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Endocrinology Vol. 148, No. 3 1206-1217
Copyright © 2007 by The Endocrine Society

A Mutant Thyroid Hormone Receptor {alpha} Antagonizes Peroxisome Proliferator-Activated Receptor {alpha} Signaling in Vivo and Impairs Fatty Acid Oxidation

Yan-Yun Liu, Robert S. Heymann, Farhad Moatamed, James J. Schultz, Daniel Sobel1 and Gregory A. Brent

Molecular Endocrinology Laboratory (Y.-Y.L., R.S.H., J.J.S., G.A.B., D.S.), Department of Pathology (F.M.), Veterans Affairs Greater Los Angeles Healthcare System, Departments of Medicine and Physiology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90073

Address all correspondence and requests for reprints to: Gregory A. Brent, Molecular Endocrinology Laboratory, Building 114, Room 230, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: gbrent{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormone regulates the balance between lipolysis and lipogenesis. We previously reported that male mice with a dominant-negative P398H mutation introduced into the TR{alpha} gene have visceral obesity, hyperleptinemia, and reduced catecholamine-stimulated lipolysis in white adipose tissue. Based on our observation of hepatic steatosis in the TR{alpha} P398H male mice, we used in vitro and in vivo models to investigate the influence of the TR{alpha} P398H mutant on peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) signaling. Wild-type TR{alpha} and the P398H mutant significantly reduced PPAR{alpha}-mediated transcription in transient transfection assays. T3 reversed the inhibition of PPAR{alpha} action by wild-type TR{alpha} but not the P398H mutant. Chromatin immunoprecipitation assays demonstrated that the P398H mutant reduces PPAR{alpha} binding to peroxisome proliferator receptor elements. In gel shift assays, the P398H mutant directly bound the peroxisome proliferator-activated receptor response element and inhibited PPAR{alpha} binding, which was not reversed by addition of retinoid X receptor. The TR{alpha} R384C and PV dominant-negative mutants are not associated in vivo with a metabolic phenotype and had reduced (PV) or absent (R384C) PPAR{alpha} inhibition compared with P398H. The metabolic phenotype of the P398H mutant mice is due, in part, to unique properties of the P398H mutant receptor interfering with PPAR{alpha} signaling. The P398H mutant is a potential probe to characterize the physiological role of thyroid hormone receptor/PPAR{alpha} interactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE PLAYS a key role in metabolic homeostasis (1, 2, 3). Thyroid hormone regulates energy expenditure, in part, by transcriptional control of specific metabolic pathway genes (4, 5, 6, 7, 8). Thyroid hormone acts through its nuclear receptors, coded by two separate genes, TR{alpha} and TRß (9, 10). The studies from thyroid hormone receptor (TR) knockout (KO) mice indicate that TR{alpha} and TRß play specific developmental and physiological roles in target tissues (12, 13, 14). In brown adipose tissue, adaptive thermogenesis involves two T3-dependent pathways. Stimulation of uncoupling protein (UCP) 1 expression is mediated by TRß (2). Augmented adrenergic sensitivity in brown adipose tissue (15) and in white adipose tissue (16) is mediated by TR{alpha}. Male mice, with a dominant-negative P398H mutation introduced into the TR{alpha} gene, have marked visceral fat accumulation and reduced sensitivity to catecholamine-stimulated lipolysis (16).

T3 influences expression of genes involved in a wide range of metabolic pathways in the liver (17): gluconeogenesis (18), glucose utilization (19, 20), glucose transport and insulin signaling (21, 22, 23, 24), lipid transport (25, 26), lipogenesis and lipolysis (27), and cell proliferation (28, 29). A well-characterized T3 target gene in liver is glycerol-3 phosphate dehydrogenase (G3PD), which converts glycerol into intermediate products in the glycolytic process. In clinical studies and animal models, hypothyroidism is associated with reduced G3PD mRNA expression and enzymatic activity (30).

TR isoform-specific regulation of hepatic gene expression was studied in TRß KO mice and TR{alpha} knockout mice treated with or without thyroid hormone and analyzed by DNA microarray analysis (31). Expression of more than 200 hepatic genes responded to T3 treatment, approximately 60% mediated by TRß and regulation of the remaining 40% mediated by TR{alpha} (31). In another analysis of liver gene expression, however, using 48 cDNA microarrays, the gene expression patterns were similar in TRß KO mice and TR{alpha} KO mice (32). Thyroid status influences expression of a number of genes involved in lipid and glucose metabolism, by direct and indirect thyroid hormone regulation. Examples of regulated genes include spot 14 (33), peroxisome proliferator-activated receptor (PPAR) (34), PPAR{gamma} coactivator-1 (PGC-1) (35), glucose transporter (Glut) (22, 23), and IGF-binding protein 2 (IGFB2) (36, 37).

Peroxisome proliferator-activated receptor (PPAR{alpha}) is a key regulator of lipid metabolism influencing expression of lipid metabolic enzymes, lipid transporters, and apolipoproteins (38, 39). PPAR{alpha} is highly expressed in metabolically active tissues including liver, heart, kidney, skeletal muscle, and brown fat (40, 41). PPAR{alpha} is activated by natural fatty acids as well as synthetic ligands, such as fibrates, and mediates the genes regulating fatty acid uptake and oxidative catabolism. PPAR{alpha}-null mice have hepatic steatosis, elevated serum cholesterol and triglycerides levels, and late-onset obesity (42). They have reduced capacity to metabolize fatty acids, depletion of glycogen, and reduced cellular uptake of lipids (43, 44).

TR and PPAR share a number of structural similarities. The P box, located in the first zinc finger of the DNA-binding domain, is fully conserved between PPAR{alpha} and TR{alpha}/TRß (34). Additionally, TR and PPAR share the heterodimerization partner, retinoid X receptor (RXR), required for optimal DNA binding and activation (45, 46). TR and PPAR{alpha} are coexpressed in many tissues, and both regulate a number of genes relevant to metabolism including malic enzyme (47), bifunctional enzymes (48, 49), uncoupling protein (UCP) 3 (50, 51), and carnitine palmitoyltransferase (CPT)-I{alpha} (52, 53). CPT-I{alpha} catalyzes the initial reaction in mitochondrial import of long-chain fatty acids for oxidation. PPAR{alpha} competes with TR for RXR, and potentially other auxiliary proteins shared by TR, resulting in an inhibition of TR action (34). A recent study reported that the TRß PV mutant receptor heterodimerizes with PPAR{gamma} in vitro and inhibits PPAR{gamma} binding to peroxisome proliferator-activated receptor response element (PPRE), independent of RXR and thyroid hormone (54). This finding was linked to the etiology of thyroid tumors observed in the TRß PV mutant mice (55, 56). The interaction between TR and PPAR may play an important role in the regulation of lipid metabolism.

Previously, we reported on the phenotype of P398H mice, generated by introducing a mutation derived from human TRß (P449H) mutation into TR{alpha}. Patients with the TRß P449H mutation manifest resistance to thyroid hormone (RTH) with elevated thyroid hormone and TSH levels in the serum and hypothyroidism at the tissue level. The P398H mice had slightly elevated T4, a 14% increase in T3, and greater than 3-fold increase in TSH in the serum (16), which resembles the RTH phenotype. We have previously shown that the P398H male mice had visceral obesity, hyperleptinemia, and marked reduction in norepinephrine-stimulated lipolysis (16). An in vitro assay showed that T3 binding capacity of TR{alpha} P398H was 50% of wild-type (wt) TR{alpha} (57).

In this study, we have used the TR{alpha} P398H mutant mouse model to demonstrate an interaction between TR{alpha} and PPAR{alpha} that influences hepatic lipid metabolism. Male mice with the TR{alpha} P398H mutation exhibited hepatic steatosis and glycogen depletion in the liver, similar to the phenotype described in PPAR{alpha}-null mice. Expression of mRNA from PPAR{alpha}-stimulated genes involved in fatty acid oxidation was significantly reduced in the TR{alpha} P398H mouse liver. We demonstrate that the abnormal hepatic lipid accumulation in TR{alpha} P398H mice is associated with a direct action of the P398H mutant receptor interfering with PPAR{alpha} binding to PPREs and reduced PPAR{alpha}-mediated gene transcription. We compared the influences of the TR{alpha} mutant receptors (P398H, R384C, and PV) on PPAR{alpha} signaling. The R384C and PV mutations were derived from corresponding human TRß mutations associated with RTH. TR{alpha} R384C retains weak T3 binding (58), and TR{alpha} PV does not bind T3 (59). Both R384C and PV mutant mice are associated in vivo with evidence of RTH but no metabolic phenotype. We demonstrate that these mutations had reduced interference with PPAR{alpha} DNA binding and signaling compared with the P398H mutant. The phenotypic differences among the TR{alpha} mutations indicate that specific domains of TR{alpha} mediate interactions with PPAR{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diet
The wt and TR{alpha} P398H mutant mice on a C57BL background were bred in normal conditions with a 12-h light, 12-h dark cycle. The mice were supplied with standard chow diet (Formula 5020 9F or 5008; LabDiet LLC, Framingham, MA). All studies were approved by the Animal Research Committee, Veterans Affairs Greater Los Angeles Healthcare System.

Liver histology
Livers were removed from wt and P398H mutant mice (at 4 months) immediately after being euthanized. The liver fragments were immediately fixed in Bouin’s solution at 4 C for 24 h and then transferred to 75% ethanol and stored at 4 C before paraffin embedding. The liver sections were stained with hematoxylin and eosin by standard procedures. The content of glycogen in the tissue specimens was determined by periodic acid-Schiff staining (PAS).

Western blots
Nuclear extracts were prepared from the liver of wt mice and P398H mice using CelLytic Nuclear Extraction kit (Sigma-Aldrich, Inc., St. Louis, MO). Nuclear proteins (15 µg/lane) were resolved in 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane. The membrane was probed with mouse monoclonal PPAR{alpha} antibody (Affinity BioReagents, Inc., Golden, CO) at 1:1500 dilution. The antigen-antibody complexes were detected by Supersignal chemiluminescence (Pierce Inc., Rockford, IL) using rabbit-antimouse Ig antibody conjugated to horseradish peroxidase (Pierce).

RNA isolation and quantitative real-time PCR
Total RNA was prepared from liver tissue using Trizol reagent (Invitrogen Inc., Carlsbard, CA). Total RNA was treated with DNaseI and purified by RNeasy kit (QIAGEN, Stanford, CA). The RNA (5 µg) was reverse transcribed using Superscript II reverse transcriptase (Invitrogen). The quantitative real-time PCR was performed as described previously (16). In brief, the test cDNA was diluted (1:5) before real-time PCR. For the standard curve of each primer set, cDNA was diluted at various ratios: 1:5, 1:25, 1:125, and 1:625. The ß-actin or glyceraldehyde-3-phosphate dehydrogenase control was diluted at ratios 1:50, 1:250, and 1:1250 and used for normalization of mRNA expression levels. The samples were performed in duplicates and triplicates for glyceraldehyde-3-phosphate dehydrogenase and ß-actin. The PCR primers are listed in Table 1Go.


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  		TABLE 1. Primer sequences and GenBank accession numbers

 
Free fatty acid (FFA) assay
Nonesterfied (or free) fatty acids in the serum were determined using an in vitro enzymatic colorimetric method and following the instruction provided in the FFA assay kit (Wako Chemicals USA Inc., Richmond, VA).

Plasmids
Reporter construct CPT-I{alpha} –4950/+1240-Luc spanning thyroid hormone response element (TRE), PPRE, and the first intron was a gift from Dr. Edward A. Park (University of Tennessee, School of Medicine). The CPT PPRE-luc reporter was constructed by site-directed mutagenesis of CPT-I{alpha} –4950/+1240-Luc TRE. The acyl-CoA-oxidase (ACO) PPRE-Luc reporter was constructed by blunt-end ligation of the double-stranded oligonucleotides (34 bp) containing ACO PPRE upstream of the SV40 promoter in pGL-3 promoter vector. The mouse PPAR{alpha} expression vector was a gift from Dr. Ronald M. Evans (Salk Institute of Biological Studies, La Jolla, CA). The TR{alpha} PV expression vector was a gift from Dr. Sheue-yann Cheng (Laboratory of Molecular Biology, Center for Cancer Research, National Institutes of Health). The expression vectors of the mouse TR{alpha} mutants P398H and R384C were constructed using site-directed mutagenesis of the TR{alpha} cDNA and sequenced to confirm correct nucleotide changes.

Cell culture and transfections
HepG2 cells were routinely cultured in MEM with 10% FBS. The HepG2 cells were plated in 12-well dishes and grown in serum-free MEM with 10% serum replacement solution (Invitrogen) for 24 h before transfection. Cells were transfected with reporter constructs and receptor expression vectors in combinations as described. T3 and PPAR{alpha} ligand (WY14346) were added in concentrations as shown. After transfection, the HepG2 cells were grown in serum-free medium with 10% serum replacement solution. Luciferase activity was determined 36 h after transfection using a luminometer. Plasmid expression control was performed by isolating RNA from transfected HepG2 cells 36 h after transfection and reverse transcribed using Superscript III reverse transcriptase. cDNA was diluted 5-fold, and 1 µl cDNA was used for testing samples and 0.5 µl cDNA for determining ß-actin by PCR (30 cycles testing samples and 22 cycles for ß-actin).

Chromatin immunoprecipitation (ChIP) assays
Liver tissue from male mice (n = 3, at 4 months) was freshly frozen in dry ice. Approximately 500 µg of frozen liver tissue was thinly sectioned. The frozen sections were immediately immersed in 1% formaldehyde made in PBS for 5 min, ground with five strokes in a homogenizer to loosen the tissue structure, left for an additional 10 min in 1% formaldehyde, and then centrifuged to remove the formaldehyde. The tissue pellet was washed twice with ice-cold PBS containing protease inhibitor cocktail (Complete Mini protease inhibitor cocktail; Roche Applied Science, Indianapolis, IN), resuspended in 3 fractions with 200 µl of ChIP lysis buffer containing protease inhibitors before sonication. The time of sonication for 200 µl of the tissue suspension was 8 x 5 sec using a 2-mm (diameter) probe with 20% power output. The rest of the process was carried out following the instructions of the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) with the exception of using protein G-agarose. Anti-TR{alpha} (5 µg) or anti-PPAR{alpha} (5 µg) (Affinity Bioreagents) antibodies were used in 200 µl of an immunoprecipitation reaction. After completion of immunoprecipitation, the chromatin/DNA complex was eluted with 2 x 0.25 ml of freshly made elution buffer (1% SDS, 0.1 M NaHCO3). Cross-linked chromatin/DNA complexes were reversed at 65 C for 4 h. DNA was recovered by phenol/chloroform extraction and ethanol precipitation, cleaned, and concentrated by MiniElute column (QIAGEN). PCR was carried out with approximately 1.5 ng input DNA or eluted (bound) DNA in each reaction. The primer sequences used in ChIP assays are shown in Table 1Go.

Gel-shift assays
Gel-shift assay techniques were as previously described (60). Briefly, wtTR{alpha}, TR{alpha} P398H, TR{alpha} R384C, TR{alpha} PV, and PPAR{alpha} proteins were synthesized by in vitro transcription/translation (TNT kit; Promega Inc., Madison, WI). Purified RXR protein was purchased from Affinity Bioreagents. For the protein-DNA binding assay, 4–5 µl of in vitro translated protein (depending on translation efficiency) was incubated with the 32P-labeled oligonucleotide-PPRE or -TRE (5,000 cpm) in a 20-µl reaction mixture containing DNA-binding buffer (60) at room temperature for 15 min. For band identification, after 15 min initial incubation, 1 µl of mouse antiserum IgG raised against mouse TR{alpha} (0.1 µg/µl) or mouse PPAR{alpha} (0.2 µg/µl) (Affinities Bioreagents) was added and incubated for an additional 10 min. Bound probe was separated from the free in 5% nondenaturing polyacrylamide gel and electrophoresed in 1x Tris-glycine buffer at constant 130 voltage for 3.5 h. The oligonucleotide sequences used in gel-shift assays are shown in Table 1Go. For translation control, protein was in vitro synthesized with [35S]methionine labeling. The synthesized products (2 µl each) were separated by 10% SDS-PAGE and shown as an autoradiograph.

Data analysis
All data are expressed as mean ± SE. Statistical analysis used ANOVA for multigroup comparison and Student’s t test for pairs with significance at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hepatic steatosis in TR{alpha} P398H mice
Males were randomly selected from different litters of the TR{alpha} P398H mice (16). The control mice (wt) were littermates of the P398H mice. The mice were fed with a standard chow diet suitable for reproduction (17.6–20.6% calories from fat). At 3–5 months of age, the liver in the TR{alpha} P398H mice was noted to be enlarged and beige in color, compared with wt mice, consistent with fat accumulation in the liver. Histological analysis of the liver sections from 3- to 5-month-old mice revealed accumulation of a large amount of fat droplets in the TR{alpha} P398H mice (Fig. 1AGo). Staining the liver sections with PAS showed glycogen depletion in the TR{alpha} P398H mice compared with wt mice (Fig. 1BGo). The fasting FFA level in the serum of the P398H mice (0.73 ± 0.15 mM) was not significantly different from wt mice (0.63 ± 0.22 mM) although lower than expected for the marked increase in visceral fat. These findings indicate that fat was being stored in the liver rather than mobilized for oxidation to produce fuel. The glycogen depletion in the P398H mice is consistent with use of glycogen for fuel production in the liver.


Figure 1
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  		FIG. 1. Histology of liver samples from male wt and TR{alpha} P398H mutant (P398H) mice. Liver tissue was obtained from mice at 3 months of age, freshly fixed, and paraffin embedded. A, Hematoxylin and eosin stain shows normal liver architecture in wt sample and extensive deposition of lipid droplets in the sample from the P398H mouse; B, PAS stain shows the presence of glycogen stores (purple) in the liver of the wt, but not P398H, mice.

 
Reduced mRNA expression of the genes involved in glycogen metabolism and fatty acid oxidation
We analyzed the mRNA expression of key enzymes involved in fatty acid oxidation in the liver. There was a significant reduction in mRNA levels for these enzymes in TR{alpha} P398H compared with wt mice, including CPT-I{alpha} reduced 85% (P < 0.001); cytochrome P450 family 4 subfamily A polypeptide 10 (CYP4A10), a PPAR{alpha} marker gene, reduced 52% (P < 0.05); thiolase reduced 56% (P < 0.01); and acyl-CoA oxidase (ACO) reduced 68% (P < 0.05). The mRNA level of acyl-CoA dehydrogenase (ACD) was reduced 23% in P398H mice but did not reach statistical significance (P < 0.15) (Fig. 2AGo).


Figure 2
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  		FIG. 2. Liver gene mRNA expression profiles and PPAR{alpha} protein levels in wt and TR{alpha} P398H mutant mice (age 3 months, n = 4). A, Fatty acid oxidation enzymes CPT-I{alpha}, ACO, CYP4A10, thiolase, and ACD; B, glycogen synthesis enzymes GS, GSK, GP, PDK2,and PDK4; C, Glut-1, -2, -4, and -10; D, PPAR{alpha} mRNA levels. Total RNA was isolated from liver of wt and P398H mice at 3 months of age (n = 4) and reverse transcribed. The quantitative real-time PCR was performed as described previously (14 ). The data were normalized for ß-actin levels. PCR results are shown as the mean value ± SE of triplicates. *, P < 0.05 compared with wt mice. E, PPAR{alpha} protein levels in wt mice and the P398H mice are shown by Western blotting. Nuclear extracts were prepared from liver (n = 3) and loaded (15 µg) in each lane. Monoclonal anti-PPAR{alpha} antibody at 1:1500 dilution was used to probe PPAR{alpha} (see Materials and Methods for details). Solid arrow indicates anti-PPAR{alpha}-probed PPAR{alpha} protein. Open arrow indicates protein loading.

 
Glycogen metabolism is allosterically controlled by glycogen synthase (GS) and glycogen phosphorylase (GP). GS activity is inhibited by GS kinase (GSK). The GP is activated by GP kinase (GPK). In the P398H mice, the mRNA level of GS was not affected, but GSK was reduced 64% (P < 0.05) (Fig. 2BGo) and GP was increased 3.6-fold (P < 0.05) (Fig. 2BGo). The liver contains two pyruvate dehydrogenase kinases (PDKs), PDK2 and PDK4, which mediate glycolysis through inhibition of pyruvate dehydrogenase complex phosphorylation. Both PDK2 and PDK4 expression are affected by thyroid hormone and PPAR{alpha}. The dominant effect on PDK2 protein level is thyroid hormone (61). Hepatic PDK4 protein is stimulated in hyperthyroid rats but not in euthyroid rats (61). PPAR{alpha} activation stimulates hepatic PKD4 and activates glycogen synthesis, which inhibits fatty acid synthesis in the liver. PDK2 mRNA levels were reduced 60% (P < 0.05), and PDK4 mRNA was reduced 32% (P < 0.056) in the P398H mice compared with the wt mice (Fig. 2BGo). The pattern of enzyme expression indicates that glycogen was used from liver at a higher rate than its synthesis, consistent with the changes in liver histology.

Intracellular glucose mobilization requires the action of glucose transporters (Glut). In the P398H mice, the mRNA levels of Glut-1, -2, and -10 in the liver were similar to the wt mice (Fig. 2CGo). Glut-4 mRNA was reduced but not statistically different from the levels in the wt mice.

We investigated the possibility that reduced PPAR{alpha}-mediated gene expression was due to reduced PPAR{alpha} protein. PPAR{alpha} mRNA levels in the liver were significantly reduced in the P398H compared with wt mice at 3 months (n = 3) (Fig. 2DGo). The level of PPAR{alpha} protein in the liver was reduced 21 ± 0.8% (P < 0.065) in P398H mice (n = 3) compared with that in the wt mice (n = 3) at 3 months (Fig. 2EGo). Although the reduction is not statistically significant, reduced PPAR{alpha} protein may also contribute to reduced ß-oxidation in the mutant mice. A significant reduction in hepatic PPAR{alpha} protein was seen in P398H compared with wt mice at 12 months of age (data not shown).

Inhibition of PPAR{alpha}-mediated CPT-I{alpha} induction by TR{alpha}
CPT-I{alpha} is a rate-controlling enzyme of ß-fatty acid oxidation regulating the import of long-chain fatty acids into mitochondria. CPT-I{alpha} activity determines cellular fatty acid oxidative flux (53). A well-characterized PPRE is located in the CPT-I{alpha} 5'-flanking region and requires the first intron for maximal induction (52, 53). A functional TRE, located 55 nucleotides upstream from the PPRE, was mutated in CPT-Luc (–4950/+1240) to eliminate TR binding at this site (Fig. 3AGo) and created CPT PPRE-Luc (contains the PPRE and first intron). We examined the effect of TR{alpha} on PPAR{alpha}-mediated transcription in transient transfection assays. PPAR{alpha} with added PPAR{alpha} agonist (WY14,346), stimulated luciferase activity 5.6-fold (Fig. 3BGo). Cotransfection with PPAR{alpha}/wtTR{alpha} reduced stimulation to 2.3-fold. Cotransfection with PPAR{alpha}/TR{alpha} P398H completely blocked PPAR{alpha} induction. Addition of T3 (50 nM) reversed inhibition by wtTR{alpha} but not the inhibition by the TR{alpha} P398H mutant.


Figure 3
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  		FIG. 3. The influence of TR{alpha} on PPAR{alpha}-mediated stimulation of CPT-I{alpha} PPRE-Luc in HepG2 cells. CPT PPRE-Luc generated by removal of the TRE from original CPT-Luc was tested for TR{alpha}/T3 stimulation of transcription activity (A) and for the effects of TR{alpha} on PPAR{alpha}-stimulated luciferase activity with (B) or without (C) cotransfection of RXR. The CPT PPRE-Luc reporter construct was transfected with PPAR{alpha} in HepG2 cells with or without PPAR{alpha} ligand (WY14634) or cotransfected with wtTR{alpha}/PPAR{alpha} or P398H /PPAR{alpha}. In each transfection, 0.1 µg reporter and each receptor were used. RXR expression vector was cotransfected into cells at various concentrations (0.1, 0.5, and 1.0 µg). Data are shown as mean value ± SE of triplicates. *, P < 0.05 compared with PPAR{alpha}/WY; **, P < 0.01 compared with PPAR{alpha}/WY.

 
TR{alpha} and PPAR{alpha} both heterodimerize with RXR for optimal DNA binding and activation. We determined whether the inhibition of PPAR{alpha} signaling by TR was due to competition for limited RXR. Cotransfection of RXR (0.1–1 µg) with PPAR{alpha} did not significantly increase luciferase expression (P < 0.067) and did not restore PPAR{alpha}-mediated induction when wtTR{alpha} or TR{alpha} P398H were present. Addition of T3 (50 nM) eliminated inhibition by wtTR{alpha} (Fig. 3CGo). In the presence of TR{alpha} P398H, however, addition of T3, RXR, or both combined could not restore PPAR{alpha}-mediated activation. The data demonstrate that P398H inhibition of PPAR{alpha}-mediated transcription is not due to limiting RXR and is more likely the result of direct inhibition.

Influence of wtTR{alpha} and TR{alpha} P398H on PPAR{alpha} binding to PPRE
To determine whether TR{alpha} P398H interacts directly with the CPT-I{alpha} PPRE and inhibits PPAR{alpha} binding to DNA, we performed ChIP assays using liver tissue from wt mice (n = 3) and TR{alpha} P398H mice (n = 3). An 85-bp PCR-amplified band representing PPAR{alpha} bound to the PPRE was observed as 20.3 ± 1.2% of input in wt mice but only 2.8 ± 1.1% in the P398H mice (Fig. 4Go, A and D), indicating minimal PPAR{alpha} binding to the CPT PPRE. The DNA samples isolated by ChIP with anti-TR{alpha} antibody were PCR amplified. The 85-bp band representing TR-bound PPRE was detected in both wt and P398H mice (Fig. 4BGo). The intensity of the 85-bp band, after subtraction from the negative control, was 4 ± 0.18% input in wt mice but was present as 13.3 ± 0.6% in the TR{alpha} P398H mice (Fig. 4Go, B and D). The P398H mutant receptor bound to the CPT-PPRE 3.3-fold greater than wtTR{alpha}.


Figure 4
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  		FIG. 4. ChIP analysis of PPAR{alpha} binding to the CPT-I{alpha} PPRE. Liver tissue from 4-month-old wt (n = 3) and P398H mutant mice (n = 3) was thinly sectioned and cross-linked. The immunoprecipitation of cross-linked chromatin was performed with anti-PPAR{alpha} or anti-TR{alpha} antibodies. Immunoprecipitation-bound fractions were reversed, purified, and PCR amplified with primer sets (Table 1Go) for the CPT PPRE (A and B) and TFE PPRE and ACO PPRE (C). Quantitation (D) was performed with a densitometer (Alpha Imager) and the associated imaging software (Alpha Innotech, San Francisco, CA). After subtraction of the negative control (IgG only), the intensity of receptor-bound DNA was expressed as percent input.

 
To confirm our findings in other PPAR{alpha}-regulated genes, we performed liver ChIP assays using PPRE sequences from the ACO and peroxisomal enoyl-CoA hydrotase/3-hydroxyacyl-CoA dehydrogenase (triple functional enzyme or TFE) genes, important in ß-fatty acid oxidation. Immunoprecipitated DNA was PCR amplified using primer sets to detect the TFE PPRE (65 bp) and ACO PPRE (82 bp). PPAR{alpha} bound to the TFE PPRE was detected as 7.3 ± 0.88% input in wt mice and 1.36 ± 0.39% in P398H mice (Fig. 4Go, C and D). PPAR{alpha} bound to the ACO PPRE as 13.5 ± 1.56% input in wt mice and 4.1 ± 0.29% in P398H mice (Fig. 4Go, C and D). In both the ACO and TFE PPREs, the reduction of TR{alpha} P398H-bound PPRE was 5.4-fold (TFE PPRE) and 3.3-fold (ACO PPRE) compared with wt TR{alpha} in liver. These results demonstrate that TR{alpha} P398H interferes with PPAR{alpha} signaling by binding to the PPRE. PPAR{alpha} binding in liver, as determined by ChIP assay, differs for various PPREs, possibly because of the differences in the PPRE sequence and promoter context.

We used gel-shift assays to better understand the influence of TR{alpha} on PPAR{alpha} binding to a PPRE. In the absence of RXR, TR{alpha} P398H-bound CPT-I{alpha} PPRE was detected clearly and trace amounts of PPAR{alpha}/DNA or wtTR{alpha}/DNA were also detected (Fig. 5Go, lanes 1–4). In the presence of RXR, PPAR{alpha} and P398H binding to CPT-I{alpha} PPRE was significantly enhanced (lanes 5 and 7). The wtTR{alpha}/RXR had little binding to DNA (compare lanes 3 and 6) and had little (if any) influence on PPAR{alpha}/RXR (compare lane 8 with added TR{alpha} in lane 9). PPAR{alpha} binding was enhanced with addition of P398H/RXR (lane 10). The PPAR{alpha}/RXR and TR{alpha} (or P398H)/RXR complexes migrated to the same position under the gel-shift conditions. We used antibodies to specifically inhibit binding of the receptor-containing complexes. Reduced intensity of binding, as seen with the PPAR{alpha}/RXR/DNA complex (lane 11), indicate that the band contained PPAR{alpha}. The wtTR{alpha} did not have significant influence on PPAR{alpha} binding because most of the complex was diminished with addition of PPAR{alpha} antibody (lane 12). In the presence of TR{alpha} P398H mutant receptor, however, anti-PPAR{alpha} antibody only slightly reduced the intensity of the DNA/receptor complex (lane 13) compared with the marked reduction and supershifted band with TR antibody (lane 15). The wt TR was only a small component of the complex (lane 14). This indicates that TR{alpha} P398H mutant, in the presence of PPAR{alpha}/RXR binds to the PPRE to a greater extent than wtTR{alpha}.


Figure 5
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  		FIG. 5. Gel shift analysis of TR{alpha} and TR{alpha} P398H mutant binding to the CPT I{alpha} PPRE. PPAR{alpha}, TR{alpha} and the TR{alpha} P398H mutant proteins were synthesized using in vitro transcription-coupled translation kit. A, Receptors were incubated with the 32P-CPT PPRE probe for 15 min either in the presence or absence of 0.2 µg RXR (Affinity Bioreagents Inc). Antibodies (1 µg) were added to the reaction and incubated for an additional 10 min. The DNA-protein complexes were resolved on a nondenaturing 5% polyacrylamide gel. The solid arrow indicates protein/DNA complexes and the open arrow indicates nonspecific binding from the lysate seen in all lanes. The supershifted band after addition of TR{alpha} antibody in lanes 14 and 15 is shown by the solid arrowhead. The efficiency of in vitro synthesized wtTR{alpha} and P398H proteins is shown in B. Proteins were synthesized with 35S-methionine, resolved on 10% SDS-PAGE, and scanned using phosphor-imager.

 
Increasing concentrations of RXR (0.15–0.3 µg per reaction) in gel-shift assays did not have a noticeable effect on the intensity of TR{alpha} P398H binding to PPRE in the presence of PPAR{alpha} (Fig. 6Go, lanes 7 and 10). In the presence of 0.3 µg RXR, PPRE-bound PPAR{alpha} was not further increased (Fig. 6Go, lanes 8 and 9) compared with 0.15 µg RXR, indicating that RXR was not a limiting factor for binding. Addition of anti-PPAR{alpha} antibody disrupted binding of PPAR{alpha} to DNA in the presence of wtTR{alpha} (lane 12). However, only a slightly reduced intensity of binding was observed when P398H was present (lane 13), indicating that the receptor/DNA complex contains only a small amount of PPAR{alpha}. These data indicate that TR{alpha} P398H binding to PPRE was not dependent on RXR. This finding is in agreement with studies of TRß PV interaction with PPAR{gamma} on the ACO PPRE, which showed that it was independent of RXR and T3 (54).


Figure 6
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  		FIG. 6. Gel-shift analysis of TR{alpha} and TR{alpha} P398H mutant binding to the CPT-I{alpha} PPRE with increasing RXR. Gel-shift conditions were the same as described in Fig. 5Go except the addition of RXR at two concentrations (0.15 and 0.3 µg). The solid arrow indicates protein/DNA complexes, and the open arrow indicates nonspecific binding from the lysate seen in all lanes. The supershifted band after addition of TR{alpha} antibody in lanes 14 and 15 is shown by the solid arrowhead.

 
Influence of PPAR{alpha} on TR{alpha} binding to a TRE
We have demonstrated that TR{alpha} influences PPAR{alpha} binding to a PPRE. To determine whether PPAR{alpha} would affect TR{alpha} binding to a TRE on the CPT-I{alpha} promoter, we performed a gel-shift assay using 32P-labeled CPT-I{alpha} TRE. PPAR{alpha} alone and PPAR{alpha}/RXR did not bind to the CPT-I{alpha} TRE (Fig. 7Go, lanes 5 and 6). In the presence of PPAR{alpha}, however, the intensity of the wtTR{alpha}/RXR complex was significantly reduced (lanes 3 and 7). This suggests that PPAR{alpha} can compete with TR{alpha}, for RXR. In the same conditions, PPAR{alpha} did not reduce the intensity of TR{alpha} P398H/RXR (lanes 4 and 8), suggesting that PPAR{alpha} was not able to compete with the TR{alpha} P398H mutant for RXR. This finding suggests that RXR is less accessible when it is part of a heterodimer with the P398H mutant receptor.


Figure 7
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  		FIG. 7. Binding of receptors to the CPT-I{alpha} TRE in a gel-shift assay. The DNA-binding reaction contained equal amounts of each in vitro synthesized PPAR{alpha}, TR{alpha} and the P398H mutant, 0.4 µg RXR protein, and labeled CPT-I{alpha} TRE probe. The reaction was incubated at room temperature for 15 min. The protein/DNA complexes were resolved in 5% PAGE. Arrows indicate nonspecific binding (a) and binding to the CPT TRE (b).

 
Differential influence of TR{alpha} mutants (TR{alpha} P398H, TR{alpha} R384C, and TR{alpha} PV) on PPAR{alpha} transactivation
Three different TR{alpha} mutant mouse models, P398H, R384C, and PV, have been characterized (16, 58, 59). Significant phenotypic similarity is seen among the three models, including embryonic or neonatal lethality and elevated thyroid hormone and TSH levels. The metabolic phenotype observed in TR{alpha} P398H mice, however, was not seen in the TR{alpha} R384C or TR{alpha} PV mutant mice. Mutations can differentially influence nuclear receptor ligand binding, coactivator/corepressor recruitment, and interaction with other nuclear receptors (62, 63, 64, 65). We compared the TR{alpha} mutants with regard to PPRE binding and the influence on PPAR{alpha}-mediated transcription using CPT PPRE-Luc and ACO PPRE-Luc reporter constructs in transient transfection assays and 32P-labeled CPT PPRE and ACO PPRE in gel-shift assays. In the absence of T3, PPAR{alpha}-mediated transcription was inhibited by wtTR{alpha} and TR{alpha} mutants compared with transfection with PPAR{alpha} alone (wtTR{alpha} reduced 46%, R384C reduced 39%, PV reduced 58%, and P398H reduced 86%); after addition of 50 nM T3, the inhibition by the R384C mutant and wtTR{alpha} was largely reversed. However, the repression by the P398H and PV mutant persisted (Fig. 8AGo). The results from transfection using ACO PPRE-Luc reporter construct showed an identical pattern to that using CPT PPRE-Luc reporter (Fig. 8BGo). In the presence of both T3 and PPAR ligand (WY 14643), the activity of ACO PPRE-Luc reporter stimulated by wtTR{alpha} and TR{alpha} R384C was similar to that with PPAR{alpha} alone. Cotransfection of TR{alpha} P398H or TR{alpha} PV, however, reduced activity 84 and 71%, compared with PPAR{alpha} alone. In comparison among the three TR{alpha} mutants, the P398H mutation was the most potent inhibitor of PPAR{alpha} signaling.


Figure 8
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  		FIG. 8. Differential influences of TR{alpha} mutants on PPAR{alpha}-mediated transactivation. Reporter constructs CPT PPRE-Luc (A) and ACO PPRE-Luc (B) were used in transient transfection assays. HepG2 cells were transfected with PPAR{alpha} alone or cotransfected with wtTR{alpha} or R384C or P398H or PV mutant receptors. For each transfection, 0.1 µg of each plasmid DNA was used. Empty vector was used to keep DNA concentration constant in each transfection. WY14634 (20 µM) and T3 (50 nM) were supplied to cells. Luciferase activity was determined using a luminometer 36 h after transfection. C, mRNA expression levels of transfected receptors in HepG2 cells.

 
To examine the relative affinity of TR{alpha} mutants for the PPRE, we performed competition assay using 32P-labeled CPT-I{alpha} PPRE to compete with cold competitor in gel-shift assays. The intensity of the receptor/DNA complex was quantified and expressed as the percentage of the baseline (PPAR{alpha}/RXR/DNA complex). Without competitor, wtTR{alpha}/RXR had only trace DNA binding (Fig. 9AGo, lanes 5 and 13). TR{alpha} R384C/RXR showed very weak binding without competitor (lane 13). In the presence of competitor, TR{alpha} R384C/RXR had no binding activity (lanes 14 and 15). The other TR mutants had detectable binding. The intensity of TR{alpha} P398H/RXR/DNA was 65% of the baseline (Fig. 9AGo, lane 9, and B). In the presence of 50x cold competitor, the DNA-bound TR{alpha} P398H/RXR was competed away completely. The intensity of TR{alpha} PV/RXR/DNA complex was 20% baseline in the absence of competitor and was not competed with the cold competitor (Fig. 9AGo, lane 17, and B). Moreover, TR{alpha} PV bound to 32P-labeled mutant CPT-I{alpha} PPRE that did not interact with other TR{alpha} receptors and PPAR{alpha} (Fig. 9AGo lanes 4, 8, 12, 16, and 20). These data indicate that TR{alpha} R384C mutant had similar properties to wtTR{alpha} in PPRE binding, and both had little influence in PPAR{alpha} binding to DNA and PPAR{alpha}-mediated transcription (Fig. 8Go). PV has significant lower affinity and specificity for PPRE compared with TR{alpha} P398H.


Figure 9
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  		FIG. 9. Comparing the specificity of wtTR{alpha} and TR{alpha} mutant binding to the CPT-I{alpha} PPRE. In vitro synthesized receptor proteins with RXR (0.2 µg) were incubated with 32P-labeled CPT-I{alpha} PPRE and competed with cold oligonucleotide at 0x, 5x, and 50x labeled probe. DNA binding and gel-shift conditions were the same as shown in Fig. 5Go. Mutant CPT-I{alpha} PPRE (sequence shown in Table 1Go) was 32P labeled and used for DNA binding as shown in lanes 4, 8, 16, and 20. Solid arrow indicates receptor/DNA and open arrow nonspecific binding. Quantification of receptor/DNA complexes (B) was done as described in Fig. 4Go. The intensity of protein/DNA complexes was compared with PPAR/RXR (100%). C, In vitro protein synthesis efficiency. TR{alpha} mutant receptors and PPAR{alpha} were labeled with [35S]methionine, and 2 µl of each translation product was resolved in 10% SDS-PAGE and scanned using a phosphorimager.

 
Similar findings were obtained when using the ACO PPRE with respect to intensive binding to PPRE by the TR{alpha} P398H compared with that by TR{alpha} PV or by TR{alpha} R384C (data not shown). The DNA binding studies from two different PPREs demonstrated that although both TR{alpha} P398H and TR{alpha} PV were able to bind the PPRE, TR{alpha} P398H had significantly higher affinity and specificity for PPRE, consistent with the results obtained from the functional assays.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The close structural similarity among members of the nuclear hormone receptor superfamily has led to the demonstration of a wide range of in vitro interactions, especially among the nuclear receptors regulating metabolism. The relevance of these interactions for normal physiology, however, remains difficult to determine. Although TR and PPAR{alpha} interactions had been previously reported (34, 45, 54), our study focused on mechanisms of interaction relevant to the hepatic gene expression profile and liver phenotype of our TR{alpha} P398H mutant mouse line (16).

The initial TR/PPAR{alpha} interaction described was the formation of TR/PPAR{alpha} heterodimers that could bind to and transactivate a DR+2 response element (45). The nature of the interaction was TR-isoform specific because PPAR{alpha} heterodimers with TRß formed complexes that induced gene expression and complexes with TR{alpha} did not induce gene expression. The potential for PPAR{alpha} to down-regulate T3-mediated gene induction was subsequently recognized (34, 46). The proposed mechanism for PPAR{alpha} down-regulation of T3 induction was competition for RXR (34, 46). This is consistent with our functional and binding studies of PPAR{alpha} and the wt TR. Mutations in the PPAR{alpha} heptad repeat region that prevent RXR interaction also impaired the ability of PPAR{alpha} to reduce TR-mediated gene induction (34).

Our observations confirm and extend the findings of PPAR{alpha} inhibition of TR signaling but, based on the hepatic gene expression profile in the TR{alpha} P398H mice, focus on inhibition of PPAR{alpha} signaling by TR{alpha}. The TR{alpha} P398H mutant interfered with PPAR target genes expression in vivo and in vitro. The TR{alpha} P398H mutant inhibited PPAR{alpha} binding to several PPREs as shown by ChIP and gel-shift assays. This inhibition was not reversed by addition of ligand or excess RXR. A number of previous studies have demonstrated that corepressor binding to TR mutants, associated with RTH, is not reversed or incompletely released in response to ligand (62, 63, 64). TR mutations that retain some T3 binding, such as P398H, can be stimulated at very high ligand concentrations (57). TR{alpha} P398H mutant interference with PPAR{alpha} signaling, however, persisted despite high ligand concentrations. These data suggest that factors in addition to reduced ligand-dependent corepressor release are involved in interference with PPAR{alpha} signaling.

The interaction of the mutant TR with PPAR{alpha} is likely to be important in producing the P398H mutant mouse metabolic phenotype, but these data do not directly address the role of TR/PPAR{alpha} interactions in physiological regulation of metabolism. The interference of PPAR{alpha} signaling by wtTR{alpha} is reversed by T3 and additional RXR, as shown in transient transfection and gel-shift assays. These findings indicate that TR modulation of PPAR{alpha} signaling may vary depending on thyroid status and with availability of response cofactors.

The PPAR{alpha}-deficient mice and the TR{alpha} P398H mice share the phenotype of reduced fatty acid oxidation producing hepatic steatosis and reduced glycogen storage in the liver (16, 43). The phenotype in both mouse models is also sexually dimorphic, with the metabolic phenotype predominantly in male mice, and both have increased obesity with age (16, 43). The in vitro data presented in this study provide a mechanistic link between TR{alpha} and PPAR{alpha} in mediating lipid metabolism in the liver.

Lipid metabolism in the liver is highly complex and involves multiple transcription factors acting at multiple sites. Another major transcription factor regulating hepatic lipid metabolism is the liver X receptor (LXR). LXR, like TR and PPAR, requires heterodimerization with RXR for gene induction. Mice deficient in LXR{alpha} have a fatty liver (65), which is caused by the accumulation of cholesterol, not by the failure of fatty acid ß-oxidation. LXR activates the gene expression of ATP-binding cassette transporter A1 (ABCA1), which modulates cholesterol efflux and mediates reverse cholesterol transport from peripheral tissues (66). LXR activates expression of sterol regulatory element-binding protein 1c (SREBP-1c), a dominant lipogenic gene regulator, whereas PPAR{alpha} and TR stimulate expression of genes regulating fatty acid ß-oxidation (67).

Several studies have shown LXR interactions with PPAR{alpha} and with TR. PPAR interacts with LXR via competition for RXR and DNA binding, as observed in gel-shift assays (68, 69). TRß represses LXR{alpha} induction by directly binding to the LXR response element (70). These studies indicate that TR may regulate metabolism by modulating PPAR, LXR, and perhaps other nuclear receptors. The relative importance of these TR interactions, and factors that modulate their influence, remain to be described. It is likely that thyroid status and dietary intake of fat influence these relationships.

The results with the TR{alpha} R384C mutant show similarities and differences from the P398H mutant. The limited ability of the R384C mutant to interfere with PPAR{alpha} signaling, compared with the P398H mutant, is consistent with the absence of a metabolic phenotype (61). The mutations, however, both retain some ligand-binding capacity. TR{alpha} PV mutant does not possess detectable ligand binding. Although TR{alpha} PV mutant shows moderate inhibition to PPAR{alpha}-mediated transcription, the PV mutation has much lower affinity and specificity for PPRE compared with P398H mutant. Because TR{alpha} PV mutant mice did not have a metabolic phenotype (60), the characteristics of specificity and affinity to PPRE may play an important role in TR{alpha} interaction with PPAR{alpha}, which may, in turn, explain the uniqueness of the P398H mutant mice with metabolic phenotype. The P398H mutant mice may provide a useful model for understanding the interaction between TR{alpha} and PPAR{alpha} signaling in regulating metabolism.

A TR mutant has recently been shown to interact with PPAR{gamma} (54, 71). Mice with the TRß PV mutations develop thyroid cancer. These mice have reduced expression of PPAR{gamma} in the thyroid. A series of in vitro studies showed that the TRß PV mutant interfered with PPAR{gamma} signaling and directly competed with PPAR{gamma} for binding to the PPRE. These findings further support a potential role for TR/PPAR interactions.

We have demonstrated the role of TR{alpha} in PPAR{alpha}-mediated fatty acid oxidation using wtTR{alpha} and TR{alpha} mutants (P398H, R384C, and PV) in both in vivo and in vitro studies. The wtTR{alpha} and TR{alpha} mutants have the ability to influence PPAR{alpha}-mediated transcription by competing for RXR (wtTR{alpha} and the R384C mutant) or by occupying PPAR{alpha} DNA-binding sites (the P398H and PV mutants). The physiological role of these interactions in metabolic regulation, and the influence of diet and thyroid status, will require additional study.


   Footnotes
 
This work was supported by a grant from the National Institutes of Health, RO1 DK67233, and medical research funds from the Department of Veterans Affairs.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 22, 2006

1 Deceased February 9, 2006. Danny was an undergraduate student with an insatiable desire for knowledge and love of science, and left us far too soon. We were all enriched by knowing him and dedicate this work to his memory. Back

Abbreviations: ChIP, Chromatin immunoprecipitation; FFA, free fatty acid; Glut, glucose transporter; GP, glycogen phosphorylase; GPK, glycogen phosphorylase kinase; GS, glycogen synthase; GSK, GS kinase; KO, knockout; LXR, liver X receptor; PAS, periodic acid-Schiff; PDK, pyruvate dehydrogenase kinase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response element; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; TR, thyroid hormone receptor; TRE, thyroid hormone response element; wt, wild-type.

Received June 20, 2006.

Accepted for publication November 15, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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