactivation mechanism of mPGES-1 gene transcription is not clear. This is a critical direction of study, however, because excess PGE2 production based on inflammatory stimuli causes inflammatory diseases such as arthritis and atherosclerosis. Liver X receptors ␣ and ␤ (LXRs) are ligand-dependent transcriptional activators belonging to a nuclear receptor superfamily [24–27]. Their ligands include cholesterol metabolites, oxysterols such as 22(R)-hydroxycholesterol (22R-HC) and 20(S)-HC, and synthetic ligands such as T0901317 and GW3965 [28–31]. In the presence of their ligands, LXRs induce target genes related to cholesterol and lipid metabolism [32–38]. Ligand activation of LXR was recently shown to play an important anti-inflammatory role. In LPS-activated macrophages, LXRs suppress the induction of a subset of inflammatory genes, including COX-2, inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), IL-1β, IL1rn, matrix metalloproteinase-9 (MMP-9), and osteopontin [39–42]. In this study, we examined the effects of LXR ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands, 22R-HC and T0901317, reduced the LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as COX-2 protein. However, LXR ligands did not influence the expression of mPGES-2 or cPGES protein. Consequently, LXR ligands suppressed the production of PGE2 . These results suggest that LXR ligands diminish PGE2 production through the inhibition of LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages. LXR ligands could potentially be developed into therapeutic drugs to control PGE2 levels in inflammatory diseases.

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2. Materials and methods 2.1. Materials 22R-HC was purchased from Sigma (St. Louis, MO). T0901317 and prostaglandin E2 EIA Kit-Monoclonal were purchased from Cayman Chemical (Ann Arbor, MI). Antibodies against mPGES-1, mPGES-2, cPGES, and COX-2 were also purchased from Cayman Chemical. Anti-␤-tubulin antibody was purchased from Sigma. Horseradish peroxidase (HRP)-conjugated mouse anti-rabbit IgG (1:10,000) (Promega, Madion, WI) or HRP-conjugated goat anti-mouse IgG (1:10,000) (Upstate, Lake Placid, NY) were purchased. The cDNAs for murine microsomal prostaglandin E synthase-1 (mPGES-1) obtained from total RNA of mouse peritoneal macrophages by reverse transcriptasepolymerase chain reaction (RT-PCR) using the following primers: mPGES-1 Fwd, 5 -agcacactgctggtcatcaagatgtac-3 ; mPGES-1 Rev, 5 -ttgtgaggtgaaggatgtcctctc-3 . The cDNA fragment of 653 bp of mPGES-1 was subcloned in the pCRIITOPO vector (Invitrogen, Tokyo, Japan) and their nucleotide sequences were confirmed as described previously [42]. 2.2. Animals Seven-week-old male C57/BL6J mice were purchased from Charles River Japan Inc. All animal experiments were conducted according to guidelines of Animal Care and Use Committee at Hiroshima University. 2.3. Preparation of mouse peritoneal macrophages Four days before use, mice were injected intraperitoneally with 2 ml of sterile 3% (w/v) thioglycolate. After sacrificing the animals, the peritoneal cavity was flushed with 5 ml of sterile PBS. The peritoneal cell suspension was carefully aspirated avoiding hemorrhage and kept at 4 ◦ C to prevent the adhesion of the macrophages to plastic. Cells were recovered by centrifugation at 200 × g for 5 min and washed three times with 10 ml of sterile ice-cold PBS. Cells were seeded at 7.6 × 104 /cm2 in DMEM (Gibco/Invitrogen Corp., Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biological Industries, Ashrat, Israel) and penicillin (100 units/ml) and streptomycin (100 ␮g/ml). After settlement for 1 h, cells were washed three times by 10 ml of PBS and adherent cells were used as peritoneal macrophages. The macrophages were seeded in DMEM supplemented with 10% heat-inactivated FCS and penicillin (100 units/ml) and streptomycin (100 ␮g/ml). All incubation procedures were performed with 5% CO2 in humidified air at 37 ◦ C. 2.4. RNA extraction and Northern blot analysis Total RNA was isolated from peritoneal macrophages using Trizol reagent (Invitrogen). Northern blotting was performed as previously described [42]. In brief, total RNA

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(20 ␮g/lane) was separated on 1% formaldehyde agarose gels, transferred to Hybond XL nylon membranes (Amersham Biosciences; Piscataway, NJ). After UV cross-linking, the membranes were prehybridized, hybridized with a specific cDNA probe for mPGES-1 or β-actin as below, washed and exposed to Hyperfirm MP (Amersham). The levels of mPGES-1 mRNA or β-actin mRNA were calculated on the basis of signals as measured by an image analyzer, BAS2000 (Fuji Photo Film Co., Tokyo, Japan). For mPGES-1 cDNA probes, a 0.65-kb insert of mPGES-1 in the pCRII-TOPO vector was digested with EcoRI. The digested cDNA fragments of mPGES-1 were separated on 1× TAE agarose gels, purified by QIAquick gel extraction kit (Qiagen, Tokyo, Japan) and labeled with [α-32 P]dCTP using Rediprime II random primer labeling system (Amersham). 2.5. Real-time quantitative RT-PCR Real-time quantitative PCR was performed as previously described [43]. In brief, total RNA was reverse-transcribed by Transcriptor Reverse Transcriptase (Roche, Diagnostics, Tokyo, Japan) with oligo(dT)18 primer. The reverse transcripts were used as templates for analysis of the gene expression levels using Mx3000P (Stratagene, La Jolla, CA) and Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) according to the manufacturer’s instructions. The real-time quantitative PCR analyses were performed using the following primers: mPGES-1 Fwd, 5 -agcacactgctggtcatcaa-3 ; mPGES-1 Rev, 5 -tccacatctgggtcactcct-3 ; β-actin Fwd, 5 -actgctctggctcctagcac-3 ; β-actin Rev, 5 -acatctgctggaaggtggac-3 . Data were expressed as percentage relative to condition of LPS stimulation alone using the CT method as detailed in manufacturer’s guidelines (Applied Biosystems). 2.6. Western blot analysis Cells were lysed in ice-cold 1× RIPA lysis buffer (Upstate) containing Complete protease inhibitor cocktail (Roche). Lysates were sonicated on ice and centrifuged at 10,000 × g for 15 min. The protein concentration of the supernatant was determined using Protein assay (Bio-Rad Laboratories, Tokyo, Japan). 6.5 ␮g of total cell lysates was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to an Immobilon-P transfer membrane (Millipore, Bedford, MA). After blocking in TBS-T buffer (20 mM Tris–HCl pH 7.6 containing 150 mM NaCl and 0.1% Tween 20) containing 5% (w/v) nonfat dry milk, blots were incubated overnight at 4 ◦ C with anti-COX-2 antibodies (1:10,000), anti-␤-tubulin antibodies (1:4000), anti-mPGES1 antibodies (1:500), anti-cPGES antibodies (1:3000) or antimPGES-2 antibodies (1:2000) as primary antibodies (Cayman) and washed with TBS-T. The blots were then incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:10,000) (Promega) or goat anti-mouse IgG (1:10,000)

(Upstate) as secondary antibodies and washed with TBST again. The blots were then incubated with the enhanced chemoluminescence (ECL) plus reagents (Amersham) and finally exposed to Hyperfilm MP (Amersham). 2.7. PGE2 assays At the end of the incubation period, the culture medium was collected, frozen in liquid nitrogen and stored at −80 ◦ C. Levels of PGE2 were determined using a PGE2 enzyme immunoassay kit from Cayman Chemical. 2.8. Statistical analysis Results were expressed as mean ± S.D. When indicated, statistical significance was calculated by analyses of variance supported by the Scheffe multiple comparisons test. Differences were considered significant at p < 0.05.

3. Results Through LXR activation, LXR ligands suppress the induction of a subset of inflammatory genes (e.g. COX-2, iNOS, IL-6, and IL-1β) that is induced by LPS in thioglycolateelicited murine peritoneal macrophages [39]. To examine whether the mPGES-1 gene is part of the subset of inflammatory genes that is suppressed by LXR ligands, such as 22R-HC or T0901317, we measured mPGES-1 mRNA levels by Northern blotting. Expression of mPGES-1 mRNA was induced by LPS (Fig. 1A (lanes 1 and 2) and B). Pretreatment of macrophages with 22R-HC significantly reduced mPGES-1 mRNA levels (Fig. 1A (lanes 2–5) and B). The inhibitory effect of 22R-HC on the expression of mPGES-1 mRNA was in a dose-dependent manner (Fig. 1A and B). In addition, pretreatment of macrophages with 0.6 ␮M, 2 ␮M, and 6 ␮M of T0901317 also reduced mPGES-1 mRNA levels (Fig. 1A (lanes 2 and 6–8) and B). Since the inhibitory effect of T0901317 seems to be saturated at the concentration of 0.6 ␮M, we further evaluated its effect on mPGES-1 expression at lower concentrations with real-time quantitative RT-PCR. T0901317 reduced the expression levels of mPGES-1 mRNA in a dose-dependent manner like 22R-HC (Fig. 1C). These results strongly suggest that the mPGES-1 gene is one of the inflammatory genes suppressed by LXR ligands. To investigate whether the LXR ligand-mediated decrease in mPGES-1 mRNA levels was accompanied by reduced protein levels of mPGES-1, we examined mPGES-1 by Western blotting. Treatment with LPS resulted in strong induction of mPGES-1 protein expression (16 kDa) in murine peritoneal macrophages (Fig. 2A and B). As expected, pretreatment with 22R-HC or T0901317 significantly reduced the LPS-induced expression of mPGES-1 protein in a dose-dependent manner as well as mPGES-1 mRNA (Fig. 2A and B). In contrast to mPGES-1 protein, expression levels of cPGES protein

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Fig. 2. (A) LXR agonists inhibit mPGES-1 protein expression in LPS-activated murine macrophages. Thioglycolate-elicited peritoneal macrophages were treated with vehicle or the indicated concentrations of 22R-HC or T0901317 for 18 h. Cells were then stimulated with or without LPS (100 ng/ml) for 18 h. Cell lysates were prepared and 6.5 ␮g of proteins were analyzed for mPGES-1 protein by Western blotting using antimPGES-1 antibody. The expression of mPGES-2 and cPGES proteins was also analyzed by Western blotting using anti-mPGES-2 and anti-cPGES antibodies. These blots are the representative of two independent experiments. (B) The inhibitory effect of lower concentrations of T0901317 on protein expression of mPGES-1, mPGES-2 and cPGES was estimated. The experiments were performed in a same manner as described in (A).

Fig. 1. LXR ligands inhibit mPGES-1 mRNA expression in LPS-activated murine macrophages. (A) Thioglycolate-elicited peritoneal macrophages were pretreated with vehicle or indicated concentration of 22R-HC or T0901317 for 18 h. Cells were then stimulated with or without LPS (100 ng/ml) for 18 h. Total RNA was isolated and total RNA (20 ␮g/lane) was analyzed by Northern blotting and hybridized with 32 P-labeled cDNA probe for mPGES-1. Arrows indicate four transcript variants of mPGES-1 [18]. (B) The relative mRNA expression of mPGES-1 was measured by an image analyzer BAS2000 after normalization with that of β-actin. The signal intensity of the third largest band of mPGES-1 was measured and is shown as percentages relative to that from the sample stimulated with LPS alone. Similar results were obtained in two additional experiments. (C) The inhibitory effect with lower concentrations of T0901317 on the expression levels of mPGES-1 mRNA was estimated using real-time quantitative RT-PCR. The experiments were performed in triplicate and the relative mRNA expression is shown as percentages relative to that from the sample stimulated with LPS alone. Data are expressed as mean ± S.D. from three independent experiments.

(23 kDa) and mPGES-2 protein (33 kDa) were constitutive and did not change in response to LPS treatment or pretreatment with 22R-HC or T0901317 (Fig. 2A and B). Since mPGES-1 is one of the terminal enzymes in the PGE2 production pathway of LPS-activated macrophages, an LXR ligand-mediated decrease in mPGES-1 mRNA and mPGES-1 protein might decrease PGE2 synthesis in LPSactivated peritoneal macrophages. To examine this possibility, we measured the quantity of PGE2 by enzyme immunoassay. PGE2 production was induced by LPS treatment in peritoneal macrophages (Fig. 3). As expected, the pretreatment of macrophages with 22R-HC reduced LPS-induced PGE2 production in a dose-dependent manner (Fig. 3). T0901317 also reduced PGE2 production in LPSactivated macrophages at the concentration more than 0.6 ␮M (Fig. 3). Finally, we examined whether the expression of COX2, another key enzyme for LPS-induced PGE2 production,

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Fig. 3. LXR agonists suppress PGE2 production in LPS-activated peritoneal macrophages. (A) Thioglycolate-elicited peritoneal macrophages were treated with vehicle or the indicated concentrations of 22R-HC or T0901317 for 18 h. Cells were then stimulated with or without LPS (100 ng/ml) for 18 h. The culture media were collected and PGE2 production was determined with enzyme immunoassay for PGE2 . Data are expressed as mean ± S.D. from three independent experiments. * p < 0.05 significantly lower than the samples with LPS alone (control).

is inhibited in murine peritoneal macrophages by 22R-HC and T0901317 using Western blotting. LPS induced protein expression of COX-2 in peritoneal macrophages (Fig. 4A (lanes 1, 2) and B (lanes 1, 2)). Pretreatment with 22R-HC and T0901317 diminished COX-2 protein expression in a dose-dependent manner (Fig. 4A (lanes 2–5) and B (lanes 2–8)). Pretreatment with 22R-HC or T0901317 alone did not induce COX-2 expression, similar to control macrophages (Fig. 4A (lanes 9, 10) and B (lane 9)). Taken together, these results suggest that LXR ligands diminish the production of PGE2 in LPS-activated macrophages via the inhibition of LPS-induced gene expression of the COX-2-mPGES-1 axis.

4. Discussion Although LPS- or inflammatory cytokine-induced PGE2 production in macrophages is one of the key markers for proinflammatory reactions, the biological function of PGE2 in inflammation remains unclear. Several groups have recently reported, based on analyses of mPGES1 knockout mice, that mPGES-1 plays important roles through PGE2 production in inflammatory pain hypersensitivity, inflammatory arthritis, and inflammatory granulation

Fig. 4. (A) LXR agonists inhibit COX-2 protein expression in LPS-activated murine macrophages. Thioglycolate-elicited peritoneal macrophages were treated with vehicle or the indicated concentrations of 22R-HC or T0901317 for 18 h. Cells were then stimulated with or without LPS (100 ng/ml) for 18 h. Cell lysates were prepared and 2 ␮g of proteins were analyzed for COX-2 protein by Western blotting using anti-COX-2 antibody. The expressions of ␤-tubulin proteins were also analyzed by Western blotting using anti-␤tubulin antibody. These blots are representative of similar results obtained from two independent experiments. (B) The inhibitory effect of lower concentrations of T0901317 on COX-2 protein expression was estimated. The experiments were performed in a same manner as described in (A).

[3,4]. Therefore, in order to treat symptoms of inflammatory disease, it may help to regulate mPGES-1 expression and diminish the production of PGE2 . In this study, we used peritoneal murine macrophage cultures as a model for inflammation, and showed that an endogenous and a synthetic LXR ligand (22R-HC and T0901317, respectively) markedly diminished mPGES-1 mRNA levels in LPSactivated macrophages. These LXR ligands also diminished mPGES-1 protein expression in LPS-activated macrophages, but not affected the expression of cPGES or mPGES-2. LXR ligands also inhibited COX-2 protein expression [39]. Finally, LXR ligands diminished PGE2 production in LPS-activated macrophages. In other words, LXR ligands suppressed PGE2 production in macrophages via inhibition of LPS-induced gene expression of the COX-2-mPGES-1 axis. These results, therefore, suggest that LXR ligands might potentially be developed into drugs to decrease the PGE2 production that causes inflammatory symptoms. Our results showed that both 22R-HC and T0901317 inhibited mPGES-1 expression. Although this strongly suggests that LXR ligands inhibit mPGES-1 expression via LXRs, the mechanism remains unclear. To confirm that the inhibitory effect of LXR ligands on mPGES-1 gene expression occurs via LXRs, analyses of macrophages derived from LXR knockout mice will be required.

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Although we were not able to explain the molecular mechanism(s) by which LXR ligands suppress the expression of mPGES-1, we can suggest some possibilities. It was recently shown that PPAR␥ and its ligands also inhibit the expression of mPGES-1 mRNA and mPGES-1 protein and diminish PGE2 production in IL-1␤-induced human synovial fibroblasts [43]. PPAR␥ ligands inhibit the DNA-binding activity of Egr-1, a transcription factor that binds specifically to the promoter region of the mPGES-1 gene and activates mPGES-1 gene transcription, and suppress the transcription of mPGES1 [44]. In a similar fashion, LXR ligands might also suppress the transcription of mPGES-1 by decreasing the transcriptional activity of Egr-1. In addition, binding elements for NF-IL6 (C/EBP␤), AP-1, GR, PR, and C/EBP␣ were also found in the mPGES-1 promoter region [21]. In particular, based on experiments with macrophages derived from NF-IL6 (C/EBP␤)-deficient mice, in which LPS-induced expression of mPGES-1 mRNA is not observed, mPGES1 transcription must be dependent at least upon NF-IL6 [18]. Therefore, LXR ligands may inhibit mPGES-1 transcription by modulating the transcriptional activity of NF-IL6. In macrophages derived from TLR4- or MyD88-deficient mice, LPS-induced expression of mPGES-1 mRNA was not observed, similar to the case with NF-IL6-knockout mice [18]. Therefore, mPGES-1 transcription is TLR4- and MyD88-dependent. NF-␬B is a major transcription activator involved in the TLR4/MyD88 signaling pathway. Thus far, no NF-␬B binding sites have been identified in the promoter region of the mPGES-1 gene, therefore, mPGES-1 transcription may be indirectly regulated by NF-␬B. Since the transcription activity of NF-␬B is inhibited by the LXR pathway in LPS-activated macrophages [39], the decrease in mPGES-1 transcription by LXR ligands might result from the inhibition of NF-␬B activity via LXR. Alternatively, IRF-3, a MyD88-independent pathway transcriptional activator, may compete with the LXR pathway. IRF-3 appears to compete with LXR for transcriptional cofactors such as CBP and p300 [45,46]. Since IRF-3 binding sites have also not been identified in the mPGES-1 promoter region, it is unlikely that competition between IRF-3 and the LXR pathway occurs on the mPGES-1 promoter. It is possible, however, that other transcription activators that are crucial for mPGES-1 gene might compete with LXR for transcription coactivation at the mPGES-1 promoter. Similar competition might explain the inhibitory effects of LXR ligands on mPGES-1 transcription. In summary, we have shown that LXR ligands suppress PGE2 production in LPS-activated macrophages by inhibiting gene expression of the COX-2-mPGES-1 axis. In the future, using LXR ligands to control levels of PGE2 might help treat the symptoms of inflammatory disease.

Acknowledgements We thank Professor Masamoto Kanno for helpful advice and critical reading of manuscript. We thank Dr. Kaoru

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