Microsomal Prostaglandin E Synthase-1 Deficiency Is Associated

Microsomal Prostaglandin E Synthase-1 Deficiency IsAssociated with Elevated PeroxisomeProliferator-activated Receptor �REGULATION BY PROSTAGLANDIN E2 VIA THE PHOSPHATIDYLINOSITOL 3-KINASE ANDAKT PATHWAY*

Received for publication, October 30, 2006, and in revised form, December 22, 2006 Published, JBC Papers in Press, December 22, 2006, DOI 10.1074/jbc.M610153200

Mohit Kapoor‡, Fumiaki Kojima‡, Min Qian§, Lihua Yang‡, and Leslie J. Crofford‡1

From the ‡Department of Internal Medicine, Rheumatology Division, University of Kentucky, Lexington, Kentucky 40536and the §Department of Ophthalmology and Visual Sciences, Division of Cell and Developmental Biology,University of Michigan Medical School, Ann Arbor, Michigan 48109

mPGES-1 (microsomal PGE synthase-1) is an inducibleenzyme that acts downstreamof cyclooxygenase (COX) and spe-cifically catalyzes the conversion of prostaglandin (PG) H2 toPGE2 under basal as well as inflammatory conditions. In thisstudy, using mouse embryo fibroblasts (MEFs) isolated frommice genetically deficient for the mPges-1 gene, we show basalelevation of peroxisome proliferator-activated receptor �(PPAR�) expression (protein and mRNA) and transcriptionalactivity associated with reduced basal PGE2. We further showthat basal mPGES-1-derived PGE2 suppresses the expression ofPPAR� through a cAMP-independent pathway involving phos-phatidylinositol 3-kinase and Akt signaling. Using specificPPAR� agonist (rosiglitazone), PPAR� ligand (15-deoxy-�12,14-PGJ2), andPPAR� inhibitor (GW9662), we confirm thatactivation of PPAR� blocks interleukin-1�-induced up-regula-tion ofCOX-2,mPGES-1, and their derivedPGE2. Furthermore,we demonstrate that up-regulation of PPAR� upon geneticdeletion of mPGES-1 is responsible for reduced COX-2 expres-sion under basal as well as interleukin-1�-stimulated condi-tions. This study provides evidence for the first time thatmPGES-1 deletion not only decreases proinflammatory PGE2but also up-regulates anti-inflammatory PPAR�, which has theability to suppress COX-2 and mPGES-1 expression and PGE2production. Thus, mPGES-1 inhibitionmay limit inflammationby multiple mechanisms and is a potential therapeutic target.

Prostaglandins (PGs)2 are formed by metabolism of arachi-donic acid by cyclooxygenases (COX) to generate an interme-

diate substrate, PGH2, which is furthermetabolized by terminalsynthases to generate specific PGs (1, 2). mPGES-1, originallyknown as microsomal glutathione S-transferase 1-like 1(MGST1-L1), is an inducible enzyme that acts downstream ofCOX and specifically catalyzes the conversion of PGH2 to PGE2(3), most prominently in inflammatory conditions (4, 5). How-ever, we have recently shown that mPGES-1 is critical for PGE2production under basal as well as inflammatory conditions (6).mPGES-1 is coordinately induced with COX-2 by inflamma-

tory stimuli in a variety of cells and tissues (4, 7). PGE2 is themost abundant PG associated with inflammatory conditions,and overproduction of PGE2 coincides with increased COX-2and mPGES-1 expression (4, 7). PGE2 exerts the majority of itsactions through a family of G protein-coupled receptors,including EP1, EP2, EP3, and EP4 (8). The effects of PGE2 viathese receptors are mediated through various downstream sig-naling pathways, including cAMP-dependent protein kinase,mitogen-activated protein kinase (MAP kinase), phosphatidyl-inositol 3-kinase (PI 3-kinase), and Akt (8–10).Inhibition of PGE2 production and action is associated with

reduction of the pain and inflammation associated with a widevariety of diseases. Nonselective and COX-2- selective nonste-roidal anti-inflammatory drugs (NSAIDs) block PGE2 produc-tion by inhibiting the activity of COX and are extensively usedto treat arthritis and other inflammatory conditions. However,side effects associated with the inhibition of COX-2 (11–13)have revived efforts to develop safer anti-inflammatory drugs.mPGES-1 is an attractive target to achievemore specific inhi-

bition of PGE2 production associated with inflammatory disor-ders while preserving production of other PGs. Specific inhibi-tors of mPGES-1 are yet not available; however, studies usingmice genetically deficient in mPGES-1 have demonstrated thatthis enzyme is a key mediator of inflammation, pain, angiogen-esis, fever, bone metabolism, and tumorigenesis (14–17). Ourstudies also demonstrate thatmPGES-1 expression is increasedin tissues and cells of various inflammatory conditions, includ-ing rheumatoid arthritis and osteoarthritis (4, 5, 18, 19). Previ-

* This work was supported by the NIAMS Grant R01 AR 049010 from theNational Institutes of Health and an Arthritis Foundation Biomedical Sci-ences grant. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 To whom correspondence should be addressed: Dept. of Internal Medicine,Rheumatology Division, Rm. J-509, KY Clinic, University of Kentucky, Lex-ington, KY 40536-0284. Tel.: 859-323-4939; Fax: 859-257-8258; E-mail:[email protected].

2 The abbreviations used are: PG, prostaglandin; mPGES-1, microsomal PGEsynthase-1; COX, cyclooxygenase; MEF, mouse embryo fibroblast; PPAR�,peroxisome proliferator-activated receptor �; PI 3-kinase, phosphatidyl-inositol 3-kinase; NSAIDs, nonsteroidal anti-inflammatory drugs; DMEM,Dulbecco’s modified Eagle’s medium; GAPDH, glyceraldehyde-3-phos-

phate dehydrogenase; WT, wild type; RT, reverse transcription; FBS, fetalbovine serum; IL, interleukin; TBS, Tris-buffered saline; MAP, mitogen-acti-vated protein; ELISA, enzyme-linked immunosorbent assay; MEK, MAPkinase/extracellular signal-regulated kinase kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 8, pp. 5356 –5366, February 23, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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ous studies have also shown that mPGES-1 null mice are resist-ant to arthritis in the models of collagen-induced arthritis andcollagen antibody-induced arthritis (14, 15).Peroxisome proliferator-activated receptor � (PPAR�) is a

member of nuclear hormone receptor superfamily of ligand-activated transcription factors that have been shown to regulateinflammatory responses and assist in the resolution of inflam-mation (20–24). Recent studies have shown a close relationshipbetween PPAR� and PGs in the regulation of inflammation(25–27). However, until now, no study has evaluated the poten-tial role of mPGES-1 in the regulation of PPAR�. We createdmouse embryo fibroblast (MEF) cell lines derived frommPGES-1 null mice and wild type (WT) littermates to facilitatethese studies. This study demonstrates for the first time thatmPGES-1 deficiency and reduced PGE2 lead to elevation ofPPAR� under basal conditions. This study further identifies keydownstream signaling targets responsible for PPAR� regula-tion by mPGES-1-derived PGE2.

EXPERIMENTAL PROCEDURES

Animals—mPGES-1 heterozygous mice on a DBA1 lac/Jbackground were obtained from Pfizer (15). Mice were housedinmicroisolator cages in a pathogen-free barrier facility, and allexperiments were performed under the approved IACUC andinstitutional guidelines.Materials—Rabbit anti-human mPGES-1 antiserum was a

gift from Dr. Per-Johan Jakobsson (Karolinska Institute, Stock-holm, Sweden). PPAR� transcription assay kit, rabbit anti-mouse COX-2 polyclonal antibody, PGE2, carbacyclin, NS-398(N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide),rosiglitazone, (15-deoxy-�12,14-PGJ2), GW9662, LY294002,ovine COX-2 standard protein, and enzyme-linked immu-nosorbent assay (ELISA) kit for PGE2 were all purchased fromCayman Chemical Co. (Ann Arbor, MI). PD98059 (selectiveinhibitor of MAP kinase kinase (MEK)), SB203580 (specificinhibitor of p38 MAP kinase), and 1L-6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate (specificAkt inhibitor) were purchased from Calbiochem. Mousemonoclonal PPAR� primary antibody was obtained from SantaCruz Biotechnology (Santa Cruz, CA). Mouse anti-human�-actin monoclonal antibody and indomethacin were obtainedfrom Sigma. Horseradish peroxidase-conjugated goat anti-rab-bit IgG and horseradish peroxidase-conjugated goat anti-mouse IgGwere obtained from Jackson ImmunoResearch.Dul-becco’s modified Eagle’s medium (DMEM) and fetal bovineserum (FBS) were from Invitrogen. Recombinant mouse IL-1�was obtained from R&D Systems (Minneapolis, MN). TRIpurewas purchased from Roche Diagnostics. The polyvinylidenedifluoride membrane and enhanced chemiluminescence (ECL)reagent were purchased from Amersham Biosciences.Preparation and Activation of Mouse Embryo Fibroblasts—

Embryoswere harvested frommPGES-1 (DBA1 lac/J) pregnantheterozygous females (E12.5) who had been mated with het-erozygous males.Whole embryos were minced and placed intoculture DMEM containing 10% FBS, 100 units/ml penicillin,and 100 �g/ml streptomycin at 37 °C under an atmosphere of5% CO2. At confluence, the cells were detached and passaged,and 3–4 passage cells were used in all experiments. MEFs were

plated into the wells of a 6-well plate at a density of 3 � 105cells/well in DMEM containing 10% FBS. Cells were starved for72 h in DMEM containing 1% FBS and then incubated with orwithout 1 ng/ml IL-1� in the presence or absence of varioustreatments for 12 h. Cell viability was determined bymeasuringmitochondrial NADH-dependent dehydrogenase activity withWST-1 assay (Dojindo Laboratories, Kumamoto, Japan).Reverse Transcription (RT)-PCR—RNA from the cells was

extracted with TRIpure reagent according to the manufactur-er’s instructions. Reverse transcription was performed accord-ing to the manufacturer’s instructions using a SuperScript pre-amplification system (Invitrogen) with 1 �g of total RNA fromthe cells as a template. Subsequent amplifications of the cDNAfragments by PCR with HotStar Taq polymerase (Qiagen,Valencia, CA) were performed using 0.5 �l of the reverse-tran-scribed mixture as a template with specific oligonucleotideprimers and cycle numbers as follows: mouse PPAR� (31cycles), sense 5�-CCT CTC CGT GAT GGA AGA CC-3� andantisense 5�-GCA TTG TGA GAC ATC CCC AC-3�; mouseGAPDH (20 cycles), sense 5�-GGGGTGAGGCCGGTGCTGAGTAT-3� and antisense 5�-CATTGGGGGTAGGAACACGGA AGG-3�. After initial denaturation at 95 °C for 15 min,PCR involved amplification cycles of 30 s at 95 °C, 30 s at 56 °C,and 45 s at 72 °C, followed by elongation for 5min at 72 °C. Theamplified cDNA fragments were resolved by electrophoresis on2% (w/v) agarose gel andwere visualized under UV light using aChemidoc apparatus (Bio-Rad) after staining of the gel withethidium bromide.Western Blotting—Cells were lysed in Tris-buffered saline

(TBS) containing 0.1% SDS, and the protein content of thelysates was determined using bicinchoninic acid (BCA) proteinassay reagent (Pierce) with bovine serum albumin as the stand-ard. Cell lysates were adjusted to equal equivalents of proteinand then were applied to SDS-polyacrylamide gels (10–20%)for electrophoresis. Next, the proteins were electroblotted ontopolyvinylidene difluoride membranes. After the membraneswere blocked in 10mMTBS containing 0.1%Tween 20 (TBS-T)and 5% skim milk, the membranes were probed for 1.5 h withthe respective antibodies (1:1000 for mPGES-1, COX-2,PPAR�, and �-actin) in TBS-T for 1.5 h. After washing themembranes with TBS-T, the membranes were incubated withhorseradish peroxidase-conjugated anti-rabbit (for mPGES-1and COX-2) or horseradish peroxidase-conjugated anti-mouse(for PPAR� and �-actin) IgG (1:10,000 dilution in TBS-T con-taining 5% skim milk) overnight at 4 °C. After further washingwith TBS-T, protein bands were visualized with an ECLWest-ern blot analysis systemusing aChemidoc apparatus (Bio-Rad).PPAR� Transcriptional Activity Assay—MEFs were plated

into the T-75 flasks at a density of 2 � 106 cells/flask in DMEMcontaining 10% FBS. Cells were starved for 72 h in DMEM con-taining 1% FBS and then incubated with or without 1 ng/mlIL-1� in the presence/absence of various treatments. Nuclearextraction and PPAR� transcriptional activity assays were per-formed using the PPAR� transcription assay kit (CaymanChemicals, Ann Arbor, MI), and procedures were followedaccording to the manufacturer’s recommendation.Measurement of PGE2 inCultureMedium—MEFswere incu-

bated for 12 h in the presence or absence of IL-1� (1 ng/ml). In

PPAR� Up-regulation in mPGES-1 Deficiency

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experiments involving treatment with indomethacin andNS-398, these compounds were added 72 h before IL-1� stim-ulation. The culture supernatantwas harvested, and concentra-tion of PGE2 was measured by ELISA (Cayman Chemical, AnnArbor MI). Assays were performed according to the manufac-turer’s recommendation.Statistical Analysis—The data are expressed as mean � S.E.

Statistical analysis was performed using Student’s t test. p �0.05 was considered statistically significant.

RESULTS

Effect of mPGES-1 Genetic Deletion on PPAR� Expression(mRNA and Protein) and Transcriptional Activity in mPGES-1WTandNullMEFs—mPGES-1WTandnullMEFs, in the pres-ence/absence of IL-1� stimulation, were analyzed for PPAR�mRNA expression (Fig. 1a), protein expression (Fig. 1b), andtranscriptional activity (Fig. 1c). Results showed that underbasal conditions, PPAR� mRNA and protein levels were signif-icantly higher (p � 0.05) in the mPGES-1 null MEFs comparedwith their WT counterparts. Similarly PPAR� transcriptionalactivity was significantly higher (p � 0.01) in mPGES-1 nullMEFs compared withWTMEFs under basal conditions. IL-1�stimulation decreased the levels of PPAR�mRNA, protein, and

transcriptional activity by near 50% in both cell types; however,PPAR� expression and transcriptional activity levels were stillsignificantly higher (p� 0.05) in themPGES-1 nullMEFs com-pared withWTMEFs. These results show that genetic deletionof mPGES-1 leads to elevation of PPAR� expression and tran-scriptional activity levels. Furthermore, proinflammatory sig-nals reduce PPAR� expression and transcriptional activity, aneffect mitigated in the absence of mPGES-1.Effect of mPGES-1 Genetic Deletion on PGE2 Levels in

mPGES-1 WT and Null MEFs—PGE2 levels were significantly(p � 0.05) higher in mPGES-1 WT MEFs compared with nullMEFs under unstimulated conditions (Fig. 2). Upon stimula-tion with IL-1�, a significant (p � 0.01) increase in the levels ofPGE2 was observed in WT MEFs; however, no change in thePGE2 levels was observed inmPGES-1 nullMEFs. These resultsclearly demonstrate that mPGES-1 is critical for PGE2 produc-tion at basal as well as stimulated conditions even though wehave shown previously that both cytosolic PGES (cPGES) andmPGES-2 are expressed in these MEFs (6).Our recent study using mPGES-1 WT and null MEFs also

showed that genetic deletion ofmPGES-1 not only blocks PGE2production but also results in the elevation of 6-keto-PGF1�

(stable breakdownproduct of prostacyclin; PGI2) under basal as

FIGURE 1. Effect of mPGES-1 deletion on PPAR� expression (mRNA and protein) and transcriptional activity in IL-1�-stimulated and unstimulatedMEFs. mPGES-1 WT and null MEFs were incubated with or without IL-1� (1 ng/ml) for 12 h. a, mRNA levels of PPAR� (30 cycles) and GAPDH (20 cycles) frommPGES-1 WT and null MEFs were determined by RT-PCR. Mean � S.E. for PPAR� expression normalized with GAPDH from n � 3 separate embryo lines is shown.*, mPGES-1 null MEFs compared with mPGES-1 WT MEFs. Cont, control. b, PPAR� and �-actin protein expressions were determined by Western blotting.Mean � S.E. for PPAR� expression normalized with �-actin from n � 5 separate embryo lines is shown. *, mPGES-1 null MEFs compared with mPGES-1 WT MEFs.c, PPAR� transcriptional activity was measured in the nuclear extracts of IL-1�-stimulated and unstimulated mPGES-1 WT and null MEFs. Open bars representmPGES-1 WT MEFs, and closed bars represent mPGES-1 null MEFs. PPAR� transcriptional activity was normalized to mg of protein for each sample. Data areexpressed as the mean � S.E. for four embryo lines. * and ** indicate statistical significance at p � 0.05 and p � 0.01, respectively.




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well as cytokine-stimulated conditions, suggesting a shuntingphenomenon within the arachidonic acid metabolic pathwayupon deletion ofmPGES-1 (6). In this study, we further showedthat mPGES-1 genetic deletion did not have any effect on theproduction pattern of other PGs such as PGD2 and thrombox-ane B2, which remained unaltered in mPGES-1 WT and nullMEFs under basal as well as cytokine-stimulated conditions.Effect of Exogenous PGE2 and Carbacyclin (Prostacyclin Ana-

logue) on PPAR� Expression and Transcriptional Activity inmPGES-1WTandNullMEFs—Todeterminewhether PGE2 oran alternate change induced by mPGES-1 deletion suppressesPPAR� expression and transcriptional activity in MEFs, exog-enous PGE2 treatment of mPGES-1 WT and null MEFs in thepresence or absence of IL-1� stimulationwas performed. Addi-tion of PGE2 under basal conditions resulted in a significantdecrease (p � 0.05) in the PPAR� expression both at mRNAand protein levels in mPGES-1 null MEFs with little or nochange observed inWTMEFs (Fig. 3, a and b). In the presenceof IL-1�, a further decrease in the PPAR� mRNA expressionlevels was observed in both mPGES-1 WT and null MEFs.PPAR� protein expression did not show any further decreasewhen PGE2 was used in combination with IL-1�.Because our previous studies using mPGES-1 WT and null

MEFs showed elevation of 6-keto-PGF1� upon genetic deletionof mPGES-1 in MEFs (6), we therefore investigated the effectsof carbacyclin (PGI2 analogue) to determine the contribution ofPGI2 versus PGE2 toward regulation of PPAR� expression inMEFs. However, we did not observe any changes in the proteinexpression levels of PPAR� in mPGES-1 WT and null MEFsupon treatment with carbacyclin (Fig. 3c).Addition of PGE2 in the absence of IL-1� stimulation signif-

icantly (p� 0.01) decreased PPAR� transcriptional activity lev-els in mPGES-1 null MEFs bringing the levels similar tomPGES-1 WTMEFs (Fig. 3d). In the presence of IL-1� stimu-lation, a further significant decrease in the levels of bothmPGES-1 WT (p � 0.05) and null MEFs (p � 0.01) wasobserved. Treatment with carbacyclin did not have any effecton PPAR� transcriptional activity in mPGES-1 WT and null

MEFs. These results suggest that deletion of mPGES-1 and asubsequent decrease in PGE2 levels (and not increased PGI2)play a key role in the differential regulation of PPAR� in MEFs.In addition, these results also suggest that IL-1� has the abilityto itself regulate the expression and transcriptional activity ofPPAR� andmay exert other intrinsic effects on PPAR� expres-sion and activity in addition to its ability to increase PGE2production.Effect of PGE2 Inhibition by NSAIDs (Indomethacin and

NS-398) on PPAR� Expression and Transcriptional Activity inmPGES-1 WT and Null MEFs—To further confirm the contri-bution of PGE2 toward differential regulation of PPAR� expres-sion and transcriptional activity, mPGES-1WT and null MEFswere treated with NSAIDs, including indomethacin (nonselec-tive COX inhibitor) and NS-398 (selective COX-2 inhibitor).Addition of indomethacin and NS-398 significantly increasedthe levels of PPAR� protein expression (Fig. 4a) and transcrip-tional activity (Fig. 4b) in mPGES-1WTMEFs only, raising thelevels similar to that of mPGES-1 null MEFs. These results fur-ther confirm that PGE2 is the key mediator involved in differ-ential regulation of PPAR� in mPGES-1 WT and null MEFs.Effect of mPGES-1 Deletion on cAMP Levels in mPGES-1WT

and Null MEFs—PGE2 has been shown to mediate some of itsdownstream effects via cAMP-dependent pathways (8). There-fore, we investigated the effect of mPGES-1 deletion and thesubsequent decrease in the levels of PGE2 on cAMP levels inmPGES-1 WT and null MEFs. Results showed that mPGES-1deletion did not have any significant effect on the levels ofcAMP in MEFs under basal conditions (Fig. 5a). We furtherinvestigated the effects of exogenous PGE2 and forskolin (adirect adenyl cyclase activator) on the levels of cAMP inmPGES-1WT and null MEFs. Treatment with PGE2 (p� 0.05)and forskolin (p � 0.01) significantly increased cAMP levels inboth mPGES-1 WT and null MEFs to a similar extent. Theseresults show that the cAMP machinery is intact in bothmPGES-1 WT and null MEFs.Effect of Forskolin on PPAR� Expression and Transcription

Activity Levels in mPGES-1 WT and Null MEFs—To delineatethe mechanism by which PGE2 regulates PPAR� expressionand transcriptional activity, we treated mPGES-1 WT and nullMEFs with forskolin in the presence/absence of PGE2, and weassessed its effect on PPAR� protein expression and transcrip-tional activity. Forskolin did not have any effect on PPAR� pro-tein expression (Fig. 5b) and transcriptional activity (Fig. 5c) inmPGES-1WTand nullMEFs in the absence of PGE2. However,PPAR�protein expression and transcriptional activitywere sig-nificantly (p� 0.01) decreasedwhen forskolinwas used in com-bination with PGE2, an effect that seems to be solely elicited byPGE2 and not forskolin. These results suggest that even thoughcAMP pathways are activated by PGE2 and forskolin in MEFs,PGE2 regulates PPAR� expression and transcriptional activityvia a signaling pathway independent of cAMP.Effect of PI 3-Kinase Inhibitor, Akt Inhibitor, p38 Inhibitor,

andMEK Inhibitor on PPAR� Expression in mPGES-1WT andNull MEFs—PGE2 is also known to mediate some of its biolog-ical responses through MAP kinase and PI 3-kinase/Akt path-ways (8). Therefore, to delineate the mechanism with whichPGE2 regulates PPAR� expression in mPGES-1 WT and null

FIGURE 2. Effect of mPGES-1 genetic deletion on PGE2 production inMEFs. Levels of PGE2 in supernatants of mPGES-1 WT and null MEFs weredetected by ELISA at 12-h post-IL-1� stimulation. Open bars representmPGES-1 WT MEFs, and closed bars represent mPGES-1 null MEFs. Data areexpressed as the mean � S.E. for four embryo lines. Cont, control. * and **indicate statistical significance at p � 0.05 and p � 0.01, respectively.




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FIGURE 3. Effect of PGE2 and carbacyclin on PPAR� expression (mRNA and protein) and transcriptional activity in mPGES-1 WT and null MEFs. mPGES-1 WTand null MEFs were incubated for 12 h with PGE2 (1 �M) or carbacyclin (1 �M) in the presence or absence of IL-1� (1 ng/ml). a, mRNA levels of PPAR� (30 cycles) andGAPDH (20 cycles) from mPGES-1 WT and null MEFs were determined by RT-PCR. Mean � S.E. for PPAR� expression normalized with GAPDH from n � 3 separateembryo lines is shown. Cont, control. *, mPGES-1 null MEFs compared with mPGES-1 WT MEFs; �, IL-�-stimulated group compared with unstimulated controls;$, IL-� � PGE2-stimulated group compared with unstimulated controls. b, PPAR� and �-actin protein expressions were determined by Western blotting. Mean � S.E.for PPAR� expression normalized with �-actin from n � 6 separate embryo lines is shown. *, mPGES-1 null MEFs compared with mPGES-1 WT MEFs; �, PGE2 treatedgroup compared with their respective untreated controls; $, IL-� � PGE2-stimulated group compared with respective unstimulated controls. c, PPAR� and �-actinprotein expressions were determined by Western blotting. Mean � S.E. for PPAR� expression normalized with �-actin from n � 3 separate embryo lines is shown. *,mPGES-1 null MEFs compared with mPGES-1 WT MEFs;�, PGE2-treated group compared with their respective untreated controls. d, PPAR� transcriptional activity wasmeasured in the nuclear extracts of mPGES-1 WT and null MEFs treated with PGE2 or carbacyclin in the absence or presence of IL-1�. Open bars represent mPGES-1 WTMEFs, and closed bars represent mPGES-1 null MEFs. PPAR� transcriptional activity was normalized to mg of protein for each sample. Data are expressed as the mean�S.E. for 3–4 embryo lines. * and ** indicate statistical significance at p � 0.05 and p � 0.01 respectively.




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MEFs, we investigated the effects of LY294002 (specific PI 3-ki-nase inhibitor), 1L-6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate (specific Akt inhibitor),SB-203580 (selective p38 MAP kinase inhibitor), and PD98059(selective MEK inhibitor) on PPAR� expression and transcrip-tional activity in mPGES-1 WT and null MEFs.Addition of PI 3-kinase or Akt inhibitors elevated the pro-

tein expression levels of PPAR� in mPGES-1WTMEFs only,raising the levels similar to that observed in mPGES-1 nullMEFs (Fig. 6, a and b). PI 3-kinase and Akt inhibitors in thepresence of PGE2 also showed elevation in the PPAR�expression in both mPGES-1 WT and null MEFs, thusreversing the inhibitory effects of PGE2 on PPAR� expres-sion. Addition of p38 MAP kinase and MEK inhibitorsshowed no effect on PPAR� protein expression in mPGES-1WT and null MEFs in the presence or absence of PGE2 (Fig.6c). These results clearly suggest that PI 3-kinase and Aktsignaling are key downstream pathways by which PPAR�expression is suppressed by PGE2 in MEFs.Effect of PPAR� on COX-2, mPGES-1, and PGE2 in MEFs—

PPAR� is an endogenous anti-inflammatory mediator knownto down-regulate key proinflammatory signals and ultimately

assists in the resolution of inflam-mation (2, 28). Recent studies haveshown that PPAR� is involved in theregulation of COX-2 and mPGES-1expression (24, 25). To further con-firmthese findings inMEFs,we inves-tigated the regulatory effects ofPPAR� on COX-2 and mPGES-1expression and PGE2 productionusing selective PPAR� agonist (ros-iglitazone), PPAR� ligand (15-deoxy-PGJ2), and PPAR� antagonist(GW9662).Because previous reports have

shown that rosiglitazone, 15-deoxy-PGJ2, and GW9662 have the abilityto affect cell viability, we thereforefirst determined the effects ofvarious concentrations of thesereagents on cell viability of MEFsusingWST-1 assay. Concentrationsof rosiglitazone (10 �M), 15-deoxy-PGJ2 (10 �M), and GW9662 (3 �M)were chosen for the subsequentexperiments as these concentra-tions did not have any effect on cellviability of MEFs (data not shown).Low levels ofCOX-2proteinwere

observed in unstimulated mPGES-1WT MEFs (Fig. 7a). Upon stimula-tion with IL-1�, a significant eleva-tion (p � 0.05) in the proteinexpression of COX-2 was observed.Pretreatmentwith rosiglitazone and15-deoxy-PGJ2 blocked up-regu-lated expression of COX-2 in IL-1�-

stimulated mPGES-1 WT. To confirm that rosiglitazone and15-deoxy-PGJ2 reduced COX-2 expression via the PPAR�pathway, we incubated mPGES-1 MEFs with rosiglitazone and15-deoxy-PGJ2 in the presence or absence of PPAR� inhibitor(GW9662). GW9662 completely reversed the inhibitory effectof rosiglitazone on COX-2 expression but showed only partialrecovery on the inhibitory effect of 15-deoxy-PGJ2 on COX-2expression in mPGES-1 WT and null MEFs. Some effects of15-deoxy-PGJ2 have been shown previously to be mediated viaalternate mechanistic pathways independent of PPAR� (26). Inthis study, partial recovery of COX-2 expression by GW9662 inthe 15-deoxy-PGJ2 group further suggests the involvement ofan alternative mechanistic pathway in addition to the PPAR�pathway by which 15-deoxy-PGJ2 blocks COX-2 expression inMEFs.We further investigated the effects of rosiglitazone and

15-deoxy-PGJ2 on mPGES-1 expression in mPGES-1 WTMEFs. Low level ofmPGES-1 protein was observed in unstimu-lated MEFs (Fig. 7a). Upon stimulation with IL-1�, significantelevation (p � 0.05) in the protein expression of mPGES-1 wasseen in MEFs as expected. Treatment with rosiglitazone and15-deoxy-PGJ2 blocked the increased expression of mPGES-1

FIGURE 4. Effect of indomethacin and NS-398 on PPAR� protein expression (a) and transcriptional activity inmPGES-1 WT and null MEFs (b). mPGES-1 WT and null MEFs were incubated with or without indomethacin (1 �M)or NS-398 (5 �M) for 12 h. a, PPAR� and �-actin protein expressions were determined by Western blotting. Mean �S.E. for PPAR� expression normalized with �-actin from n � 3 separate embryo lines is shown. Cont, control.*, mPGES-1 null MEFs compared with mPGES-1 WT MEFs; �, indomethacin-treated group compared with respec-tive unstimulated controls; $, NS-398-treated group compared with respective unstimulated controls. b, PPAR�transcriptional activity was measured in the nuclear extracts of mPGES-1 WT and null MEFs treated with indometha-cin and NS-398. Open bars represent mPGES-1 WT MEFs, and closed bars represent mPGES-1 null MEFs. PPAR�transcriptional activity was normalized to mg of protein for each sample. Data are expressed as the mean � S.E. forthree embryo lines. * and ** indicate statistical significance at p � 0.05 and p � 0.01, respectively.




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in IL-1�-stimulated mPGES-1WT and null MEFs. To confirmthat rosiglitazone and 15-deoxy-PGJ2 reduced mPGES-1expression via the PPAR� pathway, we incubated mPGES-1WT MEFs with rosiglitazone and 15-deoxy-PGJ2 in the pres-

ence or absence of PPAR� inhibitor(GW9662). GW9662 significantly(p � 0.05) reversed the inhibitoryeffects of rosiglitazone and 15-de-oxy-PGJ2 on mPGES-1 expression.These results show that increasedPPAR� expression results in down-regulation of COX-2 and mPGES-1expression under basal as well asinflammatory conditions.A significant elevation (p � 0.05)

in the levels of PGE2was observed inMEFs upon stimulation with IL-1�compared with unstimulated MEFs(Fig. 7b). Treatment with rosiglita-zone and 15-deoxy-PGJ2 signifi-cantly (p � 0.05) blocked theincreased production of PGE2 inIL-1�-stimulated mPGES-1 WTMEFs. This inhibition was recov-ered when rosiglitazone and 15-de-oxy-PGJ2 were used in the presenceof GW9662 in IL-1�-stimulatedmPGES-1 WT MEFs. These resultsshow that increased PPAR� expres-sion results in decreased PGE2 pro-duction under basal as well asinflammatory conditions.Because our results showed that

genetic deletion of mPGES-1 andresultant decrease in PGE2 produc-tion increased the levels of PPAR�in MEFs, we expected thatmPGES-1 null MEFs would havelow levels of COX-2 compared withWTMEFs. Indeed, we observed sig-nificantly higher levels of COX-2protein in mPGES-1 WT MEFscompared with null MEFs underbasal conditions (Fig. 7c). Theseresults suggest that increasedPPAR� expression as a result ofgenetic deletion of mPGES-1 couldlead to decreased COX-2 expres-sion in mPGES-1 null MEFs underbasal conditions.

DISCUSSION

This tudy using MEFs isolatedfrom mPGES-1-deficient miceclearly presents threemajor conclu-sions. First, genetic deletion ofmPGES-1 results in up-regulationof PPAR� expression and transcrip-

tional activity under basal conditions. Second, mPGES-1-de-rived PGE2 is responsible for regulation of PPAR� expressionthrough a cAMP-independent pathway involving PI 3-kinaseand Akt signaling. Third, specific activation of PPAR� blocks

FIGURE 5. a, effect of mPGES-1 deletion on cAMP levels in MEFs and effect of forskolin and PGE2 on cAMP levelsin mPGES-1 WT and null MEFs. Cont, control. b, effect of forskolin in the absence or presence of PGE2 on PPAR�protein expression. c, PPAR� transcriptional activity in mPGES-1 WT and null MEFs. a, cAMP levels in mPGES-1WT and null MEFs incubated for 12 h with or without forskolin (10 �M), PGE2 (1 �M), and combination offorskolin � PGE2 were assessed by cAMP ELISA. cAMP levels were normalized to �g of protein. Open barsrepresent mPGES-1 WT MEFs, and closed bars represent mPGES-1 null MEFs. Data are expressed as the mean �S.E. for four embryo lines. * and ** indicate statistical significance at p � 0.05 and p � 0.01, respectively.b, PPAR� protein expression was assessed in mPGES-1 WT and null MEFs treated for 12 h with or withoutforskolin (10 �M) in the absence or presence of PGE2 (1 �M). Mean � S.E. for PPAR� protein expression normal-ized with �-actin from n � 3 separate embryo lines is shown. *, mPGES-1 null MEFs compared with mPGES-1 WTMEFs; �, forskolin- � PGE2-treated group compared with respective unstimulated controls. c, PPAR� transcrip-tional activity was measured in the nuclear extracts of mPGES-1 WT and null MEFs treated for 12 h with orwithout forskolin (10 �M) in the absence or presence of PGE2 (1 �M). Open bars represent mPGES-1 WT MEFs,and closed bars represent mPGES-1 null MEFs. PPAR� transcriptional activity was normalized to mg of proteinfor each sample. Data are expressed as the mean � S.E. for three embryo lines. * and ** indicate statisticalsignificance at p � 0.05 and p � 0.01, respectively.




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IL-1�-induced up-regulation of proinflammatory COX-2,mPGES-1, and their derived PGE2 in WT MEFs, whereasincreased PPAR� in mPGES-1 null MEFs is associated withdecreased COX-2 expression under basal conditions and aftertreatment with IL-1�.Differential Regulation of PPAR� in mPGES-1 Null MEFs—

mPGES-1 is an inducible enzyme that acts downstreamof COXand specifically catalyzes the conversion of PGH2 to PGE2.Using MEFs isolated from mPGES-1-deficient mice, we haveshown previously that in the absence of mPGES-1, low levels ofPGE2 are produced under basal as well as cytokine-stimulatedconditions (6). In this study, we demonstrate for the first timethat genetic deletion of mPGES-1 leads to increased PPAR�expression and transcriptional activity by eliminating the sup-pressive effects of PGE2. PPAR� is a member of the nuclearhormone receptor superfamily of ligand-activated transcrip-tion factors that have been shown to initiate mechanisms thatinhibit inflammatory responses and assist in the resolution ofinflammation (20, 21, 29). Thus inhibition ofmPGES-1 not onlyblocks proinflammatory PGE2 production but also results inthe up-regulation of anti-inflammatory PPAR� in MEFs.

PGE2 Regulates PPAR� Expres-sion via Non-cAMP PathwayInvolving PI 3-Kinase and AktSignaling—Genetic deletion of spe-cific genes can be associated withunforeseen phenotypic changes.However, we showhere that PGE2 isresponsible for the differentialregulation of PPAR� expressionand transcriptional activity inMEFs. Exogenous PGE2 decreasedand NSAIDs (indomethacin, anonselective COX inhibitor, andNS-398, a selective COX-2 inhibi-tor) increased PPAR� under basalconditions confirming that thepresence of PGE2 down-regulatesPPAR�, whereas PGE2 depletionresults in the up-regulation ofPPAR�.

The next aim of this study was todelineate underlying signaling path-ways by which mPGES-1-derivedPGE2 regulates PPAR� expressionand transcriptional activity inMEFs. PGE2 exerts the majority ofits actions through a family of Gprotein-coupled receptors, includ-ing EP1, EP2, EP3, and EP4 viadownstream signaling pathways,including cAMP-dependent proteinkinase, MAP kinase, and PI 3-ki-nase/Akt signaling (8, 30–32).In this study we first confirmed

that the cAMP pathway was intactin mPGES-1 WT and null MEFs.However, forskolin, a direct adenyl

cyclase activator, did not lead to any change in PPAR� expres-sion and transcriptional activity in either mPGES-1WT or nullMEFs in the presence or absence of PGE2. These data suggestthat regulation of PPAR� bymPGES-1-derived PGE2 occurs viaa cAMP-independent pathway. No changes in PPAR� expres-sion were observed following inhibition of MEK or p38 MAPkinase. However, our results clearly show that PI 3-kinase andAkt inhibitors significantly up-regulated PPAR� expression inmPGES-1WTMEFs, raising the levels similar tomPGES-1 nullMEFs. PI 3-kinase and its immediate downstream effector Aktare therefore the key downstream signaling pathways by whichPPAR� expression is suppressed by PGE2. Various other stud-ies using fibroblasts and other cell types have also shown thatPGE2 mediates its regulatory effects on various downstreamtargets via PI 3-kinase and Akt signaling pathways (33, 34).Negative Regulation of Proinflammatory COX-2, mPGES-1,

and Their Derived PGE2 by PPAR�—PPAR� is an endogenousregulator known to mediate its anti-inflammatory effects bydown-regulation of proinflammatory mediators (20, 21, 29). Incontrast COX-2, mPGES-1, and their derived PGE2 are keyproinflammatory mediators involved during initiation of inflam-

FIGURE 6. Effect of PI 3-kinase inhibitor (a), Akt inhibitor (b), p38 inhibitor and MEK inhibitor on PPAR�expression in mPGES-1 WT and null MEFs (c). mPGES-1 WT and null MEFs were incubated for 12 h withLY294002 (30 �M), Akt inhibitor (30 �M), SB203580 (30 �M), and PD98059 (30 �M) in the presence or absence ofPGE2, and PPAR� protein expression was assessed by Western blotting. Mean � S.E. for PPAR� protein expres-sion normalized with �-actin from n � 3 to 5 separate embryo lines is shown. p � 0.05 is considered statisticallysignificant. Cont, control. * mPGES-1 null MEFs compared with mPGES-1 WT MEFs; �, PGE2-treated groupscompared with their respective unstimulated controls; $, LY294002- or Akt inhibitor-treated groups comparedwith their respective unstimulated controls; #, LY294002 � PGE2 or Akt inhibitor- � PGE2-treated groupscompared with their respective PGE2-stimulated groups.




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mation (1). Using specific PPAR�agonist (rosiglitazone), PPAR� ligand(15-deoxy-PGJ2), and PPAR� inhibi-tor (GW9662), we observed thatspecific activation of PPAR�blocked IL-1�-induced up-regula-tion of proinflammatory COX-2,mPGES-1, and their derived PGE2in WT MEFs. Because of the ele-vated PPAR� levels observed upongenetic deletion of mPGES-1, wetherefore expected that mPGES-1null MEFs would have lower levelsof COX-2 compared with WTMEFs. Indeed, our results showedsignificantly lower levels of COX-2protein in mPGES-1 null MEFscompared with WT MEFs underbasal conditions. These results fur-ther confirm the endogenous anti-inflammatory properties of PPAR�in down-regulating key proinflam-matory signals.Significance of mPGES-1 Inhi-

bition—PGE2 is a key proinflamma-torymediator of inflammation asso-ciated with various disease states,and increased PGE2 requires bothCOX-2 and mPGES-1 (4, 5, 18, 19,35–38). Nonselective and COX-2-selective NSAIDs reduce PGE2 pro-duction by inhibiting COX-2 activ-ity and are extensively used forreducing inflammation, pain, andfever (39). COX-2-specific NSAIDswere developed with improved gas-trointestinal safety (40–42). How-ever, recent clinical trials usingselective COX-2 inhibitors suggestthat specific inhibition of COX-2 isassociated with increased incidenceof cardiovascular events (11–13).Specific COX-2 inhibition results inloss of anti-thrombotic prostacyclin(PGI2) derived from endothelialCOX-2, which plays a key role in theregulation of thrombogenesis (43)and is a possible factor associatedwith cardiovascular side effectsobserved with the use of specificCOX-2 inhibitors.mPGES-1 specifically catalyzes

the conversion of PGH2 to PGE2,particularly during inflammation,and is an attractive target to achievemore specific inhibition of PGE2production (3). Recent studies byour group and others (6, 44, 45) have

FIGURE 7. Effect of PPAR� on COX-2 and mPGES-1 protein expression and PGE2 levels in MEFs. a, IL-1�-stimulated and unstimulated mPGES-1 WT MEFs were incubated for 12 h with PPAR� agonists, rosiglitazone (Ros, 10�M) and 15-deoxy-PGJ2 (10 �M), in the presence or absence of PPAR� antagonist GW9662 (3 �M). COX-2, mPGES-1,and �-actin protein expressions were assessed by Western blotting. Mean � S.E. for COX-2 and mPGES-1 proteinexpressions normalized with �-actin from n � 3 to 4 separate embryo lines is shown. p � 0.05 is considered statis-tically significant. Cont, control. *, IL-1� stimulation compared with unstimulated control;�, rosiglitazone � IL-1� or15-deoxy-PGJ2 � IL-1� treated groups compared with their respective IL-1�-stimulated groups; $, rosiglitazone �GW9662 � IL-1� or 15-deoxy-PGJ2 � GW9662 � IL-1�-treated groups compared with rosiglitazone � IL-1� or15-deoxy-PGJ2 � IL-1�-treated groups, respectively. b, levels of PGE2 in supernatants of mPGES-1 WT MEFs weredetected by ELISA at 12 h post-IL-1� stimulation. Closed bars represent mPGES-1 WT MEFs. Data are expressed as themean � S.E. for four embryo lines. * and ** indicate statistical significance at p � 0.05 and p � 0.01, respectively.c, COX-2 expression in IL-1�-stimulated and unstimulated mPGES-1 WT and null MEFs was determined by Westernblotting. Mean � S.E. for COX-2 protein expressions normalized with �-actin from n � 5 separate embryo lines isshown. *, untreated mPGES-1 null MEFs compared with untreated mPGES-1 WT MEFs.




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shown that genetic deletion of mPGES-1 results in diversion ofprostaglandin production from predominant PGE2 towardPGI2. In addition, we have recently shown that genetic deletionof mPGES-1 results in up-regulation of nitrite levels (stablemetabolic product of nitric oxide (NO)) (6). PGI2 and NO arekey mediators involved in maintaining vascular homeostasis(46). A recent in vivo study by Fitzgerald and co-workers (45)also showed that mPGES-1 deletion depressed PGE2 andincreased PGI2, with no effect on thrombogenesis or bloodpressure in mice. Thus, these observations suggest that inhibi-tion of mPGES-1may avoid the cardiovascular side effects seenwith inhibition of COX-2. In addition, this study shows thatgenetic deletion of mPGES-1 elevates anti-inflammatoryPPAR�. In vitro and in vivo studies suggest that PPAR� has theability to stimulate anti-inflammatory responses and assist inthe resolution of inflammation by inhibiting a broad range ofproinflammatory mediators, including IL-1�, tumor necrosisfactor-�, and nuclear transcription factor �B (20, 23, 28, 47). Inthis study we also demonstrate that PPAR� has the ability todown-regulate proinflammatory COX-2, mPGES-1, and theirderived PGE2 (Fig. 8).Although the biology of mPGES-1 and the consequences of

blocking its activity have yet to be completely delineated,mPGES-1 remains a viable target. It is not known if the efficacyof such a therapeutic strategy would equal inhibition of COXand all downstream PGs; however, data in mPGES-1 null miceand the efficacy of monoclonal antibodies against PGE2 in anarthritis model (48) offer promise. Studies to date in mPGES-1null mice and MEFs suggest that inhibiting mPGES-1 may beassociated with downstream changes, such as increased NO (6)andPPAR�, whichwould promote efficacy andpotentially limit

adverse effects associated with pharmacological inhibition ofmPGES-1.

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FIGURE 8. Schematic diagram showing consequences of mPGES-1 dele-tion on PPAR�. mPGES-1 deletion decreases proinflammatory PGE2 and itsdownstream signaling and as a result elevates anti-inflammatory PPAR�,which has the ability to suppress proinflammatory COX-2, mPGES-1, and theirderived PGE2. � represents down-regulation; � represents elevation; dottedarrows represent decrease in PGE2 signaling via EP receptors and its down-stream signaling pathways.




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Mohit Kapoor, Fumiaki Kojima, Min Qian, Lihua Yang and Leslie J. CroffordAKT PATHWAY

PROSTAGLANDIN E2 VIA THE PHOSPHATIDYLINOSITOL 3-KINASE AND : REGULATION BYγPeroxisome Proliferator-activated Receptor

Microsomal Prostaglandin E Synthase-1 Deficiency Is Associated with Elevated

doi: 10.1074/jbc.M610153200 originally published online December 22, 20062007, 282:5356-5366.J. Biol. Chem.

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Microsomal Prostaglandin E Synthase-1 Deficiency Is Associated