Proc. Nati. Acad. Sci. USAVol. 90, pp. 1440-1444, February 1993Biochemistry

Interaction of the peroxisome-proliferator-activated receptor andretinoid X receptorK. L. GEARING*, M. GOTTLICHER*t, M. TEBOUL*, E. WIDMARKO, AND J.-A. GUSTAFSSON**Department of Medical Nutrition and tCenter for Biotechnology, Karolinska Institute, Novum S-141 86 Huddinge, Sweden

Communicated by Elwood V. Jensen, October 7, 1992 (received for review July 23, 1992)

ABSTRACT The rat peroxisome-proliferator-activated re-ceptor (PPAR) was expressed in insect cells and was shown tobind to a cognate PPAR response element (PPRE) from theacyl-CoA oxidase gene. Upon purification, PPAR was no longerable to bind DNA, although binding could be restored byaddition of insect cell extracts. We investigated whether theretinoid X receptor (RXR) could supplement for this accessoryactivity. The rat RXRa cDNA was cloned and it was found thataddition of in vitro-translated RXRa to purified PPAR facili-tated binding ofPPAR to a PPRE. Furthermore, an additionalactivity, which appeared to be distinct from rRXRa, was foundin COS cell nuclear extracts that enabled binding of PPAR toa PPRE. Transient expression ofRXRa in CHO cells was foundto be essential for the response of a chloramphenicol acetyl-transferase reporter construct containing PPREs to activatorsof PPAR. These results raise the possibility of convergence ofthe PPAR and retinoid-dependent signaling pathways on pro-moters containing PPRE-like responsive elements.

The peroxisome-proliferator-activated receptor (PPAR) is amember of the nuclear receptor superfamily and is structur-ally related to the subfamily of receptors that includes thethyroid hormone receptor (TR), retinoic acid receptor(RAR), and vitamin D3 receptor (VDR)(1). PPAR has beenshown to be activated by fatty acids (2, 3) and by drugs thatinduce peroxisome proliferation such as clofibric acid,nafenopin, and WY-14,643 (4).PPAR response elements (PPREs) have recently been

identified in the 5' flanking sequences of peroxisome-proliferator-inducible genes such as the rat acyl-CoA oxidase(AOX) gene (5), encoding the key enzyme in peroxisomal,B-oxidation and the gene for cytochrome P450 CYP4A6 (6),which catalyzes w and c-1 hydroxylation of fatty acids. Theknown PPREs contain a binding motif consisting of a directrepeat (DR) of a hexanucleotide sequence [5'-TGA(A/C/T)CT] separated by a single nucleotide (DR1). This kind ofDR sequence is also found in a number of other nuclearreceptor response elements, e.g., the TR, RAR, VDR, COUPtranscription factor, and retinoid X receptor (RXR) (7, 8).Although these receptors can all recognize the same half-sitemotif, they discriminate between target elements through thearrangement and spacing of the half-site motifs. A single sitemay also be the target of several receptors that can act topositively or negatively regulate the transcription of a targetgene. For example, the DR1 is recognized by retinoid recep-tors and by homomeric and heteromeric complexes of RXRand COUP transcription factor (9, 10).

It has also been shown recently that the TR, RAR, andVDR all require an auxiliary factor (RXR) to bind to theirrespective target DNA sequences efficiently (8-14). SincePPAR is structurally related to these receptors, we wanted todetermine whether the PPAR could bind to its DR1 typeelement alone or whether it also required additional factors

such as RXR for binding. If RXR was required for DNAbinding, we wanted to test the effect of receptor activation oninducibility of a promoter containing PPREs to elucidatewhether the PPAR-activated pathways in the cell could beinfluenced by activation of RXR.

MATERIALS AND METHODS

Expression of Rat PPAR in Insect Cells. The rat PPARcDNA (2) was modified by deletion of nucleotides encodingthe first 26 amino acids and insertion of an oligonucleotide(coding strand, 5'- T CGA GGA TCC ATG CCG GAT CATCAC CAT CAC CAT CAC CGA TAT C) encoding a stretchof six histidine residues preceded by the first 3 amino acidsof the Autographa californica nuclear polyhedrosis virus(AcNPV) polyhedrin gene (Met-Pro-Asp-His-His-His-His-His-His-Arg-Tyr-Leu-Glu). The modified PPAR cDNA wasthen inserted in the baculovirus transfer vector pAcRP23 (15)and used to generate a recombinant virus containing themodified PPAR gene (Ac.PPAR) in place of polyhedrin.Spodoptera frugiperda (Sf) cells (IPLB-Sf2i)(16) used forvirus propagation were cultured in TC100 medium supple-mented with 10% (vol/vol) fetal calf serum, penicillin (100units/ml), and streptomycin (100 ,ug/ml) at 28°C.For pulse labeling of insect cell proteins, methionine-free

TC100 medium was prepared and supplemented with 4%dialyzed fetal calf serum and [35S]-methionine (12.5 ,uCi/ml;1000 Ci/mmol; 1 Ci = 37 GBq; Amersham). The mediumfrom 107 infected cells was replaced with labeling medium 24h after infection and cells were harvested 30 h after infection.

Purification ofRecombinant PPAR. Sf cells grown in mono-layers were infected with recombinant Ac.PPAR or wild-typeAcNPV at =10 plaque-forming units per cell and wereincubated at 28°C until -36 h after infection. The cells wereharvested, washed in phosphate-buffered saline, resus-pended in buffer A (25 mM potassium phosphate/i mMEDTA/2 mM dithiothreitol/1 mM phenylmethylsulfonyl flu-oride, pH 7.4 at 4°C), and disrupted by 50 strokes in a Douncehomogenizer. KCI was added to 0.4 M and the homogenatewas incubated on ice for 30 min before centrifugation for 30min at 70,000 rpm in a Beckman TL100.3 rotor. The super-natant was loaded onto a Ni2+-NTA agarose column (Diagen,Dusseldorf, F.R.G.) with 125-,ul bed volume and washedtwice with 1 ml of buffer A containing 0.4 M KCl and twicewith 1 ml of buffer A containing 0.1 M KCl. PPAR was elutedin 500 ,ul of buffer B (20 mM Hepes/1 mM EDTA/2 mMdithiothreitol/100 mM KCI/30 mM imidazole, pH 7.6). Ad-ditionally, PPAR contained in the pellet after cell disruptionwas solubilized in 6 M guanidinium hydrochloride, purifiedunder denaturing conditions on a Ni2+-NTA agarose column

Abbreviations: AcNPV, Autographa californica nuclear polyhedro-sis virus; RAR, TR, VDR, receptors for retinoic acid, thyroidhormone, and vitamin D; RXR, retinoid X receptor; PPAR, perox-isome-proliferator-activated receptor; PPRE, PPAR response ele-ment; DR, direct repeat.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(Diagen) according to the manufacturers instructions, andused for immunizing two rabbits against PPAR.

Cloning and Expression of Rat RXRa cDNA.* Rat RXRawas cloned from a livercDNA library essentially as describedfor the isolation of PPAR (2). The cDNA was subcloned inpGEM-3Z (Promega) as a Pst I fragment (nucleotides 100-1861, see Fig. 2) to give the plasmid pRRXR-T7. RXRa wastranslated in vitro using the TNT-coupled reticulocyte lysatesystem (Promega).

Preparation ofCOS Cell Nuclear Extracts. COS cells grownon 600-cm2 dishes were transfected with 80 ,tg of the pCMV-RXR expression vector using DEAE-dextran and weregrown for an additional 48 h. Cells were homogenized inhypotonic buffer (25 mM Hepes/1 mM EDTA/2 mM dithio-threitol), and nuclei were prepared by three sequential cen-trifugations at 1000 X g. Nuclear proteins were extracted byadjusting the KCl concentration to 0.4 M, and insolublematerial was removed by centrifugation for 30 min at 100,000x g. Prior to use, the salt concentration was reduced to 0.1M KCl by addition of hypotonic buffer and the precipitateformed was removed by centrifugation at 15,000 x g.DNA Binding Studies. Gel shifts were performed using in

vitro-translated receptors, baculovirus-expressed protein, orCOS cell nuclear extracts. Proteins were incubated on ice for15 min with 4 ug of poly (dI-dC) (and with unlabeledcompetitor DNAs or 1 ,ul of rabbit antiserum where indi-cated) before addition of 0.5 ng of a 32P-end-labeled PPREoligonucleotide. The reaction mixtures were incubated for afurther 10 min at 22°C before electrophoresis at 200V and 4°Cin pre-run 4% polyacrylamide/0.25 x TBE gels (1 x TBE is 90mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3).

Expression and Transactivation Analysis in CHO Cells.CHO cells were cultured in Ham's F-12 medium supple-mented with 10% fetal calf serum, penicillin (100 units/ml),and streptomycin (100 ,ug/ml; GIBCO/BRL). The plasmidpMT-PPAR was constructed by inserting the PPAR openreading frame (2) into the expression vector pMThGH (17).The expression vector pCMV-RXR was constructed by in-serting the RXR open reading frame from pRRXR-T7 as anEcoRI-HindIll fragment in the multiple cloning site ofpCMV5 (18). The reporter plasmid pACO-2-CAT was pre-pared by inserting two copies of the AOX PPRE oligonucle-otide (see Fig. lb) into the Xba I site upstream from nucle-otide -105 of the herpes simplex virus thymidine kinasepromoter in pBLCAT2 (19). Reporter plasmid (1 jig), pRSV-GAL (5 jig), and expression vector for either RXR or PPAR(10 ,ug) were transfected into CHO cells (80% confluent in21-cm2 dishes) by using 25 ,ug of Lipofectin. The cultureswere harvested 4 h after completion of transfection, dividedbetween four plates, and incubated for 36 h in the presenceor absence of all-trans-retinoic acid (10 ,uM; Sigma) and/orWY-14,643 (100,M; Chemsyn Science Labs., Lenexa, KS).

RESULTS AND DISCUSSIONExpression and Purification of PPAR. A recombinant bac-

ulovirus (Ac.PPAR) was prepared containing the modified ratPPAR cDNA sequence. A protein of 54 kDa was synthesizedin Ac.PPAR-infected cells (Fig. 1A). Fractionation of cellspulse-labeled with [35S]-methionine and harvested 30 h afterinfection showed that a minor fraction of the recombinantPPAR could be recovered in a soluble form from cytosolicextracts after mild detergent lysis (0.05% Nonidet P-40/0.1 MKCl). Most of the expressed protein copurified with thenuclear pellet and was not extractable in the presence of 0.6M KCI. Since this fraction was soluble only in the presence

tThe sequence reported in this paper has been deposited in theGenBank data base [accession no. L06482 (rat RXRa cDNA)].

A wholecells

NPV PPAR

NP40 nuclear pelletextract extract

NPV PPAR NPV PPAR NPV PPAR

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Sf-Ac.NPV (HSEiSf-Ac.PPAR eLSE1

pure PPAR from HSEpure PPAR (from LSE

imidazole buffemock purification

w w&w-

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FIG. 1. (A) Extraction of PPAR from cells infected with arecombinant baculovirus. Cells were infected with AcNPV (NPV) orAc.PPAR (PPAR) and pulse-labeled with [35S]methionine 24 h afterinfection (whole cells). Cell lysis using a mild detergent [Nonidet P-40(NP40) extract] was followed by extraction with 0.6 M KCl (nuclearextract). The residue (pellet) was also analyzed. Lanes labeledstained show Coomassie-stained molecular mass markers (lane M;66, 45, 36, 29, and 24 kDa) and PPAR after purification by Ni2+-chelate chromatography. PPAR (solid arrowhead) and polyhedrin(arrow) are indicated. (B) Gel-shift analysis ofoverexpressed PPAR.High salt (HSE) or low salt (LSE) extracts from infected cells orPPAR purified from either high salt cell extracts or low salt cellextracts were incubated in 100 mM KCI/10 mM HEPES, pH 7.6/1mM dithiothreitol/1 mM EDTA/1l% (wt/vol) glycerol with a 32pend-labeled PPRE oligonucleotide from the AOX gene (upper strand,5'-CTAGCGATATCATGACCTTTGTCCTAGGCCTC; lowerstrand, 5'-CTAGGAGGCCTAGGACAAAGGTCATGATATCG).Mock-purified material was prepared in the same way as purifiedPPAR from high salt extracts but using cells infected with AcNPV.

of 8 M urea or 6 M guanidinium hydrochloride, we assumedthat the PPAR had formed insoluble aggregates, perhapssimilar to those formed by the androgen receptor whenoverexpressed in this system (20). A smaller [35S]_methionine-labeled band is also seen in the extracts of cellsinfected with Ac.PPAR (Fig. 1A). This is probably a degra-dation product ofPPAR in this particular experiment and wasnot seen in subsequent experiments.For in vitro binding studies, the soluble PPAR present in

cell extracts prepared in 0.1 M KCI or 0.4M KCl was purifiedunder nondenaturing conditions on Ni2+-NTA agarose col-umns. Typically, 10 ,ug ofPPAR was purified from =4 x 107infected Sf cells grown in monolayers. The PPAR was >90%

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1442 Biochemistry: Gearing et al.

pure as judged by Coomassie blue staining of SDS/polyacrylamide gels (Fig. 1A).DNA Binding of PPAR. Extracts of cells infected with

Ac.PPAR were prepared in high and low salt buffers and weretested in a gel-retardation assay for the ability to bind to aPPRE from the AOX gene (Fig. 1B). PPAR present in highsalt extracts (0.4 M KCI) was able to bind to this PPRE,whereas PPAR in the low salt extracts (0.1 M KCl) was notable to bind. PPAR purified from either high or low saltextracts was also unable to bind to the PPRE. However,addition of high salt extracts from cells infected with AcNPVcould restore DNA binding to both the purified PPAR frac-tions and to the low salt cell extracts. This suggested that afactor present in insect cells was necessary for efficientbinding ofoverexpressed PPAR to its response element. Thisfactor was absent in the low salt cell extracts and, althoughinitially present in the high salt extracts, was subsequentlylost during the purification procedure.

Cloning of the Rat RXR. The behavior of purified overex-pressed PPAR appeared to be similar to that ofthe RAR whenexpressed in insect cells (12) in that nonpurified protein in cellextracts is able to bind to a DNA response element, whereasthe purified protein cannot. For RAR, an RXR-like activitywas present in the insect cells that compensated for the lackof mammalian RXR. It was of interest, therefore, to testwhether the complementing factor for DNA binding in thecase of overexpressed PPAR could also be RXR. The cDNAfor RXRa was isolated by low-stringency hybridization of arat liver cDNA library with an oligonucleotide probe derivedfrom the coding regions for the DNA-binding domain of apanel of eight nuclear receptors (2). We obtained a 2.1-kbcDNA clone encoding a 467-amino acid protein (Fig. 2) thatshows the highest homology to RXRa proteins from mouse(98.5%) (21, 22) and human (97.4%6)(23).

In vitro translation of this RXR clone produced a proteinthat was assayed in agel shift experiment for its ability to bindto a PPRE with purified PPAR (Fig. 3). Although PPAR orRXR alone were unable to bind the PPRE, together theseproteins were able to form a specific complex with this DNAelement. The appearance of this band depends on the pres-ence of RXR and is inhibited by a 10-fold excess of thespecific DNA target element, but not by a 100-fold excess ofan unrelated DNA element (Fig. 3, lanes 8-11). Furthermore,the specific complex is destroyed by a polyclonal antiserumagainst PPAR (Fig. 3, lanes 12 and 13). These data suggestthat a protein-DNA complex is formed that contains the

Proc. Natl. Acad. Sci. USA 90 (1993)

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FIG. 3. Interaction of PPAR and RXR on a PPRE. In vitro-translated RXR mixed with purified PPAR was incubated with32P-end-labeled PPRE oligonucleotide. Preincubations for 15 min onice with a 10- or 100-fold excess of nonlabeled competitor oligonu-cleotides or immune sera were carried out where indicated. Thesequence of the nonspecific oligonucleotide is 5'-GGATCCACCCT-GTCTCATGAATATGCAAATCAGGTGAG. The samples withcontrol reticulocyte lysate are identical to those with RXR except forcontaining the complementary RNA instead of the coding mRNA.The specific PPAR-RXR-PPRE complex is indicated (solid arrow-head) and the complex formed by Ac.PPAR-infected Sf cells isshown as a reference (arrow).

PPRE, PPAR, and rRXRa and indicate that rRXRa cansubstitute for the accessory factor present in Sf cells.

Interaction of PPAR with COS Cell Nucear Extrcs. Ex-periments to express receptors in COS cells revealed anactivity in COS cell nuclear extracts that supplements bindingof purified PPAR to its cognate responsive element (Fig. 4,lanes 2 and 6). However, this activity does not depend ontransfection ofthe COS cells with the RXR expression vector(Fig. 4, compare lanes 2-5 with lanes 6 and 7). The electro-phoretic mobility of the complex differs from the complexwith in vitro-translated RXR (Fig. 4, lane 11), the complex issupershifted by an anti-PPAR serum (Fig. 4, lanes 10 vs. 9)and the specific band is competed by a 10-fold excess ofnonlabeled specific DNA target element (data not shown).Increasing amounts of COS cell nuclear extracts inhibit

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I GAATTCCGAr.TAaGGACCAAGAGAGCTGGACTGATAGAACTAATrTTATlTTCCAGGGGCATTGACTTTCCAAGTTCACTTCAGAGGCAGCTrrAGTAAAGCGCGTAaAAANACAA,1 TTAAAAGAAGTrACATTAGTCCGGCTWGGATGAGTTAGTAGCAGACMaCAQCCAAACATTTCC'T&CCGCTCGACTTCTCTACCCAGGAACTCTTCATCCCTGAGCTC CAACGG1 M D T K H F L P L D P S T Q V N S S S L S S P T G,1 GTCGAGGCTCCATGGCTGCCCCCTCGCTGCACCCTTCCl GGGTCCAGGACTCGGCTCVCCTGC3GCTCCCCIGGCAGCTGCACTCGCCTATCAGCACCCTGAGTTCTCCCATCAAIN'6 R G S N A A P S L H P S L G P G L G S P L G S P G Q L H S P I S T L S S P I N G1 GCATGGGCCCACCCTTCTCGGTCATCAGCTCCCCGATQGGCCCGCACTCCATGTCGGTACCCACCACACCCACACTGGGCTTCGAGACTG GCAGCCCCCAGCTCAACTCACCCATIAACCi6 N G P P P S V I S S P N G P H S N S V P T T P T L G P E T G S P Q L N S P N N PI1 CCGTIAGCAGCAGTGAGGATATCAAMCCCCCTCTA GCCTCAAT GCGTCCTCAAGGTTCCTGCCCACCCCTCAGGAAATATGTCATCC I ICAGC TCACC16 V S S S E D I K P P L G L N G V L K V P A H P S G N N S S F T K H I C A I C G DII1 ACCGCTCCTCGCkCihkCGTl AGGTU ^AC1G Arm AGAAGCCCCCTACACC-T.(CaGTGCAQLCAAG;ACTGQm~CL6 R S S G K H Y G V Y S C E G C K G P P K R T V R K D L T Y T C R D N K b C L I D1 Akk&1 &A&G tI CTCCGCGGGAAEICCGTGCAGGAGGAGCGGCAGCGAGGCAAGGACCGGAACGAGEATGfG16 K R Q R N R C Q Y C R Y Q K C L A N G N K R E A V Q E E R Q R G K D R N E N E VI1 IGGAGTCCACCAGCAGEGCCAACGAGGACATGCCIGTAGAGAAGATTCTGGAAGCTGAGCTCGCTTTGAGCCCAAGACTGAGACATACGTCGAGGCAAACATIGGGCT6AACCCCAGCT!6 E S T S S A N E D M P V E K I L E A E L A V E P K T E T Y V E A N N G L N P S S1 CACCAAATGACCCGCACCAACA¶VIITCAAGCAGCAGACAAGCAGCTCAG CAAGAGGATCCCACACTTTNICTGCCCCCTECGACEACCAUGCATCCi6 P N D P V T N I C Q A A D K Q L F T L V E W A K R I P H P S E L P L D D Q V I LI1 IGCTCCGGGCAGGCTGGAACGAGCGCTGATT'GCCTCCTTCTCCCACCGCTCCATAGCIGTGAAAGACGGCATCCTCCTGGCCACCGGCCT&CACGTACACAGGAACAGCGCTCACAGTG16 L R A G W N E L L I A SF H R S I A V K D G I L L A T G L H V H R N S A H S A1 CTGGGGGGCCGCCATCTC ACAGGGTGCTAACGAGCTGGINTCGAAGATGCGTGACATGCAGATIGACAAGACGGAGCTGQGCTGCTIGCGCGCCATIG7TCCTCTTCAACCCTGACT16 G V G A I P D R V L T E L V S K M R D N Q N D K T E L G C L R A I V L F N P D S1 CTAAGGGGCTICCAAACCCTGCIsAGGTGGAGCGCIIAGGGAGMGGGIGTATGCAICACTAGAACGTACIGCAACA6CAAGTACCC¶IAGCGCCGQGCAGGIT1TGCCAAGCT CTGC16 K G L S N P A E V E A L R E K V Y A S L E A Y C K H K Y P E Q P G R P A K L L LI1 TCCG CGCCTGCACTGCGATCCATTGGCTCAAGIVCCTGGAGCACCTGTCTTTCAAGCTCATCGGGGATACACCCAICGACACTTTCCTCAITGAGATsIICTG11GCCCCACATC16 R L P A L R S I G L K C L E H L F PF K L I G D T P I D T F L N E M LH A P H Qi1 AAACCACCTAGGCCCGTCACCCAIT<GCCGGTCCCTTGCCCCGCCTGCACAGCIVCIt:AGCTCCAGCCCTGTCCCTGCCCTTTCT:GAIVGGCCII VGATCTITTGGGGICAGCGTi6

11

FIG. 2. Sequence of the rat RXRa cDNA and predicted amino acid sequence are given. Two potential translation start sites are underlinedat nucleotides 114 and 168, the second being in a more favorable Kozak consensus sequence. The predicted DNA binding domain is alsounderlined.

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FIG. 4. Gel-shift analysis of PPAR with COS cell nuclear ex-tracts. Proteins from the indicated sources were mixed and analyzedfor binding to the PPRE. Proteins were whole cell extract fromAc.PPAR-infected Sf cells (Sf-PPAR), purified PPAR (PPAR),0.01-1 ,g of COS cell nuclear extracts (COS-NE) after transfectionwith pCMV-RXR or mock transfection, RXR synthesized in retic-ulocyte lysate (RXR Retic.L.), reticulocyte lysate identical to RXRRetic.L. except for containing the noncoding complementary RNA,and a polyclonal antiserum against PPAR or preimmune serum. ThePPAR-dependent complexes with COS-NE (arrow) or in vitro-translated RXR (solid arrowhead) and the supershift by anti-PPARserum (bracket) are indicated.

formation of the complex depending on in vitro-translatedRXR (Fig. 4, lanes 11-14). In a similar experiment, thesimultaneous formation of the complexes with rRXRa or theCOS cell factor was also shown using extracts from non-

transfected COS cells (data not shown). Thus, the transfec-tion of RXR into COS cell does not yield any apparentdifference in the activity of the nuclear extract. This may bedue to inefficient expression of RXR or, alternatively, COScells might contain a factor that associates more tightly withRXR than PPAR does, so that RXR is unavailable forinteraction with PPAR.The nature of the endogenous COS cell factor remains

unclear, but it forms a PPAR-dependent protein-DNA com-

plex that appears distinct from that formed with in vitro-translated rRXRa, since its electrophoretic mobility is dif-ferent and the anti-PPAR serum produces a supershift signalwith the COS nuclear extract (Fig. 4, lane 10) rather thaninhibiting the specific gel-shift signal of the rRXRa-containing complex (Fig. 3, lane 13). It can not be ruled outthat the complex formed with in vitro-translated RXR con-tains additional proteins endogenously present in reticulo-cyte lysate. However, it appears unlikely that the PPAR-dependent complexes with in vitro-translated rRXRa or COScell nuclear extract differ solely in the recruitment of anadditional reticulocyte lysate factor, since addition of re-verse-primed reticulocyte lysate to Ac.PPAR-infected Sf cellextracts or to COS cell nuclear extracts mixed with purifiedPPAR does not result in formation of the slower-migratingcomplex (Fig. 3, lanes 1 and 2; Fig. 4, lanes 2 and 8). Insummary, these data indicate that PPAR can form at least twocomplexes on a PPRE together with mammalian proteins.

Transient Transfection of a PPRE-Reporter Gene Constructin CHO Cells. Since binding of PPAR to a PPRE in vitro wassupplemented by RXR, we tested whether coexpression ofRXR in cells that express PPAR could affect the activity ofa reporter gene under the control of a PPRE-containing

promoter. Preliminary experiments showed that the responseof the PPRE-containing promoter did not depend on over-expression of PPAR even when an RXR expression vectorwas cotransfected. We therefore analyzed CHO cell extractsfor the presence of endogenous PPAR and found a 55-kDaprotein that was specifically recognized by the anti-PPARserum (data not shown). Since these cells appear to expressendogenous PPAR, they were cotransfected with the RXRexpression vector and the reporter constructs. The PPRE-containing reporter was inducible by WY-14,643 if pCMV-RXR was cotransfected (Fig. 5A). The response was onlymarginal in the absence of the PPREs and the reporters werenot inducible at all in the absence of RXR. The differencebetween WY-14,643-treated and nontreated cells after trans-fection ofRXR and the pACO-2-CAT reporter was subtle butreproducible. Moreover, in each experiment the reportergene activity was analyzed in a single transfected cell poolthat was subdivided for the four treatment groups. Similardata were also obtained in another set of three experimentswith CHO cells stably overexpressing PPAR (data notshown). However, due to the high standard deviation of thenoninduced reporter gene activity in the different transfec-tion groups (Fig. 5B), it is not evident whether WY-14,643acts to induce the reporter gene or to release a PPAR- andRXR-dependent repression of the reporter gene activity. Theactivation of the reporter gene depending on PPAR and RXRis consistent with the finding that Xenopus PPAR isoformscan activate a similar reporter construct in HeLa cells (25).These cells contain constitutive levels ofRXR (12), whereasin CHO cells PPAR appears to be endogenously present andthe levels of RXR limit the response.

A

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FIG. 5. Transient expression from a PPRE-containing promoterin CHO cells. Chloramphenicol acetyltransferase (CAT) activities ofpACO-2-CAT or pBLCAT2 reporter plasmids were determined (24)after cotransfection of the rRXRa expression vector (pCMV-RXR)or a control vector lacking RXR sequences (pCMV5). The figuresshow CAT activities (±SD) or induction of normalized CAT activ-ities (±SD) from triplicate transfections. Identical results wereobtained from three additional experiments in CHO cells stablytransfected with an expression vector for PPAR. (A) Induction ofreporter genes by 100 ,AM WY-14,643 and/or 10 ,uM all-trans-retinoic acid (RA) in CHO cells in the presence (+RXR) or absence(-RXR) of transiently expressed RXR. The normalizedCAT activityis set at 1 for each transfection in the absence ofinducing compounds.(B) Basal activity of the reporter constructs in the absence ofinducing agents in the presence (+RXR) or absence (-RXR) oftransiently expressed RXR. In each experiment, the normalized CATactivity is set at 1 for the transfection of pBLCAT2 in the absence ofRXR.

Sf-PPAR +PPARlngl ! 00 10

COS-NE (.mock'COS-NE (RXRIJRXR (Retic.L

Retic.LysaveAnt serLum -

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The responsive element for RXR in the retinol-bindingprotein type II gene promoter contains four copies of thehexameric recognition half-site for retinoid/thyroid hor-mone-like receptors each spaced by 1 nucleotide (26). SincetheAOX gene PPRE is similar to the DR1 type sequence, wetested whether the pACO-2-CAT reporter gene could also beactivated by retinoids. all-trans-Retinoic acid has beenshown to activate RXR albeit at much higher concentrationsthan 9-cis-retinoic acid (23). We found that retinoic acid couldactivate the PPRE reporter construct in the presence oftransiently expressed RXR. The level of induction was com-parable to that obtained with WY-14,643 (Fig. SA). Inductionby retinoic acid does not appear to be modulated by the ligandbinding domain of PPAR, since retinoic acid concentrationsofup to 100AM do not activate a receptor chimera containingthe PPAR ligand binding domain that we have used inprevious studies (ref. 2, M.G., unpublished observations).

CONCLUSIONSThe data presented here indicate that rat PPAR requiresadditional protein factors for binding to the PPRE ofthe AOXgene. Rat RXRa can substitute for a factor endogenouslypresent in Sf cell extracts and, additionally, COS cell nuclearextracts contain an activity that forms a PPAR-dependentprotein-PPRE complex that is distinct from that produced byrat RXRa. Furthermore, PPAR and rat RXRa can interactfunctionally, in that RXR is required for the responsivenessofa promoter containing aPPRE to peroxisome proliferators.

In conclusion, DR1 type responsive elements might allowregulation of genes such asAOX by interaction with multipleprotein complexes. These homo- or heteromeric complexesmay consist of RXR and other members of the nuclearreceptor superfamily such as PPAR and the COUP transcrip-tion factor (9) or, alternatively, may contain PPAR andaccessory factors that may be distinct from RXR. Thediversity of possible protein-DNA interactions might reflecta more general role of the DR1 type responsive element inpromoters that respond to multiple signaling pathways suchas in the phosphoenolpyruvate carboxykinase gene (27) andmight provide the link that confers responsiveness to keyenzymes of peroxisome proliferation to peroxisome prolifer-ators and retinoids (28). It remains to be established, how-ever, which mechanisms determine a distinct and cell-type-specific response.

Note Added in Proof. During the review of this work, similarobservations on PPAR and RXR were published by Kliewer et al.(29). However, our data also suggest the formation of additionaldistinct protein-DNA complexes.

The contributions ofK.L.G. and M.G. should be considered equal.We thank Dan Svensson for excellent technical assistance; Qiao Lifor help with cloning RXR; Marika Ronnholm and Charlotte Wik-strom for help in preparing antibodies; Karin Chaloner (Institute ofVirology and Environmental Microbiology, Oxford) for advice onpreparing cell culture medium; Zekiye Cansu for synthesizing oli-gonucleotides; Johan Lund, Sam Okret, and Stefan Nilsson forproviding plasmid vectors; Ian Catchpole and Staffan Bohm forcritical reading of the manuscript; and Lorenz Poellinger for helpfuldiscussions. This work was supported by fellowships from InstitutNational de la Santd et de la Recherche Medicale, the SwedishMedical Research Council (M.T., CS/MCC-806), and European

Molecular Biology Organization (M.G., ALTF 426-1990), and bygrants from the Swedish Medical Research Council (13X-2819) andthe Swedish Cancer Society.

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