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Endocrinology, doi:10.1210/en.2007-0114
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Endocrinology Vol. 148, No. 12 5966-5976
Copyright © 2007 by The Endocrine Society

Regulation of Fibroblast Growth Factor Receptor-1 (Fgfr1) by Thyroid Hormone: Identification of a Thyroid Hormone Response Element in the Murine Fgfr1 Promoter

Patrick J. O’Shea, Celine J. Guigon, Graham R. Williams and Sheue-yann Cheng

Gene Regulation Section (P.J.O, C.J.G., S.C.), Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264; and Molecular Endocrinology Group (P.J.O., G.R.W.), Division of Medicine and Medical Research Council Clinical Sciences Centre, Imperial College London, Hammersmith Campus, London W12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Dr. Sheue-yann Cheng, Chief, Gene Regulation Section, Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264. E-mail: chengs{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T3 is essential for normal skeletal development, acting mainly via the TR{alpha}1 nuclear receptor. Nevertheless, the mechanisms of T3 action in bone are poorly defined. Fibroblast growth factor receptor-1 (FGFR1) is also essential for bone formation. Fgfr1 expression and activity are positively regulated by T3 in osteoblasts, and in mice that harbor a dominant negative PV mutation targeted to TR{alpha}1 or TRß, Fgfr1 expression is sensitive to skeletal thyroid status. To investigate mechanisms underlying T3 regulation of FGFR1, we obtained primary calvarial osteoblasts from wild-type and TRßPV/PV littermate mice. T3 treatment increased Fgfr1 expression 2-fold in wild-type cells, but 8-fold in TRßPV/PV osteoblasts. The 4-fold increased T3 sensitivity of TRßPV/PV osteoblasts was associated with a markedly increased ratio of TR{alpha}1:TRß1 expression that resulted from reduced TRß1 expression in TRßPV/PV osteoblasts compared with wild-type. Bioinformatics and gel shift studies, and mutational analysis, identified a specific TR binding site 279–264 nucleotides upstream of the murine Fgfr1 promoter transcription start site. Transient transfection analysis of a series of Fgfr1 promoter 5'-deletion constructs, of a mutant reporter construct, and a series of heterologous promoter constructs, confirmed that this region of the promoter mediates a TR-dependent transcriptional response to T3. Thus, in addition to indirect regulation of FGFR1 expression by T3 reported previously, T3 also activates the Fgfr1 promoter directly via a thyroid hormone response element located at positions –279/–264.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T3 ACTIONS ARE mediated by TRs, which act as ligand-dependent transcription factors. Alternative splicing of the two TR genes, Thra encoding TR{alpha} and Thrb encoding TRß, contributes to multiple protein isoforms (1). The ontogeny of TR expression is regulated in a temporospatial manner, and, consequently, the ratio of TR{alpha} and ß-isoforms expressed in individual tissues is variable. TRs bind to thyroid hormone response elements (TREs) located within the promoters of T3-responsive target genes. TREs generally comprise two hexameric half-sites, and previous studies have identified a half-site consensus sequence of G/A G G T C/G/A A (2, 3, 4, 5). Half-site hexamers are arranged either as a direct repeat separated by a spacer of four bases (DR4), a palindrome (PAL), or an inverted repeat (1). In the absence of T3, TRs repress basal target gene transcription by interacting with corepressor proteins, such as NCoR and SMRT. Corepressors possess histone deacetylase activity and modify the structure of chromatin to restrict access to the basal transcriptional machinery. Binding of T3 to the TR causes a conformational change that results in release of corepressors and the recruitment of a range of coactivators, including the steroid receptor coactivator-1 (SRC1) and CBP/p300. Coactivator proteins possess histone acetyltransferase and methyltransferase activities, which induce changes in chromatin structure to promote transcription by increasing accessibility to the transcriptional machinery (1, 6).

T3 is a principal regulator of growth and bone development (4, 7). Thyroid hormone deficiency in children causes growth arrest and delayed bone age with disruption of epiphyseal growth plate architecture, whereas treatment with T4 induces "catch-up" growth and restores bone development (8). Thyroid hormone excess during childhood accelerates bone formation, advances bone age but paradoxically causes short stature because of premature fusion of the epiphyses. Early fusion of the sutures of the skull may also result in craniosynostosis (9). Autosomal dominant resistance to thyroid hormone (RTH) is characterized by hypothalamic-pituitary-thyroid axis dysfunction and reduced tissue sensitivity to thyroid hormones. Affected individuals possess dominant-negative mutant TRß proteins. The clinical features of RTH are diverse, and include goiter, short stature, tachycardia, weight loss, deafness, hypercholesterolemia, and attention deficit hyperactivity disorder (10). Phenotypical variability in RTH is observed especially in the skeleton, and reported features include high bone turnover osteoporosis with fracture, decreased bone mineral density, craniosynostosis, and a range of vertebral and facial bone defects (11).

Recently, we analyzed skeletal development in a mouse model of RTH with a PV mutation targeted to the Thrb gene encoding TRß (12, 13, 14). The PV mutation was derived from a patient with severe RTH and short stature (15), and consists of a C insertion at codon 448 resulting in a frameshift of the 14 amino acids at the carboxy terminal end of TRß. The mutant protein has no T3 binding activity, lacks transactivation activity, and acts as a potent dominant-negative antagonist (16). Homozygous TRßPV/PV mutants display advanced endochondral and intramembranous ossification, craniosynostosis, and short stature resulting from skeletal thyrotoxicosis (12). By contrast, when the PV mutation was targeted to TR{alpha}1, heterozygous TR{alpha}1PV/+ mice exhibited severely delayed bone formation and dwarfism resulting from skeletal hypothyroidism (13).

In previous subtraction hybridization studies, we identified that Fgfr1 is a positively regulated T3 target gene in osteoblasts (17). TRßPV/PV mice with skeletal thyrotoxicosis displayed increased Fgfr1 expression in osteoblasts in vivo, whereas TR{alpha}1PV/+ mice and mice lacking TR{alpha} both had reduced Fgfr1 mRNA expression in bone and skeletal hypothyroidism (12, 13, 17). Consistent with the increased Fgfr1 expression in TRßPV/PV mice, FGFR1 activating mutations in humans result in craniosynostosis (18). These findings suggest that FGFR1 lies downstream of TR{alpha}1 and acts as an important mediator of skeletal responses to T3 during bone development. Therefore, in these studies we examined the underlying cause of skeletal thyrotoxicosis in primary osteoblasts obtained from TRßPV/PV mice and investigated the mechanism by which T3 regulates Fgfr1 gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TRßPV/PV mutant mice
Animal studies were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals, and were approved by the National Cancer Institute Animal Care and Use Committee. Wild-type and homozygous TRßPV/PV mutants were bred and genotyped as described previously (16).

Primary osteoblast isolation
Primary osteoblasts were prepared from the calvaria of 3- to 5-d-old mice. After dissection from surrounding soft tissues, calvaria were washed first in PBS, and then Hanks’ balanced salt solution containing penicillin/streptomycin/neomycin (PSN) (50, 50, and 100 µg/ml, respectively) and amphotericin B (1.5 µg/ml). Calvariae were cut into 2- to 3-mm strips and digested with 0.5% trypsin, 4 mM EDTA in PBS with PSN and amphotericin B. Osteoblasts were obtained after sequential 30-min digestions with type II collagenase (Worthington Biochemical Corp., Lakewood, NJ). Cells from the first two digestions were discarded. Cells from digestions 3–5 were pooled, centrifuged, and resuspended in {alpha}-MEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), PSN, and amphotericin B. After 4–5 h, media were replaced, and adherent cells were cultured for 7 d in differentiation {alpha}-MEM containing 10% charcoal-stripped, thyroid hormone-deprived serum (Td-media) (19), PSN, amphotericin B, ascorbic acid (50 µg/ml), and ß-glycerophosphate (5 mM), either in the absence or presence of T3 (100 nM).

RNA isolation and quantitative real-time RT-PCR
Total RNA was extracted from primary osteoblasts using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed with a SYBR Green Quantitative RT-PCR kit (Sigma-Aldrich, St. Louis, MO) to determine Fgfr1 mRNA expression and a Quantitect Quantitative RT-PCR kit (QIAGEN, Inc., Valencia, CA) to determine TR mRNA expression according to the manufacturer’s instructions using a LightCycler thermal cycler (Roche Diagnostics, Mannheim, Germany). Briefly, 2.5 µl forward primer (2 µM) and 2.5 µl reverse primer (2 µM) were added to 15 µl SYBR Green Enzyme Reaction Mix. The cycles were: 55 C for 30 min; 95 C for 30 sec; 95 C for 15 sec; 58 C for 30 sec; and 72 C for 30 sec; 65–95 C with a heating rate of 0.1 C/sec and a cooling step to 40 C. Primer sequences were: Fgfr1, 5'-GTAGCTCCCTACTGGACATCC-3' (sense) and 5'-GCATAGCGAACCTTGTAGCCTC-3' (antisense); Thra1 (TR{alpha}1), 5'-GTGACTGACCTCCGCATGAT-3' (sense) and 5'-ATCCTCAAAGACCTCCAGGAA-3' (antisense); Thrab1 (TRß1), 5'-GCAGACTTCCCCACACCTT-3' (sense) and 5'-ACAGGTGATGCAGCGATAGT-3' (antisense); and glyceraldehyde-3-phosphatase (Gapdh), 5'-ACATCATCCCTGCATCCACT-3' (sense) and 5'-GTCCTCAGTGTAGCCCAAG-3' (antisense). Amplifications using Thrb1 primers were unaffected by the TRßPV mutation, and both wild-type and mutant alleles were recognized. Quantitative data were normalized to expression of GAPDH.

EMSA
Complementary oligonucleotides containing putative TREs (pTREs) were annealed and the recess 3'-end filled with DNA polymerase (Klenow fragment) in the presence of [{alpha}-32P]deoxy-CTP to generate double-stranded DNA probes as described by Ying et al. (20). TR{alpha}1, TRß1, and TRßPV were synthesized in vitro using the TNT-quick-couple transcription/translation system (Promega, Madison, WI). Approximately 0.2 ng of each probe (3–5 x 104 cpm) was incubated with the in vitro translated protein, in the presence or absence of RXRß (2 µl), in binding buffer (Promega) for 30 min at room temperature. A monoclonal anti-TR antibody mAbC4 (C4, 1 µg), polyclonal anti-TRßPV antibody (T1, 1 µg), and irrelevant antibody MOPC (1 µg; Sigma-Aldrich) were used to determine specificity of TR binding to TRE sequences (21). DNA-bound complexes were resolved on a 5.2% polyacrylamide gel and, after electrophoresis for 2.5–3 h at 250 V, detected by autoradiography.

Transient transfection and chloramphenicol acetyltransferase (CAT) ELISA assay
CV-1 kidney fibroblasts and UMR106 osteoblasts were cultured in DMEM plus 5% FCS (UMR106) or 10% FBS plus PSN (CV-1). Cells were seeded into six-well plates (1.8–3 x 105 cells per well) 18–24 h before transfection using Lipofectamine 2000, according to manufacturer’s instructions (Invitrogen). Cells were transfected, in serum-free medium, and in the absence of antibiotics, with expression plasmids for TRß1 (pCDM8-TRß1), TR{alpha}1 (pcDNA3.1-TR{alpha}1, and pCDM8-TR{alpha}1), and TRßPV (pcDNA3.1-TRßPV), and a range of CAT-reporter gene constructs (a kind gift from Dr. Haruhiko Kouhara, Osaka University Graduate School of Medicine, Osaka, Japan) linked to 5'-deletion mutants of the Fgfr1 gene 5'-flanking region (1 µg) (22) in the pSV00CAT expression vector (23). In addition, 100 ng Renilla internal control reporter (Promega) was cotransfected, and the total DNA transfected per well was equalized using pCDM8 or pcDNA3.1 empty vector carrier DNA. In some experiments, an SRC1 (pIRES-SRC1, 1 µg) or an Sp1 expression vector (CMV-Sp1, 1 µg) was cotransfected. In further experiments, a mutant CAT reporter construct, –866/+107-M1-CAT, was generated using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturers’ instructions and used in UMR106 osteoblasts. Primer sequences were: –866/+107-M1-CAT-forward 5'-CCAAAGACCAAGTTGCAAAGTGCAACAGCAAAATTATAGCCAGGC-3' (sense); and –866/+107-M1-CAT-reverse, 5'-GCCTGGCTATAATTTTGCTGTTGCACTTTGCAACTTGGTCTTTGG-3' (antisense). In addition, the identified pTRE sequence was cloned, along with mutant TRE sequences, upstream of a heterologous thymidine kinase (TK) promoter, into the pBLCAT2 CAT-reporter plasmid (American Type Culture Collection, Manassas, VA). The pTRE5-TKpBLCAT2, M1-TKpBLCAT2 and M3-TKpBLCAT2 constructs were cotransfected (1 µg) into UMR106 osteoblasts. At 6 h after transfection, medium was replaced with DMEM plus 5% FCS (UMR106) or 10% FBS and PSN (CV-1). After 24 h, T3 or rT3 was added and incubated for a further 24 h before harvest. CAT activity was determined using a specific CAT ELISA assay (Roche Diagnostics) with values normalized to Renilla through the use of a Renilla Luciferase Assay System (Promega), and luciferase activities for two copies of a PAL TRE (24) were determined through use of the Dual Luciferase Reporter Assay (Promega).

Statistical analysis
Data were expressed as mean ± SEM. The differences between groups were examined for statistical significance using either the Student’s t test or a one-way ANOVA, followed by the Tukey’s Multiple Comparison Test as appropriate. P values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary calvarial osteoblasts were obtained from 3- to 5-d-old wild-type and homozygous mutant TRßPV/PV littermates, and cultured in the absence and presence of T3 (100 nM). Consistent with previous studies (17), T3 treatment of wild-type osteoblasts resulted in a 1.9-fold increase in Fgfr1 expression compared with untreated wild-type cells (Fig. 1Go). Basal levels of Fgfr1 expression in TRßPV/PV osteoblasts in the absence of T3 were similar to wild type, but expression was increased by 7.98-fold after T3 treatment. Thus, although basal Fgfr1 expression was similar in wild-type and TRßPV/PV osteoblasts, a 4.2-fold greater response to T3 was observed in TRßPV/PV cells, indicating their increased sensitivity to T3 (Fig. 1Go).


Figure 1
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  		FIG. 1. Quantitative real-time RT-PCR analysis of Fgfr1 mRNA expression in primary osteoblasts from wild-type (TRß+/+) and TRßPV/PV littermates in the absence (Td-media) or presence of T3 (Td-media + 100 nM T3). Differences in total RNA input were normalized by signals obtained with specific primers for Gapdh. Data are expressed as mean ± SEM and were analyzed by two-tailed Student’s t test (n = 5; *, P < 0.05).

 
We showed previously that Thra1, encoding TR{alpha}1, was expressed at approximately 12-fold higher levels than the gene encoding TRß1 (Thrb1) in RNA samples extracted from whole mouse tibia and femur (12). To determine whether this expression ratio was maintained in isolated primary osteoblasts, quantitative real-time RT-PCR studies were performed using RNA extracted from wild-type and TRßPV/PV osteoblasts and specific primers for Thra1 and Thrb1 (Fig. 2Go). Consistent with previous data (12), Thra1 was the predominantly expressed TR isoform in both wild-type and TRßPV/PV primary osteoblasts. Wild-type osteoblasts expressed Thra1 at 7-fold higher levels than Thrb1, whereas in TRßPV/PV osteoblasts, Thra1 was expressed at 18-fold higher levels relative to Thrb1 (Fig. 2CGo). The increased ratio of Thra1:Thrb1 expression in TRßPV/PV osteoblasts resulted from a 1.8-fold reduced level of Thrb1 expression in TRßPV/PV (Fig. 2BGo) compared with wild type (P < 0.05; n = 3), whereas levels of Thra1 were similar in both wild-type and TRßPV/PV osteoblasts (Fig. 2AGo). These results demonstrate that, in both wild-type and TRßPV/PV primary osteoblasts, Thra1 is the predominant TR isoform, and this is consistent with previous observations from total bone RNA, in which we demonstrated that the relative expression of Thra1 was 10-fold higher than Thrb1 (12). In Thra knockout mice and primary osteoblasts, we also demonstrated that T3 stimulates Fgfr1 expression and activity via a TR{alpha}-dependent mechanism (17). Furthermore, we showed that increased skeletal Fgfr1 expression in TRßPV/PV mice in vivo correlates with a phenotype of advanced ossification (12), whereas reduced Fgfr1 expression in TR{alpha} knockout and TR{alpha}1PV/+ mice correlates with delayed ossification (13, 17), thus indicating the physiological importance of the TR{alpha}1-FGFR1 signaling pathway in bone. The current findings are supportive of these previous data and conclusions.


Figure 2
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  		FIG. 2. Quantitative real-time RT-PCR analysis of Thra1 (A) and Thrb1 (B) mRNA expression and their relative expression ratios in primary osteoblasts from wild-type and TRßPV/PV (C) littermate mice. Differences in total RNA input were normalized by signals obtained with specific primers for Gapdh. Data are expressed as mean ± SEM and were analyzed by two-tailed Student’s t test (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
To investigate whether T3 regulates Fgfr1 expression directly, we first analyzed the murine Fgfr1 promoter region for the presence of pTRE sequences. In previous studies the murine Fgfr1 gene was found to lack TATA box or CAAT box sequences (25) and to contain a minimal promoter extending 103 nucleotides upstream of the transcription initiation site (22). In this study the 5'-flanking region of the promoter was examined for the presence of TREs up to nucleotide position –600 upstream of the transcription start site. A consensus TRE sequence (1) (Fig. 3AGo) was used to search the Fgfr1 promoter, in both sense and antisense directions, for potential hexameric half-sites. To be designated a pTRE, a half-site was required to score four of six matches against the idealized consensus. Suitable half-sites were subjected to a more stringent test using a TRE characteristic described by Farsetti et al. (26), who reported that a number of naturally occurring TREs in positive T3-responsive genes, including rat malic enzyme, rat GH, and mouse myelin basic protein, contained two pairs of either cytosine or guanine nucleotides separated by an 8-bp "spacer" (Fig. 3BGo). After these criteria were applied, sequences arranged in DR4, inverse repeat, or PAL configurations were identified as pTREs. In the 600 nucleotide 5'-flanking region of the Fgfr1 promoter, 10 pTREs were identified (Figs. 3CGo and 4Go), and probes were designed to evaluate them in EMSA studies.


Figure 3
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  		FIG. 3. Criteria applied to identify pTREs. A, Consensus hexameric half-site sequence; each pTRE was required to score four of six matches to the consensus. B, Well-defined endogenous TREs in several positively regulated T3-target genes in which the arrangement of two pairs of cytosine or guanine nucleotides separated by an 8-bp spacer is highlighted (25 ). PTREs that satisfied the consensus scoring criteria were analyzed to determine the presence of the 8-bp spacer. C, Locations of 10 pTRE containing regions in the 5'-flanking region of the mouse Fgfr1 promoter. +1, Transcription initiation site; ATG, translation initiation codon; cLys, chicken lysozyme; Ex, exon; mMBP, mouse myelin basic protein; r{alpha}MHC, rat myosin heavy chain {alpha}; rGH, rat GH; rME, rat malic enzyme.

 

Figure 4
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  		FIG. 4. Sequences of Fgfr1 promoter oligonucleotide probes used in EMSA assays to identify TR/RXR binding sites. pTRE refers to the pTRE-containing region in the Fgfr1 promoter, and the nucleotide positions of the 5'- and 3'-ends of each probe are shown. pTRE sequences and arrangements of possible TR binding site hexamers that fit the criteria in Fig. 3Go are shown. Arrows indicate orientation of hexamer. n, Spacer nucleotide.

 
EMSA studies revealed the presence of a specific TR binding site (pTRE no. 5) between positions –279/–264 in the Fgfr1 promoter (Fig. 5AGo). TR/RXR heterodimers bound specifically to this putative element. The TR/RXR complex was supershifted by co-incubation with a monoclonal anti-TR antibody mAbC4 (C4), which recognizes an epitope located in the C terminal of TRß1 (21), whereas an irrelevant antibody (MOPC), which does not recognize TRß1 (27), failed to supershift the TR/RXR complex (Fig. 5BGo). Analysis of the pTRE no. 5 sequence suggested this TR binding site was organized as a DR4 motif (Fig. 6AGo). To investigate the specificity of TR/RXR binding to this pTRE, mutational analysis was performed as shown in Fig. 6Go, B–D. The mutation strategy involved the design of three mutant probes in which C to A or T to G substitutions were included. Each probe contained mutations of two nucleotides in each half-site (Fig. 6DGo). Mutant 1 (M1) had mutations at positions 1 and 2 of each hexamer, mutant 2 (M2) at positions 3 and 4, and mutant 3 (M3) at positions 5 and 6. EMSA using an F2 probe, containing an inverted PAL TRE (28), demonstrated binding of TR{alpha}1 homodimers and TR{alpha}1/RXRß heterodimers (Fig. 6BGo). In contrast, TR{alpha}1 (Fig. 6BGo) and TRß1 (Fig. 6CGo) binding to the pTRE no. 5 sequence did not include TR/TR homodimer formation (Fig. 6BGo, compare lanes 2 and 7). TR/RXR heterodimer binding to each mutant was markedly reduced compared with binding of heterodimers to the wild-type sequence, indicating that TR/RXR binding to pTRE no. 5 was specific. Nevertheless, both the TR{alpha}1 EMSA and, after longer exposure, the TRß1 EMSA revealed that M3 retained some residual TR/RXR binding activity (Fig. 6Go, B and C), which correlated with the finding that the half-site sequences in M3 scored four of six and five of six matches with the consensus TRE (Fig. 6DGo). These data confirm the presence of a specific TR/RXR binding site, consisting of a DR4 motif, located at position –279/–264 in the murine Fgfr1 promoter.


Figure 5
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  		FIG. 5. Binding of TR to pTRE regions in the Fgfr1 promoter. A, EMSA in which each pTRE oligonucleotide probe (numbers 1–10) was incubated with reticulocyte lysate negative control (lanes 1), in vitro translated TRß1 (lanes 2), or in vitro translated TRß1/RXRß (lanes 3). B, EMSA in which oligonucleotide probe pTRE no. 5 was incubated with reticulocyte lysate (lane 1), TRß1 (lane 2), TRß1/RXRß (lane 3), TRß1 + TR antibody C4 (lane 4), TRß1/RXRß + TR antibody C4 (lane 5), TRß1 + antibody MOPC that does not bind TR, or RXR (lane 6), TRß1/RXRß + antibody MOPC that does not bind TR or RXR (lane 7). *, Binding of TRß1/RXRß complex to single-stranded probe. NS, Nonspecific binding of reticulocyte lysate to probe.

 

Figure 6
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  		FIG. 6. Mutation analysis of pTRE no. 5. A, The sequence and location of the double-stranded oligonucleotide probe containing the pTRE no. 5 DR4 is shown. Hexameric half-sites within this TRE score four of six and five of six matches with the consensus sequence shown previously. B, EMSA (14-h exposure) in which the inverted PAL TRE (F2 probe), pTRE no. 5, and three mutant pTRE no. 5 probes (M1–M3) were incubated with reticulocyte lysate (lanes 1, 6, 11, 16, and 21), TR{alpha}1 (lanes 2, 7, 12, 17, and 22), TR{alpha}1/RXRß (lanes 3, 8, 13, 18, and 23), TR{alpha}1/RXRß + TR antibody C4 (lanes 4, 9, 14, 19, and 24), TR{alpha}1/RXRß + antibody MOPC that does not bind TR, or RXR (lanes 5, 10, 15, 20, and 25). *Binding of TRß1/RXRß complex to single-stranded pTRE no. 5 probe. C, EMSA (12-h exposure) in which pTRE no. 5 and M1–M3 probes were incubated with reticulocyte lysate (lanes 1, 6, 11, and 16), TRß1 (lanes 2, 7, 12, and 17), TRß1/RXRß (lanes 3, 8, 13, and 18), TRß1/RXRß + C4 (lanes 4, 9, 14, and 19), TRß1/RXRß + MOPC (lanes 5, 10, 15, and 20). Panel on the right shows a 48-h exposure of the region containing the M3 probe. *, Binding of TRß1/RXRß complex to single-stranded probe. D, The hexameric half-site sequences of mutant pTRE no. 5 probes M1–M3 are shown together with the half-site sequences of the pTRE no. 5 DR4. The matching scores against the consensus sequence are shown for each hexamer.

 
Transient transfection experiments were performed to determine whether the TR/RXR binding site, pTRE no. 5, could mediate a T3 response and thereby act as a functional TRE. A range of CAT-reporter constructs containing 5'-deletion mutants of the Fgfr1 5'-flanking region (Fig. 7AGo) were cotransfected with TR{alpha}1 or TRß1 into CV-1 cells, which lack endogenous TRs, in the absence or presence of T3 (100 nM). Consistent with previous reports by Saito et al. (22), deletion of nucleotides –866 to –103 did not influence basal promoter activity, whereas further deletions resulted in reduced promoter activity in the absence and presence of T3 (Fig. 7Go, B and C, left-hand graphs). These data confirm the presence of a minimal promoter in the Fgfr1 gene extending to –103 nucleotides upstream of the transcription start site. Activity of this minimal promoter was not influenced by T3 or the presence of cotransfected TR{alpha}1 or TRß1 (Fig. 7Go, B and C, right-hand graphs). By contrast, activity of the full-length –866/+107 Fgfr1-CAT reporter was increased by T3 only in the presence of cotransfected TR{alpha}1 or TRß1 (P < 0.01; n = 3). These data indicate that the region of the Fgfr1 promoter between –866 and –103 mediates a TR-dependent transcriptional response to T3. In addition, when transfected into UMR106 osteoblastic cells, increasing concentrations of T3 resulted in increased activity of the –866/+107 Fgfr1-CAT reporter in the presence of TR{alpha}1 (Fig. 7DGo). Furthermore, transfection of the –866/+107-M1-CAT reporter, containing the identical mutation to that used in previous EMSAs (Fig. 6Go) in the context of the native –866/+107 Fgfr1-CAT reporter, resulted in loss of T3-induced activation in the presence of cotransfected TR{alpha}1 (Fig. 7EGo). In further studies the putative DR4 TRE sequence and M1 and M3 mutants were cloned upstream of a heterologous TK promoter in the pBLCAT2 plasmid (Fig. 8Go). Similar to results obtained with the native promoter, the activity of the pTRE5-TKpBLCAT2 was increased in UMR106 cells in response to T3 in the presence of cotransfected TR{alpha}1 (Fig. 8BGo). The magnitude of the T3-induced response was less than observed with a reporter containing two copies of a PAL TRE (24). Nevertheless, specificity of the T3-dependent increase in pTRE5-TKpBLCAT2 activity was confirmed by the lack of response to the inactive thyroid hormone metabolite rT3 (Fig. 8BGo). Furthermore, T3-induced activation was also absent when the wild-type pTRE5-TKpBLCAT2 reporter was replaced by the M1-TKpBLCAT2 reporter (Fig. 8CGo). By contrast, a 1.24-fold activation of the M3-TKpBLCAT2 mutant reporter was observed in response to T3, although this response was not statistically significant. These findings correlate with DNA binding results in EMSA studies, in which no TR/RXR binding to the M1 mutant TRE was observed, whereas some residual TR/RXR binding activity was evident on the M3 mutant element (Fig. 6Go, B and C). Together, these data identify a single TR binding site within the Fgfr promoter at position –279 to –264 (Figs. 5Go and 6Go) and indicate that pTRE no. 5 is a functional T3 response element. Unliganded TRs (ApoTRs) repress T3-target genes in the absence of T3, and this was observed with the classical PAL TRE (Fig. 8AGo). In contrast, no ApoTR effect was observed with pTRE no. 5 (Fig. 8BGo), consistent with our recent studies demonstrating that ApoTR effects on basal target gene expression are dependent on the cell type and TRE. For example, in COS-7 cells, ApoTR{alpha}1 repressed a malic enzyme TRE, but in ROS17/2.8 cells, malic enzyme expression was increased (24).


Figure 7
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  		FIG. 7. 5'-Deletion analysis of Fgfr1 promoter and transient transfection studies in CV-1 and UMR106 cells. A, Truncated reporter constructs containing deletions of the Fgfr1 promoter (between locations –866 and +107) upstream of a CAT reporter. The location of pTRE no. 5 is shown. B, Activities of Fgfr1-CAT reporter constructs relative to basal activity of full-length Fgfr1 promoter in the absence of T3. Responses of each Fgfr1 construct to T3 (100 nM) treatment in CV-1 cells in the absence (left graph) or presence of added TR{alpha}1 (right graph) are shown. C, Relative activities of Fgfr1-CAT reporter constructs in response to T3 in CV-1 cells in the absence (left graph) or presence of added TRß1 (right graph). Data are expressed as mean ± SEM and were analyzed by two-tailed Student’s t test (**, P < 0.01; two independent experiments performed in triplicate). D, Relative activities of –866/+107 Fgfr1-CAT reporter construct in response to increasing concentrations of T3 in UMR106 cells in the absence and presence of added TR{alpha}1. Data are expressed as mean ± SEM and were analyzed by two-tailed Student’s t test (*, P < 0.05; **, P < 0.01; two independent experiments performed in triplicate). E, Relative activities of the –866/+107 Fgfr1-CAT and the –866/+107-M1-CAT reporter constructs in response to T3 in UMR106 cells in the absence and presence of added TR{alpha}1. Data are expressed as mean ± SEM and were analyzed with one-way ANOVA, followed by the Tukey’s multiple comparison test (**, P < 0.01; three independent experiments performed in triplicate).

 

Figure 8
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  		FIG. 8. Evaluation of pTRE no. 5 in the context of a heterologous promoter. A, Activities of two copies of a PAL TRE luciferase reporter construct in response to 100 nM T3 in UMR106 osteoblasts in the absence and presence of TR{alpha}1. B, Relative activities of pTRE5-TKpBLCAT2 reporter construct in response to 100 nM T3 or rT3 in UMR106 cells in the absence or presence of TR{alpha}1. C, Relative activities of pTRE5-TKpBLCAT2, M1-TKpBLCAT2, and M3-TKpBLCAT2 reporter constructs in response to 100 nM T3 in UMR106 cells in the absence or presence of TR{alpha}1. Data are expressed as mean ± SEM and were analyzed with one-way ANOVA, followed by the Tukey’s multiple comparison test (*, P < 0.05; ***, P < 0.001; three independent experiments performed in triplicate).

 
Similar to both TR{alpha}1 and TRß1, EMSA studies revealed that TRßPV/RXR heterodimers also bind specifically to the pTRE no. 5 sequence (Fig. 9AGo). The TRßPV/RXR complex was supershifted by coincubation with a polyclonal anti-TRßPV antibody T1 (Fig. 9AGo, lane 10) (21), whereas an irrelevant antibody (MOPC), which does not recognize TRßPV, failed to supershift the TR/RXR complex (Fig. 9AGo, lane 11). In further studies, increasing concentrations of TRßPV at TR{alpha}1:TRßPV ratios of 20:1, 10:1, and 5:1 were investigated (Fig. 9AGo, lanes 12–17). The presence of increased TRßPV concentrations did not affect TR{alpha}1/RXR heterodimer binding to pTRE no. 5. Furthermore, when pTRE5-TKpBLCAT2 was transfected into UMR106 cells, the presence of either an 18:1 ratio of TR{alpha}1:TRßPV or a 7:1 ratio of TR{alpha}1:TRß1 did not significantly alter the T3-induced increase in reporter activity observed in the presence of TR{alpha}1 alone (Fig. 9BGo). Together, these results indicate that the presence of the dominant negative TRßPV receptor does not affect the interaction of TR{alpha}1 with the pTRE sequence or the TR{alpha}1-mediated response of the Fgfr1 TRE to T3 in osteoblastic cells.


Figure 9
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  		FIG. 9. Binding of TRßPV to pTRE no. 5 in the Fgfr1 promoter and evaluation of the effects of TRßPV on pTRE no. 5-mediated transcriptional activity. A, EMSA (3-h exposure) in which the pTRE no. 5 probe was incubated with reticulocyte lysate (lanes 1 and 7), TR{alpha}1 (lane 2), TR{alpha}1/RXRß (lanes 3 and 8), TR{alpha}1/RXRß + C4 (lane 4), TR{alpha}1/RXRß + MOPC (lane 5), TRßPV/RXRß (lane 9), TRßPV/RXRß + TRßPV antibody T1 (lane 10), TRßPV/RXRß + MOPC (lane 11). Lanes 12–17 demonstrate the influence of increasing concentrations of TRßPV on the interaction of TR{alpha}1 with pTRE no. 5 with TR{alpha}1 at constant concentration: TR{alpha}1/TRßPV/RXRß (20:1 {alpha}1:ßPV ratio) (lane 12); TR{alpha}1/ TRßPV/RXRß (20:1 {alpha}1:ßPV ratio) + T1 (lane 13); TR{alpha}1/TRßPV/RXRß (10:1 {alpha}1:ßPV ratio) (lane 14); TR{alpha}1/TRßPV/RXRß (10:1 {alpha}1:ßPV ratio) + T1 (lane 15); TR{alpha}1/TRßPV/RXRß (5:1 {alpha}1:ßPV ratio) (lane 16); TR{alpha}1/TRßPV/RXRß (5:1 {alpha}1:ßPV ratio) + T1 (lane 17). *, Binding of complexes to single-stranded probe. B, Relative activities of pTRE5-TKpBLCAT2 reporter construct in response to T3 in the presence of either TR{alpha}1, an 18:1 ratio of TR{alpha}1:TRßPV or a 7:1 ratio of TR{alpha}1:TRß1. Data are expressed as mean ± SEM and were analyzed by two-tailed Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; three independent experiments performed in triplicate).

 
In addition to TRs, Sp1 is an important transcription factor involved in the positive regulation of Fgfr1 gene expression (29). Furthermore, Sp1 has been implicated in potentiation of the activation of gene expression by TRs. In studies of T3-dependent transcription factor recruitment to the TK promoter, activation of the TR complex by T3 resulted in recruitment of Sp1 followed by further activation of critical transcription factors (30). SRC1 is a member of the p160 coactivator family that may also be involved in positive regulation of target gene expression by TRs (31). Recently, deletion of SRC1 in wild-type and TRßPV/PV mice was shown to disrupt the hypothalamic-pituitary-thyroid axis causing RTH. Furthermore, deletion of SRC1 in TRßPV/PV mice resulted in amelioration of their thyrotoxic skeletal phenotype (32), suggesting that SRC1 along with TR{alpha}1 may also be involved in mediating skeletal responses to T3. Thus, we investigated the roles of Sp1 and SRC1 in T3- and TR-dependent activation of the Fgfr1 promoter. In these experiments, Sp1 and SRC1 expression vectors (0.2 or 0.5 µg) were cotransfected along with TR{alpha}1 and the full-length –866/+107 Fgfr1-CAT reporter into CV-1 cells, and the effects of T3 treatment were evaluated (Fig. 10Go). Consistent with experiments in Fig. 7Go, no T3 responses were observed in the absence of TR{alpha}1 (Fig. 10AGo). By contrast, in the presence of TR{alpha}1, cotransfection of 0.5 µg Sp1 or SRC1 increased the T3 response by 35 and 7%, respectively (Fig. 10BGo). These preliminary data suggest that T3 induced activation of the Fgfr1 promoter is modulated by Sp1 acting in a concentration-dependent manner, whereas SRC1 may be less involved in modulating the response of Fgfr1 to T3.


Figure 10
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  		FIG. 10. Activities of full-length –866/+107 Fgfr1-CAT reporter construct (relative to basal activity) in CV-1 cells in response to T3 (100 nM) in the presence of either 0.2 or 0.5 µg cotransfected Sp1 or SRC-1. A, Data from the experiment performed in the absence of cotransfected TR{alpha}1. B, Results in the presence of TR{alpha}1. Data are expressed as mean ± SEM and were analyzed by two-tailed Student’s t test (*, P < 0.05; **, P < 0.01; experiment performed in triplicate).

 

   Discussion
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 Abstract
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 Materials and Methods
 Results
 Discussion
References
 
In these studies we demonstrate that sensitivity of Fgfr1 gene expression to T3 regulation is increased in TRßPV/PV mutant osteoblasts compared with wild-type cells. This increased sensitivity correlated with reduced levels of TRß, which resulted in an increased ratio of Thra1:Thrb1 expression in TRßPV/PV osteoblasts, even though Thra1 mRNA expression did not differ between osteoblasts obtained from mutant and wild-type mice. The reason for reduced Thrb1 mRNA expression in TRßPV/PV osteoblasts is unclear, although it may result from an effect of the PV frameshift mutation to reduce Thrb1 mRNA stability. In the physiological situation, increased expression of Fgfr1 mRNA in bone is likely to result from the high circulating T3 levels in the TRßPV/PV mouse. The basis of the increased T3 sensitivity in TRßPV/PV osteoblasts is less clear as in the primary culture system there was no difference in the amount of T3 to which cells were exposed. Importantly, the TRßPV protein does not bind T3 and has no capacity for transcriptional activation (16). Thus, in TRßPV/PV osteoblasts, there may be an increased amount of T3 available to bind TR{alpha}1. In addition, TRßPV homodimers and TRßPV/RXR heterodimers retain the ability to bind corepressors such as NCoR. Because TRßPV is unable to bind T3, corepressor complexes associated with TRßPV cannot be dissociated by hormone binding, resulting in the possible reduction of corepressor complexes available for binding to unliganded TR{alpha}1. Consistent with previous findings (12, 13, 14, 17), we conclude that T3 induction of Fgfr1 expression in osteoblasts is mediated predominantly by TR{alpha}1. In this study, Thra1 was expressed at 7-fold higher levels than Thrb1 in osteoblasts. Furthermore, we previously demonstrated that Thra1 is expressed at 10-fold higher levels than Thrb1 in RNA extracted from total bone (12). Consistent with findings in TRßPV/PV mice, we also recently showed that skeletal Fgfr1 mRNA expression in TRß–/– mice is increased relative to wild-type animals (33). However, it is important to note that all these studies do not exclude a role for TRß in T3-mediated stimulation of Fgfr1 mRNA expression, particularly in tissues in which TRß is more predominant such as in the liver. However, in the skeleton our results indicate that TR{alpha}1 is predominantly responsible for mediating effects of T3 on Fgfr1 expression.

In studies to investigate the mechanism of T3-dependent regulation of Fgfr1 gene expression, we identified a TR/RXR binding site that binds TR{alpha}1, TRß1, and TRßPV at position –279/–264 in the Fgfr1 promoter. We demonstrated the specificity of this binding site in EMSA experiments by mutational analysis and the use of specific anti-TR antibodies to supershift TR/RXR complexes. Analysis of Fgfr1 promoter 5'-deletion constructs in transfection studies in CV-1 cells were consistent with gel shift studies, and indicated that the region of the Fgfr1 promoter that binds TR/RXR complexes also mediates TR-dependent transcriptional responses to T3. These results were confirmed when Fgfr1-CAT reporter constructs were transfected into the UMR106 osteoblast cell line, and when the pTRE sequence was removed from within its native promoter context and tested ahead of a heterologous TK promoter. The presence of TRßPV in both EMSA and transfection studies provided novel insight into the actions of this dominant negative receptor in osteoblastic cells (Fig. 9Go). Our studies show that in TRßPV/PV mutant primary osteoblasts, Thra1 is expressed at 18-fold higher levels than ThrbPV (Fig. 2Go). However, at this 18:1 expression ratio, we demonstrated that TRßPV did not interfere with the T3-induced increase of pTRE5-TKpBLCAT2 reporter activity (Fig. 9BGo). Furthermore, in EMSAs, an increased concentration of TRßPV in a 5:1 ratio of TR{alpha}1:TRßPV compared with a 20:1 ratio also failed to interfere with binding of TR{alpha}1 to the TRE. Previously, we reported in vivo, that PV can compete with wild-type TRs for binding to TREs and that in a TRß-predominant tissue such as the liver, this PV-competition results in inhibition of positively regulated T3-target genes (34). In contrast, in heart, a TR{alpha}1-predominant tissue, reduced expression of TRßPV led to an inability of the protein to exert any dominant negative action. Similar to the heart, bone is a TR{alpha}1-predominant tissue (14), and our present studies provide further evidence to indicate that differential expression of TR isoforms between tissues determines the extent of the dominant negative activity exerted by TRßPV.

In all the transfection studies, the magnitude of T3-mediated transcriptional activation of the Fgfr1 promoter was small (1.2- to 1.4-fold) and contrasted with the 8-fold increase in Fgfr1 mRNA expression observed after T3 treatment of TRßPV/PV osteoblasts. This apparent discrepancy is probably because the T3-mediated transcriptional induction at the pTRE is only partially responsible for the overall stimulation of Fgfr1 by T3. Interestingly, similar levels of T3 response were observed in both CV1 and UMR106 cells (Fig. 7Go), suggesting that the mechanism of transcriptional activation may be similar in both cell types, although the possible influence of cell-specific cofactors on T3-mediated stimulation of Fgfr1 expression cannot be excluded. Thus, in addition to T3, various other proteins, including coactivators, corepressors, and a range of transcription factors that may act as protein/protein interaction partners, e.g. Sp1 and AP-1 (35, 36), may play important, as yet undetermined, roles in the regulation of Fgfr1 stimulation by T3.

In previous studies we showed that the T3-induced increase in Fgfr1 expression in osteoblasts was independent of Fgfr1 mRNA half-life and that the transcriptional response to T3 involved an intermediary factor (17). These studies used the relatively insensitive methods of Northern and Western blotting to demonstrate that Fgfr1 expression was regulated by T3 indirectly. In the present studies, we have identified and characterized a TRE within the Fgfr1 promoter that binds TR proteins specifically and mediates direct T3 and TR-dependent activation of gene expression. Together, these studies indicate that T3-dependent regulation of Fgfr1 is achieved via both direct and indirect mechanisms. At present it is unknown whether these mechanisms act independently or whether they may be linked to fine-tune control of Fgfr1 expression by T3.

To examine this issue further, we investigated the role of two transcription factors, Sp1 and SRC1, which are known to be involved in the modulation of T3 action. In preliminary experiments, cotransfection of either Sp1 or SRC1 resulted in approximately 50% reduced levels of basal reporter gene activity in the absence or presence of T3. This phenomenon was independent of the reporter construct studied and was not affected by increasing the concentration of either Sp1 or SRC1 (Fig. 10Go). These findings suggest that overexpression of either Sp1 or SRC1 results in nonspecific transcriptional interference that may result from sequestration of the basal transcription machinery ("squelching"). Nevertheless, and despite the observed squelching, cotransfection of Sp1 resulted in an increase in the TR-dependent Fgfr1 promoter response to T3 of 35%. Therefore, we propose that Sp1 interacts positively with TR{alpha}1 in the control of Fgfr1 gene expression.

Activation of gene expression by T3 is a multistep process involving the release of corepressor and the recruitment of SRC1/p160 coactivator after binding of T3 to the TR. Subsequently, an exchange of coactivators occurs resulting in recruitment of a large coactivator complex (TRAP/DRIP/ARC), which acts as a bridge to facilitate communication between liganded TR and the transcription machinery (31). TRs interact directly with a variety of coactivator proteins, which include Sp1, AP2, CREB, or Pit-1, depending on the context of the target gene promoter (36). Kim et al. (30) studied the TK promoter and proposed a model in which unliganded TR suppresses recruitment of transcription factors, including Sp1, via interactions with corepressor proteins in the absence of T3. After exposure to T3, liganded TR was proposed to stabilize TR/Sp1 complexes, possibly via DNA looping or interaction with additional proteins, and promote recruitment of core transcription factors, including TBP, TFIIB, and Cdk7. Sp1 may also interact indirectly with TR via intermediary bridging factors to stabilize the transcriptional environment and promote gene activation (3, 37, 38). Together, these studies provide convincing evidence that TR and Sp1 signaling pathways converge in the regulation of gene expression by T3. In these studies we have identified a new site for interaction between TR and Sp1 at the Fgfr1 promoter.

Like T3, Sp1 is an important regulator of bone turnover and bone mass. Sp1 plays a key role in the control of basal transcription of the receptor activator of nuclear factor {kappa} B ligand (RANKL) gene in osteoblasts (39) and may regulate osteoblast/osteoclast interactions via its effects on RANKL expression. Furthermore, Sp1 stimulates macrophage colony stimulating factor gene expression (40), an essential factor required along with RANKL for osteoclastogenesis. Intriguingly, a few studies are emerging to show that T3 also stimulates expression of RANKL and several cytokines involved in macrophage colony stimulating factor-dependent osteoclastogenesis, including IL-1{alpha}, IL-6, and prostaglandin E2 (41, 42, 43). Together with our identification of a positive interaction between TR and Sp1 that augments T3-dependent activation of the Fgfr1 promoter, these new findings suggest that Sp1 may play a physiological role in mediating the effects of T3 on osteoblast/osteoclast communications that ultimately regulate bone maintenance. The importance of Sp1 activity in the regulation of bone mass is further underscored by the identification of a functional polymorphism at an Sp1 site in the human COL1A1 promoter associated with reduced bone mineral density and osteoporotic fracture (44, 45).

In conclusion, we have identified a novel TRE at position –279/–264 in the murine Fgfr1 gene promoter that is responsible, in part, for the regulation of FGFR1 expression by T3. Preliminary analysis of T3- and TR-dependent expression mediated by this element has identified a functional interaction between TR and Sp1, which may represent a new pathway involved in the regulation of bone turnover by T3.


   Footnotes
 
This work was supported by European Union FP6 Marie Curie Outgoing International Fellowship (to P.J.O. and G.R.W.) and by the Intramural Research Program of the National Cancer Institute, National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 30, 2007

Abbreviations: CAT, Chloramphenicol acetyltransferase; DR4, direct repeat-4; FBS, fetal bovine serum; FGFR1, fibroblast growth factor receptor-1; GAPDH, glyceraldehyde-3-phosphatase; M1, mutant 1; M2, mutant 2; M3, mutant 3; PAL, palindrome; PSN, penicillin/streptomycin/neomycin; pTRE, putative thyroid hormone response element; RANKL, receptor activator of nuclear factor {kappa} B ligand; RTH, resistance to thyroid hormone; SRC1, steroid receptor coactivator-1; TK, thymidine kinase; TRE, thyroid hormone response element.

Received January 25, 2007.

Accepted for publication August 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cheng SY 2000 Multiple mechanisms for regulation of the transcriptional activity of thyroid hormone receptors. Rev Endocr Metab Disord 1:9–18[CrossRef][Medline]
  2. Brent GA, Williams GR, Harney JW, Forman BM, Samuels HH, Moore DD, Larsen PR 1992 Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements. Mol Endocrinol 6:502–514[Abstract/Free Full Text]
  3. Chen JL, Attardi LD, Verrijzer CP, Yokomori K, Tjian R 1994 Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators. Cell 79:93–105[CrossRef][Medline]
  4. Bassett JH, Harvey CB, Williams GR 2003 Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol 213:1–11[CrossRef][Medline]
  5. Yen PM 2001 Physiological and molecular basis of thyroid hormone action. Physiol Rev 81:1097–1142[Abstract/Free Full Text]
  6. Smith CL, O’Malley BW 2004 Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25:45–71[Abstract/Free Full Text]
  7. Bassett JH, Williams GR 2003 The molecular actions of thyroid hormone in bone. Trends Endocrinol Metab 14:356–364[CrossRef][Medline]
  8. Rivkees SA, Bode HH, Crawford JD 1988 Long-term growth in juvenile acquired hypothyroidism: the failure to achieve normal adult stature. N Engl J Med 318:599–602[Abstract]
  9. Segni M, Leonardi E, Mazzoncini B, Pucarelli I, Pasquino AM 1999 Special features of Graves’ disease in early childhood. Thyroid 9:871–877[Medline]
  10. Weiss RE, Refetoff S 2000 Resistance to thyroid hormone. Rev Endocr Metab Disord 1:97–108[Medline]
  11. Weiss RE, Refetoff S 1996 Effect of thyroid hormone on growth. Lessons from the syndrome of resistance to thyroid hormone. Endocrinol Metab Clin North Am 25:719–730[CrossRef][Medline]
  12. O’Shea PJ, Harvey CB, Suzuki H, Kaneshige M, Kaneshige K, Cheng SY, Williams GR 2003 A thyrotoxic skeletal phenotype of advanced bone formation in mice with resistance to thyroid hormone. Mol Endocrinol 17:1410–1424[Abstract/Free Full Text]
  13. O’Shea PJ, Bassett JH, Sriskantharajah S, Ying H, Cheng SY, Williams GR 2005 Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor {alpha}1 or ß. Mol Endocrinol 19:3045–3059[Abstract/Free Full Text]
  14. O’Shea PJ, Bassett JH, Cheng SY, Williams GR 2006 Characterization of skeletal phenotypes of TR{alpha}1 and TRß mutant mice: implications for tissue thyroid status and T3 target gene expression. Nucl Recept Signal 4:e011
  15. Parrilla R, Mixson AJ, McPherson JA, McClaskey JH, Weintraub BD 1991 Characterization of seven novel mutations of the c-erbA ß gene in unrelated kindreds with generalized thyroid hormone resistance. Evidence for two "hot spot" regions of the ligand binding domain. J Clin Invest 88:2123–2130[Medline]
  16. Kaneshige M, Kaneshige K, Zhu X, Dace A, Garrett L, Carter TA, Kazlauskaite R, Pankratz DG, Wynshaw-Boris A, Refetoff S, Weintraub B, Willingham MC, Barlow C, Cheng S 2000 Mice with a targeted mutation in the thyroid hormone ß receptor gene exhibit impaired growth and resistance to thyroid hormone. Proc Natl Acad Sci USA 97:13209–13214[Abstract/Free Full Text]
  17. Stevens DA, Harvey CB, Scott AJ, O’Shea PJ, Barnard JC, Williams AJ, Brady G, Samarut J, Chassande O, Williams GR 2003 Thyroid hormone activates fibroblast growth factor receptor-1 in bone. Mol Endocrinol 17:1751–1766[Abstract/Free Full Text]
  18. Ornitz DM, Marie PJ 2002 FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev 16:1446–1465[Free Full Text]
  19. Kitagawa S, Obata T, Willingham MC, Cheng SY 1987 Thyroid hormone action: induction of morphological changes and stimulation of cell growth in rat pituitary tumor GH3 cells. Endocrinology 120:2591–2596[Abstract/Free Full Text]
  20. Ying H, Suzuki H, Zhao L, Willingham MC, Meltzer P, Cheng SY 2003 Mutant thyroid hormone receptor ß represses the expression and transcriptional activity of peroxisome proliferator-activated receptor {gamma} during thyroid carcinogenesis. Cancer Res 63:5274–5280[Abstract/Free Full Text]
  21. Bhat MK, McPhie P, Ting YT, Zhu XG, Cheng SY 1995 Structure of the carboxy-terminal region of thyroid hormone nuclear receptors and its possible role in hormone-dependent intermolecular interactions. Biochemistry 34:10591–10599[CrossRef][Medline]
  22. Saito H, Kouhara H, Harada T, Miyake S, Sugiyama H, Kishimoto T, Sato B 1994 Mapping of a transcription element critical for expression of the fibroblast growth factor receptor 1 gene. Biochem Biophys Res Commun 198:1020–1026[CrossRef][Medline]
  23. Araki E, Shimada F, Shichiri M, Mori M, Ebina Y 1988 pSV00CAT: low background CAT plasmid. Nucleic Acids Res 16:1627[Free Full Text]
  24. Harvey CB, Bassett JH, Maruvada P, Yen PM, Williams GR 2007 The rat thyroid hormone receptor (TR) {delta}ß3 displays cell-, TR isoform-, and thyroid hormone response element-specific actions. Endocrinology 148:1764–1773[Abstract/Free Full Text]
  25. Saito H, Kouhara H, Kasayama S, Kishimoto T, Sato B 1992 Characterization of the promoter region of the murine fibroblast growth factor receptor 1 gene. Biochem Biophys Res Commun 183:688–693[CrossRef][Medline]
  26. Farsetti A, Desvergne B, Hallenbeck P, Robbins J, Nikodem VM 1992 Characterization of myelin basic protein thyroid hormone response element and its function in the context of native and heterologous promoter. J Biol Chem 267:15784–15788[Abstract/Free Full Text]
  27. Lin HM, Kaneshige M, Zhao L, Zhang X, Hanover JA, Cheng SY 2001 An isoform of branched-chain aminotransferase is a novel co-repressor for thyroid hormone nuclear receptors. J Biol Chem 276:48196–47205[Abstract/Free Full Text]
  28. Zhu XG, McPhie P, Lin KH, Cheng SY 1997 The differential hormone-dependent transcriptional activation of thyroid hormone receptor isoforms is mediated by interplay of their domains. J Biol Chem 272:9048–9054[Abstract/Free Full Text]
  29. Parakati R, DiMario JX 2005 Dynamic transcriptional regulatory complexes, including E2F4, p107, p130, and Sp1, control fibroblast growth factor receptor 1 gene expression during myogenesis. J Biol Chem 280:21284–21294[Abstract/Free Full Text]
  30. Kim MK, Lee JS, Chung JH 1999 In vivo transcription factor recruitment during thyroid hormone receptor-mediated activation. Proc Natl Acad Sci USA 96:10092–10097[Abstract/Free Full Text]
  31. Harvey CB, Williams GR 2002 Mechanism of thyroid hormone action. Thyroid 12:441–446[CrossRef][Medline]
  32. Kamiya Y, Zhang XY, Ying H, Kato Y, Willingham MC, Xu J, O’Malley BW, Cheng SY 2003 Modulation by steroid receptor coactivator-1 of target-tissue responsiveness in resistance to thyroid hormone. Endocrinology 144:4144–4153[Abstract/Free Full Text]
  33. Bassett JH, O’Shea PJ, Sriskantharajah S, Rabier B, Boyde A, Howell PG, Weiss RE, Roux JP, Malaval L, Clement-Lacroix P, Samarut J, Chassande O, Williams GR 2007 Thyroid hormone excess rather than thyrotropin deficiency induces osteoporosis in hyperthyroidism. Mol Endocrinol 21:1095–1107[Abstract/Free Full Text]
  34. Zhang XY, Kaneshige M, Kamiya Y, Kaneshige K, McPhie P, Cheng SY 2002 Differential expression of thyroid hormone receptor isoforms dictates the dominant negative activity of mutant ß receptor. Mol Endocrinol 16:2077–2092[Abstract/Free Full Text]
  35. Gonzalez-Manchon C, Ayuso MS, Parrilla R 1999 AP-1 and T3RE cis elements operate as a functional unit in the transcriptional control of the human malic enzyme gene. Gene 226:111–119[CrossRef][Medline]
  36. Voz ML, Peers B, Wiedig MJ, Jacquemin P, Belayew A, Martial JA 1992 Transcriptional regulation by triiodothyronine requires synergistic action of the thyroid receptor with another trans-acting factor. Mol Cell Biol 12:3991–3997[Abstract/Free Full Text]
  37. Baniahmad A, Ha I, Reinberg D, Tsai S, Tsai MJ, O’Malley BW 1993 Interaction of human thyroid hormone receptor ß with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone. Proc Natl Acad Sci USA 90:8832–8836[Abstract/Free Full Text]
  38. Hoey T, Weinzierl RO, Gill G, Chen JL, Dynlacht BD, Tjian R 1993 Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators. Cell 72:247–260[CrossRef][Medline]
  39. Liu J, Yang H, Liu W, Cao X, Feng X 2005 Sp1 and Sp3 regulate the basal transcription of receptor activator of nuclear factor {kappa} B ligand gene in osteoblasts and bone marrow stromal cells. J Cell Biochem 96:716–727[CrossRef][Medline]
  40. Srivastava S, Weitzmann MN, Kimble RB, Rizzo M, Zahner M, Milbrandt J, Ross FP, Pacifici R 1998 Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1. J Clin Invest 102:1850–1859[Medline]
  41. Miura M, Tanaka K, Komatsu Y, Suda M, Yasoda A, Sakuma Y, Ozasa A, Nakao K 2002 A novel interaction between thyroid hormones and 1,25(OH)(2)D(3) in osteoclast formation. Biochem Biophys Res Commun 291:987–994[CrossRef][Medline]
  42. Siddiqi A, Burrin JM, Wood DF, Monson JP 1998 Tri-iodothyronine regulates the production of interleukin-6 and interleukin-8 in human bone marrow stromal and osteoblast-like cells. J Endocrinol 157:453–461[Abstract]
  43. Tanabe N, Maeno M, Suzuki N, Fujisaki K, Tanaka H, Ogiso B, Ito K 2005 IL-1 {alpha} stimulates the formation of osteoclast-like cells by increasing M-CSF and PGE2 production and decreasing OPG production by osteoblasts. Life Sci 77:615–626[CrossRef][Medline]
  44. Ralston SH, Uitterlinden AG, Brandi ML, Balcells S, Langdahl BL, Lips P, Lorenc R, Obermayer-Pietsch B, Scollen S, Bustamante M, Husted LB, Carey AH, Diez-Perez A, Dunning AM, Falchetti A, Karczmarewicz E, Kruk M, van Leeuwen JP, van Meurs JB, Mangion J, McGuigan FE, Mellibovsky L, del Monte F, Pols HA, Reeve J, Reid DM, Renner W, Rivadeneira F, van Schoor NM, Sherlock RE, Ioannidis JP 2006 Large-scale evidence for the effect of the COLIA1 Sp1 polymorphism on osteoporosis outcomes: the GENOMOS study. PLoS Med 3:e90
  45. Grant SF, Reid DM, Blake G, Herd R, Fogelman I, Ralston SH 1996 Reduced bone density and osteoporosis associated with a polymorphic Sp1 binding site in the collagen type I {alpha} 1 gene. Nat Genet 14:203–205[CrossRef][Medline]



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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals