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Endocrinology Vol. 145, No. 12 5515-5524
Copyright © 2004 by The Endocrine Society

Unliganded Thyroid Hormone Receptor-ß1 Represses Liver X Receptor {alpha}/Oxysterol-Dependent Transactivation

Kotaro Kawai, Shigekazu Sasaki, Hiroshi Morita, Takeshi Ito, Sadako Suzuki, Hiroko Misawa and Hirotoshi Nakamura

Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan

Address all correspondence and requests for reprints to: Shigekazu Sasaki, M.D., Ph.D., Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan. E-mail: sasakis{at}hama-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The thyroid hormone receptor (TR) and liver X receptor (LXR)-{alpha} are members of the nuclear hormone receptor family and are ligand-dependent transcription factors. Among the promoter target genes, TR and LXR recognize the T3 response element and LXR response element (LXRE), respectively. Because T3 response elements and LXREs have similar configurations, referred to as direct repeat 4, we investigated the possibility of cross-talk between the two ligand-dependent signal transduction pathways. We found that TRß1, a major isoform of TR in the liver, binds and transactivates LXREs derived from the mouse mammary tumor virus long-terminal repeat and the promoter of the sterol regulatory element binding protein 1c. Moreover, unliganded TRß1 suppresses promoter activity driven by LXR{alpha} and its ligand, whereas transactivation by T3-bound TRß1 is not affected by LXR{alpha} in the presence or absence of oxysterols. Gel shift, mammalian two-hybrid, and glutathione S-transferase pull-down assays demonstrated the direct binding of TRß1 to these LXREs and revealed that the interaction between TRß1 and corepressors is important to the unliganded TR-mediated suppression of LXR{alpha}-transactivation. Our findings suggest that T3 and TR influence lipid metabolism regulated by oxysterol/LXR{alpha} at the transcriptional level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE RECEPTORS (TRs) and liver X receptors (LXRs) are the major regulators for the energy homeostasis and belong to a subclass of nuclear hormone receptors that ligand-dependent transcription factors. Oxysterols including 22(R)-hydroxycholesterol [22(R)-HC] were identified as the natural ligand of LXRs (1). In conjunction with retinoid X receptor (RXR), TRs and LXRs form a heterodimer on specific DNA sequences referred to as the T3 response element (TRE) and LXR response element (LXRE), respectively. TREs and LXREs have similar configurations; they consist of a pair of direct repeats of half-sites (typically AGGTCA) spaced by four random bases designated as direct repeat four (DR4) (2, 3). In the presence of cognate ligands, these heterodimers associate with coactivators including members of the p160 protein family, CREB-binding protein/p300 and p300/CREB-binding protein -associating factor (4). These cofactors have histone acetyl transferase activity, and the acetylation of lysine residues of core histones relaxes the folding of DNA to facilitate the transcription of target genes (5).

TRs are encoded by two genes (TR{alpha} and TRß) and are expressed as several isoforms (TR{alpha}1, TRß1, and TRß2). Whereas the expression of TRß2 is restricted to the pituitary and hypothalamus, TR{alpha}1 and TRß1 are ubiquitously expressed. In addition to T3-dependent transactivation, the unliganded TR/RXR heterodimer recruits corepressors including the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoic acid and TRs (SMRT) to suppress the transcription of target genes (silencing). In patients with resistance to thyroid hormone (RTH), many natural mutations in the TRß gene have been identified. The interaction of TR with corepressors plays a role in the dominant-negative effect of mutant TRs found in RTH patients (6).

LXRs are also encoded by two genes (LXR{alpha} and ß). Whereas LXRß is ubiquitously expressed, the transcription of LXR{alpha} is regulated by another nuclear receptor, peroxisome proliferator-activated receptor-{gamma}2 and expressed in metabolic organs including the liver, kidneys, intestine, and adipose tissue (7). The establishment of a LXR{alpha} null mouse line suggests that this receptor plays a role in the metabolism of cholesterol and fatty acids by stimulating the expression of proteins involved in lipid metabolism (8). For example, LXR{alpha}/22(R)-HC activates the expression of cholesterol 7{alpha}-hydroxylase (9), which is the rate-limiting enzyme in bile acid biosynthesis. In the ATP-binding cassette transporter 1 (ABC1) gene, oxysterol-bound LXR{alpha} enhances promoter activity, and the ABC1 protein facilitates the translocation of cholesterol from peripheral tissues to apolipoprotein A1 (7). The expression of cholesterol ester transfer protein, which transports the cholesterol ester from high-density lipoprotein to triglyceride-rich lipoproteins, is also up-regulated by LXR{alpha} in the presence of oxysterols (10). Recently two functional LXREs were identified in the promoter region of sterol regulatory element binding protein (SREBP)-1c (8, 11), which enhances the expression of genes that encode acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA and FAS synthesizes fatty acids using acetyl-CoA as a primer and malonyl-CoA as elongating units. Because the expression of ACC and FAS is stimulated by T3 (12, 13), both oxysterol/LXR and T3/TR facilitate fatty acid synthesis. However, a mutual relationship of these ligand-dependent signaling pathways in FAS has not been verified.

Due to the sequence homology between the TRE and LXRE, we speculated the possible mutual cross-talk between the two receptors and investigated the effect of T3/TR on transcriptional regulation mediated by oxysterol/LXR{alpha}. We report here that TRß1 recognizes LXREs derived from deleted mouse mammary tumor virus promoter (dMTV-LXRE) and SREBP-1c promoter (SREBP1c-LXRE). TRß1 enhances the thymidine kinase (tk) promoter fused to dMTV-LXRE and SREBP-1c promoter in a T3-dependent fashion at a magnitude comparable with that by oxysterol/LXR{alpha}. Unliganded TRß1 suppresses the oxysterol/LXR{alpha}-dependent transcriptional activity of the dMTV-LXRE and SREBP-1c promoters, whereas LXR{alpha} dose not affect transactivation driven by T3/TRß1 in the presence or absence of oxysterol. Aside from the direct binding of the TRß1/RXRß heterodimer to dMTV-LXRE and SREBP1c-LXRE, the preferential association of corepressors NCoR and SMRT with TRß1 plays a role in TRß1 predominant cross-talk. These findings suggest that in organs in which both receptors are coexpressed, the T3/TR pathway may have profound effects on oxysterol/LXR-dependent gene regulation including that of SREBP-1c.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
The human LXR{alpha} expression plasmid, pCMX-LXR{alpha}, a luciferase reporter gene containing the tk promoter fused to three copies of the LXRE derived from dMTV long-terminal repeat [tk-(LXRE)3-luciferase (Luc)] (3), luciferase gene constructs containing a 2.6-kb and a 186-bp fragment of the mouse SREBP-1c promoter (pBP1c-2.6-Luc and pBP1c-186-Luc, respectively) (11), and expression plasmid for wild-type human TRß1, pCMX-TRß1 (14), were described elsewhere. Expression plasmids for human TRß1s, which have mutations in their zinc finger (C127S) (15), the CoR-box (AHT) (16), NCoR interacting surface (C309K) (17), and the RXR interacting surface (L428R) (18), were previously described.

Cell culture and transient transfection
A monkey kidney cell line, CV1 cell, a human embryonic kidney cell line, 293T cell, and a human hepatoma cell line, HepG2 cell, were grown in DMEM containing 10% fetal bovine serum, penicillin G (100 U/ml), and streptomycin (100 mg/ml) at 37 C under 5% CO2-95% air. CV1 cells were trypsinized and seeded at a density of 2 x 105 cell/well in 6-well plates. Using the calcium-phosphate technique, the cells were cotransfected with 500 ng of (LXRE)3-tk-Luc, 1 µg ß-galactosidase expression vector (pCH111, a modified version of pCH110, Pharmacia, Piscataway, NJ) and expression plasmids for the receptors. The total amount of transfected plasmid was adjusted to 2 µg/well by adding empty vector (pCMX). After transfection for 20 h, the medium was replaced with fresh DMEM containing 10% dextran-coated charcoal-stripped (DCC) fetal bovine serum (10% DCC serum) in the presence or absence of ligands. After 24 h incubation, the cells were harvested and luciferase activity was measured. 293T cells were trypsinized and seeded in 6-well plates at a density of 2 x 105 cells/well. After 12 h, the medium was replaced with the serum-free fresh DMEM and the cells were transfected with 400 ng pBP1c-2.6-Luc, 1 µg pCH111, and expression plasmids for the receptors in the quantities as indicated in the figure legends using the lipofection method. After transfection for 5 h, the medium was replaced with fresh DMEM containing 10% DCC serum in the presence or absence of ligands, and the cells were harvested after incubation for an additional 24 h. Luciferase activity was measured following the manufacturer’s protocol. Transfection efficiencies were normalized by a ß-galactosidase assay. For interassay control, we performed the transfection with Rous sarcoma virus (RSV)-Luc (20 ng/well), the magnitude of which was adjusted to 100.

Immunoblotting
Cultured CV1 and 293T cells were transfected with wild-type or mutant human TRß1 expression plasmids (5 µg per 10-cm dish). They were then harvested and lysed in lysis buffer containing 125 mM Tris-HCl (pH 7.6) and 0.5% of Triton X-100. The suspensions were centrifuged and the precipitations were boiled at 100 C for 5 min in sample buffer containing 3.0% sodium dodecyl sulfate, 10% glycerol, 2.5% ß-mercaptoethanol, 5 mM Tris-HCl (pH 7.8), 0.75 mM MgCl2, and 5 mM KCl. The samples were next fractionated on a 10% SDS-PAGE and transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA). The membrane was incubated for 1 h at room temperature in blocking solution (5% nonfat milk in PBS). After washing with PBS containing 0.05% Tween 20 three times for 5 min, it was incubated with the anti-TR monoclonal antibody (1:1000, Affinity Bioreagents, Inc., Golden, CO.) for 1 h at room temperature. It was then incubated with horseradish peroxidase-conjugated antimouse IgG (1:1000, Promega, Madison, WI) for 1 h at room temperature, and the antibody-protein complexes were visualized using ECL detection reagents (Amersham Pharmacia Biotech, Little Chalfont, UK).

Gel shift assay
Oligonucleotides for dMTV-LXRE (sense, 5'-AGCTTCCAGGGTTTAAATAAGTTCATCTAG-3') (3) and SREBP1c-LXRE (sense, 5'-AGCTCGCTGGGGTTACTGGCGGTCACTGTA-3') (11) were labeled with {gamma}-32P-ATP using T4 polynucleotide kinase (TOYOBO, Tokyo, Japan). The receptor proteins were in vitro translated using a TNT T7 quick coupled transcription/translation system (Promega). The {gamma}32P-labeled probes and in vitro-translated receptors were incubated for 30 min on ice in 20 µl of binding buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.5 µg/ml poly (dI-dC), 0.1% Triton X-100, and 1 mg/ml nonfat dry milk. The DNA-protein complexes were resolved on 5% polyacrylamide gel at 100 V for 2.5 h at 4 C. The gel was dried and visualized using a BAS-1000 auto radiography system (Fuji Film, Tokyo, Japan). For Scatchard analysis, constant amounts of in vitro-translated receptor proteins (3 µl each) were incubated with the different concentrations of {gamma}32P-labeled probes (0.14, 0.28, 0.56, 1.13, 2.25, and 4.50 nM). Quantitation of free and bound DNA was performed with BAS-1000. The ratio of bound to free DNA was plotted vs. the molar concentration of bound DNA.

Mammalian two-hybrid assay
Using the calcium-phosphate technique, CV1 cells in 6-well plates were cotransfected with 300 ng of luciferase reporter gene containing (UAS)-tk promoter, 1 µg ß-galactosidase expression plasmid, 15 ng of the expression plasmid for hTRß1 fused with VP16 transactivating domain, 15 ng of the plasmid for SMRT fused with GAL-4 DNA binding domain, and 60 ng of receptor expression vector. To adjust the total DNA amount to 2 µg/well, pCMX was added. After incubation for 24 h, the cells were harvested and luciferase activities were measured. The luciferase activity was normalized with ß-galactosidase activity.

RNA isolation, cDNA synthesis, and PCR
HepG2 cells cultured in 10% DCC serum were incubated with 22(R)-HC and/or T3 for 96 h. The cells were harvested and total RNA was purified by the acid guanidium thiocyanate-phenol-chloroform extraction method (19). For the first-strand cDNA synthesis, 2 µg total RNA was mixed with random hexanucleotide and 200 U Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). The cDNA for SREBP-1c was amplified with the forward primer (5'-GCGGAGCCATGGATTGCAC-3') and the reverse primer (5'-CTCTTCCTTGATACCAGGCCC-3'), to generate a 313-bp product. ß-Actin cDNA was amplified with the forward primer (5'-GGGCATGGGTCAGAAGGATT-3') and the reverse primer (5'-GAGGCGTACAGGGATAGCAC-3') to generate a 302-bp product. PCR amplification was carried out using a DNA thermal cycler (PerkinElmer Cetus, Norwalk, CT) under the following conditions: denaturation at 95 C for 1 min, annealing at 62 C for 1 min, extension at 72 C for 2 min, and the final extension at 72 C for 4 min. We determined the cycle number for each primer set so that the specific product was amplified during the exponential phase of the amplification. Based on preliminary studies (data not shown), ß-actin cDNA was diluted 400-fold, and 26 cycles were employed for the amplification of SREBP-1c and diluted ß-actin cDNA. The PCR products were subjected to electrophoresis on a 1.4% agarose gel and stained with ethidium bromide. Densitometry was performed using Densitograph software (AE-6900 MF, ATTO, Tokyo, Japan). The densities of amplified SREBP-1c cDNA were measured by semiquantitative evaluation by normalization with the density of the ß-actin band.

Glutathione-S-transferase (GST) pull-down assay
Receptor proteins were generated in vitro using a TNT T7 quick coupled transcription/translation system in the presence of 35S-methionine (TRAN35S-LABEL, ICN Biochemicals, Inc., Costa Mesa, CA). Bacterially expressed GST fusion protein that has a receptor interacting domain (ID-I, amino acids 1164–1399) of mouse NCoR (20) was immobilized on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech, Buckinghamshire, UK). The beads were washed once with binding buffer [25 mM HEPES (pH 7.6), 2 mM dithiothreitol, 20 mM NaCl, 20% glycerol, 2 mg/ml BSA, 0.3 mM phenyl methylsulfonylfloride, 1.0 µg/ml leupeptin, and 2.0 µg/ml aprotinin], followed by equilibrating with binding buffer overnight at 4 C. Twenty microliters of beads (packed volume) were incubated at 4 C for 3 h with 20 µl of 35S-labeled receptor proteins. The beads were washed twice with 1 ml of binding buffer containing 0.1% Triton X-100 and once with 1 ml of binding buffer containing 50 mM Tris-HCl (pH 8.0). Eluted proteins were boiled and resolved by SDS-PAGE and were visualized using a BAS-1000 auto radiography system (Fuji Film, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TRß1 activates transcription of dMTV-long-terminal repeat (LTR) and SREBP-1c promoter in the presence of T3
To investigate possible cross-talk between TR and LXR on the LXRE, tk-(LXRE)3-Luc (3) that has three copies of the LXRE derived from dMTV-LTR and the expression plasmids for human TRß1 or human LXR{alpha} were cotransfected into CV1 cells. As previously reported (3), the tk-(LXRE)3-Luc reporter was transactivated by LXR{alpha} and 22(R)-HC. T3/TRß1 also transactivated the tk-(LXRE)3-Luc reporter in a dose-dependent manner without LXR{alpha} and 22(R)-HC (Fig. 1CGo). This suggests that TRß1 is able to transactivate LXREs derived from dMTV-LTR, although the concentration of T3 required for the induction was higher than typical TRE. The SREBP-1c promoter has two functional LXREs (11). We cotransfected the reporter plasmid containing SREBP-1c promoter, pBP1c-2.6-Luc (11), together with wild-type TRß1 expression plasmid into 293T cells. In the absence of LXR{alpha} and 22(R)-HC, TRß1 transactivated the pBP1c-2.6-Luc reporter in a T3-dependent manner (Fig. 1DGo). The reporter plasmid that has a single LXRE, pBP1c-186-Luc (11), was also stimulated by T3 and TRß1 (Fig. 1EGo). We examined whether T3/TRß1 synergistically stimulates transactivation on the LXRE by 22(R)-HC/LXR{alpha} and found that T3/TRß1 did not exhibit such activity in the presence of pBP1c-2.6-Luc or tk-(LXRE)3-Luc (data not shown). Without TRs and LXR{alpha}, neither tk-(LXRE)3-Luc nor pBP1c-2.6-Luc was stimulated even in the presence of cognate ligand (data not shown).



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  		FIG. 1. T3 and TRß1 activate the promoters that possess LXRE. A, Schematic representation of the tk-(LXRE)3-Luc plasmid. B, Schematic representation of the pBP1c-2.6-Luc and pBP1c-186-Luc plasmids. C, The tk-(LXRE)3-Luc plasmid (500 ng/dish) was cotransfected into CV1 cells with pCMX-LXR{alpha} or TRß1 (50 ng/dish) in the presence of cognate ligands. D, The pBP1c-2.6-Luc plasmid (400 ng/dish) was cotransfected into 293T cells with pCMX-LXR{alpha} or TRß1(200 ng/dish) in the presence of cognate ligands. E, The pBP1c-186-Luc plasmid (400 ng/dish) was cotransfected into 293T cells with pCMX-LXR{alpha} or TRß1(200 ng/dish) in the presence of cognate ligands. After incubation for 24 h with 0–5 µM of 22(R)-HC or 0–1.0 µM of T3, the cells were harvested and luciferase activity was measured and normalized by ß-galactosidase activity. For the interassay control, 50 ng/dish of RSV-Luc were transfected, and its magnitude was adjusted to 100. Each transfection was conducted in duplicate, and data represent the mean ± SD of more than four experiments. *, P < 0.05 vs. without ligand.

 
Transactivation of LXR{alpha}/22(R)-HC is suppressed by unliganded TRß1, whereas transactivation by TRß1 is not affected by unliganded LXR{alpha}
Because unliganded TRß1 has a strong silencing effect, we investigated the influence of unliganded TRß1 on promoter activity driven by LXR{alpha} and 22(R)-HC. In the absence of T3, the activation of tk-(LXRE)3-Luc and pBP1c-2.6-Luc induced by LXR{alpha} and 22(R)-HC was suppressed by the expression of an equal amount of TRß1 (Fig. 2Go, A and B). Figure 2Go, C and D, show the dose response of expressed TRß1. Even a small amount (10 ng/dish) of TRß1 had an inhibitory effect on transactivation by LXR{alpha} and 22(R)-HC. Conversely, increasing amounts of unliganded LXR{alpha} did not suppress the T3/TRß1-induced luciferase activity of tk-(LXRE)3-Luc and pBP1c-2.6-Luc (Fig. 3Go, A and B). Taken together, unliganded TRß1 has a silencing effect on the transactivation of the LXRE by LXR{alpha}/22(R)-HC, whereas LXR{alpha} does not affect transactivation by T3/TRß1.



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  		FIG. 2. Unliganded TRß1 inhibits the transcriptional activity of LXR{alpha} on the LXRE. A, The tk-(LXRE)3-Luc plasmid (500 ng/dish) was cotransfected into CV1 cells with pCMX-LXR{alpha} alone (solid bar, 50 ng/dish) or with pCMX-LXR{alpha} and TRß1 (hatched bar, 50 ng/dish each). B, The pBP1c-2.6-Luc plasmid (400 ng/dish) was cotransfected into 293T cells with pCMX-LXR{alpha} alone (solid bar, 200 ng/dish) or with pCMX-LXR{alpha} and TRß1 (hatched bar, 200 ng/dish each). C, The tk-(LXRE)3-Luc plasmid (500 ng/dish) and pCMX-LXR{alpha} (50 ng/dish) were cotransfected into CV1 cells with increasing amounts of pCMX-TRß1 (0–50 ng/dish) in the absence (solid bar) or presence (hatched bar) of 22(R)-HC. D, The pBP1c-2.6-Luc plasmid (400 ng/dish) and pCMX-LXR{alpha} (200 ng/dish) were cotransfected into 293T cells with increasing amounts of pCMX-TRß1 (0–200 ng/dish) in the absence (solid bar) or presence (hatched bar) of 22(R)-HC. After incubation with 0–5 µM of 22(R)-HC for 24 h, the cells were harvested, and luciferase activity was measured using the same protocol as in Fig. 1Go. Each transfection was conducted in duplicate, and data represent the mean ± SD of more than four experiments. *, P < 0.05 vs. without 22(R)-HC. #, P < 0.05 vs. without TRß1.

 


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  		FIG. 3. LXR{alpha} does not inhibit the transcriptional activity of TRß1 on the LXRE. A, The tk-(LXRE)3-Luc (500 ng/dish) and pCMX-TRß1 (50 ng/dish) were cotransfected into CV1 cells with increasing amounts of pCMX-LXR{alpha} (0–250 ng/dish). B, The pBP1c-2.6-Luc (400 ng/dish) and pCMX-TRß1 (200 ng/dish) were transfected into 293T cells with increasing amounts of pCMX-LXR{alpha} (0–400 ng/dish). Twenty hours after transfection, the medium was replaced with fresh DMEM in the absence (solid bars) or presence (hatched bars) of T3 (1 µM). After the cells were incubated for 24 h and harvested, luciferase activity was measured using the same protocol as in Fig. 1Go. Each experiment was conducted in duplicate, and data represent the mean ± SD of more than four experiments. *, P < 0.05 vs. without T3.

 
Mutant TRß1s, which do not form heterodimers with RXRs on the TREs or interact with corepressors, are unable to repress the transactivation of LXRE by LXR{alpha}/22(R)-HC
To map the domain of TRß1, which plays a role in the inhibitory effect on transactivation by LXR{alpha}, the effect of various mutants TRß1s were examined. Previously we reported a mutant TRß1, F451X, which has a strong dominant-negative effect on transactivation by wild-type TR and T3 (21). When F451X was cotransfected with LXR{alpha}, the activation of tk-(LXRE)3-Luc and pBP1c-2.6-Luc by 22(R)-HC was remarkably inhibited (Fig. 4Go, B and D, upper panels). However, the transcriptional activity of tk-(LXRE)3-Luc and pBP1c-2.6-Luc by LXR{alpha} and its ligand was not affected by L428R (18), for which interaction with RXR was disrupted, or C127S (15), which cannot bind to TRE (Fig. 4Go, B and D, upper panels). AHT (16) and C309K (17) are mutant TRß1s that have no corepressor binding activity. As shown in Fig. 4Go, C and D (upper panels), these mutants lost their inhibitory effect. The expression of mutant TRß1 was comparable with that of wild-type TRß1 (Fig. 4Go, B–D, lower panels). Collectively, these findings indicate that stable heterodimer formation with RXR on the LXRE and corepressor interaction are essential for unliganded TRß1 to inhibit the transactivation of the LXRE by LXR{alpha} and 22(R)-HC.



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  		FIG. 4. The effect of TRß1 mutation on the transcriptional activity of LXR{alpha} and 22(R)-HC. A, Schematic representation of the mutant TRß1s. B and C, upper panels, The tk-(LXRE)3-Luc plasmid (500 ng/dish) and pCMX-LXR{alpha} (50 ng/dish) were cotransfected into CV1 cells with 50 ng/dish of expression plasmids for wild or mutant TRß1s. D, upper panel, The pBP1c-2.6-Luc plasmid (400 ng/dish) and pCMX-LXR{alpha} (200 ng/dish) were cotransfected into 293T cells, respectively, with 200 ng/dish of expression plasmids for wild or mutant TRß1s. Twenty hours after transfection, the medium was replaced with fresh DMEM in the absence (solid bars) or presence (hatched bars) of 22(R)-HC. Luciferase activity was measured using the same protocol as in Fig. 1Go. The experiments were repeated four times, and the data were expressed as the mean ± SD. *, P < 0.05 vs. without T3. The expression of mutant TRß1s in 293T and CV-1 cells are shown in the lower panels. Whole-cell extracts of CV1 cells (B and C) or 293T cells (D) transfected with mutant TRß1 expression plasmids were resolved by 10% SDS-PAGE. The blot was proved with an anti-TRß1 antibody followed by chemiluminescent detection.

 
TRß1 binds to LXREs with an affinity similar to LXR{alpha}
The T3/TRß1-dependent activation of LXREs lead us to hypothesize that unliganded TRß1 may compete for DNA binding with LXR{alpha}, resulting in interference with 22(R)-HC-dependent transactivation. In a gel mobility shift assay, 32P-labeled dMTV-LXREs or SREBP1c-LXREs were incubated with in vitro-translated TRß1 or LXR{alpha} in the presence of RXR (Fig. 5Go, A and B). TRß1 and LXR{alpha} formed a heterodimer with RXR on these LXREs. The binding was specific for the LXRE because it was lost by a 50-fold molar excess of unlabeled specific oligo DNA. When an increasing amount of TRß1 was added to the binding reaction of LXR{alpha}/RXRß with SREBP1c-LXRE, the specific band for the receptor-LXRE interaction migrated from the position of LXR{alpha}/RXRß to that of TRß1/RXRß (Fig. 5CGo)



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  		FIG. 5. TRß1/RXRß heterodimer binds to dMTV-LXRE and SREBP-1c-LXRE. Three microliters of in vitro-translated LXR{alpha}, TRß1, and RXRß protein generated from rabbit reticulocyte lysates were incubated with 32P-raidolabeled dMTV-LXRE (A) or SREBP1c-LXRE probe (B). Competitions were performed using a 50-fold molar excess amount of unlabeled specific or nonspecific oligonucleotides. C, In the presence of constant amount of LXR{alpha} and RXRß (3 µl each), 32P-labeled SREBP1c-LXRE probe was incubated with 0–3 µl of TRß1.

 
The difference in LXRE binding affinities between TRß1 and LXR{alpha} was examined by a Scatchard analysis, plotting the ratio between receptor-bound and free DNA against the amount of receptor-bound DNA obtained from gel shift assay data (Fig. 6AGo). As shown in Fig. 6Go, B and C, the dissociation constant (Kd) for the LXR{alpha}/RXR heterodimer on dMTV-LXRE was comparable with that for the TRß1/RXR heterodimer (3.23 and 3.86 nM, respectively). On SREBP1c-LXRE, the Kd for the LXR{alpha}/RXR heterodimer was also similar to that of the TRß1/RXR heterodimer (5.05 and 4.37 nM, respectively, data not shown). As illustrated in Fig. 7Go, A and B, not only F451X but also AHT and C309K bound to the LXRE, although these mutant TRß1s lack the inhibitory effect of oxysterol/LXR{alpha} as seen by the reporter assay (Fig. 4Go, C and D). Therefore, heterodimer formation alone is not sufficient to explain the inhibitory effect of unliganded TRß1.



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  		FIG. 6. The binding affinity of the LXR{alpha}/RXRß and TRß1/RXRß heterodimers to dMTV-LXRE. A, Gel mobility shift assays were performed as described in Fig. 5Go. Three microliters of LXR{alpha} (B), TRß1 (C), and RXRß protein generated with rabbit reticulocyte lysates were incubated with 4.50, 2.25, 1.13, 0.56, 0.28, and 0.14 nM of 32P-raidolabeled dMTV-LXRE. The ratio of bound to free DNA was plotted vs. the molar concentration of bound DNA in the reaction mixture, and the value of the Kd was calculated. All numerical values were obtained by computer quantitation as described in Materials and Methods.

 


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  		FIG. 7. Mutant TRß1s, C309K, AHT, and F451X form a heterodimer with RXR on the LXRE of dMTV and SREBP-1c. 32P-raidolabeled dMTV-LXRE (A) or SREBP-1c-LXRE probe (B) was incubated with 3 µl of in vitro-translated LXR{alpha}, wild-type TRß1, C309K, AHT, F451X, and RXR protein generated with rabbit reticulocyte lysates. Gel mobility shift assays were performed as described in Fig. 5Go.

 
The interaction of TRß1 with corepressor is more potent than that of LXR{alpha}
The silencing effects of unliganded receptors are closely related to corepressor interactions. When examined, TRß1, but not LXR{alpha}, reduced the reporter activity (Fig. 8AGo). We then measured the affinity of receptors with a corepressor, SMRT, using a mammalian two-hybrid assay, in which the Gal4-SMRT fusion protein interacts with the TRß1-VP16 chimeric protein in the absence of T3. Luciferase activities mediated by the interaction between Gal4-SMRT and TRß1-VP16 should be diminished by coexpression of a receptor protein if it interacts with SMRT. When the TRß1 expression plasmid was cotransfected in the absence of T3, it did reduce the luciferase activity (Fig. 8BGo). However, an equal amount of LXR{alpha} did not affect the reporter activity. The binding affinity between the receptors and another corepressor, NCoR, was further examined by a GST pull-down assay. The 35S-labeled receptors were incubated with bacterially expressed GST-NCoR fusion protein possessing the receptor-interacting domain (20), and the binding fraction was analyzed by SDS-PAGE. As seen in Fig. 8CGo, the binding between TRß1 and NCoR was remarkable and LXR{alpha} interacted very weakly with NCoR. These data suggest that the interaction of TRß1 with corepressors is much stronger than that of LXR{alpha}.



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  		FIG. 8. TRß1 has a greater affinity with corepressors than LXR{alpha}. A, The tk-(LXRE)3-Luc plasmid (500 ng/dish) was cotransfected into CV1 cells with or without pCMX-LXR{alpha} (50 ng/dish) or TRß1 (50 ng/dish). After incubation for 24 h in the absence of 22(R)-HC or T3, the cells were harvested and luciferase activity was measured. B, The expression plasmids for TRß1-VP-16 (15 ng/dish), Gal4-SMRT (15 ng/dish), and 300 ng of the Gal4-responsive reporter gene, UAS-tk-Luc, were cotransfected into CV1 cells with pCMX-TRß1 or LXR{alpha} (60 ng/dish). Luciferase activity was measured using the same protocol as in Fig. 1Go. The experiment was repeated four times, and the data were expressed as the mean ± SD. *, P < 0.05. C, The binding affinity of LXR{alpha} and TRß1 to immobilized GST-mouse NCoR fusion protein or GST alone. 35S-raidolabeled receptors were in vitro translated using rabbit reticulocyte lysates and incubated with immobilized GST-mouse NCoR or GST alone in the absence of ligand.

 
The induction of SREBP-1c mRNA by 22(R)-HC in HepG2 cells is enhanced by T3
To evaluate the in vivo function of TRß1 in the transcriptional regulation of SREBP-1c gene activated by LXR{alpha}/22(R)-HC, RT-PCR was performed to estimate the expression of SREBP-1c in HepG2 cells, which possess endogenous LXR{alpha} (22) and TRß1 (23). In the absence of the ligand, SREBP-1c mRNA was barely detected in HepG2 cells, whereas it was induced by the addition of 22(R)-HC (Fig. 9Go, A and B). SREBP-1c mRNA was also induced by T3 and the addition of T3 enhanced the effect of 22(R)-HC. These results provided in vivo evidence that T3/TRß1 as well as LXR{alpha} regulates transcription of the SREBP-1c gene.



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  		FIG. 9. The endogenous expression of SREBP-1c mRNA in HepG2 cells is enhanced by TR/T3. A, RT-PCR was performed on SREBP-1c and ß-actin mRNA isolated from HepG2 cells treated with or without 22(R)-HC and/or T3 (1 µM). B, SREBP-1c mRNA expression was evaluated by semiquantitative RT-PCR. The densitometric units of the PCR products were normalized against ß-actin. The experiments were repeated five times, and the data were expressed as the mean ± SD. *, P < 0.05.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TRß1 is able to recognize the LXREs of dMTV and SREBP-1c
In this study, we demonstrated that the TRß1/RXR heterodimer recognizes the dMTV-LXRE and SREBP-1c-LXRE, whose configurations belong to DR4 and that TRß1 stimulates them in a T3-dependent manner. Compared with the typical TRE, the higher concentration of T3 was required for the transactivation of the LXREs that we used. Because the optimal DNA sequences of half-site in LXRE is somewhat different from AGGTCA, we speculate that such difference may influence the T3-dependent transactivation function of TR-RXR heterodimer (24). The gel shift assay suggested the direct binding of TRß1 to these LXREs. Willy et al. (3) tested the effect of T3-bound TR on dMTV-LXRE. Because the natural ligand for LXR{alpha} was not identified at that time, they evaluated RXR-specific ligands such as 9cis-retinoic acid and methoprene, which enhanced strongly LXR{alpha}/RXR-mediated transactivation of LXRE. They defined LXR{alpha}/RXR as a novel mediator for retinoid signaling but did not mention the effect of T3/TR in detail (3). We showed here that the transactivation of LXREs derived from dMTV and the SREBP-1c promoter by T3/TR is comparable with that by LXR{alpha} bound to its natural ligand, 22(R)-HC. In addition to mouse mammary tumor virus and SREBP-1c, similar cross-talk may occur on the LXREs in other oxysterol-target genes such as ABC1 and cholesterol ester transfer protein. Conversely, the LXR/RXR heterodimer was not reported to bind to DR4-type TRE in the malic enzyme promoter (25). Quack et al. (26) recently proposed that in the DR4-type hormone response element, the sequence of two nucleotides 5'-flanking the downstream half-site and those flanking the upstream half-site may direct receptor specificity. The receptor specificity on the DR4-type element should be characterized in the future.

Mechanisms responsible for the T3/TRß1 dominant regulation of LXREs
Unliganded TRß1 suppresses transactivation by LXR{alpha} (Fig. 2Go), whereas LXR{alpha} dose not suppress transactivation by T3/TRß1 in the presence or absence of 22(R)-HC (Fig. 3Go and data not shown). Studies using mutant TRß1s, C127S and L428R, suggest that the stable heterodimer formation of TRß1 and RXR on LXREs is essential for this silencing (Fig. 4Go). In a gel shift assay, we found that the TRß1/RXR heterodimer binds to these LXREs (Fig. 5Go). However, heterodimer formation on LXRE is not sufficient to explain the stronger inhibitory effect of TRß1 because the affinity of LXR{alpha} for LXREs is comparable with that of TRß1 (Fig. 6Go). The polarity of LXR-RXR heterodimer on dMTV-LXRE has been reported to be same as that of TR-RXR (24). We found that the inhibition of oxysterol/LXR{alpha} transactivation on LXREs was absent in AHT and C309K, which lack interaction with corepressors. Horlein et al. (16) suggested that suppressive effect of NCoR depends on the receptor species because unliganded TR exhibits the robust silencing effect, whereas LXR does not mediate repression. In agreement with this, we found that unliganded TRß1 but not LXR{alpha} exhibited a silencing effect on dMTV-LXREs (Fig. 8AGo). Although Hu et al. (27) and Wagner et al. (28) recently reported that LXR{alpha} and -ß bind corepressors with various affinities, they did not directly compare the affinity of these receptors with that of TRß1. Our mammalian two-hybrid assay (Fig. 8BGo) and GST pull-down assay (Fig. 8CGo) showed that unliganded TRß1 binds to the corepressors with a greater affinity than unliganded LXR{alpha}. The stronger affinity of TRß1 for corepressor is more apt to recruit the Sin3-histone deacetylase complex to LXRE, leading the chromatin inactivation (29).

Thyroid hormone may influence fatty acid metabolism through transcriptional regulation of the SREBP-1c gene
A recent study (30) revealed that SREBP-1c plays a role in the transcriptional regulation of fatty acid synthesis and energy metabolism. Insulin enhances the expression of SREBP-1c, suggesting that glucose metabolism influences fatty acid synthesis (31, 32). Transgenic mice that overexpress SREBP-1c in the liver exhibit increased expression of ACC and FAS (30). Cholesterol metabolism also modulates fatty acid synthesis because LXR{alpha} binds to the LXRE in the SREBP-1c promoter and transactivates it in the presence of oxysterols including 22(R)-HC (8, 11). SREBP-1c provides a platform for the regulation of fatty acid metabolism by monitoring energy metabolism. T3/TR is also a regulator of energy metabolism. Fatty acid synthesis in the liver is elevated in hyperthyroid rats as compared with hypothyroid rats (33). Cachefo et al. (34) reported that the plasma concentration of fatty acids and the level of lipogenesis in the liver is higher in hyperthyroid patients than in hypothyroid patients. Here we showed that T3 stimulates fatty acid synthesis presumably through transactivation of the SREBP-1c gene. Although such an effect on fatty acid synthesis by T3/TRß1 is indirect, these two ligand-dependent signals are not necessarily independent. They have a close relationship though T3/TR dominant cross-talk on the LXRE in the SREBP-1c promoter. In addition, thyroid hormones directly transactivate ACC genes through functional TRE on ACC (35).

The NH2-terminal segment of SREBPs are cleaved from the membrane when cells are depleted of sterol (36), and cleaved SREBPs activate expression of SREBP-1c (37). To exclude the influence of serum cholesterol that is directly influence by thyroid hormones in animals, we employed a human hepatoma cell line, HepG2, and found that not only 22(R)-HC but also T3 induces SREBP-1c mRNA.

Unliganded TR may have a silencing effect on transcriptional regulation by LXR{alpha} and oxysterols
In addition to TR/T3-dependent stimulation of promoters that have LXREs, unliganded TRß1 exhibits a strong silencing effect on LXR{alpha}/oxysterol. Such a silencing effect on fatty acid synthesis may not be detected in animals that lack endogenous TR. In fact, Gullberg et al. (38) reported that after the administration of high-cholesterol chow, TRß knockout mice but not wild mice exhibit enhanced expression of the CYP7A gene, the promoter of which has a functional LXRE and is stimulated by 22(R)HC (9). Further studies on fatty acid synthesis in TR knockout mice or patients with RTH are intriguing.


   Acknowledgments
 
We are grateful to the following researchers for providing the plasmids: Dr. H. Shimano (Tsukuba University, Ibaraki, Japan) for pBP1c-2.6-Luc and pBP1c-186-Luc; Dr. D. J. Mangelsdorf (University of Texas, Dallas, TX) for tk-(LXRE)3-Luc; Dr. Kazuhiko Umesono (Kyoto University, Kyoto, Japan) for pCMX-hTRß1, GAL4-SMRT and TRß1-VP16; Akira Kakizuka (Kyoto University, Kyoto, Japan) for pCH111; and Takashi Nagaya (Nagoya University, Nagoya, Japan) for pRSV-C127S.


   Footnotes
 
This work was supported in part by a Health Sciences Research grant (to H.N.) and a Grant-in-Aid for Scientific Research (to H.M., S.S., and H.N.) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.

Abbreviations: ABC1, ATP-binding cassette transporter 1; ACC, acetyl-CoA carboxylase; DCC, dextran-coated charcoal; dMTV, deleted mouse mammary tumor virus promoter; DR4, direct repeat four; FAS, fatty acid synthase; GST, glutathione-S-transferase; Kd, dissociation constant; LTR, long-terminal repeat; Luc, luciferase; LXR, liver X receptor; LXRE, LXR response element; NCoR, nuclear receptor corepressor; 22(R)-HC, 22(R)-hydroxycholesterol; RSV, Rous sarcoma virus; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; SMRT, silencing mediator of retinoic acid; SREBP, sterol regulatory element binding protein; tk, thymidine kinase; TR, thyroid hormone receptor; TRE, T3 response element.

Received March 24, 2004.

Accepted for publication August 9, 2004.


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 Discussion
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