Inhibition of Peroxisome Proliferator Signaling Pathways by Thyroid Hormone Receptor
COMPETITIVE BINDING TO THE RESPONSE ELEMENT*

(Received for publication, February 12, 1996, and in revised form, January 16, 1997)

Takahide Miyamoto §, Atsuko Kaneko , Tomoko Kakizawa , Hiroki Yajima , Keiju Kamijo , Rieko Sekine , Kunihide Hiramatsu , Yutaka Nishii , Takashi Hashimoto and Kiyoshi Hashizume

From the Department of Geriatrics, Endocrinology and Metabolism,  Department of Biochemistry, Shinshu University School of Medicine, Matsumoto 390, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Peroxisome proliferators (e.g. clofibric acid) and thyroid hormone play an important role in the metabolism of lipids. These effectors display their action through their own nuclear receptors, peroxisome proliferator-activated receptor (PPAR) and thyroid hormone receptor (TR). PPAR and TR are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related nuclear receptor superfamily. The present study focused on the convergence of the effectors on the peroxisome proliferator response element (PPRE). Transcriptional activation induced by PPAR through a PPRE was significantly suppressed by cotransfection of TR in transient transfection assays. The inhibition, however, was not affected by adding 3,5,3'-triiodo-L-thyronine (T3). Furthermore, the inhibition was not observed in cells cotransfected with retinoic acid receptor or vitamin D3 receptor. The inhibitory action by TR was lost by introducing a mutation in the DNA binding domain of TR, indicating that competition for DNA binding is involved in the molecular basis of this functional interaction. Gel shift assays revealed that TRs, expressed in insect cells, specifically bound to the 32P-labeled PPRE as heterodimers with the retinoid X receptor (RXR). Both PPAR and TR bind to PPRE, although only PPAR mediates transcriptional activation via PPRE. TR·RXR heterodimers are potential competitors with PPAR·RXR for binding to PPREs. It is concluded that PPAR-mediated gene expression is negatively controlled by TR at the level of PPAR binding to PPRE. We report here the novel action of thyroid hormone receptor in controlling gene expression through PPREs.


INTRODUCTION

Peroxisomes are cytoplasmic organelles that are important in mammalian lipid homeostasis (1). The structurally diverse xenobiotic peroxisome proliferators (PPs),1 such as clofibrate, nafenopin, and WY-14,643 stimulate the proliferation of peroxisomes (2-5) and cause tumorigenic transformation of hepatic cells in rodents (6, 7). Some of these compounds have been used in man as hypolipidemic agents. PPs have been shown to induce peroxisomal and microsomal enzymes involved in lipid metabolism through activation of the peroxisome proliferator-activated receptor (PPAR) (8, 9). The PPAR is a member of the nuclear receptor superfamily of ligand-dependent transcriptional factors and is structurally related to the subfamily of receptors that includes the thyroid hormone receptor (TR), retinoic acid receptor (RAR), and vitamin D3 receptor (VDR) (10). To date, three subtypes of PPARs have been identified in amphibians, rodents, and humans, PPARalpha , -beta , and -gamma (8, 9, 11-14). Further investigation revealed that natural fatty acids are also potent activators of PPARalpha (14, 15), although no direct interaction of PPARalpha with either PPs or fatty acids has been described so far. Recently, ligands for PPARgamma have been identified that are potent inducers of adipogenesis in vivo. These include thiazolidine diones, a class of anti-diabetic drugs, and the arachidonic acid derivative 15-deoxy-D12,14-prostaglandin J2 (16-18).

PPARs regulate gene expression by binding to DNA sequence elements termed PPAR response elements (PPRE). PPREs have recently been identified in the 5'-flanking sequences of peroxisome proliferator-inducible genes such as the rat acyl-CoA oxidase (aox) gene (19, 20), and the gene for cytochrome P450 CYP4A6 (21). The product of the former is the key enzyme in peroxisomal beta -oxidation and that of latter catalyzes omega  and omega -1 hydroxylation of fatty acids. PPREs are composed of two direct AGG(A/T)CA repeats separated by a single nucleotide (DR1), which is similar to previously described retinoid X response elements (22). These direct repeat motifs are also found in a number of other nuclear receptor response elements, e.g. the TRE, RARE, VDRE (22). Each receptor can recognize the same half-site motif. However, they discriminate between target elements through the spacing between the half-site motifs.

It is well established that heterodimerization with retinoid X receptor (RXR) strongly enhances binding of the TR, RAR, and VDR to their cognate response elements (23-26). Like other members of this subfamily, it has been demonstrated that PPAR binds to the PPRE by forming a heterodimer with RXR (27, 28). Therefore, it is possible that PPAR may exhibit promiscuous cross-talk with other members of the nuclear receptor family (29). Thyroid hormone is another effector that influences lipid metabolism including fatty acid beta -oxidation (30-33). TRs and PPAR appear to play an important role in lipid metabolism, and their signaling pathways might be coupled.

Based on the considerations described above, we focused on the convergence of TRalpha 1 and PPARalpha signaling pathways on PPREs. We have examined the suppressive effects of TRalpha 1 expression on PPARalpha -mediated transcriptional activation of peroxisome proliferator response genes. We demonstrated that TRalpha 1 negatively regulates PPARalpha action on PPREs through competition for DNA binding, and this negative regulation occurs in a ligand (T3) -independent manner.

In addition, we present evidence that PPRE (DR1) is a high affinity binding site for TRalpha 1 but not a functional response element for TRalpha 1. TRalpha 1 cannot activate PPRE in the presence of T3, despite its specific and high affinity binding to the element as a heterodimer with RXR. These results imply that binding to DNA is necessary but not sufficient for T3-dependent transcriptional regulation by TR.


MATERIALS AND METHODS

cDNA Isolation and Plasmids Constructions

Total RNA was extracted from rat liver using a guanidinium thiocyanate method. A cDNA pool was made by reverse transcriptase and (dT)17 primer. Reverse transcriptase-polymerase chain reaction technique was applied to amplify the rat PPARalpha cDNA using reported primers as follows: 5'-ATGGTGGACACAGAGAGCCCCATCTGTCCT-3' as sense primer and 5'-TCAGTACATGTCTCTGTAGATCTCTTGCAA-3' as antisense primer (14). The nucleotide sequence of the isolated rat PPARalpha cDNA was confirmed by sequencing (34). Fig. 1 illustrates the plasmid constructs used in this study. Full-length rat PPARalpha was inserted into the BamHI site of pCMV expression plasmid using BamHI linkers. Human TRalpha 1 and mutant TR were also expressed under the control of the CMV promoter (pCDM) (35, 36). The TRalpha 1 DNA binding domain mutant (DBD mutant) was made by introducing a cysteine to serine substitution at amino acid 73 using a site-directed mutagenesis system (pSELECT vector, Promega) as described previously (36). RARalpha and VDR cDNA are kind gifts from Dr. R. M. Evans (The Salk Institute, La Jolla, CA) and Dr. B. W. O'Malley (Baylor College, TX), respectively. The coding sequences of RARalpha or VDR was amplified by PCR and inserted into the BamHI site of pCMV expression vector. PPRE-TK-luciferase reporter plasmid harbors three copies of PPRE from the aox promoter in front of the TK promoter (27). The reporter employing the native promoter for aox (27) is kindly provided by Dr. R. M. Evans. The DR4-TK-luciferase reporter plasmid contains one copy of the DR4-TRE sequence, 5'-GGATCCAGGTCACAGGAGGTCAGGATCC-3'.


Fig. 1. Construction of expression vector and luciferase reporter used in this study. Full-length rPPARalpha was inserted into the BamHI site of pCMV expression plasmid using BamHI linker. TRalpha 1 and mutant TRs were also expressed under the control of CMV promoter (pCDM) (26). PPRE-TK-luciferase reporter plasmid harbors three copies of PPRE from the aox promoter in front of the TK promoter (19). DR4-TK-luciferase reporter contains one copy of DR4-TRE.
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Cell Culture and Transfection

COS1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transfected by the calcium phosphate precipitation technique as described previously (36). Transfections were performed in 24-well plates. In general, each dish received 250 ng of reporter construct, 0.8-50 ng of expression vector, 50 ng of beta -galactosidase expression vector (pCH110, Pharmacia Biotech Inc.) to monitor the efficiency of transfection and, if necessary, carrier DNA (pBluescript, Stratagene) to reach a total of 450 ng of DNA. Twenty hours after transfection, the medium was replaced by that containing T3 (10-7 M) or clofibric acids (10-3 M), and an additional 24 h later, cells were harvested and assayed for beta -galactosidase and luciferase activity (36, 37).

beta -Galactosidase and Luciferase Assays

beta -Galactosidase was measured by the method previously described (38). Luciferase assays were performed using the PicaGene Luciferase Assay System (Toyo Inki, Tokyo). Cells were harvested by adding 50 µl/well Cell Culture Lysis Reagent buffer. Samples were centrifuged (12,000 × g) at 4° C for 10 min, and the supernatant was retained for assay. Luciferase assays were performed by adding 30 µl of cell extract to 100 µl of Luciferase Assay Reagent. The reactions were performed at room temperature and assessed using Lumat LB9501 (Berthold Japan K.K., Tokyo, Japan) and expressed as relative light units. Luciferase activities were corrected for the beta -galactosidase activity present. Assays were conducted in triplicate, and data represent the mean ± S.E. of more than three individual experiments.

Gel Mobility Shift DNA Binding Assay

Gel mobility shift assays were carried out as described in several reports (38). In standard conditions, synthetic oligonucleotides representing each strand of sequences were purified by polyacrylamide gel electrophoresis, eluted, and annealed. Double-stranded oligonucleotides were radiolabeled with [32P]dCTP (>3300 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) by fill-in reactions using Klenow large fragment DNA polymerase. Labeled probes were separated from unincorporated nucleotides by centrifugation through a Sephadex G-25 column, which was equilibrated with 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, and 150 mM NaCl. Radiolabeled probes (10 fmol, 20,000-30,000 cpm) were then incubated with binding proteins in 30 µl of reaction mixture containing 10 mM KPO4 (pH 8.0) buffer, 1 mM EDTA, 80 mM KCl, 1 µg of poly(dI-dC), 1 mM dithiothreitol, 0.5 mM MgCl2, 5 µg of bovine serum albumin, and 10% glycerol. These reactions were incubated for 30 min at room temperature and analyzed on a 5% nondenaturing polyacrylamide gel in TAE buffer. Electrophoresis was performed at a constant voltage of 200 V at 4 °C in the same buffer. Gels were dried under vacuum and autoradiographed for 6-12 h at room temperature. Complexes were quantified by densitometric scanning of autoradiographs and by liquid scintillation counting of excised gel slices. Both methods gave essentially identical results. The sequences of the probes used in this study are listed as follows: PPRE 5'-gatccTGACCTTTGTCCTg-3' for sense strand; 5'-gatccAGGACAAAGGTCAg-3' for antisense strand; DR4 5'-gatccAGGTCACAGGAGGTCAg-3' for sense strand; 5'-gatccTGACCTCCTGTGACCTg-3' for antisense strand. The source of PPRE and DR4 are from aox gene and rat malic enzyme gene, respectively.


RESULTS

TRalpha 1 Inhibits the PPAR Action on PPRE but RAR or VDR Do Not

In a transient transfection system using COS1 cells, coexpression of TRalpha 1 suppressed the PPRE-TK luciferase activity induced by PPARalpha in the presence of clofibric acid, whereas equivalent amounts of the empty expression vector did not inhibit PPARalpha -mediated transcription (Fig. 2). In the absence of PPRE, TK-luciferase reporter was not affected by cotransfection of PPARalpha or TRalpha 1 in the presence of clofibric acid or T3, respectively (Fig. 2). Cotransfection of increasing amounts of TRalpha 1 expression vector showed that the inhibitory effect of TRalpha 1 on PPRE occurred at doses of TRalpha 1 similar to those necessary for activation of a T3 response element (DR4-TRE) (Fig. 3), suggesting a physiological role of TRalpha 1 in negative control of gene expression through PPRE. Then we tested the specificity of the inhibitory effect among other members of the nuclear receptor family. Expression plasmids for RARalpha or VDR were cotransfected with the expression vector for PPARalpha and PPRE-TK-luciferase reporter construct. As shown in Fig. 4, RARalpha or VDR did not inhibit the trans-activation of PPRE-TK-luciferase reporter by PPARalpha , whereas TRalpha 1 effectively blocked the transcriptional activation of the reporter.


Fig. 2. TRalpha 1 inhibits PPAR action on PPRE. COS1 cells were transfected with either TK-luciferase (TKluc) or PPRE-TK-luciferase reporter plasmid (250 ng). Twenty five ng of expression plasmid consisting of either the parental CDM expression vector or 12.5 ng of TRalpha 1 expression vector and 12.5 ng of the parental CDM vector or a combination of PPAR and TRalpha 1 expression vectors (12.5 ng each) was cotransfected. Cells were treated with either dimethyl sulfoxide as vehicle or 10-3 M clofibrate in the presence or absence of 10-7 M T3, as indicated. Cell extracts were assayed for luciferase activity. All luciferase activity was corrected for transfection efficiency by measuring beta -galactosidase activity. Normalized luciferase activity was expressed as fold induction relative to untreated cells. Assays were conducted in triplicate, and data represent the mean ± S.E. of five individual transfection experiments. Error bars are indicated.
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Fig. 3. Dose dependence of TR expression plasmid for PPAR inhibition and TRE activation. COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) and 12.5 ng of pCMV-PPAR expression vector (A) or DR4-TK-luciferase reporter plasmid (250 ng) (B). Indicated amounts of pCDM-TRalpha 1 expression plasmid were cotransfected. Cells were treated with vehicle or 10-3 M clofibric acid (A) or T3 (10-3 M) (B). Normalized luciferase activity was expressed as fold induction relative to untreated cells. Assays were conducted in triplicate, and data represent the mean of three individual experiments.
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Fig. 4. Effect of RAR and VDR on PPAR activity. COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) and 12.5 ng of pCMV-PPAR. Indicated parental expression vector or receptor expression vector (12.5 ng each) was cotransfected. Cells were treated with either dimethyl sulfoxide as vehicle or 10-3 M clofibrate in the presence or absence of 10-3 M of T3 for TR, or 10-3 M of all trans-retinoic acid (RA) for RAR, or 10-3 M of 1-25-OH vitamin D3 (VitD3) for VDR. Cell extracts were assayed for luciferase activity. All luciferase activities were corrected for transfection efficiency by measuring beta -galactosidase activities. Normalized luciferase activity was expressed as fold induction relative to untreated cells. Assays were conducted in triplicate, and data represent the mean ± S.E. of three individual experiments.
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Negative Effect of TRalpha 1 Was Not Reversed by Coexpression of RXRalpha

Chu et al. (39) reported a similar inhibition of TR on the PPAR-regulated peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA hydrogenase gene. They showed that inhibition of this gene by TR was through titration of limiting amounts of RXR. To test this possibility, RXRa expression plasmid was cotransfected into COS1 cells in which inhibitory activity of TRalpha 1 occurred. As shown in Fig. 4, the inhibitory effect of TRalpha 1 is not obliterated by cotransfection of expression plasmid for RXRalpha , suggesting that inhibition is not due to squelching of the cofactor, RXR. This in vivo study indicates that sequestering of RXR is not responsible for the inhibitory effect of TRalpha 1.

Introduction of a Mutation in the DNA Binding Domain of TRalpha 1 Eliminates the Inhibitory Effect of TR on PPAR

To define the mechanism for inhibitory effect of TRalpha 1 on PPARalpha action, we examined the contribution of the DNA binding domain of TRalpha 1 to this inhibition. A mutation introduced into the P box in the DNA binding domain (DBD) of the TRalpha 1 was designed to prevent its binding to DNA (TRalpha 1 DBD mutant) (36). In transient cotransfection assay, as shown in Fig. 5, the DBD mutant did not show an inhibitory effect on PPARalpha , indicating that competition for DNA binding is involved. These results indicate that TRalpha 1 regulates PPARalpha -mediated transcriptional activation of genes containing PPRE through competing binding to PPRE.


Fig. 5. Mutation in DNA binding domain of TRalpha 1 restored the inhibition of PPAR. COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) and 12.5 ng of pCMV-PPAR. Parental expression vector or receptor expression vector was cotransfected as indicated. Cells were treated with either dimethyl sulfoxide as vehicle or 10-3 M clofibrate in the presence or absence of 10-7 M T3. Cell extracts were assayed for luciferase activity. All luciferase activities were corrected for transfection efficiency by measuring beta -galactosidase activities. Normalized luciferase activity was expressed as fold induction relative to untreated cells. Assays were conducted in triplicate, and data represent the mean ± S.E. of four individual experiments.
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Expression of TRalpha 1 DBD Mutant Protein

Expression of wild type and DBD mutant proteins, as determined by Western blot, has been previously reported (36). Expression of the wild type and mutant proteins was examined by transfection of expression plasmids into COS1 cells. T3 binding capacity of protein expressed by pCDalpha 1 DBD mutant was, as expected, equivalent to wild type and was not influenced by cotransfection of PPAR expression vector (Table I). It is logical to consider that the lack of inhibition by the DBD mutant receptor is not due to the amounts of mutant protein present in COS1 cells in transient transfection assays.

Table I.

Protein expression of TRa1 and its DBD mutant in COS1 cells

TRalpha 1 or TRalpha 1 DBD mutant expression vector (2 µg) were transfected into COS1 cells in 10-cm plates with or without PPAR expression vector (5 µg) using the calcium phosphate precipitation method. Twelve hours after transfection, the medium was changed, and cells were incubated another 36 h. Cell extracts were prepared, and T3 binding assays were performed as described previously (61). T3 binding is shown as specific binding per total T3 added, corrected for beta -galactosidase activity.
Minus PPAR PPAR PPAR + clofibric acid (+)

TRalpha 1 31.4  ± 4.1 30.0  ± 3.4 33.2  ± 4.8
TRalpha 1(BDB)mut 32.5  ± 3.5 33.4  ± 4.6 34.2  ± 4.2

TR Modulates PP-dependent Transcriptional Activation by PPAR of the aox Gene

We tested whether TRalpha 1 can inhibit PPARalpha activity on a native promoter as well as an heterogeneous promoter (TK promoter). We confirmed transcriptional regulation by TR of the gene containing a PPRE, using a rat acyl-CoA oxidase (aox) gene promoter. aox-luciferase reporter plasmid was activated by PPARalpha in the presence of clofibric acid. This activation was completely suppressed by cotransfection of TRalpha 1 expression plasmid (Fig. 6). These results are similar to those observed when PPRE-TK luciferase was used.


Fig. 6. TR modulates PP-dependent transcriptional activation by PPAR in aox gene. COS1 cells were transfected with the aox promoter in a luciferase reporter plasmid. Twenty five ng of expression plasmid consisting of either the parental CDM expression vector or 12.5 ng of receptor and 12.5 ng of the parental CDM vector or a combination of PPAR and TRalpha 1 expression vector (12.5 ng each) was cotransfected. Cells were treated with either dimethyl sulfoxide as vehicle or 10-3 M clofibrate in the presence or absence of 10-7 M T3. Cell extracts were assayed for luciferase activity. All luciferase activities were corrected for transfection efficiency by measuring beta -galactosidase activities. Normalized luciferase activity was expressed as fold induction relative to untreated cells. Assays were conducted in triplicate, and data represent the mean ± S.E. of three individual experiments.
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Binding of TRalpha 1 to PPAR and TRE

To test the ability of TRalpha 1 to bind to PPRE in vitro, gel mobility shift assay was performed using TRalpha 1 and RXRalpha expressed in Sf9 insect cells. The results of these experiments employing TRalpha 1, RXRa, and 32P-labeled probes are shown in Fig. 7. TRalpha 1 plus RXRa produced a retarded heterodimeric band with PPRE, and increasing amounts of unlabeled probe displaced the binding, as observed when a classical TRE (DR4) was utilized as a probe. To achieve high concentrations of DNA, we diluted a fixed amount of the particular radioactive probe with increasing amounts of unlabeled DNA and approximately corrected the specific activity in calculations. Bound and free complexes were quantified by a densitometric analysis. The TRalpha 1·RXRalpha binding to DNA increased with increasing concentrations of DNA and approached saturation. Scatchard plots of data obtained using the PPRE and DR4 probes could be interpreted as a straight line and a single biomolecular reaction. The Ka was calculated from the slope of the Scatchard plots. The Ka values for binding of TRalpha 1·RXRalpha heterodimers to PPRE and TRE (DR4) are (2.2 × 109 M-1) and (2.5 × 109 M-1), respectively.


Fig. 7. TR homodimers and heterodimers with RXR can bind to PPRE(DR1). Ability of TR to bind DR4-TRE and DR1-PPRE from aox gene was tested. Sf9 cells extracts containing 50 fmol of TRalpha 1 were incubated with 20 fmol of 32P-labeled DR1-PPRE (A) or DR4-TRE (B). Increasing amounts (0, 200, 400, 1000-2000 fmol) of each unlabeled probe were coincubated. Reactions were incubated for 30 min at room temperature and analyzed on a 5% nondenaturing polyacrylamide gel in TAE buffer. Electrophoresis was performed at a constant voltage of 200 V at 4 °C in the same buffer. Gels were dried under vacuum and autoradiographed for 6-12 h at room temperature. The results shown are representative of three experiments.
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DISCUSSION

It is well documented that hypolipidemic drugs, such as clofibrate, induce peroxisome proliferation in rodent liver and increase the activity of enzymes involved in peroxisomal beta -oxidation of fatty acids (1-5). Regulation of the expression of genes involved in lipid metabolism by hypolipidemic drugs and hormones is of great physiological and clinical interest. In this paper we show that TRalpha 1 negatively regulates PPRE containing genes by competing for DNA binding with PPARalpha . TRalpha 1 inhibits the binding of PPARalpha to aox-PPRE, resulting in the suppression of peroxisome proliferator-dependent activation by PPARalpha . This is a novel mechanism of actions of TR, to regulate gene expression through the DR1 motif (PPRE).

To date, several enzymes, which are involved in peroxisomal beta -oxidation, have been shown to be regulated by PPAR through a PPRE in the promoter region. These include the peroxisomal fatty acid acyl-CoA oxidase (9, 19, 20), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme) (40, 41), the liver fatty acid binding protein (42), and the rabbit P450 4A6 fatty acid omega -hydroxylase (21). Our results strongly suggest that these enzymes might be modulated by TRs through convergence of PPAR signaling pathways.

The mechanism for negative regulation of PPARalpha -mediated transcription by TRalpha 1 was clarified in this paper. In general, three different mechanisms are proposed for inhibition of transcription factors: 1) competition for binding to a response element, 2) formation of inactive heterodimers, and 3) squelching a cofactor. It has been reported that PPARalpha is able to modulate TRbeta 1 activity by forming TRbeta 1·PPARalpha heterodimers (29) or by competing for heterodimerization with RXR (43). In this study, the importance of DNA binding activity of TRalpha 1 for negative regulation of PPARalpha was demonstrated using an artificial mutant receptor (TRalpha 1 DBD mutant). A mutation at a base coding for a cysteine residue in the P box (44) of the first zinc finger of the DNA binding domain in TRalpha 1 destroyed binding to DNA. This artificial DBD mutant TRalpha 1 reveals no inhibitory effect on PPARalpha action in transient transfection assay, whereas wild type TRalpha 1 acts as a strong suppressor of PPARalpha (Fig. 2). Previously, we confirmed by Western blotting that both wild type TRalpha 1 and TRalpha 1 DBD mutant are expressed at similar levels in COS1 cells when identical amounts of expression plasmids are transfected (36). It is important to know whether the TRalpha 1 DBD mutant is appropriately expressed in COS1 cells, since differences in suppression of PPAR could be due to expression of different amounts of wild type TRalpha 1 or TRalpha 1 DBD mutant in COS1 cells in the transient transfection assay. In this paper, we reconfirmed the similar expression levels of wild type and DBD mutant TRalpha 1 by T3 binding analysis (Table I). We have now shown that DNA binding is required for inhibition of PPARalpha activity by TRalpha 1.

A second possible inhibitory mechanism is formation of inactive TRalpha 1·PPARalpha heterodimers. Interestingly, weak suppression of PP-dependent transcription by PPARalpha was observed when an excess amount of TRalpha 1 DBD mutant was cotransfected (Fig. 5). The suppression was weak but statistically significant. We speculate that this inhibition by the DBD mutant TR could be mediated by formation of inactive heterodimers. The DBD mutant receptor could form TRalpha 1 DBD mutant-PPARalpha heterodimers and decreased the number of functional PPARalpha s. Indeed, Bogazzi et al. (29) reported that TRbeta 1 and PPARalpha form heterodimers in solution (29), resulting in the inhibition of transcriptional activation by TRbeta 1. TRalpha 1·PPARalpha heterodimers may be inactive in PP-dependent trans-activation on PPRE (DR1). In fact, introduction of a second mutation into the TRalpha 1 DBD mutant (TRalpha 1DBD+9th heptad mutant), adds an artificial mutation in the 9th heptad region of TRalpha 1, and this TR has no inhibitory effect on PPARalpha signaling even when present in excess amounts (data not shown). The 9th heptad region is thought to be a domain important for dimer formation with partner proteins. This formation of inactive heterodimers is possibly involved in inhibition of PPARalpha by TRalpha 1. This could explain the inhibition seen with excess amounts of the TRalpha 1 DBD mutant, which still retains the activity for dimerization, although the inhibition is weaker than by wild type TRalpha 1 (Fig. 5).

It must be noted that a much higher concentration of TR is required for inhibition by TRalpha 1 DBD mutant than by wild type TRalpha 1. These results indicate that a supraphysiological concentration of TR is required for inhibition of PPAR activity by forming inactive TR·PPAR heterodimers. Inhibition through DNA binding competition occurs at a lower concentration of TRalpha 1 than inhibition through formation of inactive TRalpha 1·PPAR heterodimers. Therefore, the mechanism of competition for DNA binding appears to be most important in the physiological situation.

A third possible mechanism is a squelching effect. Recently Chu et al. (39) reported inhibition by TR of the PPAR-regulated peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene. Their findings indicated that inhibition of this gene by TR was ligand-dependent and through titration of limiting amounts of RXR. This finding is in agreement with reports by Juge-Aubry et al. (43) and Hunter et al. (45). Their observations appear to conflict with our data. However, at least on a PPRE from the acyl-CoA oxidase gene, coexpression of RXRalpha cannot reverse the inhibitory effect of TRalpha 1 (Fig. 4), suggesting that inhibition by TRalpha 1 was not mediated through the sequestration of limiting amounts of RXR by TRalpha 1. Further evidence to support this notion is the absence of inhibitory effect by VDR and RAR. In cotransfection studies, neither VDR nor RAR influences PPAR activity, regardless of their capacity to heterodimerization with RXR. Differences between their data and ours may be due to use of PPREs from different genes. Our observations suggest that competition for DNA binding must be the main mechanism for inhibition of PPARalpha by TRalpha 1 on the PPRE from acyl-CoA oxidase gene.

To confirm that inhibition occurred under physiological conditions, it was important to titrate the dose of TR expression plasmid necessary for inhibition of PPARalpha . We compared the titration curve of TRalpha 1 expression plasmid which was necessary for inhibition of PPARalpha on PPRE with that necessary for T3-dependent trans-activation of TRE. Experiments with cotransfection of an increasing amount of TRalpha 1 expression vector showed that the inhibitory effect by TRalpha 1 on PPRE occurred with a dose of TRalpha 1 similar to that necessary for activation of the T3 response element. This suggests a physiological role for TRalpha 1 in controlling gene expression through the PPRE in vivo.

We confirmed the high affinity binding of TRalpha 1 to a PPRE in gel shift assays. Umesono et al. (46) have shown that VDR, TR, and RAR specifically activate transcription of genes containing AGGTCA direct repeats with spacings of 3, 4, and 5 base pairs, respectively. Thus, the ability of a receptor to recognize, discriminate, and bind to variants of the AGGTCA core sequence is critical to its function. Analysis of natural PPAR response elements has shown that PPARalpha can bind to core elements with a spacing 1 base pair (27). The results of DNA binding experiments clearly show that TRalpha 1·RXRa heterodimers can bind to PPRE(DR1) as well as to a classical TRE(DR4). TRalpha 1, however, cannot activate the PPRE in the presence of T3, even with high affinity binding as a heterodimer with RXRalpha .

The convergence of retinoid and PPAR signaling pathways has been analyzed by several groups (27, 28, 47, 48), and it has been shown that both PPARalpha and RXRalpha stimulate the acyl-CoA oxidase gene through PPARalpha ·RXRalpha heterodimers that bind PPREs. Therefore, most probably PPARalpha ·RXR heterodimers are competed on PPRE by TRalpha 1·RXR heterodimers, resulting in the suppression of PP-dependent transcriptional activation.

Several lines of evidence suggest direct competition of nuclear receptors for target DNA sequence. The estrogen receptor and thyroid hormone receptor each bind to a palindromic estrogen response element, although only the estrogen receptor mediates transcriptional activation via this sequence (49). We show in this paper that both the PPARalpha and TRalpha 1 molecule bind to PPRE (DR1), although only the PPARalpha mediates transcriptional activation through PPRE, and TRalpha 1 inhibits the PPARalpha activity on PPRE. These results suggest that DNA binding, even it is specific and with high affinity in vitro, is not enough for trans-activation. The DNA sequence of the regulatory element itself contains information regulating trans-activation by TRs.

Kurokawa et al. (50) demonstrated that RXR·RAR heterodimers bind to DR1 motifs but do not activate transcription, whereas they bind in the opposite polarity on natural RAREs containing DR5 motifs, where they are functional. Thus, RAR·RXR heterodimers also seem to be potential competitors with PPAR·RXR for binding to PPREs which are also DR1 motifs. However, our results demonstrated that RARalpha does not influence the PPARalpha mediated trans-activation of PPRE (Fig. 4). Furthermore, the PPRE luciferase reporter was not activated by cotransfection of RXRalpha in the presence of 9-cis-retinoic acid, whereas RXRE, which also consists of a DR1 motif, was activated by RXRalpha in the presence of 9-cis-retinoic acid (data not shown). Differences of the flanking sequence or the sequence between the hexamers might discriminate between PPRE and RXRE. Thus, we can suppose that RAR·RXR or RXR·RXR dimers are not able to bind to PPRE so efficiently as PPAR·RXR or TR·RXR heterodimers.

Results in this study strongly suggest that alteration of TR expression level influences the transcriptional activity of genes that are regulated by PPAR via PPREs. Several conditions that alter the TR expression are reported. For example, fasting decreased the maximal T3 binding capacity (51-53) and increased the fatty acid turnover. Our results possibly connect the relationship between fasting and activated fatty acid metabolism. PPAR activity may be released from suppression by TR due to decreased number of TR during fasting, resulting in the increased transcriptional levels of enzymes regulating fatty acid beta -oxidation such as aox gene. Furthermore, TR expression is regulated by hormones (54) and strictly controlled during ontogeny and development (55-58). TRs exert their effects on lipid metabolism through convergence of PPAR signaling pathways. Recently, PPAR has shown to be involved in the activation of the adipocyte-specific AP2 gene through PPRE (59, 60), and PPARgamma plays an important role in differentiation of adipocytes. It is possible that TR might regulate the differentiation of adipocytes through controlling PPAR-mediated transcription.

In conclusion, we demonstrate the remarkable potential of TRalpha 1 to compete with PPARalpha signaling pathway regulating lipid metabolism, cell growth, and differentiation. Nuclear receptors appeared to have a great diversity of actions and promiscuous interaction. We presented further evidence for cross-talk among nuclear receptor signaling pathways.


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.
   Contributed equally to this work and should be considered first authors.
§   To whom correspondence should be addressed: Dept. of Geriatrics, Endocrinology and Metabolism, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390 Japan. Tel.: 81-263-37-2686; Fax: 81-263-37-2710.
1   The abbreviations used are: PPs, peroxisome proliferators; PPAR, peroxisome proliferator-activated receptor; TR, thyroid hormone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; VDR, vitamin D3 receptor; AOX, acyl-CoA oxidase; PPRE, peroxisome response element; TRE, thyroid hormone response element; TK promoter, herpes simplex thymidine kinase promoter; T3, 3,5,3'-triiodo-L-thyronine; DBD, DNA binding domain; CMV, cytomegalovirus.

Acknowledgments

We thank Dr. R. M. Evans (The Salk Institute for Biological Studies, La Jolla, CA) for PPRE-TK-luciferase, aox-luciferase reporter plasmid, and the RARalpha cDNA. We also thank Dr. L. J. DeGroot (The University of Chicago) for the gift of the TRalpha 1 DBD mutant expression vector and critical reading of this manuscript and Dr. B. W. O'Malley (Baylor College, TX) for the VDR cDNA.


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