©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Orphan Nuclear Hormone Receptor LXR Interacts with the Peroxisome Proliferator-activated Receptor and Inhibits Peroxisome Proliferator Signaling (*)

(Received for publication, February 14, 1996)

Kenji S. Miyata (1)(§) Shannon E. McCaw (1) Hansa V. Patel (1) Richard A. Rachubinski (2)(¶) John P. Capone (1)(**)

From the  (1)Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada and (2)Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The yeast two-hybrid system was used to isolate novel cellular factors that interact with the mouse peroxisome proliferator-activated receptor alpha (PPARalpha). One of the interacting clones isolated encoded LXRalpha, a recently described human orphan nuclear hormone receptor. LXRalpha bound directly to PPARalpha, as well as to the common heterodimerization partner 9-cis-retinoic acid receptor (RXRalpha). LXRalpha did not form a DNA binding complex with PPARalpha on synthetic hormone response elements composed of direct repeats of the TGACCT consensus half-site or on naturally occurring peroxisome proliferator response elements (PPREs) or LXRalpha response elements. However, LXRalpha inhibited binding of PPARalpha/RXRalpha heterodimers to PPREs, and coexpression of LXRalpha in mammalian cells antagonized peroxisome proliferator signaling mediated by PPARalpha/RXRalpha in vivo. These findings identify a novel partner for PPARalpha and suggest that LXRalpha plays a role in modulating PPAR-signaling pathways in the cell.


INTRODUCTION

Peroxisome proliferator-activated receptors (PPARs) (^1)are recently described members of the ligand-activated nuclear hormone receptor superfamily, which includes receptors for steroids, vitamin D, and thyroid and retinoid hormones(1) . PPARs have been shown to regulate a broad spectrum of genes involved in lipid metabolism, cellular growth, and differentiation(2) . Consequently, there is a great deal of interest in understanding their specificity and mechanisms of action. PPARs were originally identified as factors that mediate transcriptional responses to peroxisome proliferators, a broad class of xenobiotic chemicals that include fibrate hypolipidemic drugs and other nongenotoxic rodent hepatocarcinogens(3, 4) . Subsequently, PPARs were shown to be differentially activated by a variety of long chain fatty acids and lipid-like compounds(5) , suggesting that fatty acids or fatty acid derivatives serve as physiological activators. PPARs exist in a variety of pharmacologically distinct subtypes and isoforms that are differentially expressed and which mediate distinct patterns of tissue-specific gene expression(4, 6, 7, 8) . For example, mouse (m) PPAR triggers adipogenesis in cultured cells (8) and is selectively activated by 15-deoxy-Delta-prostaglandin J2, a recently identified high affinity ligand of this PPAR subtype(9, 10) .

PPARs activate expression of target genes by recognizing peroxisome proliferator response elements (PPREs) composed of TGACCT-related direct repeats that are spaced by one nucleotide (DR1)(11, 12) . Specific DNA binding is manifested through heterodimerization with the 9-cis-retinoic acid receptor, RXRalpha(13, 14) , another member of the nuclear hormone receptor superfamily that also serves as a heterodimerization partner for thyroid hormone, retinoic acid, and vitamin D receptors(1) . The involvement of PPARs in multiple and diverse cellular functions suggests that these receptors may be integrated with other cellular signaling pathways, in addition to the well characterized RXRalpha pathway. Indeed, the reciprocal modulation of thyroid hormone and peroxisome proliferator-responsive genes through cross-talk between thyroid hormone receptors and PPARs has recently been demonstrated(15, 16, 17) . Moreover, it has been reported that rat PPARalpha heterodimerizes with the thyroid hormone receptor(18) , although this conclusion remains controversial(15) . Unraveling the pleiotropic functions of PPARs requires identification of the full spectrum of factors that interact with PPARs. In this report, we used the yeast two-hybrid system (19) to isolate novel factors that interact with mPPARalpha. One mPPARalpha-interacting factor isolated was identified as LXRalpha, a recently described human orphan nuclear hormone receptor that appears to be involved in a novel retinoid signaling pathway(20) . LXRalpha inhibited the binding of mPPARalpha/RXRalpha to PPREs in vitro and antagonized transcriptional activation by mPPARalpha in vivo. Our findings demonstrate that nuclear receptors other than RXRalpha bind directly to PPARalpha and may play a role in modulating the cellular functions of this receptor.


MATERIALS AND METHODS

Two-hybrid Library Screening

Yeast two-hybrid vectors expressing full-length human RXRalpha and mPPARalpha as fusions to the GAL4 DNA-binding domain (GBD-RXRalpha and GBD-mPPAR, respectively) and RXRalpha fused to the GAL4 activation domain (GAD-RXRalpha) have been described (21) . GBD-mPPAR was not suitable for two-hybrid library screening, since it induced a low level of constitutive activity of the beta-galactosidase reporter gene when expressed alone in yeast(21) . We therefore constructed a modified vector, GBD-NDeltamPPAR (missing the amino-terminal 83 amino acid codons of mPPARalpha) that was devoid of this intrinsic activation function but remained capable of interacting with RXRalpha (see Table 1). This derivative was used as bait in the two-hybrid system. Saccharomyces cerevisiae strain HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4- 542, gal80-538, LYS::GAL1-GAL1-HIS3, URA3::GAL4CyC1-lacZ) harboring GBD-NDeltamPPAR was subjected to electroporation (22) with a HeLa cell cDNA library constructed in the GAD vector pGADGH (Clontech). Transformants were plated onto synthetic complete media plates lacking histidine, leucine, and tryptophan and containing 25 mM 3-amino-1,2,4-triazole. His, leu, trp colonies were assayed for expression of the beta-galactosidase reporter gene by agarose overlay assay using 0.2% (v/v) Triton X-100 as a permeabilization agent. Library plasmids were rescued by electroporation into Escherichia coli ElectroMAX DH10B (Life Technologies, Inc.), and isolated plasmids were used to retransform S. cerevisiae HF7c. Candidate clones were tested for interaction against GBD-NDeltamPPAR and to GBD-RXRalpha and also to irrelevant fusion proteins and the empty fusion vector so as to eliminate false positives (23) and to ensure that the his and beta-galactosidase phenotypes were dependent on the presence of both the respective GAD-cDNA library vectors and GBD-NDeltamPPAR. Positive clones were recovered and subjected to partial 5`- and 3`-sequence analysis. Two clones contained the same partial cDNA that encoded a novel member of the nuclear hormone receptor superfamily, subsequently shown to be nearly identical to the recently described human LXRalpha (20) . One of these clones (GAD-SM1; encoding amino acid residues 61-447 of LXRalpha) was selected for further analysis. The remaining positive clones were not related to LXRalpha or to other nuclear receptors and will be the focus of future studies.



Cloning a Full-length LXRalpha cDNA

The 1.2-kilobase pair (kbp) insert of GAD-SM1 was labeled with [alpha-P]dATP by random priming and used to probe, under high stringency conditions, a 5`-stretch gt11 human liver cDNA library (Stratagene). The largest hybridizing clone contained a 1.7-kbp insert. The cDNA was sequenced in both directions and shown to contain the entire 447-amino acid long open reading frame corresponding to the published sequence of LXRalpha(20) .

In Vitro Transcription/Translation

In vitro expression vectors for mPPARalpha and human RXRalpha have been described (14) . The entire open reading frame of LXRalpha was amplified from the human liver cDNA library plasmid by the polymerase chain reaction (forward primer, 5`-GCGCGGATCCGGTACCATGTCCTTGTGGCTGGGGGC; reverse primer, 5`-GCGCGGATCCGATATCTCATTCGTGCACATCCCAGATC; initiator codon is underlined) and cloned into the BglII site of the SP6 transcription vector pSPUTK. Transcription of the different cDNAs, followed by translation in rabbit reticulocytes, was performed using a coupled system (Promega), as described previously (14, 24) .

Protein Binding Assays

Maltose-binding protein (MBP)-mPPARalpha and MBP-RXRalpha fusion protein expression vectors were constructed in pMAL-2c (New England Biolabs), and the fusion proteins were purified by affinity chromatography according to the manufacturer's instructions. Purified MBP-RXRalpha, MBP-mPPARalpha, and MBP proteins (2 mg/ml in column buffer (20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 20% (v/v) glycerol)) were adsorbed to amylose resin (4.5 mg/ml settled resin) and resuspended as a 50% slurry in column buffer. Twenty µl of slurry was adjusted to 500 µl with column buffer containing 0.2% (v/v) Nonidet P-40 and 2% bovine serum albumin (BSA) and incubated with 1-5 µl of programmed reticulocyte lysate for 30 min at room temperature with continuous mixing. Beads were washed sequentially with 20 volumes of column buffer containing 0.2% (v/v) Nonidet P-40 and 2% BSA; column buffer containing 0.1% (v/v) Nonidet P-40 and 2% BSA; and column buffer alone. Bound material was eluted from beads by boiling in SDS sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis.

Gel Retardation Analysis

Electrophoretic mobility shift assays were carried out as before(14, 24) . Synthetic oligonucleotide probes corresponding to the rat fatty acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) PPREs have been described(14) . The following double-stranded oligonucleotides containing TGACCT direct repeats spaced by 0-5 nucleotides (DR0-DR5) were synthesized (for each, only the top strand is shown; TGACCT direct repeats are underlined; single-stranded BamHI compatible ends are in lower case): DR0, gatcTTCTGACCTTGACCTGG; DR1, gatcTTCTGACCTCTGACCTGG; DR2, gatcTTCTGACCTCCTGACCTGG; DR3, gatcTTCTGACCTCCTTGACCTGG; DR4, gatcTTCTGACCTCCTGTGACCTGE; DR5, gatcTTCTGACCTCCTGGTGACCTGG. The LXRalpha response element (LXRE) (20) was synthesized by annealing 5`-GATCCTTGCGGTTCCCAGGGTTTAAATAAGTTCATCTA and the complementary strand 5`-GATCTAGATGAACTTATTTAAACCCTGGGAACCGCAAG.

Transient Transfections and Measurement of Luciferase Activity

mPPARalpha and RXRalpha expression plasmids and the AOx-PPRE luciferase reporter plasmid (pAOx(X2)luc) have been described(14) . The mammalian expression vector for LXRalpha was constructed by cloning the LXRalpha cDNA into pRc/CMV (Invitrogen). Reporter plasmids containing two tandem copies of the synthetic DR4 or DR1 direct repeat elements (pDR4(X2)luc and pDR1(X2)luc, respectively) were constructed by cloning the corresponding double-stranded oligonucleotides described above into the BglII site of the enhancerless SV40 promoter/luciferase expression vector pGL2 (Promega). Transfection of BSC40 cells and measurement of luciferase activity were carried out as described before (12, 14, 24) . Where indicated, the peroxisome proliferator Wy-14,643 or the RXRalpha ligand 9-cis-retinoic acid was added to final concentrations of 10 and 10M, respectively (from 100 times stocks in dimethyl sulfoxide). Control cells were treated with vehicle alone.


RESULTS AND DISCUSSION

Interaction Cloning of PPAR-interacting Factors

Yeast harboring GBD-NDeltamPPARalpha were used to screen a HeLa cell cDNA library fused to the GAL4 transactivation domain. Of 5 times 10^5 independent transformants screened, 9 colonies remained blue in color and his upon clonal purification. Retransformation of rescued plasmids confirmed that all 9 candidate clones conferred the his and beta-gal phenotypes only in the presence of GBD-NDeltamPPAR. Two positive clones, designated GAD-SM1 and GAD-KM1, contained overlapping sequences of the same cDNA that partially encoded a previously undescribed member of the nuclear hormone receptor superfamily. Interestingly, the same clone was isolated in an independent screen for RXRalpha-interacting factors (data not presented), demonstrating that this factor also interacts with RXRalpha (Table 1). A 1.7-kbp cDNA was isolated from a gt11 human liver cDNA library using GAD-SM1 as a probe and shown to contain an open reading frame encoding a 447-amino acid polypeptide. After this work was completed, Willy and co-workers (20) reported the cloning of a cDNA encoding a novel orphan nuclear hormone receptor using low stringency screening of a human liver cDNA library. The cDNA for this receptor, designated LXRalpha, is essentially identical at both the nucleotide and deduced amino acid sequence level to the cDNA clone isolated here by mPPARalpha interaction cloning. The only differences noted in the deduced polypeptide are a phenylalanine in place of the leucine at position 192 and a histidine in place of the leucine at position 414. LXRalpha was shown to interact with RXRalpha(20) , consistent with our two-hybrid analysis results. By convention, we will refer to our mPPARalpha-interacting receptor as LXRalpha.

LXRalpha Interacts with mPPARalpha and RXRalpha in Vitro

To confirm that LXRalpha physically interacts with mPPARalpha and RXRalpha, protein binding assays were carried out using immobilized MBP fusion proteins and [S]methionine-labeled LXRalpha synthesized in vitro. Labeled LXRalpha bound to both MBP-mPPARalpha (Fig. 1a, lane 4) and MBP-RXRalpha (Fig. 1b, lane 6) but not to the control MBP beads (Fig. 1a, lane 2; Fig. 1b, lane 3). A labeled luciferase control protein did not bind to beads complexed with MBP (Fig. 1, a and b, lanes 1), MBP-mPPARalpha (Fig. 1a, lane 3), or MBP-RXRalpha (Fig. 1b, lane 4), demonstrating the specificity of the observed interactions. Quantitative analysis indicated that LXRalpha bound approximately 10-fold more efficiently to RXRalpha vis à vis mPPARalpha. LXRalpha thus binds directly to both mPPARalpha and RXRalpha in vitro.


Figure 1: LXRalpha interacts with mPPARalpha and RXRalpha in vitro. [S]Methionine-labeled LXRalpha synthesized in vitro was incubated with immobilized MBP-mPPARalpha, MBP-RXRalpha, and MBP, as indicated. Bound radiolabeled protein was analyzed by polyacrylamide gel electrophoresis. Labeled luciferase was used as a negative control. NDeltaRIP1 in panel b is a truncated version of LXRalpha that is missing 60 amino-terminal amino acids. This derivative retains the ability to interact with RXRalpha (lane 5) and mPPARalpha (not shown).



LXRalpha Binds to Direct Repeat Hormone Response Elements Cooperatively with RXRalpha but Not with mPPARalpha

To identify potential mPPARalpha/LXRalpha DNA-binding sites, we synthesized a series of oligonucleotides that contained TGACCT direct repeats separated by 0-5 nucleotides (DR0-DR5) and tested them in electrophoretic mobility shift assays with in vitro translated LXRalpha, RXRalpha, and mPPARalpha alone or in combination. Binding studies were also carried out with natural PPREs from the HD and AOx genes and with a natural LXRE previously identified in the mouse mammary tumor virus promoter(20) . As shown in Fig. 2a, mPPARalpha, LXRalpha, and RXRalpha on their own did not bind to any of the elements tested. However, LXRalpha bound cooperatively with RXRalpha to a DR4 element, the LXRE, and to a lesser extent, to a DR5 element, as reported previously (20) . As expected, mPPARalpha/RXRalpha heterodimers bound preferentially to a DR1 element as well as to the AOx- and HD-PPREs. In contrast, cooperative binding of LXRalpha and mPPARalpha was not observed on any of these synthetic direct repeat or natural response elements. Whether there exist natural and specific DNA target sites recognized by mPPARalpha/LXRalpha heterodimers remains to be determined.


Figure 2: LXRalpha binds cooperatively with RXRalpha but not with mPPARalpha to synthetic or natural response elements. Electrophoretic mobility shift assays were carried out by incubating in vitro synthesized LXRalpha, mPPARalpha, and RXRalpha (1 µl of programmed reticulocyte lysate), as indicated, with synthetic DR0-DR5 direct repeat response element, AOx-PPRE, HD-PPRE, or LXRE probes (panel a). Panel b, the HD- and AOx-PPREs were incubated with constant amounts of RXRalpha and mPPARalpha (1 µl of programmed reticulocyte lysate each) and increasing amounts of LXRalpha (0.5-4 µl of programmed reticulocyte lysate). Protein concentration in each reaction was normalized with unprogrammed reticulocyte lysate as appropriate.



LXRalpha Inhibits Binding of mPPARalpha/RXRalpha Heterodimers to PPREs

Coincubation of LXRalpha with RXRalpha and mPPARalpha resulted in a decrease in the binding of mPPARalpha/RXRalpha to the HD- and AOx-PPREs (Fig. 2a, compare lanes 5 and 8). Similarly, mPPARalpha reduced binding of LXRalpha/RXRalpha to DR4 and DR5 elements and to the LXRE (compare lanes 7 and 8). Coincubation with increasing amounts of LXRalpha resulted in a progressive decrease in the binding of mPPARalpha/RXRalpha heterodimers to the HD- and AOx-PPREs (Fig. 2b). Since LXRalpha binds directly to mPPARalpha and to RXRalpha, this inhibition is likely the result of LXRalpha sequestering one, or both, of these receptors into non-binding complexes. Reciprocally, mPPARalpha can competitively inhibit the binding of LXRalpha/RXRalpha to the LXRE and to synthetic DR target sites through the formation of both LXRalpha/mPPARalpha and mPPARalpha/RXRalpha heterodimers.

LXRalpha Antagonizes Peroxisome Proliferator-mediated Signaling in Vivo

-To investigate the effect of LXRalpha on peroxisome proliferator-mediated signaling in vivo, a luciferase reporter plasmid containing the AOx-PPRE (pAOx(X2)luc) was cotransfected along with LXRalpha, RXRalpha, and mPPARalpha expression vectors into BSC40 cells, and luciferase activity was monitored. LXRalpha and mPPARalpha individually or in combination had little effect on reporter gene expression either in the presence or absence of the peroxisome proliferator Wy-14,643 (Fig. 3a) or the RXRalpha ligand 9-cis-retinoic acid (not shown). However, LXRalpha potently inhibited induction mediated by mPPARalpha/RXRalpha. As shown in Fig. 3b, cotransfection with increasing amounts of the LXRalpha expression plasmid led to a progressive reduction in drug-independent and -dependent induction of the AOx-PPRE reporter gene construct by mPPARalpha/RXRalpha. The inclusion of 9-cis-retinoic acid in the transfections along with Wy-14,643 had little effect on LXRalpha-mediated repression. Similar findings were obtained using an HD-PPRE luciferase reporter gene construct (data not presented). Thus, LXRalpha antagonizes transactivation by mPPARalpha/RXRalpha in vivo.


Figure 3: LXRalpha antagonizes mPPARalpha/RXRalpha-mediated transcriptional activation by peroxisome proliferators. Panel a, the pAOx(X2)luc reporter plasmid was cotransfected into BSC40 cells with LXRalpha, mPPARalpha, and RXRalpha expression plasmids (0.5 µg each) in the absence or presence of the peroxisome proliferator Wy-14,643, as indicated, and luciferase activity was measured. Panel b, effect of increasing amounts of LXRalpha expression vector in the presence of constant amounts of mPPARalpha and RXRalpha expression vectors, with and without 9-cis-retinoic acid and Wy-14,643. Panel c, activation of a DR4 reporter gene construct by LXRalpha/RXRalpha. Transfections were carried out with LXRalpha and RXRalpha expression plasmids and with luciferase reporter gene constructs that contained a synthetic DR4 element (pDR4(X2)luc) or a DR1 element (pDR1(X2)luc), as indicated. Luciferase activity (±S.D.) in panels a and b is the average (corrected against the beta-galactosidase internal reference) from three independent transfections, each carried out in duplicate. The values shown were normalized to the value obtained with cotransfected RXRalpha and mPPARalpha expression plasmids in the presence of Wy-14,643, which was taken as 100%. In panel c, the values are from duplicate transfections (values did not vary by more than 15%) and were normalized to the value obtained with the respective reporter gene alone, which was taken as 1.



Control transfections demonstrated that LXRalpha was functionally expressed under these conditions. Thus, co-expression of LXRalpha and RXRalpha activated expression of a reporter gene that contained a synthetic DR4 (pDR4(X2)luc) response element but not a DR1 (pDR1(X2)luc) response element (Fig. 3c). Activation of the DR4 reporter gene by LXRalpha/RXRalpha was increased in the presence of 9-cis-retinoic acid but was not dependent upon its presence. Willy and co-workers (20) have also demonstrated that LXRalpha interacts with endogenous RXRalpha to mediate transcriptional activation through DR4 elements; however, under these conditions, activation was observed only in the presence of RXR-specific ligands. Our findings indicate that LXRalpha also activates transcription in the absence of RXR-specific ligands when RXRalpha is co-expressed. This is consistent with previous findings that LXRalpha homologs isolated from rat (RLD-1, rUR) and human (NER, hUR) (25, 26, 27) also activate transcription via DR4 target sites in the absence of added RXR-specific ligands.

In summary, we have shown that PPARalpha can interact directly with other members of the nuclear hormone receptor superfamily in addition to RXRalpha, suggesting that combinatorial receptor interactions involving PPARs are more extensive than previously anticipated. Moreover, our findings indicate that both LXRalpha and PPARs play a broader physiological role in the convergence of distinct receptor signaling pathways. Since LXRalpha binds to the two identified components that are necessary for peroxisome proliferator responsiveness, there is potential for complex and diverse effects on both retinoid and peroxisome proliferator signaling pathways. The physiological importance of LXRalpha and related receptors in PPAR signaling is not known at present. Thus far, we have demonstrated that LXRalpha is a negative regulator of PPAR-mediated activation of peroxisome proliferator-responsive genes. Whether PPAR/LXRalpha heterodimers may also positively regulate gene expression awaits the definition of natural high affinity binding sites and the identification of potential LXRalpha ligands and target genes.


FOOTNOTES

*
This work was supported by grants from the Heart and Stroke Foundation of Ontario and from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Studentship from the Natural Sciences and Engineering Research Council of Canada.

Medical Research Council of Canada Scientist. To whom correspondence may be addressed: Dept. of Anatomy and Cell Biology, University of Alberta, Medical Sciences Bldg. 5-14, Edmonton, Alberta T6G 2H7, Canada. Tel.: 403-492-9868; Fax: 403-492-9278; rrachubi{at}anat.med.ualberta.ca.

**
Senior Scientist of the National Cancer Institute of Canada. To whom correspondence may be addressed: Dept. of Biochemistry, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22774); Fax: 905-522-9033; caponej{at}fhs.csu.mcmaster.ca.

(^1)
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; mPPAR, mouse PPAR; GBD, GAL4 DNA-binding domain; GAD, GAL4 activation domain; BSA, bovine serum albumin; AOx, fatty acyl-CoA oxidase; DR, direct repeat; HD, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; kbp, kilobase pair; LXRE, LXRalpha response element; MBP, maltose-binding protein; PPRE, peroxisome proliferator response element; RXRalpha, 9-cis-retinoic acid receptor.


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