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Originally published In Press as doi:10.1074/jbc.M200743200 on March 4, 2002

J. Biol. Chem., Vol. 277, Issue 22, 19649-19657, May 31, 2002
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T0070907, a Selective Ligand for Peroxisome Proliferator-activated Receptor gamma , Functions as an Antagonist of Biochemical and Cellular Activities*

Gary Lee, Fabienne Elwood, John McNally, Jennifer Weiszmann, Michelle Lindstrom, Kate Amaral, Motonao Nakamura, Shichang Miao, Ping Cao, R. Marc Learned, Jin-Long Chen, and Yang LiDagger

From Tularik Inc., South San Francisco, California 94080

Received for publication, January 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma  (PPARgamma (NR1C3)) plays a central role in adipogenesis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. In a search for novel non-TZD ligands for PPARgamma , T0070907 was identified as a potent and selective PPARgamma antagonist. With an apparent binding affinity (concentration at 50% inhibition of [3H]rosiglitazone binding or IC50) of 1 nM, T0070907 covalently modifies PPARgamma on cysteine 313 in helix 3 of human PPARgamma 2. T0070907 blocked PPARgamma function in both cell-based reporter gene and adipocyte differentiation assays. Consistent with its role as an antagonist of PPARgamma , T0070907 blocked agonist-induced recruitment of coactivator-derived peptides to PPARgamma in a homogeneous time-resolved fluorescence-based assay and promoted recruitment of the transcriptional corepressor NCoR to PPARgamma in both glutathione S-transferase pull-down assays and a PPARgamma /retinoid X receptor (RXR) alpha -dependent gel shift assay. Studies with mutant receptors suggest that T0070907 modulates the interaction of PPARgamma with these cofactor proteins by affecting the conformation of helix 12 of the PPARgamma ligand-binding domain. Interestingly, whereas the T0070907-induced NCoR recruitment to PPARgamma /RXRalpha heterodimer can be almost completely reversed by the simultaneous treatment with RXRalpha agonist LGD1069, T0070907 treatment has only modest effects on LGD1069-induced coactivator recruitment to the PPARgamma /RXRalpha heterodimer. These results suggest that the activity of PPARgamma antagonists can be modulated by the availability and concentration of RXR agonists. T0070907 is a novel tool for the study of PPARgamma /RXRalpha heterodimer function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptor gamma  (PPARgamma 1 (NR1C3)) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcription factors (1, 2). At least two PPARgamma isoforms exist, gamma 1 and gamma 2, resulting from transcription from two different promoters upstream of the PPAR gene (3, 4). PPARgamma 2 possesses 30 additional amino acids at its amino terminus. PPARgamma 1 is expressed broadly in many tissues, whereas PPARgamma 2 is expressed predominantly in adipose tissue. Both "gain of function" and "loss of function" studies strongly support a critical role for PPARgamma in adipocyte gene expression and differentiation (5).

Like other members of the NHR superfamily, PPARgamma binds to a DNA-response element (PPAR-response element or PPRE) upstream of the coding regions of target genes and forms a heterodimeric complex with one of the three retinoid X receptor (RXR) proteins (1). Binding of ligands to PPARgamma causes conformational changes in the receptor, in particular to alpha -helix 12 (H12), which is located at the carboxyl-terminal end of the protein and forms part of the transcriptional activation function (AF-2). When agonists bind to PPARgamma , H12 along with H3, H4, and H5 form a charge clamp and a hydrophobic pocket that allows the recruitment of coactivator protein complexes that are essential for transcriptional activation of PPARgamma target genes (6). Although PPARgamma , in isolation, is capable of binding to transcriptional corepressor proteins NCoR and SMRT in the absence of ligand, PPARgamma does not interact with these corepressors in the context of the RXR heterodimer nor does the PPARgamma /RXR heterodimer repress transcription of PPARgamma target genes, unlike heterodimers of RXR with thyroid hormone receptor or retinoic acid receptor (7). Two explanations for the difference in PPARgamma /corepressor interaction on and off DNA have been offered. The orientation of PPARgamma and RXR on a PPRE could simply inhibit the binding of corepressor (8). Alternatively, PPARgamma may be unable to stabilize a conformation of RXR that is permissive for corepressor interaction; unlike TR and retinoic acid receptor, PPARgamma is unable to interact with H12 from RXR (9). Because other NHR antagonists stabilize the interaction of corepressors with their cognate receptors, PPARgamma antagonists or inverse agonists would be useful tools to study PPARgamma /corepressor interaction. One way to test these hypotheses is to study the effects of a PPARgamma antagonist or inverse agonist on corepressor binding. This avenue has not yet been explored.

Both natural and synthetic ligands have been reported for PPARgamma (reviewed in Ref. 10). Naturally occurring compounds that have been reported to bind PPARgamma include a number of fatty acids and eicosanoid derivatives such as 9- or 13-hydroxyoctadienoic acid and prostaglandin derivative 15-deoxy-Delta 13,14-prostaglandin J2. The most widely used synthetic agonists of PPARgamma are members of a class of antidiabetic agents known as thiazolidinediones (TZDs), including rosiglitazone, troglitazone, and pioglitazone. More recently, a series of tyrosine-based PPARgamma agonists exemplified by GI262570 have been shown to be among the highest affinity PPARgamma ligands described thus far. Synthetic partial agonists identified include GW0072 (11), L-764406 (12), 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (13), and Fmoc (N-(9-fluorenyl)methoxycarbonyl)-L-leucine (14). Several synthetic antagonists have also been described, these include bisphenol A diglycidyl ether (15), GW9662 (10), and PD068235 (16). However, relatively little is known about how these compounds affect PPARgamma /RXR heterodimer function.

Here, we describe a novel, potent, and selective PPARgamma ligand, T0070907. By using a variety of biochemical and cell-based assays, we demonstrate that T0070907 is a PPARgamma antagonist. Our studies suggest that T0070907 modulates the interaction of PPARgamma with cofactor proteins by affecting the conformation of helix 12 of the PPARgamma ligand-binding domain (LBD). Finally, our studies reveal a functional asymmetry between the effects of PPARgamma and RXR ligands on the activity of the permissive PPARgamma /RXRalpha heterodimer.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- All PPARgamma and RXRalpha proteins produced by in vitro translation and used in gel mobility shift assay were expressed from pET28b-based plasmids (Novagen, Madison, WI) containing inserts cloned into NcoI and NotI sites. For PPARgamma , the following constructs were made: hPPARgamma 1, full-length human PPARgamma 1; hPPARgamma 1 Delta H12 (amino acids 1-461 of hPPARgamma 1); hRXRalpha , full-length human RXRalpha ; hRXRalpha Delta H12 (amino acids 1-443 of hRXRalpha ). The construct used to produce human NCoR (hNCoR) protein from the baculovirus expression system was made by inserting hNCoR DNA encoding amino acids 1948-2440 into the BamHI and NotI sites of the pFASTBAC HTa vector (Invitrogen). GST-PPARgamma LBD was constructed by inserting hPPARgamma 1 DNA encoding amino acids 175-471 into the BamHI site of pGEX-2TK vector (sequence at the junction is 5'-GGATCCCATATG-hPPARgamma 1 amino acid 175).

Mass Spectrometry-- After incubation with 10 µM T0070907 for 4 h at room temperature in 50 mM Tris, pH 7.9, 50 mM KCl, 1 mM EDTA, GST-PPARgamma (12 µg) was purified on an SDS-polyacrylamide gel. The excised gel fragment containing PPARgamma was digested with trypsin at 37 °C for 12 h without reduction and alkylation in 100 mM ammonium bicarbonate using an enzyme/substrate ratio of 1:50 (w/w). Analysis of covalent binding of T0070907 to PPARgamma was performed with a Voyager-DETM MALDI-TOF Mass Spectrometer (Perspective Biosystems, Framingham, MA) and an EsquireTM Nano-electrospray Tandem Mass Spectrometer (Bruker Daltonik, Billerica, MA).

The MALDI matrix was prepared by mixing alpha -cyano-4-hydroxy-trans-cinnamic acid (40 mg/ml in acetone), nitrocellulose (20 mg/ml in acetone), and 2-propanol at a ratio of 2:1:1 (v/v/v). An aliquot (0.5 µl) of the sample/matrix was spotted and mixed on a MALDI sample plate. After drying completely, samples were washed with 3 µl of 5% formic acid and then with Milli-Q water (Millipore, Bedford, MA). The MALDI-TOF was operated in reflectron mode with delay extraction. The mass spectrometer was calibrated externally using des-Arg-bradykinin (m/z 904.4681), angiotensin I (m/z 1296.6853), and Glu-fibrinopeptide B (m/z 1570.6774) (17).

The in-gel tryptic digest was desalted and concentrated prior to analysis by nano-ESI-MS/MS. The in-gel digests were extracted twice with 10 µl of a 50% acetonitrile, 5% trifluoroacidic acid solution. All extracts were pooled and dried to 5 µl with a SpeedVac. An additional 15 µl of a 0.1% acidic acid solution was added, and a 10-µl sample was loaded into a ZiptipTM (Millipore, Bedford, MA) with C18 resin for desalting. After washing the column with 1% trifluoroacidic acid (in H2O), 5 µl of 50% acetonitrile, 0.1% acidic acid was used to elute the sample into a nanospray needle. On the basis of the MALDI-TOF mass analysis results, the tandem mass spectrometric sequencing was only acquired on selected precursor ions (18).

Homogeneous Time-resolved Fluorescence (HTRF) Assay-- HTRF assays were performed as described previously (19) with the following modifications. Reaction conditions were as follows: a 100-µl reaction volume contained 50 mM Tris, pH 7.9, 50 mM KCl, 1 mM EDTA, 0.5 mM 2-mercaptoethanol, 0.1 mg/ml bovine serum albumin, 800 ng/ml anti-GST-(Eu)K antibody (PerkinElmer Life Sciences), 1 ng/µl GST-PPARgamma , 1.5 µg/ml streptavidin conjugated with allophycocyanin (Streptavidin-APC, PerkinElmer Life Sciences), 200 nM biotin-peptide, and 5 µl compound of interest in dimethyl sulfoxide (Me2SO) as indicated in the figure legends. GST-PPARgamma /anti-GST-(Eu)K (20 µl) and biotin-peptide/streptavidin (20 µl) were incubated separately for 1 h at room temperature before being combined with the remaining components, and the complete mixture was incubated for an additional 1 h at room temperature. Reactions were carried out in 96-well plates (black polypropylene, Whatman), and fluorescence was measured on an LJL Analyst (LJL BioSystems, Sunnyvale, CA). Data were expressed as the ratio of the emission intensities at 665 and 620 nm multiplied by a factor of 1000.

Corepressor Recruitment Assay (Pull-down Assay)-- Purified GST-PPARgamma fusion protein (15 µg) was incubated with 10 µl of glutathione-Sepharose beads (50% slurry in GST binding buffer, Amersham Biosciences) in GST binding buffer (20 mM HEPES, pH 7.7, 100 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.01% Nonidet P-40, 2 mM dithiothreitol, 10% glycerol) for 90 min at room temperature. After washing 5 times (5 min each wash) with 1 ml of binding buffer, the bead-bound GST-PPARgamma protein was incubated with 7.5 µl of [35S]methionine-labeled in vitro translated hNCoR protein (TNT T7 Rabbit Reticulocyte Lysate Translation System, Promega Corp., Madison, WI) and the indicated ligand concentration in a final volume of 300 µl at room temperature for 2 h. After washing with binding buffer as indicated above, the bound protein was eluted with 20 µl of 2× SDS buffer at 95 °C, separated on a 10% SDS-PAGE, and analyzed by autoradiography.

Ligand Binding Assay-- To determine the binding affinity of T0070907 to the PPARs, scintillation proximity assay (SPA) was performed as described (12, 20) with the following modifications. A 90-µl reaction contained SPA buffer (10 mM K2HPO4, 10 mM KH2PO4, 2 mM EDTA, 50 mM NaCl, 1 mM dithiothreitol, 2 mM CHAPS, 10% (v/v) glycerol, pH 7.1), 50 ng of GST-PPARgamma (or 150 ng of GST-PPARalpha , GST-PPARdelta ), 5 nM 3H-labeled radioligands, and 5 µl of T0070907 in Me2SO. After incubation for 1 h at room temperature, 10 µl of polylysine-coated SPA beads (at 20 mg/ml in SPA buffer) were added, and the mixture was incubated for 1 h before reading in Packard Topcount. [3H]Rosiglitazone was used for PPARgamma , and [3H]GW2433 (21) was used for PPARalpha and PPARdelta .

Gel Mobility Shift Assay (GMSA)-- Our GMSAs were similar to previous studies (22, 23) with the following modifications. The sequence of the DNA probe used in the GMSAs was derived from the PPRE of the acyl-CoA oxidase gene (5'-AGCTGGACCAGGACAAAGGTCACGTTCAGCT-3'). In vitro translated PPARgamma (0.5 µl) and RXRalpha (0.5 µl) were incubated in 20 mM Tris, pH 8.0, 1 mM EDTA, 50 mM KCl, 0.05% Nonidet P-40, 10% glycerol, 2 mM dithiothreitol, 50 µg/ml poly(dI-dC), labeled probe (typically 40,000 cpm per reaction), various ligands, and with or without baculovirus-expressed NCoR (5 µg) (final volume, 20 µl) for 30 min at room temperature. Reaction mixtures were loaded on a 5% (38:2)polyacrylamide nondenaturing gel in 1× TGE (50 mM Tris, pH 8.5, 40 mM glycine, 2 mM EDTA) and separated in 1× TGE by electrophoresis at 4 °C. Gels were dried prior to autoradiography.

Transient Transfection and 3T3L1 Differentiation Assay-- Luciferase reporter assays were carried out following transient transfection of HEK293 cells using GenePORTER2 reagent (GTS Inc., San Diego, CA) according to the manufacturer's protocol. 3T3-L1 preadipocytes were cultured and induced to differentiate as described (24) with the following modifications. 3T3-L1 cells were grown to confluence in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and induced to differentiate with 0.25 µM dexamethasone, 0.5 mM isobutylmethylxanthine, and 1 µg/ml insulin. Medium was replaced 2 days post-induction (and every 2-3 days thereafter) with Dulbecco's modified Eagle's medium, 10% fetal bovine serum supplemented with 1 µg/ml insulin.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

T0070907 Is a Novel and Selective PPARgamma Ligand-- In a search for novel non-TZD ligands for PPARgamma , T0070907 was identified to bind PPARgamma with high affinity, capable of displacing [3H]rosiglitazone with an apparent Ki of 1 nM as shown in Fig. 1. Furthermore, T0070907 shows high selectivity among PPAR subtypes with a >800-fold preference for PPARgamma over PPARalpha and PPARdelta . In competition with the PPARalpha and PPARdelta co-ligand [3H]GW2433 (21), T0070907 has an apparent Ki of 0.85 µM to PPARalpha and 1.8 µM to PPARdelta (Fig. 1B).


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  		Fig. 1.   Chemical structure of T0070907 and analysis of its binding to PPARs. A, chemical structure of T0070907. B, the affinities of T0070907 for the various PPAR subtypes were determined with an SPA assay. [3H]GW2433 (5 nM) was used as a ligand for PPARalpha and PPARdelta , and [3H]rosiglitazone (5 nM) was used for PPARgamma .

T0070907 Is a Specific Potent PPARgamma Antagonist in Transient Transfection Assays-- The effect of T0070907 on the transcriptional activity of PPARgamma in a cell-based reporter gene assay was examined. HEK293 cells were transiently transfected with an expression construct that contained the PPARgamma LBD fused to the Gal4-DNA binding domain, together with a luciferase reporter gene under the transcriptional control of the Gal4 upstream activating sequence (Gal4-UAS). As shown in Fig. 2A, rosiglitazone activated transcription up to 20-fold, whereas T0070907 has no effect (or perhaps even a slight inhibitory effect) on basal transcription. In addition, T0070907 is a potent inhibitor (IC50 value in the nM range) of PPARgamma transactivation in the presence of rosiglitazone (Fig. 2A). This inhibition is not due to cytotoxicity as the concentration required to kill 50% of cells is greater than 10 µM (data not shown). The specificity of T0070907 was also examined in cell-based reporter gene assays. HEK293 cells were transiently transfected with a GAL4-UAS reporter and expression constructs encoding the LBDs of PPARalpha , PPARdelta , the farnesoid X receptor, the liver X receptor alpha , the liver X receptor beta , or pregnane X receptor fused to the Gal4-DNA binding domain. As shown in Fig. 2A, T0070907 at 1 µM has no effect on the transcriptional activity of any other receptor besides PPARgamma . These results demonstrate that T0070907 is a PPARgamma -specific antagonist.


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  		Fig. 2.   T0070907 is a specific potent PPARgamma antagonist. A, effects of T0070907 on transcriptional activities of the LBDs of PPARgamma , PPARalpha , PPARdelta , farnesoid X receptor, liver X receptor alpha , liver X receptor beta , or pregnane X receptor gamma  fused with Gal4 protein on Gal4-UAS-luciferase reporter gene expression in HEK293 cells. B, effects of T0070907 on dexamethasone, 3-isobutyl-1-methylxanthine, and insulin-induced (DEX/MIX/INS) 3T3-L1 cell differentiation. Top panel, 3T3-L1 differentiation protocol. Cells were stained with Nile Red and photographed under light microscope. DMSO, Me2SO.

T0070907 Blocks Hormone-mediated Differentiation of the Adipogenic Cell Line 3T3-L1-- We next investigated whether T0070907 could block the induction of adipogenesis by various treatments of the adipogenic cell line 3T3-L1. As shown in Fig. 2B, the standard treatment of dexamethasone, 3-isobutyl-1-methylxanthine, and insulin promoted lipid accumulation in 3T3-L1 cells. In contrast, lipid accumulation in these cells was completely inhibited when cells were treated with both 1 µM T0070907 and the differentiation mixture. Similar inhibitory effects of T0070907 were observed when adipogenesis was induced by treatment with the PPARgamma agonist, rosiglitazone (data not shown).

T0070907 Covalently Modifies PPARgamma on Cys313-- To understand the mechanism by which T0070907 antagonizes PPARgamma function, its binding properties were first examined. That rosiglitazone was unable to displace T0070907 prebound to PPARgamma suggested that the binding of T0070907 was irreversible (data not shown). To verify the covalent nature of the interaction between T0070907 and PPARgamma , and to identify the site of covalent attachment, we performed proteolytic mapping studies via mass spectrometry. The covalent binding of T0070907 (mass of 277.7 Da) to PPARgamma would result in a mass change of the modified tryptic peptide(s) by 241.1 Da. By comparing the tryptic digests of PPARgamma with and without T0070907 treatment, a candidate peptide containing the T0070907 attachment site (amino acids 272-279, IFQGCQFR, m/z 998.49 Da) was identified based on its mass shift to m/z 1239.56 (data not shown). The precise binding site on this peptide was determined with ESI-tandem mass spectrometry (Fig. 3A). The calculated dominant y- and b-ion fragments of this peptide are shown at the top of Fig. 3A, with the ions observed in the mass spectrum underlined. The mass difference between the y3- and y4-ions (m/z 344.1) identified Cys313 as the site of modification by T0070907. In addition, several double-charged y-ions and small internal fragment ions obtained also confirmed this conclusion.


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  		Fig. 3.   T0070907 covalently binds to PPARgamma at Cys313. A, nano-ESI-MS/MS spectrum of tryptic fragment modified by T0070907. The calculated y- and b-ions of this peptide are shown at the top of the figure, with ions observed in the mass spectrum underlined. Cys313 is indicated by *. B, wt or C313S mutant PPARgamma proteins were incubated with [3H]T0070907 and subsequently separated on an SDS-polyacrylamide gel, which was then subjected to autoradiography. Upper panel, autoradiography of the SDS-polyacrylamide gel. [3H]T0070907 bound covalently only to wt PPARgamma . Lower panel, Coomassie Blue-stained same gel showing equal loading of wt and mutant PPARgamma proteins. C, purified GST-PPARgamma mixed with whole-cell extract (WCE) derived from HEK293 cells was incubated with 2 µM [3H]T0070907 and subsequently separated on an SDS-polyacrylamide gel, which was then subjected to autoradiography. Upper panel, autoradiography of the gel. Only GST-PPARgamma was preferentially modified by [3H]T0070907. Lower panel, Coomassie Blue staining of the same gel.

To confirm the importance of Cys313 in T0070907 binding to PPARgamma , a mutant PPARgamma was constructed in which Cys313 was converted to a serine residue, and the corresponding recombinant GST-PPARgamma LBD (C313S) fusion protein was expressed and purified. [3H]T0070907 was first incubated with either wild (wt) type PPARgamma or the C313S mutant as described, and the reaction mixtures were separated by SDS-PAGE. As shown in Fig. 3B, [3H]T0070907 was only able to modify the wild type protein (upper panel, autoradiograph), although equal amounts of wild type and C313S mutant PPARgamma were added to each reaction (lower panel, Coomassie staining). Thus, Cys313 is necessary for the binding of T0070907 to PPARgamma .

The specificity of the covalent modification was examined in a whole-cell extract (WCE) made from HEK293 cells. Purified GST-PPARgamma LBD fusion protein (Fig. 3C) was mixed with the WCE and 2 µM [3H]T0070907 and then separated on an SDS-PAGE gel. As shown in Fig. 3C, the exogenously added PPARgamma was preferentially modified by [3H]T0070907 (upper panel, autoradiograph), despite the presence of many other proteins in the WCE (lower panel, Coomassie staining).

T0070907 Behaves as an Inverse Agonist of PPARgamma LBD in Vitro-- By using the homogeneous time-resolved fluorescence (HTRF) technology, we developed an assay to study the effects of PPARgamma ligands on the interaction of PPARgamma with fragments of coactivator or corepressor proteins. Reporter peptides of ~20 amino acids in length were synthesized from sequences derived from various coactivator and corepressor proteins (Table I) (25, 26). The effects of various ligands on PPARgamma binding to this collection of peptides are shown in Fig. 4A. The patterns that emerged from this peptide profiling have allowed us to distinguish between different functional classes of PPARgamma ligands. First, known PPARgamma agonists such as rosiglitazone, troglitazone, and the GSK compound GI262570 (27) showed very similar peptide profiles. Rosiglitazone and troglitazone, which both belong to the TZD chemical class, were more similar to each other than to tyrosine-based GI262570. GI262570 recruited additional peptides (peptides 11, 19, 23, and 27), suggesting perhaps that PPARgamma adopted a slightly different conformation when bound to GI262570, compared with the conformation assumed by the receptor when bound to the TZD compounds. In contrast, the novel PPARgamma ligand T0070907 shows a unique peptide profile (Fig. 4A), exclusively promoting recruitment of peptides derived from corepressor proteins NCoR and SMART (peptides 2 and 3, respectively). Furthermore, compared with the Me2SO control, T0070907 seems to suppress the basal interactions between PPARgamma and coactivator-derived peptides.

                              
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Table I
List of amino acid sequences and sources of peptides used in the HTRF peptide profiling assay
The LXXLL motif is in bold. Peptides are tagged on the N termini with biotin.


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  		Fig. 4.   T0070907 is an inverse agonist of the GST-PPARgamma LBD in vitro. A, HTRF peptide profiling of T0070907, rosiglitazone, troglitazone, and GI262570. All compounds were tested at 1 µM, and peptides (x axis) were in numerical order as listed in Table I. Corepressor-derived peptides 2 and 3 are shown in black. DMSO, Me2SO. B, dose-response of rosiglitazone and T0070907 in the presence and absence of 1 µM rosiglitazone in an HTRF assay with GST-PPARgamma LBD and peptide 1 (DRIP205). C, same as in B, except that the HTRF peptide is 2 (NCoR). D, dose-responses of rosiglitazone and T0070907 in a GST pull-down assay that measures the interaction between GST-PPARgamma and NCoR protein. An arrow indicates the position of the NCoR protein.

In order to confirm these results, more extensive titration and competition experiments were carried out with two peptides derived from a representative coactivator and a representative corepressor (peptides 1 and 2). As shown in Fig. 4, B and C, rosiglitazone promoted the dose-dependent recruitment of peptide derived from coactivator DRIP205 to PPARgamma , while suppressing the interaction between PPARgamma and a peptide derived from corepressor NCoR. In contrast, T0070907 suppressed the interaction between PPARgamma and the coactivator-derived peptide in the absence of ligand, while promoting the recruitment of the NCoR-derived peptide to PPARgamma . T0070907 also effectively antagonized the effects of rosiglitazone in a dose-dependent manner.

To confirm independently these observations by using an alternative nonfluorescence-based format, the effects of T0070907 on PPARgamma /NCoR interactions were examined using a GST pull-down assay. As shown in Fig. 4D, rosiglitazone suppresses the interaction between the GST-PPARgamma LBD and NCoR in a dose-dependent fashion, with an IC50 consistent with its binding affinity to PPARgamma . On the other hand, T0070907 promoted a dramatic increase in NCoR binding to GST-PPARgamma consistent with the results observed in the HTRF assay.

Effects of T0070907 and LGD1069 on PPARgamma and RXRalpha Heterodimer in GMSAs-- By having shown that T0070907 strongly promotes recruitment of NCoR to the PPARgamma LBD in both HTRF and pull-down assays, we next used a GMSA to examine whether this could also occur in the context of the PPARgamma /RXRalpha heterodimer. As shown in Fig. 5A, in vitro translated PPARgamma and RXRalpha can bind simultaneously to a PPRE-containing DNA fragment derived from the promoter of acyl-CoA oxidase gene (lane 2). This shift in fragment mobility is absolutely dependent on the presence of both PPARgamma and RXRalpha (data not shown), indicating the proper formation of a functional PPARgamma /RXRalpha heterodimer under these conditions. Whereas NCoR could not bind efficiently to the PPARgamma /RXRalpha heterodimer in the absence of ligand (compare lanes 2 and 3, and similar to Ref. 9), T0070907 was able to promote a significant increase in the recruitment of NCoR to the heterodimeric complex (compare lanes 3 and 4).


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  		Fig. 5.   Effects of T0070907 on PPARgamma /RXRalpha heterodimer in a GMSA. A, effects of T0070907 on the recruitment of NCoR to the wt PPARgamma /wt RXRalpha and PPARgamma Delta H12/wt RXRalpha heterodimers. B, effects of T0070907 on the recruitment of NCoR to the wt PPARgamma /RXRalpha Delta H12 and PPARgamma Delta H12/RXRalpha Delta H12 heterodimer complexes. C, effects of LGD1069 on T0070907-induced recruitment of NCoR to the wt PPARgamma /RXRalpha Delta H12, PPARgamma Delta H12/RXRalpha Delta H12 heterodimer complexes. D, effects of T0070907 on LGD1069-induced recruitment of SRC-1 to the PPARgamma /RXRalpha heterodimer complex. Arrows indicate the positions of free probe and various complexes.

To ensure that this increased NCoR recruitment was the result of T0070907 binding to PPARgamma and to understand the nature of the PPARgamma conformational changes associated with T0070907 binding, we next investigated the effects of deleting H12 from both receptors on the binding of NCoR to the heterodimer. The deletion of PPARgamma H12 (PPARgamma Delta H12) increased the basal interaction of NCoR with the heterodimer; however, T0070907 did not provide further enhancement of binding (Fig. 5A, lanes 5-7). In contrast, the PPARgamma wt/RXRalpha Delta H12 heterodimer responded to T0070907 and almost all PPARgamma wt/RXRalpha heterodimer could be super-shifted to form the PPARgamma /RXRalpha /NCoR complex in the presence of the antagonist (Fig. 5B, lanes 2-4). Complexes containing H12 deletions in both PPARgamma and RXRalpha interacted very efficiently with NCoR in the absence of ligand, but as in the case of the PPARgamma wt/RXR Delta H12 complex, T0070907 had almost no effect on NCoR recruitment (Fig. 5B, lanes 5-7).

The allosteric effects between PPARgamma and RXRalpha were studied next by examining the effects of simultaneous treatments of RXRalpha agonist and PPARgamma antagonist on the recruitment of coactivator and corepressor proteins to the heterodimer. Because the RXRalpha Delta H12-containing heterodimers interacted much more strongly with NCoR than the wild type-containing heterodimers (Fig. 5B), we examined the effects of an RXRalpha agonist, LGD1069 (28), on T0070907-induced NCoR recruitment to PPARgamma wt/RXRalpha Delta H12 and PPARgamma Delta H12/RXRalpha Delta H12 complexes. Strikingly, the addition of LGD1069 dramatically inhibited NCoR binding to both pairs of heterodimer complexes (Fig. 5C, lanes 5-10 and lanes 14-19). Importantly, LGD1069 was not able to inhibit completely corepressor binding to the PPARgamma Delta H12/RXRalpha Delta H12 complex as it was in the PPARgamma wt/RXRDelta H12 complex. To ensure that the effect of LGD1069 on NCoR recruitment was not due to blocking T0070907 from binding to the PPARgamma /RXRalpha heterodimer, experiments were performed during which PPARgamma was treated with T0070907 for an extended period prior to the addition of other components of the GMSA reaction mixture and LGD1069 to saturate all available binding sites on PPARgamma . No difference on the recruitment of NCoR to the PPARgamma /RXRalpha heterodimer was observed with or without preincubation with T0070907 (data not shown).

The effects of T0070907 binding to PPARgamma on the ability of RXRalpha to interact with coactivators were also studied. In the presence of the RXRalpha agonist LGD1069, the coactivator protein SRC-1 can be recruited to the PPARgamma /RXRalpha heterodimer, as evidenced by the super-shifted complex observed in GMSAs (Fig. 4D, lanes 5-10). This super-shifted complex was not observed in the presence of rosiglitazone (BRL, lane 4) or in reactions containing an RXR H12 mutant (23) suggesting that SRC-1 was specifically recruited to the RXRalpha subunit. When added to the GMSA reaction mixture together with LGD1069, T0070907 seems to have a modest effect on formation of the PPARgamma /RXRalpha /SRC-1 super-shifted complex induced by LGD1069 (compare lanes 5-10 and lanes 12-17).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have identified a specific, high affinity PPARgamma antagonist, T0070907, that blocks PPARgamma activity in both biochemical and cell-based assays. T0070907 is highly selective for PPARgamma over PPARalpha , PPARdelta , other NHRs, and proteins in an HEK293 WCE. Proteolytic mapping indicated that T0070907 irreversibly modifies PPARgamma on Cys313 in helix 3 of the LBD, a residue that is conserved in all three PPAR subtypes. This indicates that other residues in the binding pocket confer the specific binding of T0070907 to PPARgamma . Interestingly, this is also the site of covalent modification by L-764406, a PPARgamma partial agonist that was described previously (12).

T0070907 functions as a PPARgamma antagonist in cell-based assays. It effectively blocked TZD-induced transactivation by the GAL4-PPARgamma LBD, as well as adipogenesis in 3T3-L1 cells treated with a differentiation mixture. Overall, these results further support the important role for PPARgamma in fat cell differentiation. The antagonist properties of T0070907 were also demonstrated in a variety of in vitro biochemical assays using the PPARgamma LBD. T0070907 suppressed agonist-induced interactions between the PPARgamma LBD and coactivator-derived peptides and promoted recruitment of corepressor-derived peptide in HTRF assays (Fig. 4). The effect of T0070907 on assembly of corepressor NCoR/PPARgamma complex was also observed in the pull-down assay (Fig. 4D).

Previous studies (29-31) have suggested that corepressors bind to a hydrophobic groove on NHR LBDs formed by H3, H5, and H6. This binding site partially overlaps with that utilized by coactivators. NHR agonists disrupt the interaction between NHR LBDs and corepressors, and it is believed that the conformation of H12 stabilized by agonists partially occludes the corepressor binding site. In the unliganded state, H12 of NHR LBDs is thought to exist in multiple conformations, including the agonist-bound conformation. Consistent with this hypothesis, H12 is inhibitory for NCoR binding to most NHRs. Mutations and deletions of H12 from either PPARgamma or RXRalpha significantly increase the recruitment of NCoR to the PPARgamma /RXRalpha heterodimer (9, 32) (Fig. 5). T0070907 can also promote NCoR recruitment to PPARgamma /RXRalpha heterodimer, but T0070907 can only promote recruitment of NCoR to complexes containing wt PPARgamma but not to complexes containing PPARgamma Delta H12. These results suggest that the effect of T0070907 on the heterodimer is indeed mediated through PPARgamma and that T0070907 induced NCoR recruitment requires H12. Indeed, T0070907 treatment or the deletion of H12 stabilize NCoR recruitment to comparable extents (Fig. 5) suggesting that T0070907 most likely acts on PPARgamma by preventing H12 from adopting the agonist-bound conformation. The deletion of RXRalpha H12 domain has a synergistic effect with either T0070907 treatment or PPARgamma Delta H12 on the recruitment of NCoR to PPARgamma /RXRalpha heterodimer (Fig. 5B). Two NHR-binding motifs are present on NCoR protein (29-31), the deletion of H12 from RXRalpha together with either T0070907 treatment or the deletion of H12 from PPARgamma perhaps allows the cooperative binding of both motifs to PPARgamma /RXRalpha heterodimer.

In order to dissect the contributions of the PPARgamma and RXRalpha to NCoR recruitment to the heterodimer, the effects of simultaneous treatment with T0070907 and LGD1069 were determined. Notably, LGD1069 dramatically inhibited the T0070907-mediated increase in NCoR recruitment to the PPARgamma wt/RXRalpha Delta H12 and PPARgamma Delta H12/RXRalpha Delta H12 heterodimers in a dose-dependent manner (Fig. 5C). Recent x-ray crystallographic studies of the apo-RXRalpha LBD (unliganded), the holo-RXRalpha LBD (agonist bound), and a PPARgamma /RXRalpha heterodimer (each bound to agonist) suggest possible molecular mechanisms for these effects. The rosiglitazone-bound PPARgamma /9-cis-retinoic acid-bound RXR heterodimer interface which is largely composed of residues from H10 and H11 of both receptors contains several important salt bridges. In particular, a salt bridge formed between the carboxylic acid of Tyr477 from PPARgamma H12 and Lys431 from RXRalpha H10 stabilizes the positioning of H12 from PPARgamma in the agonist-bound conformation (33). In addition, comparison of the apo- and holo-RXRalpha LBD structures reveal that ligand-binding triggers several large conformational changes. For example, H11, which partially fills the ligand binding pocket in the apo-RXRalpha structure, moves out of the binding pocket and rotates by ~180° around its own axis upon binding of 9-cis-retinoic acid, allowing H10 and H11 to form an almost continuous helix (34). Although the structure of a PPARgamma /apo-RXRalpha heterodimer has not yet been described, these structural results suggest that LGD1069 binding could lead to significant alterations in the PPARgamma /RXRalpha heterodimer interface. Given that Lys431 is located near the site of the conformational changes involving H10 and H11, RXR agonists could also influence the stability of the Tyr477/Lys431 salt bridge and hence the positioning of PPARgamma H12. Thus, we suggest that LGD1069 binding inhibits binding of NCoR to the wild type heterodimer (with or without T0070907) by orienting PPARgamma and RXRalpha such that binding of NCoR is disfavored and by stabilizing PPARgamma H12 in the agonist-bound conformation. Consistent with this view, the effect of LGD1069 on T0070907-induced recruitment of NCoR is more potent on the PPARgamma wt/RXRalpha Delta H12 heterodimer than on PPARgamma Delta H12/RXRalpha Delta H12 heterodimer. In addition, a residual amount of NCoR remained on PPARgamma Delta H12/RXRalpha Delta H12 heterodimer even at the highest LGD1069 concentrations (Fig. 5C), and LGD1069 was also ineffective in preventing NCoR binding to PPARgamma Delta H12/RXRalpha wt heterodimer complexes (data not shown).

The effect of T0070907 on LGD1069-induced recruitment of coactivator SRC-1 was also tested. Whereas the T0070907-induced NCoR recruitment to PPARgamma /RXRalpha heterodimer can be almost completely reversed by the simultaneous treatment with RXRalpha agonist LGD1069, the effects of T0070907 on LGD1069-induced coactivator recruitment to the PPARgamma /RXRalpha heterodimer are more modest by comparison. These results suggest that RXRalpha agonists may have a greater influence on the conformation of the PPARgamma /RXRalpha heterodimer than do PPARgamma antagonists, and more importantly, PPARgamma antagonist activity could be modulated by the availability and concentration of RXRalpha agonist. The in vivo relevance of these effects is the focus of our current and future studies.

    ACKNOWLEDGEMENTS

We thank Merrill Ayres, Mitch Hull, Tim Hoey, Jonathan Houze, Jowell Jo, Xiao Hong Liu, Miki Rich, Heather Webb, and Haoda Xu for technical support and generous gifts of reagents. We also thank Andrew Shiau, Jurgen Lehmann, Hui Tian, and Zhulun Wang for helpful discussions, and Kelly LaMarco for editing this manuscript.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Tularik Inc., Two Corporate Dr., South San Francisco, CA 94080. Tel.: 650-825-7524; Fax: 650-825-7400; E-mail: yli@tularik.com.

Published, JBC Papers in Press, March 4, 2002, DOI 10.1074/jbc.M200743200

    ABBREVIATIONS

The abbreviations used are: PPARgamma , peroxisome proliferator-activated receptor gamma ; RXR, retinoid X receptor; TZD, thiazolidinedione; NHR, nuclear hormone receptor; PPRE, PPAR-response element; HTRF, homogeneous time-resolved fluorescence; LBD, ligand-binding domain; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectroscopy; GMSA, gel mobility shift assay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate]; Gal4-UAS, Gal4 upstream activating sequence; wt, wild type; hPPARgamma 1, full-length human PPARgamma 1; h, human; ESI, electrospray ionization; SPA, scintillation proximity assay; WCE, whole-cell extract.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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