J Biol Chem, Vol. 275, Issue 8, 5754-5759, February 25, 2000

A Dominant-negative Peroxisome Proliferator-activated Receptor  (PPAR) Mutant Is a Constitutive Repressor and Inhibits PPAR-mediated Adipogenesis^*

Mark Gurnell^§, John M. Wentworth^§, Maura Agostini, Maria Adams, Trevor N. Collingwood, Claudia Provenzano^¶, Paul O. Browne, Odelia Rajanayagam, Thomas P. Burris, John W. Schwabe^**, Mitchell A. Lazar^¶, and V. Krishna K. Chatterjee

From the  Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom, the ^¶ Division of Endocrinology, Diabetes, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the  R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869, and the ^** Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The nuclear receptor peroxisome proliferator-activated receptor  (PPAR) promotes adipocyte differentiation, exerts atherogenic and anti-inflammatory effects in monocyte/macrophages, and is believed to mediate the insulin-sensitizing action of antidiabetic thiazolidinedione ligands. As no complete PPAR antagonists have been described hitherto, we have constructed a dominant-negative mutant receptor to inhibit wild-type PPAR action. Highly conserved hydrophobic and charged residues (Leu⁴⁶⁸ and Glu⁴⁷¹) in helix 12 of the ligand-binding domain were mutated to alanine. This compound PPAR mutant retains ligand and DNA binding, but exhibits markedly reduced transactivation due to impaired coactivator (cAMP-response element-binding protein-binding protein and steroid receptor coactivator-1) recruitment. Unexpectedly, the mutant receptor silences basal gene transcription, recruits corepressors (the silencing mediator of retinoid and thyroid receptors and the nuclear corepressor) more avidly than wild-type PPAR, and exhibits delayed ligand-dependent corepressor release. It is a powerful dominant-negative inhibitor of cotransfected wild-type receptor action. Furthermore, when expressed in primary human preadipocytes using a recombinant adenovirus, this PPAR mutant blocks thiazolidinedione-induced differentiation, providing direct evidence that PPAR mediates adipogenesis. Our observations suggest that, as in other mutant nuclear receptor contexts (acute promyelocytic leukemia, resistance to thyroid hormone), dominant-negative inhibition by PPAR is linked to aberrant corepressor interaction. Adenoviral expression of this mutant receptor is a valuable means to antagonize PPAR signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptor  (PPAR),¹ an orphan member of the nuclear hormone receptor family, was first characterized as a transcription factor that regulates adipocyte-specific gene expression (1) and induces preadipocyte differentiation (2), but is now recognized to have a central role in other biological processes. PPAR mediates inhibition of inflammatory cytokine production (interleukin-6 and tumor necrosis factor ) from monocytes (3), and receptor activation by oxidized low density lipoprotein-derived ligands promotes macrophage foam cell formation (4). PPAR activation promotes colonic neoplasia (5), but inhibits the growth of breast cancer cells (6). Thiazolidinediones (TZDs), a novel class of antidiabetic agent that act as insulin sensitizers in vivo, bind PPAR with high affinity (7), and prostaglandin J2 (8) and fatty acids have been proposed to be natural ligands. PPAR regulates target gene transcription as a heterodimer with the retinoid X receptor, and this heterodimeric complex has been shown to be activated synergistically by TZDs and RXR-specific ligands (9). However, no complete synthetic or natural PPAR antagonists have been described hitherto. We have therefore generated a dominant-negative PPAR mutant to inhibit wild-type receptor action.

In keeping with other members of the nuclear receptor superfamily, PPAR exhibits a modular structure consisting of a central DNA-binding domain, an amino-terminal activation domain, and a carboxyl-terminal ligand-binding domain (LBD) that encompasses a strong ligand-dependent transactivation (AF-2) function. The extreme C terminus of the PPAR LBD forms an amphipathic -helix that can also be delineated in a number of other nuclear receptors. There is striking conservation of hydrophobic (leucine) and negatively charged (glutamic acid) residues within this motif, and mutational analyses in the estrogen (10), thyroid (11, 12), and retinoic acid (13) receptors have shown that they are critical for ligand-dependent transactivation and the recruitment of nuclear receptor coactivators (14). Resistance to Thyroid Hormone is associated with diverse thyroid hormone  (TR) receptor mutations that inhibit the action of their wild-type counterparts in a dominant-negative manner (15). We have previously described a natural mutation of the conserved hydrophobic residue (Leu⁴⁵⁴) in the AF-2 domain of TR that exhibits strong dominant-negative activity and is associated with marked refractoriness to thyroid hormone action in vivo (16).

Here, we describe the mutation of homologous hydrophobic and charged residues (L468A and E471A) in PPAR. The compound mutant receptor exhibits impaired transcriptional activation and coactivator recruitment. In addition, it silences basal transcription by recruitment of corepressors and is a potent dominant-negative inhibitor of wild-type PPAR action. Adenoviral expression of this mutant receptor in human preadipocytes inhibits thiazolidinedione-induced target gene transcription and cellular differentiation, providing direct evidence for the role of PPAR in adipogenesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Constructs-- Full-length human PPAR1 and PPAR2 cDNAs were cloned by reverse transcription-polymerase chain reaction from total human preadipocyte RNA and cloned into the pcDNA3 expression vector (Invitrogen). The L468A/E471A PPAR double mutant was generated by site-directed mutagenesis of the wild-type receptor and verified by sequencing. FLAG epitope-tagged PPAR was constructed by placing the peptide sequence MDYKDDDDK in frame upstream of the first methionine of wild-type and mutant PPAR1. DNA sequences encompassing residues 173-475 of the wild-type and mutant PPAR LBDs were cloned into pSG424 (12), pGEX4T (Amersham Pharmacia Biotech), and AASV (12) to yield Gal4-PPAR LBD, GST-PPAR LBD, and VP16-PPAR LBD fusions, respectively. WT, L454A-TR LBD, and P214R-TR LBD expression constructs were generated by cloning EcoRI/EcoRI fragments from corresponding pSG424-TR fusions into pCMX (17). Gal4-NCoR contains the nuclear receptor interaction domains (amino acids 2276-2454) of murine NCoR (18) fused in frame to the Gal4 DBD in pSG424, and Gal4-SMRT consists of the 468 C-terminal amino acids of SMRT fused in frame to the Gal4 DBD in pCMX (19). PPARETKLUC (8); UASTKLUC (12); pSG5-PPAR and CRBPIITKLUC (20); MALTKLUC, RSV-TR1, and RSV-RXR (15); RAR2TKLUC and RSV-RAR1 (21); mSiah2 (22); and pCMXNCoR (18) have been described previously.

Protein-Protein Interaction Assays-- Bacterially expressed GST fusion proteins were immobilized on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) and incubated with in vitro translated ³⁵S-labeled CBP or SRC-1 (16) in 50 mM Tris-HCl, 0.1 M KCl, 0.14 M NaCl, 0.5% Nonidet P-40, and 10% glycerol (pH 8.0) with or without 10 µM BRL49653 for 1 h at room temperature. Following four washes with 20 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, and 0.5% Nonidet P-40 (pH 8.0), bound coactivators were resolved by SDS-polyacrylamide gel electrophoresis. Coomassie staining verified equal loading of GST-PPAR LBD fusion proteins prior to autoradiography.

Hormone and DNA Binding Assays-- Hormone binding assays were performed using bacterially expressed GST-PPAR LBD fusion proteins and the PPAR-specific radioligand ¹²⁵I-SB236636 in a modification of a previously described filter binding assay (23). DNA binding was assessed as described previously (15) using in vitro translated WT PPAR1, L468A/E471A PPAR1, and human RXR and oligonucleotide duplexes encoding the acyl-CoA oxidase PPARE (24).

Transfection Assays-- Calcium phosphate-mediated transient transfection was performed in 24-well cultures of 293EBNA and JEG-3 cells. Each well was cotransfected with 50-100 ng of receptor expression vector, 500 ng of reporter vector, and 100 ng of the internal control plasmid Bos--gal. Cells were harvested and assayed as described previously (15).

Immunoprecipitation and Western Blot Analysis-- 293T cells were grown to 60% confluence in 10-cm dishes, transfected with 7.5 µg of FLAG-tagged WT PPAR or FLAG-tagged L468A/E471A PPAR, and cultured in Dulbecco's modified Eagle's medium and 10% fetal bovine serum with or without 1 µM BRL49653. The following day, cells were lysed in ice-cold buffer (50 mM Tris-HCl, 0.15 M NaCl, 5 mM EDTA, 0.5% Nonidet P-40, Roche Molecular Biochemicals protease inhibitor mixture, 250 mM Na₃PO₄, and 0.1 mM okadaic acid (pH 8.0)) with or without 10 µM BRL49653. Following centrifugation at 12,000 × g for 10 min at 4 °C, the supernatants were precleared with protein A beads and incubated with goat polyclonal anti-SMRT antibody (N-20, Santa Cruz Biotechnology). For Western blot analysis, detection was performed with anti-SMRT and mouse monoclonal anti-FLAG (Research Diagnostics, Inc.) antibodies.

Preadipocyte Culture-- Preadipocytes (isolated from human breast adipose tissue by collagenase digestion) were cultured in serum-containing medium, and differentiation was induced using serum-free medium ± 1 µM BRL49653 as described previously (25). b-DNA assays (26) for adipocyte P2 (aP2) expression were performed at 24 h, whereas morphologic assessment and glycerol-3-phosphate dehydrogenase activity determination (25) were performed on day 10.

Adenovirus Construction and Expression-- Recombinant type 5 adenoviruses (Ad5) expressing GFP (AdGFP) or GFP and full-length L468A/E471A PPAR1 (Ad_m) were generated using an ADENO-QUEST kit (Quantum Biotechnologies, Montreal, Canada). JEG-3 cells cultured in 24-well plates were infected with recombinant virus following calcium phosphate transfection by addition of 1.6 × 10⁷ plaque-forming units/well. Primary human preadipocytes grown in 6- or 96-well plates were infected with 9.6 × 10⁷ or 0.8 × 10⁷ plaque-forming units/well, respectively, 2 days prior to induction of differentiation. Comparable viral infection efficiency was verified by fluorescence microscopy.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The transcriptional activity of WT and mutant L468A/E471A PPAR was assayed by cotransfection of receptor expression vectors together with a reporter gene (PPARETKLUC) containing three copies of the PPARE from the acyl-CoA oxidase gene linked to the thymidine kinase promoter and luciferase (Fig. 1). Cells transfected with the WT receptor exhibited a strong ligand-dependent transcriptional response following exposure to increasing concentrations of the thiazolidinedione BRL49653. In comparison, the mutant receptor showed negligible transcriptional activity even at the highest concentration of ligand. However, ligand binding assays with bacterially expressed WT or mutant GST-PPAR LBD fusion proteins and the radiolabeled thiazolidinedione ¹²⁵I-SB236636 (27) indicated that the mutant receptor retained significant ligand binding (WT K_d = 45 ± 12 nM; L468A/E471A K_d = 200 ± 60 nM), suggesting that this did not account for its transcriptional inactivity. Likewise, DNA binding assays performed using WT or mutant PPAR, retinoid X receptor, and radiolabeled PPARE showed comparable formation of heterodimeric complexes (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   L468A/E471A PPAR is a constitutive repressor with impaired transactivation and potent dominant-negative activity. 24-well plates of 293EBNA cells were transfected with 500 ng of PPARETKLUC reporter gene, 100 ng of Bos--gal control plasmid, and either 100 ng of receptor expression vector (pcDNA3 (), WT PPAR1 (), or L468A/E471A PPAR1 ()) or 100 ng each of the WT PPAR1 and L468A/E471A PPAR expression vectors (). Transcriptional activity in response to increasing doses of ligand (BRL49653) is expressed relative to the WT maximum (100%). Inset, in contrast to the constitutive activity of WT PPAR1, L468A/E471A PPAR1 represses basal transcription.

By analogy with the effect of homologous mutations in TR (16), we hypothesized that the interaction of the L468A/E471A PPAR mutant with transcriptional coactivator proteins might be altered. In a protein-protein interaction assay using bacterially expressed GST-PPAR LBD fusion proteins, the WT receptor showed strong ligand-dependent recruitment of ³⁵S-labeled CBP and SRC-1 proteins (Fig. 2). In contrast, ligand-dependent coactivator recruitment by the L468A/E471A PPAR mutant was abolished.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   L468A/E471A is impaired for coactivator recruitment. 35S-Labeled CBP (lane 1) and SRC-1 (lane 7) were incubated with GST (lanes 2 and 8) or GST-PPAR LBD fusion proteins (WT PPAR (lanes 3, 4, 9, and 10) and L468A/E471A PPAR (lanes 5, 6, 11, and 12)) in the absence or presence of 10 µM BRL49653 as shown.

To assess dominant-negative inhibition by the L468A/E471A PPAR mutant, cells were transfected with the WT receptor plus an equal amount of mutant receptor and increasing concentrations of BRL49653 (Fig. 1). In the presence of the L468A/E471A mutant, reporter gene activation was markedly attenuated (~50% of the WT response) at all ligand concentrations, whereas the transcriptional response to WT plus further WT receptor was unchanged (data not shown).

Cells transfected with empty expression vector (pcDNA3) showed a small but significant response, reflecting transcriptional activation mediated by low levels of endogenous PPAR (unpublished Western blotting data not shown) in 293 cells (Fig. 1). In comparison, cells transfected with the L468A/E471A mutant exhibited even lower transcriptional activity, presumably reflecting dominant-negative inhibition of endogenous WT receptor (Fig. 1).

Other members of the nuclear receptor family (e.g. TR and RAR) are able to silence basal gene transcription in the absence of ligand by binding corepressor proteins such as NCoR (18) and SMRT (19). Furthermore, corepressor recruitment has been shown to be essential for dominant-negative inhibition by natural TR mutants (28). We therefore examined the properties of the unliganded L468A/E471A PPAR mutant. In comparison to empty pcDNA3 vector, the WT receptor exhibited moderate (~5-fold) constitutive basal activation, whereas the PPAR mutant showed striking silencing of basal transcription (pcDNA3 = 1.0; L468/E471 = 0.25) (Fig. 1, inset). To further substantiate that this silencing is PPAR-mediated, cells were transfected with vectors expressing either the Gal4 DBD alone or linked to the L468A/E471A PPAR mutant LBD. Again, marked repression of basal transcription was observed (Gal4 = 1.0; Gal4-PPAR mutant = 0.15) (Fig. 3a), suggesting that PPAR might interact with corepressors in vivo.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   a, unliganded repression by L468A/E471A PPAR is mediated by corepressors (CoR) that interact with TR. 293EBNA cells were transfected with 500 ng of UASTKLUC, 100 ng of Bos--gal control plasmid, and 50 ng of expression vectors encoding Gal4 DBD (Gal4), Gal4 DBD-L468A/E471A PPAR LBD (468/471), TR LBD (WTTR), or mutant TR LBDs defective in coactivator (L454A) or corepressor (P214R) recruitment and cultured in the absence or presence of T3 as shown. b, basal repression by L468A/E471A PPAR is mediated by NCoR. 293EBNA cells were transfected with reporter, internal control, and Gal4 expression vectors as described for a together with 500 or 1000 ng of NCoR expression vector in the absence or presence of 500 ng of the NCoR-depleting construct mSiah2 as shown. c, L468A/E471A PPAR demonstrates impaired corepressor release in mammalian two-hybrid assays. 293EBNA cells were transfected with 500 ng of UASTKLUC, 100 ng of Bos--gal control plasmid, 50 ng of expression vector encoding the Gal4 DBD fused to the nuclear receptor interaction domains of SMRT (Gal4-SMRT) and NCoR (Gal4-NCoR), and 50 ng of expression vector encoding VP16 or VP16 fused to the LBD of WT PPAR (VP16 WT) or L468A/E471A PPAR (VP16 468/471) and cultured in 0, 10, 100, or 1000 nM BRL49653 as shown. UAS, upstream activating sequence; TK, thymidine kinase.

The ligand-binding domain of TR mediates interaction with NCoR or SMRT when unliganded, and the addition of T₃ promotes corepressor dissociation and coactivator recruitment (18, 19). When cotransfected with the Gal4-PPAR mutant, the unliganded TR LBD relieved transcriptional silencing by the mutant receptor, and this effect was reversed by the addition of T₃ (Fig. 3a). In contrast, coexpression of a mutant (P214R) TR LBD with impaired corepressor binding (29) did not affect basal repression by the Gal4-PPAR mutant (Fig. 3a). Cotransfection of a mutant (L454A) TR LBD that exhibits impaired hormone-dependent corepressor release (30) also relieved silencing by the Gal-PPAR mutant, but this persisted following the addition of T₃ (Fig. 3a).

To specifically address the role of individual corepressors in mediating silencing by the Gal4-PPAR mutant, we examined the effects of mSiah2, a novel protein that has been shown to target the corepressor NCoR for proteasomal degradation (22). Cotransfected mSiah2 significantly impaired transcriptional silencing by the Gal4-PPAR mutant (Fig. 3b). Furthermore, coexpression of NCoR was able both to enhance basal repression by the mutant receptor and to restore silencing in the presence of mSiah2 in a dose-dependent manner (Fig. 3b).

To examine the association of WT and mutant PPAR with corepressor in vivo, cells were cotransfected with an SMRT expression vector together with FLAG epitope-tagged full-length WT or L468A/E471A PPAR in the absence and presence of BRL49653. Following immunoprecipitation with an SMRT antibody, PPAR complexed with corepressor was quantitated by Western blotting (Fig. 4). In the absence of ligand, both WT and mutant PPAR bound SMRT, with greater quantitative binding by the mutant receptor. The addition of 10 µM BRL49653 ligand resulted in complete dissociation of SMRT from the WT receptor, whereas the L468A/E471A mutant retained significant corepressor binding, suggesting that ligand-dependent release of corepressor from the PPAR mutant might be impaired. We tested this hypothesis in a mammalian two-hybrid assay using the Gal4-NCoR (residues 2276-2454) or Gal4-SMRT (residues 982-1448) fusion together with the VP16 construct with WT or mutant PPAR LBD. In the absence of ligand, both WT and mutant PPAR were recruited comparably to Gal4-SMRT, whereas interaction of the L468A/E471A mutant with the Gal4-NCoR fusion was slightly enhanced. In contrast to a dose-dependent dissociation of the WT receptor from corepressors following the addition of ligand, the release of the PPAR mutant from both SMRT and NCoR was markedly impaired (Fig. 3c).

View larger version (79K): [in this window] [in a new window]   Fig. 4.   WT and L468A/E471A PPAR recruit SMRT in vivo, and ligand-dependent release by L468A/E471A PPAR is impaired. 293T cells, cultured in 10-cm plates, were transfected with 7.5 µg of expression vectors encoding SMRT and FLAG epitope-tagged WT or L468A/E471A PPAR. Whole cell lysates were treated with polyclonal anti-SMRT antibody in the absence or presence of 10 µM BRL49653 as shown, and precipitates were Western-blotted and probed with anti-FLAG antibody. The dual band corresponds to phosphorylated and unphosphorylated forms of PPAR. Cell lysates were also Western-blotted and probed with anti-FLAG and anti-SMRT antibodies to verify comparable transfection efficiencies of PPAR and SMRT, respectively. IP, immunoprecipitate.

To introduce the L468A/E471A PPAR mutant into primary cells and tissues, we have constructed a recombinant adenovirus expressing the mutant receptor as well as GFP. In the first instance, we tested the ability of PPAR mutant adenovirus (Ad_m) to inhibit the action of transfected nuclear receptors. Mutant adenoviral infection of cells blocked ligand-dependent transactivation by both human PPAR1 and PPAR2 isoforms, whereas receptor-mediated activation was unaffected in cells infected with control adenovirus expressing GFP alone (AdGFP) (Fig. 5, a and b). Furthermore, the mutant receptor adenovirus only partially inhibited PPAR-mediated signaling (Fig. 5c) and was unable to block ligand-dependent transactivation by human RAR (Fig. 5d), human RXR (Fig. 5e), or human TR (Fig. 5f).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   An Ad5 adenovirus expressing L468A/E471A PPAR (Adm) selectively impairs WT PPAR-dependent transcription. JEG-3 cells were transfected with 500 ng of PPARETKLUC (a-c), RAR2TKLUC (d), CRBPIITKLUC (e), or MALTKLUC (f) reporter plasmid, 100 ng of Bos--gal internal control, and 50 ng of expression vector encoding WT human PPAR1 (a), WT human PPAR2 (b), murine PPAR (c), human RAR1 (d), human RXR (e), or human TR1 (f). Following media change, cells were infected with no virus (nil) or with AdGFP or Adm in the absence or presence of 1 µM BRL49653 (a and b), 10 µM WY14643 (c), 10 µM all-trans-retinoic acid (RA) (d), 1 µM RXR-specific ligand LG100268 (e), or 100 nM T3 (f) as shown. DMSO, dimethyl sulfoxide.

PPAR plays a central role in murine preadipocyte differentiation (2). We have shown previously that thiazolidinediones promote the differentiation of cultured human preadipocytes (25) and therefore tested the effect of the PPAR mutant adenovirus on this process. Following infection with mutant receptor adenovirus, TZD-induced differentiation of cells into lipid-laden adipocytes was markedly inhibited compared with cells infected with GFP adenovirus (Fig. 6c). The degree of differentiation was assessed quantitatively by measurement of glycerol-3-phosphate dehydrogenase enzyme activity (25) and aP2 mRNA accumulation (26) normalized to cell number as described previously. Thiazolidinedione induction of aP2 mRNA (Fig. 6a) and glycerol-3-phosphate dehydrogenase activity (Fig. 6b) was significantly reduced following infection with PPAR mutant adenovirus compared with uninfected or GFP virus-infected cells. Thus, the L468A/E471A PPAR mutant is capable of blocking responses mediated by endogenous wild-type PPAR.

View larger version (81K): [in this window] [in a new window]   Fig. 6.   a, Adm impairs BRL49653-mediated induction of aP2 mRNA in human preadipocytes. Preadipocytes were grown to confluence in 96-well plates, infected with no virus (nil) or with 0.8 × 107 plaque-forming units/well AdGFP or Adm, and harvested at 24 h. aP2 values were standardized against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene. Results represent the combined data from four independent experiments. b and c, Adm inhibits BRL49653-induced preadipocyte differentiation. b, human preadipocytes from breast tissue were infected with no virus or with 9.6 × 107 plaque-forming units/well AdGFP or Adm and cultured in differentiation medium ± 0.1 µM BRL49653. At 10 days, cells were harvested, and glycerol-3-phosphate dehydrogenase activity/µg of protein was determined. Results represent the means ± S.E. of four independent experiments. c, phase-contrast photographs (magnification × 200) of oil red O-stained preadipocytes infected with AdGFP or Adm as shown and grown for 10 days in differentiation medium supplemented with 0.1 µM BRL49653.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The carboxyl terminus of a number of nuclear receptors, including PPAR, contains a C-terminal amphipathic -helix that is required for ligand-dependent transcriptional activation (AF-2) function (10, 11, 13). We have mutated conserved hydrophobic (Leu⁴⁶⁸) and negatively charged (Glu⁴⁷¹) residues in the putative AF-2 domain of PPAR to alanine. Functional studies indicate that transcriptional activation by this compound mutant is severely impaired even in the presence of saturating concentrations of thiazolidinedione ligand sufficient to overcome its modestly reduced ligand binding affinity (Fig. 1). Protein-protein interaction assays indicate negligible ligand-dependent recruitment of CBP and SRC-1 coactivators by the PPAR mutant, accounting for its functional impairment. The crystal structure of the PPAR LBD in complex with the interaction domain of the coactivator SRC-1 (31) reveals that Leu⁴⁶⁸ and Glu⁴⁷¹ are both situated at the receptor coactivator interface. The side chain of Glu⁴⁷¹ is oriented such that it makes several hydrogen bonds with the backbone amino groups of the coactivator helix, with the negative charge of the carboxyl complementing the positive end of the helix dipole. Leu⁴⁶⁸ is situated at the bottom of the hydrophobic cleft into which the coactivator helix binds and as a result is completely buried between hydrophobic residues on helix 3 and others on the coactivator helix. It is clear that the impairment of coactivator binding seen in functional studies with this mutant is entirely consistent with the role of these residues in the structure of the receptor-coactivator complex.

The L468A/E471A PPAR mutant was also able to inhibit the action of its WT counterpart in a dominant-negative manner (Fig. 1). Mutations in the C-terminal AF-2 domain of other nuclear receptors also generate mutant proteins with strong dominant-negative activity; for example, this region is deleted in the oncogene v-erbA, a potent inhibitor of TR and RAR action (32). We (16) and others (33) have described powerful dominant-negative amphipathic -helix TR mutants in the syndrome of resistance to thyroid hormone.

A subset of nuclear receptors including TR and RAR have been shown to repress basal transcription in the absence of ligand by recruitment of corepressor proteins such as NCoR (18) and SMRT (19). However, the role of corepressors in PPAR signaling remains unclear. Whereas PPAR can interact weakly with NCoR and SMRT in vitro, the WT receptor exhibits negligible transcriptional repression in vivo (24) (Fig. 1, inset), although mitogen-activated protein kinase-dependent phosphorylation has been shown to inhibit AF-2 function via SMRT recruitment (34). In contrast, our observations indicate that the L468A/E471A PPAR mutant is a potent transcriptional repressor. Repression is exhibited by both the full-length mutant receptor (Fig. 1) as well as a Gal4-PPAR LBD fusion (Fig. 3a), indicating that its silencing function is independent of N-terminal domain phosphorylation. Coexpression of the unliganded TR LBD attenuates repression by the PPAR mutant, suggesting that this function is mediated by shared cellular factors (35), and a TR LBD mutant (P214R) that is defective for corepressor binding fails to inhibit repression. Evidence that NCoR partly mediates silencing by the PPAR mutant is provided by the observation that coexpression of mSiah2, which targets NCoR for proteasomal degradation (22), also attenuates repression (Fig. 3b). Co-immunoprecipitation experiments (Fig. 4) demonstrate that the L468A/E471A PPAR mutant interacts with SMRT in vivo, suggesting that this corepressor may also mediate transcriptional silencing. In addition to enhanced corepressor binding, the L468A/E471A PPAR mutant also exhibits impaired ligand-dependent corepressor release (Fig. 3c), indicating a role for helix 12 of PPAR in corepressor dissociation as has been documented with other nuclear receptors (30, 33, 36). Our observation that the dominant-negative PPAR mutant is a powerful repressor is consonant with the properties of other nuclear receptors, including TR mutants in Resistance to Thyroid Hormone (28), PML-RAR in acute promyelocytic leukemia (37), and v-erbA (38). Furthermore, corepressor recruitment has been shown to be required for dominant-negative inhibition (28). Our finding that the PPAR mutant is a strong repressor raises the question as to why WT PPAR appears to lack silencing activity. The crystal structure of the apo-PPAR LBD indicates that it is possible for helix 12 to adopt the same conformation as the liganded receptor-coactivator complex. It is therefore likely that, in the absence of ligand, PPAR is still able to recruit coactivator, albeit less efficiently than the holoreceptor. Abolition of such coactivator binding in the L468A/E471A PPAR mutant facilitates corepressor binding, unmasking transcriptional repression.

Finally, we have used a recombinant adenovirus expressing the PPAR mutant to block endogenous wild-type receptor action. It is well established that PPAR is a key mediator of adipogenesis. Using a recombinant PPAR mutant adenovirus that selectively inhibits thiazolidinedione-dependent PPAR activation but not other nuclear receptor signaling pathways (Fig. 5), we have been able to inhibit human preadipocyte differentiation and induction of aP2, a PPAR target gene, in response to thiazolidinediones (Fig. 6). These results provide compelling evidence that TZDs act directly via PPAR to promote human preadipocyte differentiation. Our results also validate the utility of the PPAR mutant in investigating receptor action in vivo. In contrast to a chemical antagonist, the dominant-negative mutant receptor can be used to selectively inhibit thiazolidinedione-dependent PPAR action in particular tissues. For example, the relative importance of insulin-sensitizing effects of TZDs in adipocytes versus skeletal muscle could be investigated by generating transgenic mice with the L468A/E471A PPAR mutant cDNA linked to tissue-specific promoters to target mutant receptor expression.

	FOOTNOTES

^* This work was supported in part by the Wellcome Trust (to V. K. K. C. and M. G.), The Raymond and Beverly Sackler Foundation (to M. G.), The Cambridge Commonwealth Trust (to J. W.), and the Francis and Augustus Newman Foundation (to J. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 01223 336842; Fax: 01223 336846; E-mail: kkc1@mole.bio.cam.ac.uk.

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor ; TZD, thiazolidinedione; RXR, retinoid X receptor; LBD, ligand-binding domain; AF-2, activation function-2; TR, thyroid receptor; WT, wild-type; GST, glutathione S-transferase; NCoR, nuclear corepressor; DBD, DNA-binding domain; SMRT, silencing mediator of retinoid and thyroid receptors; RSV, Rous sarcoma virus; RAR, retinoic acid receptor; mSiah2, murine Siah2; CBP, cAMP-response element-binding protein-binding protein; SRC-1, steroid receptor coactivator-1; PPARE, peroxisome proliferator-activated response element; aP2, adipocyte P2; Ad5, adenovirus type 5; GFP, green fluorescent protein; T₃, triiodothyronine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES