©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Peroxisome Proliferator-activated Receptor Mediates Cross-talk with Thyroid Hormone Receptor by Competition for Retinoid X Receptor
POSSIBLE ROLE OF A LEUCINE ZIPPER-LIKE HEPTAD REPEAT (*)

(Received for publication, March 15, 1995; and in revised form, May 16, 1995)

Cristiana E. Juge-Aubry (1) Agnieszka Gorla-Bajszczak (1) Agns Pernin (1) Thomas Lemberger (2) Walter Wahli (2) (3) Albert G. Burger (1) Christoph A. Meier (1) (4)(§)

From the  (1)Thyroid Unit, Division of Endocrinology, University Hospital of Geneva, CH-1211 Geneva, Switzerland, the (2)Institut de Biologie Animale, University of Lausanne, CH-1015 Lausanne, Switzerland, (3)Glaxo Institute for Molecular Biology, CH-1228 Plan-les-Ovates, Switzerland, and the (4)Clinique de Mdecine II, Department of Medicine, University Hospital of Geneva, CH-1211 Geneva, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The peroxisome proliferator-activated receptors (PPAR) and thyroid hormone receptors (TR) are members of the nuclear receptor superfamily, which regulate lipid metabolism and tissue differentiation. In order to bind to DNA and activate transcription, PPAR requires the formation of heterodimers with the retinoid X receptor (RXR). In addition to activating transcription through its own response elements, PPAR is able to selectively down-regulate the transcriptional activity of TR, but not vitamin D receptor. The molecular basis of this functional interaction has not been fully elucidated. By means of site-directed mutagenesis of hPPAR we mapped its inhibitory action on TR to a leucine zipper-like motif in the ligand binding domain of PPAR, which is highly conserved among all subtypes of this receptor and mediates heterodimerization with RXR. Replacement of a single leucine by arginine at position 433 of hPPAR (L433R) abolished heterodimerization of PPAR with RXR and consequently its trans-activating capacity. However, a similar mutation of a leucine residue to arginine at position 422 showed no alteration of heterodimerization, DNA binding, or transcriptional activation. The dimerization deficient mutant L433R was no longer able to inhibit TR action, demonstrating that the selective inhibitory effect of PPAR results from the competition for RXR as well as possibly for other TR-auxiliary proteins. In contrast, abolition of DNA binding by a mutation in the P-box of PPAR (C122S) did not eliminate the inhibition of TR trans-activation, indicating that competition for DNA binding is not involved. Additionally, no evidence for the formation of PPAR:TR heterodimers was found in co-immunoprecipitation experiments. In summary, we have demonstrated that PPAR selectively inhibits the transcriptional activity of TRs by competition for RXR and possibly non-RXR TR-auxiliary proteins. In contrast, this functional interaction is independent of the formation of PPAR:TR heterodimers or competition for DNA binding.


INTRODUCTION

The peroxisome proliferator-activated receptors (PPAR)()are a novel subfamily of the steroid/thyroid hormone nuclear receptor proteins involved in the ligand-inducible regulation of lipid metabolism, adipose tissue differentiation, and possibly hepatocarcinogenesis in rodents(1) . Their closest relatives in the superfamily are the type II nuclear hormone receptors, such as the retinoic acid, vitamin D, and thyroid hormone receptors. In particular, the P-box of the first zinc-finger responsible for DNA-binding specificity is fully conserved between hPPAR and the L-triiodothyronine (T)-receptors (TR) 1 and 1 (Fig. 1A), reflecting the preferential binding of both receptors to differently spaced AGGTCA half-sites(2) . Additionally, a high degree of sequence conservation is found between a putative leucine zipper motif in the ligand-binding domain of PPAR and TR(3) . This motif is very highly conserved among various species and PPAR subtypes (Fig. 1B). Besides the structural homology, TRs and PPARs require heterodimerization with the retinoid X receptor (RXR) for optimal DNA binding and both receptors are co-expressed in brain, liver, and adipocytes, where they are involved in the regulation of lipid metabolism(1) . We and others have recently reported that the PPAR is able to modulate TR activity either positively or negatively, depending on the T-response element (TRE)(4, 5) . Although it has been suggested that rTR is able to form heterodimers with rPPAR in solution, it has not been demonstrated whether this interaction quantitatively accounts for the observed transcriptional changes. Here we examined the mechanism by which hPPAR structurally and functionally interacts with TR and RXR. We show that hPPAR is an efficient competitor for RXR and most likely for other TR-auxiliary proteins (TRAPs), thereby specifically inhibiting TR activity by disrupting the formation of TR:RXR heterodimers. A series of point mutations in hPPAR allowed the mapping of a region that is indispensable for this cross-talk to a carboxyl-terminal leucine zipper-like motif, which is highly conserved among all subtypes (PPAR, , , and ) of this receptor.


Figure 1: Amino acid sequence comparison of hPPAR with hTR1 and other PPARs. A, the first zinc fingers of the DNA-binding domain of hPPAR and hTR1 were aligned, showing a 100% sequence conservation of the P-box (I indicates identical amino acids). The position of the point mutation in hPPAR-C122S changing a cysteine to a serine residue is indicated. B, the heptad repeat motif (boxed) of the leucine-zipper in the dimerization domain of hPPAR, corresponding to the ninth heptad repeat in the TR, was aligned with hTR1 and all currently available PPAR sequences. The heptad repeats are formed by hydrophobic amino acids with leucine or other hydrophobic amino acids (e.g. Ile, Val, Ala, Met, or Phe) at positions 1 and 8 and hydrophobic or charged amino acids with hydrophobic side chains (e.g. Arg and Gln) in the fifth position(3, 22, 23) . This motif is well conserved between hPPAR and hTR1 and even more so within the PPAR subtypes even across different species. The two hPPAR mutations L433R and L422R changing leucine to arginine residues are indicated (prefixes: h = human, r = rat, m = mouse, cg = hamster, and x = Xenopus).




MATERIALS AND METHODS

Construction of Plasmids and Site-directed Mutagenesis

The pSG5-hPPAR plasmid was kindly provided by Dr. F. Gonzalez(6) . The pBL2-BFE-CAT containing the peroxisome proliferator-response element (PPRE) from the bifunctional enzyme (BFE) promoter (position -2950 to -2925) as well as the pSG5-VDR vector for the human vitamin D receptor and the pBL2-DR3-CAT with a DR3 vitamin D response element were generously provided by Dr. C. Carlberg(7, 8) . The pSV2-hTR1 and pMTV-TRElap-CAT plasmids are described elsewhere(9, 10) . Mutant hPPAR (pSG5-hPPAR-L433R and -L422R) were created by the polymerase chain reaction-mediated splice donor site overlap extension method and by subsequently replacing the HindIII/XbaI fragment of pSG5-hPPAR with the mutated polymerase chain reaction product(10) . The clones were verified by dideoxy sequencing to rule out spurious mutations. hPPAR-L433R and -L422R have a T to G point mutation in codons 433 and 422 at nucleotide positions 1514 and 1400, respectively, changing a leucine to arginine. pSG5-hPPAR-C122S has a point mutation, replacing a C by a G at nucleotide position 581, changing codon 122 from cysteine to serine. This mutant polymerase chain reaction fragment was used to replace the wild-type AvaI/AvaI fragment in pSG5-hPPAR.

Preparation of Antibodies

Rabbit polyclonal anti-TR1 antibodies were raised against a synthetic peptide corresponding to the unique hTR1 amino acid sequence 61-81 as described by Falcone et al.(11) . The peptide was coupled to the maleimide-activated keyhole limpet hemocyanin (Pierce) for immunization. Fifty µg of protein was injected with Specol (Central Veterinary Institute, Lelystad, the Netherlands). The specificity of the antibody was confirmed by immunoprecipitation of [S]methionine-labeled in vitro translated hTR1, hTR2, and hTR1 as already described(12) . To prepare an anti-PPAR antibody the cDNA encoding the 101 first amino acids of the mouse PPAR were cloned into the pQE-9 bacterial expression vector. The expressed polypeptide was purified on a Ni-NTA-agarose column under native conditions according to the manufacturer's instructions (Quiagen, Hilden, Germany), and injected subcutaneously into KOBU rabbits. After the primary injection (200 µg of polypeptide with Freund's adjuvant), the rabbits were boosted 4 times (200 µg/boost). The serum was collected 10 days after the final boost.

In Vitro Transcription/Translation of Receptors

[S]Methionine-labeled and unlabeled receptors were synthesized using the rabbit reticulocyte lysate transcription/translation kit TnT/T7 (Promega, Madison, WI) according to the manufacturer's instructions. The labeled receptors were analyzed for appropriate size by electrophoresis on a 12.5% sodium dodecyl sulfate-polyacrylamide gel and quantitated by the trichloroacetic acid precipitation method as described(10) .

Co-immunoprecipitation of Receptors in Solution

In vitro translated [S]methionine-labeled and unlabeled receptors were brought to a final volume of 20 µl with EMSA binding buffer and incubated with the appropriate rabbit polyclonal antibodies or preimmune sera overnight at 4 °C. Complexes were precipitated with 50 µl of protein A-agarose (slurry 50%) (Boehringer Mannheim, Germany) previously washed with phosphate-buffered saline containing 0.3% Tween 20, 0.5 mM methionine, and 1% bovine serum albumin. Samples were incubated for 1 h at 4 °C with regular shaking. After microcentrifugation the pellet was washed 4 times with 1 ml of phosphate-buffered saline containing 0.3% Tween 20 and 0.5 mM methionine. 50 µl of SDS-PAGE denaturing sample buffer was added to the final pellet and boiled for 5 min at 94 °C. The supernatant was subjected to electrophoresis on a 12.5% polyacrylamide gel.

Electrophoretic Gel Mobility Shift Assay (EMSA)

Single stranded oligonucleotides were synthesized by Microsynth (Balgach, Switzerland) and annealed with the complementary strands. The following sequences were used(7, 10) : TRE-LAP, 5`-AAGGGGATCCAGCTTGACCTGACGTCAGGTCAAGTC-3`; and PPRE (BFE), 5`-AGGGCTTTGACCTATTGAACTATTACCTAC-3`. The ends were filled in using Taq polymerase (Promega, Madison, WI) in the presence of [-P]dCTP (Amersham, United Kingdom). In vitro translated receptors were incubated with 20,000 cpm of the labeled double stranded oligonucleotides in the presence of 2 µg of poly[d(I-C)] and EMSA binding buffer (50 mM KCl, 20 mM Hepes, 20% glycerol, 0.05% Nonidet P-40, 10 mM -mercaptoethanol, pH 7.5) added to a final volume of 25 µl. Incubation was performed at room temperature for 20 min and the mixture was then loaded on a 5% polyacrylamide gel. The electrophoresis was performed at 4 °C and 300 V for 80 min.

Cell Culture and Transfection Studies

HepG2 cells were plated 24 h before transfection in modified Eagle's medium containing 10% (v/v) hormone-depleted fetal calf serum(13) , penicillin (100 units/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml) in 6-well plates at a density of 0.5 10 cells/well. The medium was changed 4 h before transfection. Using the calcium-phosphate method (CellPhect kit, Pharmacia Biotech Inc., Piscataway, NJ) the cells were transfected with the appropriate plasmids. Eighteen hours later the plates were washed once with phosphate-buffered saline, and fresh medium was added together with 500 nM of either L-triiodothyronine, the peroxisome proliferator, and arachidonic acid analogue ETYA or 1,25(OH)-vitamin D (VD). After another 24 h the cells were harvested, lysed, and the chloramphenicol acetyltransferase activity determined in the extract as described(14) . Chloramphenicol acetyltransferase activity was normalized for the protein concentration as measured by the Coomassie Blue method. Experiments were performed in triplicate and repeated two to four times.


RESULTS

hPPAR Selectively Inhibits hTRs

We have previously reported an inhibitory effect of hPPAR on hTRs in HeLa cells(5) . In contrast to the dominant negative effect of mutant hTR1 from kindreds with thyroid hormone resistance, this effect was observed on all types of TREs, i.e. palindromic, inverted palindromic, and direct repeat arrangements of the half-sites. The present experiments were performed in the human hepatocarcinoma cell line HepG2 representing a tissue of high expression of both TR and PPAR(15, 16) . Fig. 2A shows the dose dependent inhibition by PPAR of ligand-dependent transcription of hTR1 when assessed on a TRE-LAP. Similar results were obtained on reporters containing a DR+4 or TRE-PAL (data not shown). However, this inhibitory effect of hPPAR was specific for hTR, since no alteration of vitamin D-dependent transactivation was observed on a DR+3 element in the presence of increasing doses of hPPAR (Fig. 2B). Similarly, no change in the transcriptional activity of hTR1 on a TRE-LAP was present when increasing amounts of VDR were transfected (Fig. 2C).


Figure 2: Selective inhibition of hTR1, but not hVDR, by hPPAR. A, transfection of increasing amounts of hPPAR inhibits the T-induced transcriptional capacity of hTR1. HepG2 cells were transfected with 1 µg of pMTV-TRElap-CAT, 0.2 µg of pSG5-hRXR and pSV2-hTR1 each, as well as 0, 0.2, 0.6, or 1.8 µg of pSG5-hPPAR as indicated. The amount of pSG5 promoter sequences was kept constant by adding empty pSG5 vector where necessary. Cells were treated with hormone, lysed, and chloramphenicol acetyltransferase (CAT) activity determined as described under ``Materials and Methods.'' B, transfection of increasing amounts of hPPAR does not alter the VD-induced transcriptional capacity of hVDR. HepG2 cells were transfected with 1 µg of pBL2-DR3-CAT, 0.2 µg of pSG5-hRXR and pSG5-hVDR each, as well as 0, 0.2, 0.6, or 1.8 µg of pSG5-hPPAR as indicated. C, transfection of increasing amounts of hVDR does not alter the T-induced transcriptional capacity of hTR1. HepG2 cells were transfected with 1 µg of pMTV-TRElap-CAT, 0.2 µg of pSG5-hRXR and pSV2-hTR1 each, as well as 0, 0.2, 0.6, or 1.8 µg of pSG5-hVDR as indicated.



hPPAR Inhibits the Formation of TR:RXR Heterodimers without Forming PPAR:TR Heterodimers in Solution

Co-immunoprecipitation experiments of [S]methionine-labeled hPPAR with unlabeled hTR1 in the presence of an anti-hTR1 antibody did not show evidence for the formation of relevant quantities of PPAR:TR heterodimers in solution (Fig. 3A, lane 3). However, the precipitation of S-labeled hRXR after incubation with cold hTR1 demonstrates the ability of the anti-hTR1 antibody to precipitate hTR1 also in its heterodimerized state. Similarly, when using an anti-PPAR antiserum together with [S]methionine-labeled hTR1 and cold hPPAR, no PPAR:TR heterodimers were detected (Fig. 4A, lane 9). In order to test for inhibitory mechanisms other than the formation of TR:PPAR heterodimers which we were unable to detect, we performed EMSA on a radiolabeled TRE-LAP (Fig. 3B). The addition of a 1-4-fold excess of hPPAR did not inhibit the formation and DNA binding of hTR1 homodimers, compatible with the lack of formation of TR:PPAR heterodimers (Fig. 3B, lanes 2-5). However, hPPAR was able to substantially reduce the formation of TR:RXR heterodimers already at equimolar receptor concentrations, suggesting that hPPAR competes with high affinity for this TRAP (lane 7). At excess amounts of hPPAR, the TR:RXR complex was almost completely abolished, and the reappearance of TR:TR homodimers could be observed (lanes 8 and 9). This suggests that PPAR competes for RXR, rather than for DNA binding. However, for reasons that have not been further investigated, the intensity of the homodimeric band in lane 9 does not equal that observed in the absence of PPAR and RXR in lane 2. It is nevertheless likely that the still visible DNA-bound TR:RXR complex as well as possibly unbound TR:RXR heterodimers sufficiently lower the free TR1 concentration to reduce the cooperative formation of homodimers. The dimeric nature of the TR1 complexes on this element was established previously by our and other groups(5, 17, 18) . The composition of the homo- and heterodimeric complexes of hTR1 on TRE-LAP was confirmed by quantitatively supershifting both bands with the anti-TR1 antiserum (data not shown).


Figure 3: hPPAR competes with hTR1 for hRXR without the formation of PPAR:TR heterodimers. A, co-immunoprecipitation of hTR1 and hPPAR with an anti-TR1 antibody. While this antibody precipitates TR:RXR heterodimers (lane 2), no evidence for the formation of heterodimers between TR and PPAR wild-type (lane 3) or mutants (lanes 4-6) is present. 6 fmol of in vitro translated [S]methionine-labeled wild-type (WT) or mutant hPPAR (hPPAR) were incubated with 4 fmol of hTR1 and 2 µl of anti-hTR1 antibody (lanes 2-6) or preimmune serum (lane 1). After precipitation with protein A-agarose, samples were run on SDS-polyacrylamide gel electrophoresis. B, effect of hPPAR wild-type on TR:TR homo- and TR:RXR heterodimer binding on TRE-LAP as analyzed by EMSA. While no effect of increasing amounts of in vitro translated hPPAR on the TR:TR homodimer was present (lanes 2-5) the formation of TR:RXR heterodimers was strongly reduced (lanes 6-9). At a 2- and 4-fold excess of PPAR, a homodimeric TR:TR band reappeared. [P]TRE-LAP was incubated either with unprogrammed reticulocyte lysate (lane 1) or in vitro translated hTR1 (lanes 2-8) as described under ``Materials and Methods.'' Numbers represent molar ratio of receptors.




Figure 4: Leucine residue 433 of hPPAR mediates the interaction with hRXR. A, interaction of wild-type and mutant hPPAR with hRXR assessed by co-immunoprecipitation with an anti-PPAR antibody. While the hPPAR wild-type (lanes 1 and 2), -C122S (lanes 5 and 6), and L422R (lanes 7 and 8) were able to form heterodimers with RXR, the L433R mutant completely lost its ability to interact with RXR (lanes 3 and 4). Lane 9 confirms the absence of PPAR:TR heterodimers with an anti-PPAR antibody when [S]methionine-labeled hTR1 (hTR1) was used instead of hRXR. 4 fmol of [S]methionine-labeled hRXR (hRXR) are incubated with 6 fmol of in vitro translated wild-type or mutated PPARs (lanes 1-8) and 2 ml anti-PPAR antiserum (lanes 2, 4, 6, 8, and 9). When no antiserum was added, volume is replaced by preimmune serum (lanes 1, 3, 5, and 7). Immune complexes were precipitated by protein A-agarose and electrophoresed. B, binding of wild-type and mutant hPPAR to the PPRE from BFE in EMSA. In the absence of RXR, neither the wild-type nor mutant PPARs were able to bind to DNA (lanes 3-6). Upon addition of RXR, PPAR wild-type and the control mutant L422R were capable of interacting with the BFE element (lanes 7 and 9), whereas the heptad repeat (L433R, lane 8) and P-box (C122S, lane 10) mutants showed no detectable binding. In all incubation mixtures, the total volume of reticulocyte lysate was kept constant and completed with unprogrammed reticulocyte lysate where necessary.



The Inhibitory Effect of PPAR on TR Activity Requires Its Heterodimerization with RXR

In order to test directly whether hPPAR competes for RXR, rather than for DNA binding, three mutant hPPAR were created as illustrated in Fig. 1. Mutation C122S is located in the P-box at the base of the first zinc-finger (Fig. 1A). The amino acid sequence alignment of the ligand-binding domains of hTR1 and hPPAR revealed the presence of a highly conserved leucine zipper-like heptad repeat, corresponding to the ninth heptad repeat in hTR1 (Fig. 1B)(3, 19) . In order to test its function in the context of hPPAR, the distal leucine was mutated to an arginine (L433R). To exclude a nonspecific effect of this mutation on the secondary or tertiary protein structure of PPAR, a control mutation was created 11 amino acids amino-terminally (L422R).

The three hPPAR mutants were characterized with respect to their ability to interact with RXR in solution, to bind to DNA, and to transactivate through a PPRE and to modulate TR activity. Co-immunoprecipitation experiments with the anti-PPAR antibody detected the presence of PPAR:RXR heterodimers in solution when hPPAR-wild-type, or the -C122S and -L422R mutants where used. In contrast, the Leu to Arg mutation at position 433 abolished the heterodimerization with hRXR (Fig. 4A). In addition, like the wild-type hPPAR, none of the mutants was able to heterodimerize with hTR1 (Fig. 3A). When analyzed in an EMSA using the PPRE from the BFE promoter, no binding to DNA was observed when wild-type or mutant hPPAR was used alone (Fig. 4B, lanes 3-6). However, only the wild-type and hPPAR-L422R mutant bound to DNA when RXR was present (Fig. 4B, lanes 7 and 9). The hPPAR-C122S was expected to be unable to bind to DNA due to its mutation in the DNA-binding domain (lane 10), while the hPPAR-L433R was incapable of interacting with DNA due to its defect in heterodimerizing with RXR (lane 8). Taken together, these results suggest that the distal leucine residue 433 of the minimal leucine zipper motif in the ligand-binding domain of PPAR is required for the heterodimerization with RXR, while the cysteine residue 122 in the P-box is necessary for binding to DNA without affecting the heterodimerization with RXR. The control mutation hPPAR-L422R close to this putative leucine zipper heptad repeat altered neither the formation of heterodimers nor DNA binding. The panel of mutant hPPAR was tested in a transient transfection system for the capacity to transactivate as well as to modulate TR activity. When tested on the PPRE from the BFE promoter, only wild-type hPPAR and L422R showed ligand-induced transcriptional activity, as anticipated from the EMSA experiments (Fig. 5). The inhibitory effect of hPPAR-L433R and -C122S in the presence of ETYA on the basal activity of the reporter gene was not further analyzed here.


Figure 5: Transcriptional capacity of mutant hPPAR on the PPRE from bifunctional enzyme. HepG2 cells transiently transfected with the pBL2-BFE-CAT reporter gene and treated with the peroxisome proliferator ETYA showed no evidence for functionally active endogenous PPAR activity(-). Upon transfection of pSG5-hPPAR (PPAR-WT) or the control mutant pSG5-hPPAR-L422R (L422R), a severalfold increase in chloramphenicol acetyltransferase (CAT) activity was observed. In contrast, the hPPAR mutants deficient in heterodimerization (L433R) or DNA binding (C122S) showed no transcriptional activity. Cells were transfected with 1 µg of reporter plasmid, 0.2 µg of pSG5-hRXR, and 0.2 µg of wild-type (WT) or mutant pSG5-hPPAR as indicated. The amount of pSG5 promoter sequences was kept constant by adding empty pSG5 vector where necessary. Cells were treated with ETYA, lysed, and chloramphenicol acetyltransferase activity determined as described under ``Materials and Methods.''



In order to test the ability of the mutant PPARs to inhibit ligand-induced TR transactivation, co-transfection experiments with hTR1 and mutant hPPAR were performed as shown in Fig. 6, A-C. While the leucine zipper mutant hPPAR-L433R lost its ability to down-regulate TR activity (Fig. 6A), the DNA binding-deficient mutant hPPAR-C122S (Fig. 6B) and the control mutant hPPAR-L422R (Fig. 6C) remained efficient inhibitors of TR-dependent transcription. These data demonstrate that the leucine residue at position 433 of hPPAR is essential for mediating cross-talk with hTR by a mechanism involving heterodimerization with RXR.


Figure 6: Heterodimerization of hPPAR with hRXR, but no DNA binding, is required for the inhibition of hTR1 activity. HepG2 cells were transiently transfected with constant amounts of pSV2-hTR1 (0.2 µg), pSG5-hRXR (0.2 µg), and the pMTV-TRElap-CAT reporter (1 µg) as described under ``Materials and Methods.'' The marked inhibition of hTR1 activity observed with hPPAR wild-type (Fig. 2) is completely abolished when the pSG5-hPPAR-L433R mutant is used at increasing doses (0.2, 0.6, and 1.8 µg) as shown in panel A. However, the same mutation located 11 amino acids upstream (pSG5-hPPAR-L422R) as well as the DBD mutant pSG5-hPPAR-C122S were still able to inhibit the T-induced transactivation by hTR1, as shown in panels C and B, respectively. CAT, chloramphenicol acetyltransferase.




DISCUSSION

Besides sharing structural homologies, and the ability to heterodimerize with RXR, TR and PPAR are both involved in the regulation of lipid metabolism. We now show that PPAR is able to selectively inhibit ligand-induced TR activity by competing for TRAPs, particularly RXR. In addition, our data suggest that a highly conserved leucine zipper-like heptad repeat in the ligand-binding domain may be required for PPAR to dimerize with RXR. However, despite a similar control mutation located 11 amino acids amino-terminally (L422R), the possibility of protein misfolding in the mutation L433R cannot be completely excluded. Nevertheless, immunocytochemical studies of transfected HepG2 cells show that all the PPAR mutants are expressed similarly to the wild-type protein (data not shown). Other mechanisms possibly accounting for the modulatory action of PPAR on TR, such as the formation of PPAR:TR heterodimers and/or the competition for DNA binding, were also addressed in the present study. In contrast to what has been reported for rTR and rPPAR(4) , we found no evidence for the formation of significant amounts of hPPAR:hTR1 heterodimers. While the formation of PPAR:TR heterodimers may exist, it is not likely to be quantitatively important as is already evident from the initial report. The present results obtained with the P-box mutant C122S show that competition for DNA binding is also not a major mechanism for the inhibition of TR action by PPAR. Nevertheless, the hPPAR-C122S was a somewhat less efficient inhibitor of thyroid hormone action, although its dimerization with RXR was preserved and binding to a TRE-LAP has been excluded by gel shift assays. This characteristic of the mutant has not been analyzed further, but possible explanations are differences in expression levels, post-translational modifications, or heterodimer stability.

The modulation of TR transactivation by PPAR is highly specific, since the ligand-dependent VDR activity was not altered by PPAR. However, since the transcriptional activity of VDR depends also on RXR on the vitamin D-response element used, PPAR would be expected to also inhibit the formation of VDR:RXR heterodimers, unless VDR has a higher affinity for RXR than PPAR. Indeed, our EMSA data suggest that PPAR also decreases the binding of VDR:RXR heterodimers to DNA, but to a lesser extent than TR:RXR heterodimers (data not shown). This together with the observation that the transfection of excess amounts of RXR does not restore full TR activity in the presence of PPAR argues in favor of PPAR competing and dimerizing with as yet uncharacterized non-RXR TRAPs(5) . However, this putative TRAP is expected to interact with PPAR by means of a similar dimerization region as RXR, since hPPAR-L433R eliminates the inhibitory effect of PPAR on TR.

A modulatory effect of PPAR on thyroid hormone signaling in cell cultures has been recently reported(20) , suggesting a physiological importance for this mechanism. In humans, the syndrome of thyroid hormone resistance, which is due to dominant negative mutations in one hTR1 allele, also clearly demonstrates the ability of a nuclear factor to modulate thyroid hormone action in a dominant negative manner in vivo and in vitro(21) . In addition, TRs and PPARs are co-expressed in many tissues, such as liver and brain, and both receptors regulate similar metabolic steps in lipid metabolism, such as the ones controlled by malic enzyme and bifunctional enzyme. The modulatory effect of PPAR on TR depends on the PPAR protein levels as well as its affinity for RXR, the former being controlled by glucocorticoids and the latter possibly by phosphorylation as demonstrated for other nuclear receptors(15) .

In summary, we have demonstrated that PPAR is able to selectively inhibit the transcriptional activity of TRs by competing for RXR and possibly also for a specific, but as yet unidentified non-RXR TRAP. In addition, the results suggest that a highly conserved leucine zipper-like motif in the ligand-binding domain of PPAR may be necessary, although not be sufficient, for PPAR to dimerize with RXR.


FOOTNOTES

*
This work was supported by Grants 32-33568.92 (to C. A. M.), 3200-037536.93 (to A. G. B.), and 3100-030807.91/2 (to W. W.) from the Swiss National Science Foundation, and Grant FOR 407 from the Swiss Cancer League (to C. A. M.). 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.

§
To whom correspondence should be addressed. Present address: Endocrine Div., Massachusetts General Hospital, Boston, MA 02114. Tel.: 617-726-3966; Fax: 617-726-7543.

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; T, L-triiodothyronine; TR, T receptor; RXR, retinoid X receptor; TRE, T-response element; TRAP, TR-auxiliary proteins; PPRE, peroxisome proliferator-activator response element; BFE, bifunctional enzyme; VDR, vitamin D receptor; VD, 1,25(OH)-vitamin D; EMSA, electrophoretic gel mobility shift assay; ETYA, 5,8,11,14-eicosatetraynoic acid.


ACKNOWLEDGEMENTS

We are grateful to Drs. F. Gonzalez (National Cancer Institute, Bethesda, MD) and C. Carlberg (University Hospital Geneva) for providing the PPAR and VDR expression and reporter plasmids, respectively. We also thank Prof. B. Desvergne (Institut de Biologie Animale, University of Lausanne) for helpful discussions.


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