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Endocrinology, doi:10.1210/en.2006-1248
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Endocrinology Vol. 148, No. 4 1764-1773
Copyright © 2007 by The Endocrine Society

The Rat Thyroid Hormone Receptor (TR) {Delta}ß3 Displays Cell-, TR Isoform-, and Thyroid Hormone Response Element-Specific Actions

Clare B. Harvey, J. H. Duncan Bassett, Padma Maruvada, Paul M. Yen and Graham R. Williams

Molecular Endocrinology Group (C.B.H., J.H.D.B., G.R.W.), Division of Medicine and Medical Research Council (MRC) Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom; Molecular Regulation and Neuroendocrinology Section (P.M., P.M.Y.), Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and Cancer Biomarkers Research Group (P.M.), Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; and Endocrinology Division (P.M.Y.), Department of Medicine, Johns Hopkins Bayview Medical Center, Baltimore, Maryland 21224

Address all correspondence and requests for reprints to: Graham Williams, Molecular Endocrinology Group, MRC Clinical Sciences Centre, Clinical Research Building 5th Floor, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom. E-mail: graham.williams{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The THRB gene encodes the well-described thyroid hormone (T3) receptor (TR) isoforms TRß1 and TRß2 and two additional variants, TRß3 and TR{Delta}ß3, of unknown physiological significance. TRß1, TRß2, and TRß3 are bona fide T3 receptors that bind DNA and T3 and regulate expression of T3-responsive target genes. TR{Delta}ß3 retains T3 binding activity but lacks a DNA binding domain and does not activate target gene transcription. TR{Delta}ß3 can be translated from a specific TR{Delta}ß3 mRNA or is coexpressed with TRß3 from a single transcript that contains an internal TR{Delta}ß3 translation start site. In these studies, we provide evidence that the TRß3/{Delta}ß3 locus is present in rat but not in other vertebrates, including humans. We compared the activity of TRß3 with other TR isoforms and investigated mechanisms of action of TR{Delta}ß3 at specific thyroid hormone response elements (TREs) in two cell types. TRß3 was the most potent isoform, but TR potency was TRE dependent. TR{Delta}ß3 acted as a cell-specific and TRE-dependent modulator of TRß3 when coexpressed at low concentrations. At higher concentrations, TR{Delta}ß3 was a TRE-selective and cell-specific antagonist of TR{alpha}1, -ß1, and -ß3. Both TRß3 and TR{Delta}ß3 were expressed in the nucleus in the absence and presence of hormone, and their actions were determined by cell type and TRE structure, whereas TR{Delta}ß3 actions were also dependent on the TR isoform with which it interacted. Analysis of these complex responses implicates a range of nuclear corepressors and coactivators as cell-, TR isoform-, and TRE-specific modulators of T3 action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE (T3) ACTIONS are mediated by nuclear receptors that function as ligand-inducible transcription factors. The T3 receptor (TR) genes THRA and THRB are conserved in all vertebrates and encode TR{alpha} and TRß proteins (1, 2, 3). TRs bind to discrete thyroid hormone response elements (TREs) of varying sequence and arrangement located within the promoter regions of T3-responsive target genes (4, 5). Unliganded apoTRs bind to TREs as homodimers or in heterodimer complexes with retinoid X receptors (RXR), whereas liganded TRs bind DNA as RXR/TR heterodimers (2). ApoTRs inhibit basal transcription of T3 target genes by interacting preferentially with corepressor proteins, such as nuclear receptor corepressor (NCoR), that form additional complexes with histone deacetylases leading to repression of gene transcription. T3 binding results in a conformational change in the TR leading to release of NCoR and the recruitment of coactivator proteins, such as steroid receptor coactivator-1 (SRC-1), that possess histone acetyl transferase activity and reverse the histone deacetylation associated with basal repression (1, 2, 3, 5). Subsequent recruitment of a large transcription factor complex known as vitamin D receptor interacting protein/TR-associated protein (DRIP/TRAP) to the TR/SRC-1 coactivator leads to binding and stabilization of RNA polymerase II and hormone-dependent activation of transcription (5, 6, 7).

The well-described TR{alpha}1, -ß1, and -ß2 isoforms bind DNA and T3 and act as functional apo- and liganded TRs, whereas TR{alpha}2 does not bind T3 and acts as a weak antagonist in vitro (1, 2, 3, 5). The various TR isoforms are expressed in temporospatial-specific patterns during development (8, 9) and in distinct ratios in adult tissues (5), and studies of TR-knockout and mutant mice have indicated specific roles for TR{alpha} and TRß as well as functional redundancy (10, 11). For example, TR{alpha} mediates important T3 actions during heart, bone, and intestinal development and controls basal heart rate and body temperature in adults (12, 13, 14, 15, 16, 17, 18, 19), whereas TRß mediates T3 action in liver (20) and is responsible for regulation of the hypothalamic-pituitary-thyroid axis (21, 22). Detailed analysis of TRß indicates that TRß1 is expressed in most tissues, whereas TRß2 is restricted to the hypothalamus, pituitary, cochlea, and retina (23, 24, 25). Studies of TRß and TRß2 knockout mice indicate that TRß1 is essential for development of auditory function, whereas TRß2 is not required (22, 26, 27), but that TRß2 alone is essential for development of M-cone photoreceptors (28). In contrast, both TRß1 and TRß2 are necessary for regulation of the hypothalamic-pituitary-thyroid axis (27, 29). We recently cloned two additional rat TRß isoforms, TRß3 and TR{Delta}ß3 (30). TRß3 is a functional T3 receptor, whereas TR{Delta}ß3 lacks a DNA-binding domain but retains T3-binding activity and acts as a potent antagonist in vitro (30). Although TRß3 and {Delta}ß3 are expressed widely, their actions have not been characterized in detail, and their physiological importance is unknown.

Resistance to thyroid hormone (RTH) is an autosomal dominant but heterogeneous condition caused by a large number of described mutations of THRB, which result in expression of dominant-negative TRß proteins that inhibit T3-target gene expression in a wide range of tissues by several possible mechanisms (31, 32). In vitro analyses of mutant TRs have revealed the mutant receptors fail to mediate a transcriptional response to T3 but also interfere with wild-type TR{alpha} and TRß function. Full and potent dominant-negative activity of mutant TRs requires them to retain the ability to bind DNA and to form homodimers and RXR/TR heterodimers (32). The precise mechanism resulting in dominant-negative activity has not been determined, but mutant TRs that fail to interact with coactivators (33, 34) or are defective in T3-induced release of corepressors (35, 36) have been identified in RTH patients. These findings suggest that dominant-negative activity in RTH is mediated by transcriptionally inactive complexes that contain mutant TRs and bind to TREs (32).

The aims of these studies were to determine whether TRß3 and -{Delta}ß3 are conserved among vertebrate species and to characterize their functional activities in comparison with known TRs. The mechanism of TR{Delta}ß3 action was particularly investigated because, in contrast to RTH mutant TRs, it lacks a DNA-binding domain (30). Thus, TRß3 and -{Delta}ß3 were studied in two cell types, and T3 responses on four TREs were characterized. Mechanisms of action were studied by generating TRß1, -ß3, and -{Delta}ß3 mutants with impaired T3 binding, heterodimerization, NCoR release, or coactivator interaction activities.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TRß1, -ß3, and -{Delta}ß3 mutants
A TRß3 mutant (TRß3mut), in which the in-frame TR{Delta}ß3 AUG start codon at position 103 of TRß3 is mutated to CTG, was described previously (30). Four well-characterized TRß1 mutants (CGG->CAG to generate R243Q, TCC->TAC for S314Y, TTG->GCG for L454A, and CTG->CGG for L428R) and equivalent TRß3 (R172Q, S243Y, L383A, and L357R) and TR{Delta}ß3 (R70Q, S141Y, L281A, and L255R) mutants were generated by site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit; Stratagene, Amsterdam, The Netherlands) and sequenced. TRß1 R243Q is a naturally occurring RTH mutant that has impaired ability to release NCoR (36, 37, 38, 39). T3-binding activity of R243Q in solution is similar to wild-type TRß1, but when bound to DNA with NCoR, T3 binding is impaired significantly (36, 37). A 5- to 10-fold increased T3 concentration can overcome the dominant-negative activity of R243Q and restore normal transactivation function by inducing release of NCoR (36, 38). TRß1 S314Y is a dominant-negative RTH mutant that fails to bind T3 and exhibits no response to hormone (40). TRß1 L454A contains an artificial mutation in the activation function-2 domain. This mutant binds T3 and DNA normally and interacts with retinoid X receptors (RXR) but fails to interact with coactivators, resulting in no response to T3 (34, 41, 42). TRß1 L428R contains an artificial mutation in the dimerization domain, which results in impaired RXR heterodimerization but preserved homodimerization activity and an inability to bind T3 and no transactivation response (43, 44, 45).

Cell culture and transfections
Rat osteoblastic osteosarcoma ROS 17/2.8 cells were maintained in Ham’s F12 medium plus 5% fetal calf serum (FCS). Monkey kidney fibroblast COS-7 cells were cultured in DMEM plus 5% FCS. For transfections, ROS 17/2.8 or COS-7 cells were seeded in six-well plates (105 cells per well) containing Ham’s F12 or DMEM plus 5% charcoal-stripped FCS (CSS) (46) and transferred to serum-free medium before transfection using Lipofectamine PLUS (Invitrogen-Life Technologies, Inc., Paisley, UK) as described (30). Cells were transfected with 500 ng luciferase reporter gene driven by a thymidine kinase promoter controlled by TREs from the rat malic enzyme (ME) or {alpha}-myosin heavy chain (MHC) genes (47), by two copies of a palindromic TRE (PAL) (48) or a synthetic TRE containing a repeat of the hexanucleotide sequence AGGTCA separated by direct repeat + 4 (DR4) (Fig. 1Go), 40–200 ng TR plasmid (wild-type or mutant TR{alpha}1, TR{alpha}2, TRß1, TRß3, or TR{Delta}ß3) (30, 49, 50, 51), 100 ng Renilla internal control reporter (Promega, Southampton, UK), and pCDM8 empty vector carrier DNA to a total of 1.5 µg DNA per well. After 3 h, 1 ml 10% CSS medium was added and cells were incubated for 24 h. Transfected cells from each well were split into four in a 24-well plate containing 5% CSS medium without or with T3 (10–10–10–6 M) and incubated for 48 h. Reporter gene activities were determined as described (30). and luciferase activity was normalized to Renilla activity before analysis of responses to T3. Expression of transfected TRß1, TRß3, and TR{Delta}ß3 proteins was analyzed by Western blotting as described (30) using a monoclonal antibody (MA1–215; Affinity Bioreagents, Cambridge BioScience, Cambridge, UK) against TRß1 amino acids 235–414, which are conserved in TRß3 and TR{Delta}ß3.


Figure 1
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  		FIG. 1. Comparison of TRE structures in reporter genes used for transfection experiments. 2xPAL shows two copies of the palindromic TRE included in the PAL reporter; rMHC shows the rat MHC gene TRE located at nucleotide position –139 to –159 upstream of the transcription start site in the native gene; rME shows the rat ME gene TRE located at position –287 to –260; DR4 shows the synthetic direct repeat TRE included in the DR4 reporter (4 47 48 ). Solid arrows indicate orientations of consensus and near-consensus hexamer sequences (in bold) that bind TR proteins in gel-shift studies and that have been shown to be required to mediate maximal T3 responses in transfections. Dashed arrows indicate sequences (in bold) required for T3 responses in transfections but that interact only weakly with TRs in gel shifts (4 ).

 
Subcellular localization of TRß1, TRß3, and TR{Delta}ß3 proteins
Plasmid vectors encoding various green fluorescent protein (GFP)-TR fusion proteins were constructed as described (52). HeLa cells were maintained in DMEM with 10% FCS, antibiotics (100 U/ml penicillin and streptomycin and 0.5 ml gentamycin; Invitrogen) and 2 mM L-glutamine. For transient transfections, HeLa cells were cultured in six-well plates on coverslips at the density of 1 x 105 cells per well and transfected with various GFP-TR isoforms (100 ng/well) in serum-free medium using Lipofectamine PLUS according to the manufacturer’s instructions. The cells expressing various GFP-TR fusion proteins were viewed under a Leica TCS SP laser scanning confocal microscope mounted on a DMIRBE inverted epifluorescent microscope equipped with x63 magnification using a 1.4 numerical aperture oil immersion lens (Leica, Heidelberg, Germany). The GFP was excited at 488 nm from an air-cooled fiber-coupled argon laser at less than 10% of maximal power. GFP fluorescence was visualized using a 1.0 Airy unit pinhole and analyzed as described (53).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The TRß3/{Delta}ß3 locus is detected only in rats but not in other species
In the rat, TRß3/{Delta}ß3 locus exons A and B lie directly 5' to the first common exon of TRß (exon 3) with no intervening introns (30). To investigate the possible presence of TRß3 and TR{Delta}ß3 in other vertebrates, we identified genomic DNA sequences 5' to the first common TRß exon in eight species using Ensembl (http://www.ensembl.org/index.html) and Entrez nucleotide (http://www.ncbi.nlm.nih.gov) searches (Fig. 2Go). Open reading frames of between 25 and 133 bp were identified immediately upstream of an invariant splice site, termed the changing point (54). In-frame ATG codons, as previously identified in rat (GenBank accession no. AF239916.1), were also present in mouse (GenBank AC154626), dog (Ensembl no. ENSCAFG00000005741), and chicken (Ensembl ENSGALG00000011294) sequences but not in human (GenBank AC093927), chimpanzee (Ensembl ENSPTRG00000014697), macaque (Ensembl ENSMMUG00000000067), or zebra fish (GenBank BX927163). However, none of these ATG codons were positioned within a favorable Kozak translation initiation sequence context (55, 56). Blast searches (http://www.ncbi.nlm.nih.gov/BLAST) using these 5' sequences identified the previously published rat TRß3 and TR{Delta}ß3 sequences but no additional TRß transcripts or expressed sequence tags. Furthermore, amino acid sequence searches (rpsblast) using predicted sequences derived from the upstream open reading frames did not identify protein homology or conserved domain structures. Comparison of the predicted amino acid sequences upstream of the common TRß protein revealed 50% identity between rat and mouse but no homology between rat and dog or chicken. Thus, TRß3 may be present only in rodents and is not found in primates or other vertebrates. The lack of murine TRß3 or TR{Delta}ß3 expressed sequence tags, however, suggests that expression from this locus is unique to rats.


Figure 2
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  		FIG. 2. A, Schematic representation of the rat TRß3 locus. The first TRß common exon (exon 3) is shown as a black box, and its splice acceptor site is termed the changing point. B, The open reading frame that lies 5' of exon 3 and in frame with the common TRß coding sequence is shown in eight species. The common exon 3 sequence is underlined, and an arrow indicates the changing point. In-frame stop codons are shown in bold and ATG codons in bold underlined text. C, Predicted amino acid sequences of in-frame open reading frames identified 5' to exon 3. The changing point is shown by the arrow, and the first three amino acids encoded by exon 3 are underlined. Predicted methionine amino acids are shown in bold, and the dash at the beginning of each open reading frame indicates the location of an in-frame stop codon.

 
TRß3 exerts cell- and TRE-specific actions
COS-7 and ROS 17/2.8 cells were transfected with PAL, ME, MHC, or DR4 reporters and increasing concentrations (0–200 ng) of TR{alpha}1, -{alpha}2, -ß1, -ß3, or -{Delta}ß3 to optimize the TR concentration for additional studies. T3 responses of each TRE increased with increasing concentration of TR{alpha}1, -ß1, or -ß3, and the maximum response in both cell lines was seen after addition of 160 ng receptor (data not shown). Responses mediated by TR{alpha}1 (7.9 ± 1.2-fold in COS-7; 7.7 ± 1.0-fold in ROS 17/2.8), TRß1 (4.9 ± 0.3-fold in COS-7; 3.3 ± 1.0-fold in ROS 17/2.8), or TRß3 (11.0 ± 1.1-fold in COS-7; 7.2 ± 1.0-fold in ROS 17/2.8) were greatest on the PAL TRE in both cell types. TR{alpha}1 and -ß3 mediated a greater T3 induction of PAL than TRß1 in both cell types, whereas TRß3 was also more potent than TR{alpha}1 in COS-7 cells (Fig. 3Go). In contrast, T3 responses mediated by each receptor on the ME, MHC, and DR4 TREs were similar in both cell types, although responses of all elements tended to be lower in ROS 17/2.8 compared with COS-7 cells. TR{alpha}2 and -{Delta}ß3 did not mediate reporter gene responses on any TRE in either cell type (Fig. 3Go and data not shown).


Figure 3
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  		FIG. 3. COS-7 or ROS 17/2.8 cells transfected with TR{alpha}1, -ß1, -ß3, or -{Delta}ß3 (160 ng) and a luciferase reporter controlled by either an ME, MHC, PAL, or DR4 TRE. T3 induction of each element mediated by each receptor in both cell lines is shown. Luciferase activity was normalized to activity of a Renilla internal control vector, and results are expressed as mean T3 induction ratio (± SEM), calculated by dividing normalized luciferase activities after T3 treatment by basal values (n = 3–5 experiments, three to six replicates per experiment; ANOVA followed by Tukey’s multiple comparison post hoc tests: **, P < 0.01, ***, P < 0.001 vs. T3 induction of PAL by TRß1 or TRß3 in COS-7 cells; ##, P < 0.01 vs. T3 induction of PAL by TR{alpha}1 or TRß3 in ROS 17/2.8 cells; ^, P < 0.05; ^^^, P < 0.001 vs. T3 induction mediated by TRß1; +, P < 0.05 vs. T3 induction mediated by TRß3).

 
In the absence of T3, unliganded apoTRs bind corepressors and inhibit basal target gene expression. An exchange of cofactors occurs after addition of T3, and ligand-bound TRs interact with coactivators to stimulate target gene expression. Thus, the response to T3 is a two-stage process; T3 activation of gene transcription follows relief of apoTR-mediated repression (1, 2, 3, 5), and the T3 induction ratio mediated by TRs is calculated by dividing reporter gene activity in the presence of T3 by reporter gene activity in the absence of T3. To investigate whether the differing activities of TR{alpha}1, -ß1, or -ß3 result from differences in apoTR-mediated repression, TRE responses to each receptor were determined in the absence of T3. ApoTR{alpha}1 repressed ME by 40 ± 13% in COS-7 cells (P < 0.05) but increased its expression by 40 ± 23% in ROS 17/2.8 cells (P < 0.05), indicating cell-specific activity of apoTR{alpha}1 on the ME element. ApoTRß1 repressed PAL by 37 ± 16% in COS-7 cells (P < 0.05) and 36 ± 20% in ROS 17/2.8 cells (P < 0.05) and repressed ME by 36 ± 8% in ROS 17/2.8 cells (P < 0.01). ApoTRß3 repressed DR4 expression by 36 ± 11% in ROS 17/2.8 cells (P < 0.05) only. ApoTR{Delta}ß3 repressed PAL by 18 ± 4% in ROS 17/2.8 cells (P < 0.01) and MHC by 32 ± 12% in COS-7 cells (P < 0.05). Thus, effects of unliganded apoTRs on basal gene expression were dependent on the cell type and TRE and contribute to the complexity of cell- and gene-specific responses to T3. ApoTRß3, however, had the weakest effect on basal gene transcription, indicating that TRß3 activity results predominantly from T3-mediated effects rather than actions of the unliganded aporeceptor.

TR{Delta}ß3 is coexpressed at low concentrations along with TRß3 from a single transcript and acts as a TRE-selective modulator of TRß3 action
In previous studies, in vitro transcription-translation of TRß3 cDNA resulted in expression of a 45-kDa TRß3 protein together with a 32.5-kDa TR{Delta}ß3 protein originating from an in-frame AUG codon, whereas transcription-translation of TRß1 resulted in a single 55-kDa TRß1 protein (30). In the current studies, transfection of COS-7 cells with TRß3 similarly resulted in coexpression of a low relative concentration of TR{Delta}ß3, whereas transfection with TRß1 resulted only in expression of TRß1 (Fig. 4Go, A and B).


Figure 4
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  		FIG. 4. A, Diagram of TRß3 and TRß3mut mRNAs and translated TRß3 and TR{Delta}ß3 proteins in comparison with TRß1 protein. TRß3 mRNA contains a 584-nucleotide 5'-untranslated region, an open reading frame at nucleotide 585 that encodes TRß3 (390-amino-acid protein, predicted molecular mass, 44.6 kDa), an in-frame open reading frame at nucleotide 894 that encodes TR{Delta}ß3 (288-amino-acid protein, predicted molecular mass, 32.8 kDa), and a stop codon at position 1757. In TRß3mut mRNA, the AUG codon at nucleotide 894 is mutated to CUG so that only TRß3 protein (in which methionine at position 103 is replaced by leucine) is translated. TRß1 is a 461-amino-acid protein of predicted molecular mass 52.6 kDa that shares 100% identity with TRß3 apart from the first 94 amino acids of TRß1, which are replaced by 23 different amino acids in TRß3 (30 ). B, Extracts prepared from COS-7 cells transfected with 20 ng empty vector (CON) or TRß1, -ß3, or -{Delta}ß3 were analyzed by Western blotting using a TRß-specific MA1–215 antibody that recognizes all TRß isoforms. Cells transfected with TRß1 express a single 55-kDa protein. Transfection of TRß3 results in coexpression of 45- and 32.5-kDa ß3 and {Delta}ß3 proteins. Transfection with TR{Delta}ß3 results in expression of a single 32.5-kDa protein. C, COS-7 cells transfected with either TRß3 or TRß3mut cDNA (0–200 ng), in which the AUG at position 894 that initiates translation of TR{Delta}ß3 was mutated to CTG. Responses of PAL or MHC luciferase reporters to TRß3 ({diamondsuit}) or TRß3 mut ({blacksquare}) are plotted. Reporter gene activity was normalized to Renilla, and results are shown as luciferase activity in the absence or presence of T3 relative to activity under each condition in the absence of cotransfected receptor, which was normalized to a value of 1. Values less than 1 in the absence of T3 indicate repression by unliganded aporeceptor; values greater than 1 after addition of hormone indicate T3 response. Results are also expressed as mean T3 induction ratios (± SEM), calculated by dividing relative luciferase activities after T3 treatment by basal values (n = 3–5 experiments, three to six replicates per experiment; ANOVA followed by Tukey’s multiple comparison post hoc tests: *, P < 0.05; **, P < 0.01 response to TRß3 vs. TRß3mut).

 
To investigate the effect of TR{Delta}ß3 on activity of ß3, COS-7 cells were transfected with either a wild-type TRß3 cDNA (from which both TRß3 and TR{Delta}ß3 are coexpressed) or a TRß3mut cDNA (in which the in-frame AUG codon at position 103 is mutated to prevent coexpression of TR{Delta}ß3) (30).

Coexpression of TRß3 and TR{Delta}ß3 proteins in cells transfected with increasing concentrations of wild-type TRß3 cDNA in the absence of T3 resulted in reduced activity of the PAL element only after transfection of the highest concentration of plasmid (200 ng). By contrast, there was a concentration-dependent reduction in activity of MHC in the absence of T3 (Fig. 4CGo), and similar effects were seen with ME (not shown). These findings indicate that coexpressed TR{Delta}ß3 protein cooperates with TRß3 protein to repress basal gene expression in the absence of T3 and that the MHC and ME TREs were more sensitive to this effect than PAL (Fig. 4CGo and data not shown).

T3 responses in the absence of TR{Delta}ß3 (cells transfected with TRß3mut cDNA) on the MHC and ME TREs were greater than responses in the presence of TR{Delta}ß3 (cells transfected with TRß3 cDNA) (Fig. 4CGo and data not shown), indicating that low concentrations of TR{Delta}ß3 protein also inhibit TRß3-mediated T3 responses on these elements. By contrast, the T3 response of PAL in the absence of TR{Delta}ß3 (cells transfected with TRß3mut cDNA) was much lower than the response in the presence of TR{Delta}ß3 (cells transfected with TRß3 cDNA). Thus, transcriptional repression and activation were both impaired in the absence of TR{Delta}ß3 protein, whereas in the presence of TR{Delta}ß3, basal transcription was repressed but T3 responsiveness was enhanced. These data indicate that low levels of coexpressed TR{Delta}ß3 protein potentiate T3 activation of the PAL element by the TRß3 receptor (Fig. 4CGo).

TR{Delta}ß3 exerts dominant-negative cell-,TRE-, and TR-specific actions
To determine whether TR{Delta}ß3 actions were concentration dependent or whether they differed between cell types, on distinct TREs, or in the presence of different TR isoforms, COS-7 or ROS 17/2.8 cells were transfected with a PAL, ME, MHC, or DR4 reporter and an optimized concentration of TR together with increasing concentrations of TR{Delta}ß3 (Fig. 5Go). In ROS 17/2.8 cells, TR{Delta}ß3 did not influence T3 responses mediated by TR{alpha}1, -ß1, or -ß3 on any TRE. In COS-7 cells, TR{Delta}ß3 repressed TR{alpha}1 activity on PAL [up to 2.3-fold (57%) repression], ME [up to 5.6-fold (82%) repression], and MHC [up to 2.8-fold (64%) repression] but not on DR4. TR{Delta}ß3 also repressed TRß1 on PAL [up to 5.6-fold (82%) repression] and ME [up to 2.4-fold (59%) repression] but not on MHC or DR4. TR{Delta}ß3 also repressed TRß3 on ME [up to 2.5-fold (61%) repression] but not on PAL, MHC, or DR4. Thus, TR{Delta}ß3 acted as a TR isoform-specific and TRE-selective dominant-negative antagonist in COS-7 cells.


Figure 5
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  		FIG. 5. COS-7 or ROS 17/2.8 cells transfected with TR{alpha}1, -ß1, or -ß3 (160 ng) and a PAL, ME, MHC, or DR4 reporter together with an increasing concentration of TR{Delta}ß3. TR{Delta}ß3-mediated dominant-negative activity at each element in the presence of each TR isoform in both cell lines is shown. Luciferase activity was normalized to activity of a Renilla internal control vector. T3 induction ratios mediated by each TR in the absence of cotransfected TR{Delta}ß3 were normalized to a value of 1. The mean fold dominant-negative activity was obtained by calculating the reciprocal of the T3 induction ratio in the presence of increasing concentrations of TR{Delta}ß3 (± SEM). Values greater than 1 show the degree of repression mediated by TR{Delta}ß3 and indicate its dominant-negative activity (n = 3–5 experiments, three to eight replicates per experiment; two-tailed paired Student’s t tests: *, P < 0.05; **, P < 0.01-fold dominant-negative activity mediated by TR{Delta}ß3 in COS-7 vs. ROS 17/2.8 cells).

 
The mechanism of action of TRß3 is similar to TRß1 in COS-7 cells
To investigate the mechanism of TRß3 and -{Delta}ß3 action in comparison with the known activity of TRß1, four well-characterized TRß1 (R243Q, S314Y, L454A, and L428R) and equivalent TRß3 (R172Q, S243Y, L383A, and L357R) and TR{Delta}ß3 (R70Q, S141Y, L281A, and L255R) mutants were generated. Mutant M1 (R243Q) has impaired release of NCoR, and its dominant-negative activity can be overcome by increased concentrations of T3 (36, 37, 38, 39). M2 (S314Y) does not bind or respond to T3 and acts as a dominant-negative antagonist (40). M3 (L454A) binds T3 but fails to respond because it does not interact with coactivators (34, 41, 42). M4 (L428R) interacts poorly with RXR, fails to bind T3, and does not respond to hormone (43, 44, 45).

Activities of each of the TRß1 and TRß3 mutants were determined on PAL, ME, and MHC in COS-7 cells to compare their functional properties. There were no significant differences in the activities of TRß1 and TRß3 M1 mutants in response to saturating concentrations of T3 (100 nM) compared with the responses of wild-type TRß1 and TRß3 on each TRE (Fig. 6AGo). Treatment of TRß1 or TRß3 M1 with a lower concentration of T3 revealed impaired activities in response to 1 nM T3 on the PAL TRE (TRß1, 2.69 ± 0.60 vs. 1.21 ± 0.16, P < 0.05; TRß3, 2.36 ± 0.48 vs. 1.08 ± 0.18, P < 0.05; T3 induction ratio mediated by wild-type TR vs. M1 mutant in response to 1 nM T3, two-tailed unpaired Student’s t tests; n = 4–7), whereas activities of wild-type ß1 and ß3 and M1 mutants were not different in the presence of saturating concentrations of ligand. Similar findings were obtained using the ME and MHC TREs (data not shown). The TRß1 M1 mutant releases NCoR only in the presence of higher concentrations of T3 compared with wild-type receptor (36, 37, 38, 39), and these data indicate the TRß3 M1 mutant acts similarly. The TRß1 and -ß3 M2, M3, and M4 mutants failed to respond to T3 on each of the three TREs studied. All effects of the four mutations on TR activity resulted from blockade of the T3 response rather than by an effect on apoTR activity (data not shown).


Figure 6
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  		FIG. 6. A, COS-7 cells transfected with TRß1 or TRß3 (160 ng) or mutant TRs (ß1 M1 = R243Q, M2 = S314Y, M3 = L454A, M4 = L428R; ß3 M1 = R172Q, M2 = S243Y, M3 = L383A, M4 = L357R) and a ME, MHC, or PAL reporter. T3 induction of each element mediated by each receptor or mutant is shown. Luciferase activity was normalized to Renilla and results expressed as mean T3 induction ratio (± SEM), calculated by dividing normalized luciferase activities after T3 treatment by basal values (n = 5–10 experiments, three replicates per experiment; ANOVA followed by Tukey’s multiple comparison post hoc tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001 induction mediated by mutant TR vs. wild type). B, COS-7 cells transfected with TRß1 or TRß3 (160 ng) and a PAL or ME reporter in the absence or presence of 240 ng wild-type TR{Delta}ß3 or mutant TR{Delta}ß3 ({Delta}ß3 M1 = R70Q, M2 = S141Y, M3 = L281A, M4 = L255R). Luciferase was normalized to Renilla, and T3 induction ratio mediated by TR in the absence of cotransfected {Delta}ß3 was normalized to a value of 1. Responses are shown as the mean T3 induction ratio mediated by TRß1 or TRß3 in the absence (–) or presence of wild-type or mutant TR{Delta}ß3 (± SEM) (n = 3–5 experiments, six to eight replicates per experiment; two-tailed paired Student’s t tests: *, P < 0.05 repression mediated by TR{Delta}ß3 mutant vs. wild-type TR{Delta}ß3). WT, Wild type.

 
Differential dominant-negative inhibition of TRß1 and TRß3 by wild-type and mutant TR{Delta}ß3
TR{Delta}ß3 mutants were employed to investigate the mechanism of dominant negative activity of this TR isoform (Fig. 6BGo). In contrast to wild-type TR{Delta}ß3, TR{Delta}ß3 mutant M1 did not inhibit the activities of wild-type TRß1 or TRß3 on either the PAL or ME TRE. The TR{Delta}ß3 mutants M2, M3, and M4, however, inhibited wild-type TRß1 activity on both TREs to a similar degree as the level of inhibition mediated by wild-type TR{Delta}ß3. In contrast, TR{Delta}ß3 mutants M2, M3, and M4 failed to inhibit wild-type TRß3 on PAL but inhibited its activity on ME. None of the TR{Delta}ß3 mutants affected apoTRß1 or apoTRß3 activities (data not shown).

TR{alpha}1, TRß1, TRß3, or TR{Delta}ß3 proteins are predominantly localized to the nucleus in the absence and presence of T3
To determine whether T3 affected the subcellular localization of TRs, HeLa cells were transfected with GFP-tagged TR{alpha}1, TRß1, TRß3, or TR{Delta}ß3 cDNAs in the absence or presence of T3, and localization of expressed proteins were determined by fluorescence confocal microscopy (Fig. 7Go). These studies revealed that TR{alpha}1 was exclusively localized in the nucleus in the absence and presence of T3. Treatment with T3, however, resulted in a change from a homogeneous nuclear distribution of TR{alpha}1 to a punctate pattern after addition of T3. Similar findings have been observed previously for TRs and other nuclear receptors, suggesting that ligand-induced intranuclear reorganization of nuclear receptors may be an important process required for hormone responsiveness (52, 53, 57, 58, 59). TRß1, -ß3, and -{Delta}ß3 proteins were also localized predominantly in the nucleus in the absence of T3, although a small fraction of receptor was also present in the cytoplasm.


Figure 7
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  		FIG. 7. HeLa cells transfected with GFP-tagged TR{alpha}1, TRß1, TRß3, or TR{Delta}ß3 constructs (100 ng/well) in the absence or presence of T3 (10 nM). Transfected cells were examined using a confocal fluorescence microscope, and GFP staining reveals the effect of T3 on the cellular localization of TR isoforms.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TR{alpha}1, TRß1, and TRß3 activities were highest on the PAL TRE and similar on the ME, MHC, and DR4 elements in both COS-7 and ROS 17/2.8 cells. The increased responsiveness of PAL is likely to be due to inclusion of two copies of the TRE in the reporter construct. TRß3 mediated the greatest T3 response on PAL and was more potent than both TR{alpha}1 and TRß1 in both cell types. The TRß3>TR{alpha}1>TRß1 hierarchy of potencies, however, was not evident for activation of ME, MHC, and DR4 elements. Consequently, the reduced T3 sensitivities of ME, MHC, and DR4 resulted in a failure to discriminate between the relative potencies of TR{alpha}1, TRß1, and TRß3 on these elements. This suggests that differences in activities of TR isoforms may only be apparent on certain genes that contain more highly responsive TREs. In this model, the sequence and arrangement of the TRE would determine T3 sensitivity of a particular target gene and provide a mechanism to account for TR isoform selectivity, whereas less responsive genes would lack such selectivity.

Analysis of apoTR- and ligand-induced TR actions also revealed differences between TR isoforms. ApoTR{alpha}1 repressed basal activity of ME in COS-7 cells but activated the same element in ROS 17/2.8 cells, whereas no differences were observed on other TREs. These divergent effects on ME expression in COS-7 and ROS 17/2.8 cells suggests the two cell types express a different repertoire of cofactors that interact with TR{alpha}1 in the absence of T3. Alternatively, the same cofactor could be modified differently in the two cell types, for example by methylation, to alter its functional properties from that of a coactivator to a corepressor (60). By contrast, the similar actions of apoTRß1 and apoTRß3 in both cell types suggest that cofactors interacting with TRß in COS-7 and ROS 17/2.8 cells are not functionally distinct. Thus, apoTR{alpha}1 mediated responses that discriminate between TREs and cell types, whereas apoTRß1 and apoTRß3 did not. Studies of TR{alpha}/SRC-1 and TRß/SRC-1 double-knockout mice have revealed that SRC-1 interacts differently with TR{alpha} and TRß in the pituitary (61), with data supporting the hypothesis that a functional interaction occurs between TR{alpha} and SRC-1 in the absence of ligand, whereas in the presence of T3, SRC-1 interacts functionally with TRß. This study indicates that TR-interacting cofactors can also discriminate between TR isoforms.

TRß3 is coexpressed with TR{Delta}ß3 from a single TRß3 mRNA transcript. Deletion of the TR{Delta}ß3 initiation codon in TRß3mut results in expression of TRß3 alone and allows investigation of TR{Delta}ß3 action. Basal repression of ME and MHC was absent after transfection of TRß3mut and the response to T3 was increased. These findings indicate that coexpressed TR{Delta}ß3 modulates basal activity of ME and MHC in the absence of ligand and inhibits TRß3-mediated T3 activation in the presence of ligand. TR{Delta}ß3 lacks a DNA-binding domain but binds T3 with equal affinity to TRß3 (30), indicating TR{Delta}ß3 is functional in the absence and presence of T3 and its actions are independent of DNA binding. Inhibition of the activities of TRß3 on the ME and MHC elements by TR{Delta}ß3 is likely, therefore, to result from sequestration of cofactors that are necessary for the actions of apo- and liganded TRß3 (1, 2, 3, 5, 7). The actions of TR{Delta}ß3 at the PAL TRE, however, were different. Absence of basal repression after transfection of TRß3mut and a reduced response of PAL to liganded TRß3 indicates that coexpressed TR{Delta}ß3 also inhibits basal expression of PAL but surprisingly increases TRß3-mediated T3 activation. This finding suggests the sequence or arrangement of PAL influences the activity of TR{Delta}ß3 even though TR{Delta}ß3 lacks a DNA-binding domain. Previous studies indicate that RXR/TR heterodimers adopt different conformations when bound to different TREs, and RXR/TR interactions with TREs are also modulated by cofactors (43, 62, 63, 64). Thus, RXR/TRß3, when bound to PAL in the presence of T3, may interact with a different coactivator complex compared with when it is bound to ME or MHC. Such subtle TRE- or tissue-specific alteration of TR action (65, 66, 67) could influence the activity of TR{Delta}ß3. In this model, interaction of TR{Delta}ß3 with distinct coactivators would result in specific modifications that alter how coactivators interact with RXR/TRß3/TRE complexes, thereby accounting for divergent effects of TR{Delta}ß3 on TRß3 function that are determined by TRE structure. Although our experiments suggest a functional role for coexpressed TR{Delta}ß3 in modulating TRß3 action, it is important to consider that some of the observed effects after transfection of TRß3mut could result from increased TRß3 expression rather than a lack of TR{Delta}ß3. Nevertheless, in Fig. 4BGo, it is clear that the ratio of coexpressed TRß3 to TR{Delta}ß3 is very high after transfection of intact TRß3. Thus, as in previous studies with in vitro translated TRß3 and TRß3mut (30), transfection of an equivalent concentration of TRß3mut would have a negligible effect on the total amount of expressed TRß3, supporting the view that coexpressed TR{Delta}ß3 regulates TRß3 activity.

Activity of TR{Delta}ß3 was examined further by cotransfecting increasing concentrations of TR{Delta}ß3 with TR{alpha}1, TRß1, or TRß3 in either COS-7 or ROS 17/2.8 cells. TR{Delta}ß3 displayed cell type-, TRE-, and TR isoform-selective antagonist actions. TR{Delta}ß3 was inactive in ROS 17/2.8 cells, suggesting TR{Delta}ß3-interacting cofactors are not expressed or that ROS 17/2.8 cells express an inhibitor that prevents TR{Delta}ß3 interaction with cofactors. Similarly, TRE- and TR isoform-selective actions of TR{Delta}ß3 are likely due to sequestration of discrete cofactor complexes that differentially interact with RXR/TR{alpha}1, RXR/TRß1, or RXR/TRß3 on specific TREs. To address mechanisms underlying the complex actions of TR{Delta}ß3, well-described TRß1 mutants with defective NCoR interaction (36, 37, 38, 39), T3 binding affinity (40), coactivator interaction (34, 41, 42), or RXR heterodimerization activity (43, 44, 45) were synthesized in TR{Delta}ß3. Comparison of the activities of the TR{Delta}ß3 mutants with the activity of wild-type TR{Delta}ß3 on PAL and ME TREs suggests that TR{Delta}ß3 inhibition of TRß1 on both elements and TRß3 on ME requires TR{Delta}ß3 to interact fully with NCoR but does not require interactions between TR{Delta}ß3 and T3, SRC-1, or RXR. In contrast, TR{Delta}ß3 inhibition of TRß3 on PAL requires TR{Delta}ß3 to interact with T3 and all these cofactors. These results suggest further that TRE structure can determine TR{Delta}ß3 activity indirectly via modification of cofactor interactions.

It is noteworthy that TRß3 and TR{Delta}ß3 have restricted patterns of expression in vivo (30). In the current studies, TRß3 acted mainly as an activator in response to T3, whereas TR{Delta}ß3 was mainly a repressor. Thus, tissues such as kidney and liver that express high levels of TRß3 would be expected to be more sensitive to T3 than spleen and lung, which express mainly TR{Delta}ß3 (30). In contrast, cerebral cortex and heart express similar levels of TRß3 and TR{Delta}ß3. The adult cerebral cortex is regarded as being largely unresponsive to T3, whereas heart is a classical T3 target tissue. Interestingly, TR{Delta}ß3 expression was markedly reduced in heart from thyroidectomized rats, and TRß3 expression was increased, whereas no effect of thyroid status on the TRß3:{Delta}ß3 ratio was found in cerebral cortex (30). These findings suggest that TR{Delta}ß3 may inhibit T3 responses in cerebral cortex and heart in euthyroid animals but that down-regulation of TR{Delta}ß3 and up-regulation of TRß3 in cardiac muscle after thyroidectomy may account in part for increased T3 sensitivity of the hypothyroid heart (68). Changes in the relative levels of TRß3 and TR{Delta}ß3 in other tissues in response to alterations of thyroid status (30) may also contribute to the diverse effects of hypothyroidism and thyrotoxicosis by modulating tissue-specific interactions among TR isoforms and cofactors.

In summary, these studies demonstrate that TR actions are highly specific. Combinatorial interactions between TR{alpha}1, TRß1, or TRß3 and TR{Delta}ß3 isoforms, individual TREs, and cell-specific cofactors result in enormous potential for modification of T3 responses and a high level of complexity. All the TR isoforms studied, including TR{Delta}ß3, were mainly localized in the nucleus in the absence and presence of T3, indicating that fine tuning of T3 action is predominantly a nuclear event. Although the ratios of expressed TR isoforms and available target gene TREs in individual cell types are important determinants of T3 action, these studies also implicate a range of TR-interacting nuclear cofactors as key cell- and TRE-specific modulators of T3 action. A major challenge for the future will be to identify and characterize them.


   Acknowledgments
 
We are grateful to Liza Jinadu for valuable technical assistance.


   Footnotes
 
This work was supported by: Medical Research Council (MRC) Career Establishment Grant (G9803002) and Wellcome Trust Project Grant (50570) to G.R.W. and an MRC Clinician Scientist Fellowship to J.H.D.B.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 11, 2007

Abbreviations: CSS, Charcoal-stripped FCS; DR4, direct repeat + 4; FCS, fetal calf serum; GFP, green fluorescent protein; ME, malic enzyme; MHC, {alpha}-myosin heavy chain; NCoR, nuclear receptor corepressor; PAL, palindromic TRE; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; SRC-1, steroid receptor coactivator-1; TR, T3 receptor; TRE, thyroid hormone response element.

Received September 13, 2006.

Accepted for publication January 2, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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P. J. O'Shea, C. J. Guigon, G. R. Williams, and S.-y. Cheng
Regulation of Fibroblast Growth Factor Receptor-1 (Fgfr1) by Thyroid Hormone: Identification of a Thyroid Hormone Response Element in the Murine Fgfr1 Promoter
Endocrinology, December 1, 2007; 148(12): 5966 - 5976.
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