Contrasting Skeletal Phenotypes in Mice with an Identical Mutation Targeted to Thyroid Hormone Receptor
1 or ß
Patrick J. OShea,
J. H. Duncan Bassett,
Srividya Sriskantharajah,
Hao Ying,
Sheue-yann Cheng and
Graham R. Williams
Molecular Endocrinology Group (P.J.O., J.H.D.B, S.S., G.R.W.), Division of Medicine and Medical Research Council Clinical Sciences Centre, Imperial College London, Hammersmith Campus, London, W12 0NN, United Kingdom; and Gene Regulation Section (H.Y., S.-y.C.), Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264
Address all correspondence and requests for reprints to: Graham R. Williams, Molecular Endocrinology Group, 5th Floor Clinical Research Building, Medical Research Council Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom. E-mail: graham.williams{at}imperial.ac.uk
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ABSTRACT
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Thyroid hormone (T3) regulates bone turnover and mineralization in adults and is essential for skeletal development. Surprisingly, we identified a phenotype of skeletal thyrotoxicosis in T3 receptor ßPV (TRßPV) mice in which a targeted frameshift mutation in TRß results in resistance to thyroid hormone. To characterize mechanisms underlying thyroid hormone action in bone, we analyzed skeletal development in TR
1PV mice in which the same PV mutation was targeted to TR
1. In contrast to TRßPV mice, TR
1PV mutants exhibited skeletal hypothyroidism with delayed endochondral and intramembranous ossification, severe postnatal growth retardation, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed closure of the skull sutures. Skeletal hypothyroidism in TR
1PV mutants was accompanied by impaired GH receptor and IGF-I receptor expression and signaling in the growth plate, whereas GH receptor and IGF-I receptor expression and signaling were increased in TRßPV mice. These data indicate that GH receptor and IGF-I receptor are physiological targets for T3 action in bone in vivo. The divergent phenotypes observed in TR
1PV and TRßPV mice arise because the pituitary gland is a TRß-responsive tissue, whereas bone is TR
responsive. These studies provide a new understanding of the complex relationship between central and peripheral thyroid status.
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INTRODUCTION
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THE ACTIONS OF THYROID hormone (T3) are mediated mainly by nuclear T3 receptors (TRs), which act as ligand-inducible transcription factors (1). Several TR
and TRß isoforms are expressed in temporospatial patterns during development and in differing ratios in adult tissues (1, 2, 3). In the skeleton, TRs are expressed in growth plate chondrocytes, osteoblasts, and bone marrow stromal cells, and T3 is essential for skeletal development, linear growth, and bone mineralization (4, 5). Childhood hypothyroidism causes growth arrest, delayed skeletal maturation, and epiphyseal dysgenesis, and T4 replacement induces catch-up growth (6). Autosomal dominant resistance to thyroid hormone (RTH), which results from mutant TRß proteins, also impairs skeletal development causing short stature and bone dysplasias (7, 8). Childhood thyrotoxicosis accelerates growth and advances bone age but induces short stature due to premature fusion of the growth plates. In severe cases, craniosynostosis can result from early closure of the skull sutures and may be associated with neurological deficits (9). Thus, euthyroidism is essential for normal bone development, and the skeleton is exquisitely sensitive to changes in thyroid status.
RTH is characterized by reduced responsiveness of target tissues to circulating T4 and T3. At the level of the hypothalamic-pituitary-thyroid axis this results in the classical biochemical features of RTH, in which negative feedback control of TSH production and secretion is impaired. The resulting inappropriately high levels of TSH drive increased T4 and T3 production, and a new equilibrium set point of high circulating concentrations of T4 and T3 with elevated levels of TSH is established. Mutations in the THRB gene cause RTH and result in the expression of mutant TRß proteins that are unable to respond normally to T3, usually because of reduced binding affinity for T3 or reduced transcriptional activation capabilities. The mutant TRß proteins also interfere with the actions of normally expressed wild-type TR
and ß and thus act as dominant-negative antagonists that disrupt transcription of T3-regulated genes (10). The clinical syndrome of RTH is variable, resulting from both the direct effects of the mutant receptors and the consequences of elevated thyroid hormone concentrations. In addition, the differing ratios of TR
and TRß proteins that are expressed in individual tissues further complicate the syndrome. In tissues such as the heart, in which TR
predominates, the presence of tachycardia in RTH is likely to be due to the effects of elevated thyroid hormone levels acting via TR
. In contrast, in tissues such as the liver, in which TRß predominates, the presence of hypercholesterolemia in RTH may result from a combination of local tissue hypothyroidism and the direct dominant-negative actions of mutant TRß proteins in hepatocytes. Thus, the spectrum of clinical features in RTH is broad and can include reduced weight, cardiac disease, hypercholesterolemia, tachycardia, hearing loss, attention-deficit hyperactivity disorder, decreased IQ, and dyslexia (10, 11, 12). This complex spectrum reflects the presence of thyrotoxicosis in some T3-target tissues but evidence of hypothyroidism in others.
A wide variety of bone phenotypes has been described in RTH, and this is probably because objective studies of the skeleton and growth are available in only a small minority of patients. Features include stippled epiphyses with scattered calcification in the growth plate, high bone turnover osteoporosis and fracture, reduced bone density, craniosynostosis, and various defects of facial bone and vertebral development (7). Growth retardation and short stature have been estimated to occur in 26% of patients with variably delayed bone age in up to 47% (7, 8), although bone age has also been shown to be advanced by more than 2 SDs in two families, and lesser degrees of advancement have also been documented (7).
In a previous study, we characterized skeletal development in mutant mice with a PV mutation targeted to the TRß gene locus (13). The PV mutation was derived from a patient with severe RTH and consists of a C insertion at codon 448, which produces a frameshift of the carboxyl-terminal 14 amino acids of TRß1. The mutant TRßPV protein cannot bind T3, fails to transactivate T3 target genes in vitro, and is a potent dominant-negative antagonist (14). TRßPV mice have very high levels of circulating T4, T3, and TSH (14), and we showed they display a phenotype of skeletal thyrotoxicosis (13). We also demonstrated that TR
1 mRNA is expressed at 12-fold higher concentrations than TRß1 in bone, suggesting that the phenotype of skeletal hyperthyroidism in TRßPV mice results from increased T3 levels acting via TR
1 (13). To investigate this hypothesis and determine whether TR
1 is functionally predominant in bone, we studied mice carrying the PV mutation targeted to TR
1 (15). The mutant TR
1PV protein also acts as a potent dominant-negative antagonist that interferes with transcriptional activities of the wild-type TR
and TRß receptors (15). In keeping with other mice with dominant-negative RTH mutations in TR
1 (11, 16), heterozygous TR
1PV/+ mice display only a modest degree of thyroid failure. There were small increases in TSH (1.7-fold) and T3 (1.15-fold) levels in TR
1PV/+ mice, but no change in circulating T4 concentrations, and the homozygous mutation was lethal (15). Characterization of bone development in biochemically euthyroid TR
1PV/+ mice, therefore, enabled us to investigate mechanisms of T3 action in bone and determine whether TR
1 is the major functional TR expressed in the skeleton.
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RESULTS
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TR
1PV/+ Mice Exhibit Delayed Ossification and Postnatal Growth Retardation
Analysis of bone lengths in TR
1PV/+ mice revealed severe and persistent postnatal linear growth impairment (Fig. 1
). No sexually dimorphic influences of the TR
1PV mutation on bone growth or development were observed. TR
1PV/+ tibias were 1525% shorter than tibias from wild-type littermates at all postnatal ages examined. In contrast, no difference was observed between embryonic d 17.5 (E17.5) and postnatal d 1 (P1) wild-type and TR
1PV/+ mice. TR
1PV/+ ulnas were also markedly shorter than wild-type (1214% reduction), although the magnitude of growth impairment was less than in the tibia (Fig. 1
). This degree of postnatal growth impairment in TR
1PV/+ long bones was much more severe than documented in TRßPV/+ and TRßPV/PV mice (13).

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Fig. 1. Growth of Wild-Type (WT) and TR 1PV/+ Mice
A, Graph showing mean tibia lengths (mm) in mice between E17.5 (2.5 d before birth) and 7 wk. B, Graph showing mean ulna lengths (mm) in mice between E17.5 and 7 wk. Significance of differences between WT and TR 1PV/+ at each age was calculated by Students t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Total numbers of animals examined per group were: WT E17.5, n = 6; neonate P1, n = 3; P14, n = 6; P21, n = 4; P28, n = 4; P49, n = 10; TR 1PV/+ E17.5, n = 7; neonate P1, n = 12; P14, n = 8; P21, n = 4; P28, n = 4; P49, n = 8.
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Analysis of skeletal preparations from E17.5 and P1 TR
1PV/+ mice confirmed that bone lengths in mutant mice did not differ from wild type before birth, and the appearance of rib cages and vertebrae from wild-type and TR
1PV/+ mice was similar (Fig. 2A
). In these preparations, alizarin red stained ossified bone pink and alcian blue 8GX stained cartilage blue. Nevertheless, analysis of limbs in E17.5 mice revealed a small delay in endochondral ossification in the ulna and radius of the forelimb and in the tibia and fibula of the hindlimb that was evident in all TR
1PV/+ mice examined (reduced alizarin red staining in these regions in TR
1PV/+ mice [(n = 7) compared with wild-type (n = 6), arrowed in Fig. 2A
]. Similar findings were present in neonatal mice (wild-type n = 3; TR
1PV/+ n = 12; data not shown). These observations are in contrast with findings in TRßPV/PV mice, in which E17.5 and P1 skeletons displayed advanced endochondral ossification and were larger than wild-type littermates (13). Examination of the skull in E17.5 and neonatal mice revealed further differences between wild-type and TR
1PV/+ mice. There was no difference in anterior-posterior and biparietal skull dimensions, but the fontanelles in TR
1PV/+ mice were larger and cranial sutures wider than in wild-type littermates [E17.5: 37.9 ± 2.5 vs. 20.8 ± 0.9 (P < 0.001); P1: 9.5 ± 0.8 vs. 4.3 ± 1.2 (P < 0.05), area of open fontanelles and sutures expressed as percentage of total skull area in TR
1PV/+ vs. wild-type mice], indicating delayed fontanelle closure and suture fusion (Fig. 2
). Intramembranous bone deposited in frontal and parietal bones of the TR
1PV/+ skull was also more porous and stained less intensely (Fig. 2B
). These data demonstrate normal growth dimension, but markedly delayed intramembranous ossification of the skull in TR
1PV/+ mice and contrast with findings in TRßPV/PV mice, in which advanced ossification of the skull with craniosynostosis was demonstrated (13).
Postnatal linear growth in TR
1PV/+ and TRßPV/PV mice was examined in detail (Fig. 3
). Tibias from TR
1PV/+ mice were 15%, 17%, 20%, and 16% shorter than wild type at ages 2, 3, 4, and 7 wk, respectively, whereas tibias from TRßPV/PV mice were 11% shorter at 2 wk and only 2% shorter at 4 wk reflecting the accelerated growth spurt between these ages in TRßPV/PV mice (13). Growth impairment in TR
1PV/+ mice was accompanied by reduced ossification of the secondary tibial epiphyses, a finding not seen in TRßPV/PV mice, but which persisted in TR
1PV/+ mice at 3 and 4 wk. Furthermore, hindlimb paws from TR
1PV/+ mice at 3 and 7 wk revealed persistently impaired endochondral bone formation with delayed formation of secondary ossification centers in metatarsal bones at 2 wk and the presence of open metatarsal growth plates at 7 wk (Fig. 3
). In contrast, epiphyseal ossification and metacarpal and metatarsal growth plate closure was advanced in 3 wk-old TRßPV/PV mice (13). These data indicate that postnatal linear growth impairment in TR
1PV/+ mice was associated with delayed endochondral ossification, whereas in TRßPV/PV mice it resulted from advanced bone development.
Endochondral ossification in the proximal tibia was analyzed in histological studies in TR
1PV/+ mice (Fig. 4
). In wild-type mice the proximal tibia secondary ossification center was already established with formation of bone trabeculae within the epiphysis at 2 wk. Between 2 and 7 wk there was progressive narrowing of the growth plate with increased epiphyseal trabecular bone deposition as endochondral ossification and bone maturation continued. In contrast, in TR
1PV/+ mice endochondral ossification was markedly delayed. TR
1PV/+ tibias were smaller in all dimensions, and development of the secondary ossification center was initiated at 3 wk, a time at which this process was well advanced in wild-type littermates. Formation of an organized growth plate, and subsequent growth plate narrowing during the progression of endochondral ossification, was also markedly delayed. The histological features in 7-wk TR
1PV/+ mice were similar to those in 3- to 4-wk wild-type mice, indicating that endochondral ossification was delayed by up to 4 wk in mutants (Fig. 4A
). Delayed endochondral ossification in TR
1PV/+ mice was associated with reduced deposition of calcified trabecular bone, as evidenced by reduced von Kossa staining of undecalcified sections of the tibia in 2-wk-old mutants (n = 3) compared with wild type (n = 3) (Fig. 4B
). In particular, mineralization of trabecular bone in the secondary epiphysis in wild-type mice was already established by 2 wk of age, whereas in TR
1PV/+ mice, staining in this region was absent. A smaller reduction in von Kossa staining was evident in trabecular bone in the region of the metaphysis in TR
1PV/+ mice. In addition, cortical bone deposition in the tibial diaphysis was reduced by approximately 50% in 2-wk TR
1PV/+ mice compared with wild-type littermates (Fig. 5
). These data demonstrate that postnatal growth impairment in TR
1PV/+ mice is associated with a 3- to 4-wk delay in bone formation, reduced trabecular bone mineralization, and impaired cortical bone deposition. The findings contrast with those in TRßPV/PV mice, in which advanced ossification and increased trabecular bone mineralization were evident (13).
To investigate mechanisms underlying delayed ossification in TR
1PV/+ mice, measurements of specific regions in the growth plate were performed (Fig. 6
). Histological studies enabled the reserve (RZ), proliferative (PZ), and hypertrophic (HZ) zones of growth plates to be identified (13, 17, 18, 19). In situ hybridization was performed to determine the expression of collagen II, a marker of proliferating chondrocytes (20), and allow measurement of growth plate dimensions (Fig. 6A
). Between 2 and 4 wk there was progressive narrowing of the growth plate in wild-type mice that was due to the normal proportionate narrowing in each of the RZ, PZ, and HZ regions (Fig. 6B
). The growth plate continued to narrow in wild-type mice between 4 and 7 wk, but at a slower rate than in younger animals reflecting growth plate maturation and its imminent quiescence as linear growth tails off toward adulthood (Fig. 6C
). In TR
1PV/+ mice, measurement of specific regions of the growth plate were not possible until animals reached 7 wk of age because, before that time, formation of the proximal tibial growth plate was incomplete (Fig. 4
). At 7 wk the growth plate in TR
1PV/+ mice was significantly wider than in 7-wk wild-type littermates (P < 0.001) but did not differ in width when compared with growth plates from 4-wk wild-type mice. The finding that the width of the growth plate in 4-wk wild-type mice was similar to the width observed in 7-wk old TR
1PV/+ mice suggested that endochondral ossification was delayed by about 3 wk in TR
1PV/+ mice. Thus, comparisons of individual growth plate zones between 4-wk wild-type and 7-wk TR
1PV/+ mice were made to investigate why ossification was delayed in TR
1PV/+ mutants. These comparisons revealed that the PZ and HZ regions in TR
1PV/+ mice were narrower than in wild-type (5% and 10%, respectively; P < 0.05) but the RZ width was similar to wild type (Fig. 6B
). Taken together, these data confirm that endochondral ossification in TR
1PV/+ mice is delayed by approximately 34 wk and suggest this delay is due to impaired transition of immature RZ chondrocytes into the PZ, resulting in proportionally reduced numbers or dimensions of proliferating and hypertrophic chondrocytes. The data contrast with findings in TRßPV/PV mice, in which disproportionate and accelerated narrowing of the PZ and HZ regions accounted for premature growth plate quiescence by 4 wk of age (13).

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Fig. 6. Analysis of Growth Plate Dimensions in Wild-Type (WT) and TR 1PV/+ Mice
A, In situ hybridization for collagen II expression in proliferative zone of the upper tibia growth plate of a 3-wk WT mouse (x200 magnification). RZs, PZs, and HZs of the growth plate are indicated along with the regions of the secondary epiphysis (E) and primary spongiosum (PS). B, Graph showing relative widths of the RZ, PZ, and HZ regions and total growth plate heights (RZ+PZ+HZ) of 2-, 3-, and 4-wk WT mice compared with 7-wk TR 1PV/+ mice. Mean growth plate height and zone width measurements (µm) ± SEM were obtained from two to three animals per group (three to four differing levels of section examined for each growth plate) by taking four separate measurements across each growth plate section examined. Data were analyzed by Students t test to determine the differences in width of the growth plate zones and the total growth plate height between 7-wk TR 1PV/+ mice and WT animals aged 2, 3, and 4 wk. C, Graph showing the decline in total growth plate height (µm) ± SEM with age in WT mice aged between 2 and 7 wk (n = 3 per group). NS, Nonsignificant (P = 0.63; P = 0.71).
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TR
1PV/+ Mice Exhibit Skeletal Hypothyroidism
We previously identified that fibroblast growth factor receptor-1 (FGFR1) is a T3-target gene in bone. Skeletal FGFR1 expression was reduced in TR
-null (TR
0/0) mice, which display a hypothyroid skeletal phenotype (19), but was increased in TRßPV/PV mice (13). In contrast, comparison of 3-wk-old wild-type mice with 7-wk-old TR
1PV/+ mice (equivalent ages of growth plate maturation, Figs. 4
and 6
) revealed that FGFR1 mRNA expression in both chondrocytes and osteoblasts was markedly reduced in TR
1PV/+ mice (Fig. 7
). These data demonstrate that TR
1PV/+ mice display severe skeletal hypothyroidism.

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Fig. 7. In Situ Hybridizations (x100 Magnification) for FGFR1 in Tibial Growth Plates from 4-wk Wild-Type (WT) and 7-wk TR 1PV/+ Mice
The extent of the PZs and HZs are shown. Arrows indicate increased FGFR1 staining in osteoblasts lining trabecular bone surfaces within the secondary epiphysis in WT mice compared with TR 1PV/+ mice.
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GH and IGF-I Signaling in the Growth Plate Is Impaired in TR
1PV/+ Mice, but Increased in TRßPV Mice
In view of the impaired transition of chondrocytes from RZ to PZ in TR
1PV/+ mice, we investigated further by examining GH/IGF-I signaling in the growth plate. The GH/IGF-I pathway is initiated by GH, which activates the GH receptor (GHR) in growth plate chondrocytes. GH either acts directly on growth plate chondrocytes to regulate their proliferation and differentiation or stimulates local production of IGF-I, which subsequently acts in a paracrine manner to stimulate the IGF-I receptor (IGF-IR). IGF-I also exerts GH-independent actions on growth plate chondrocytes (21). GHR stimulation results in activation of a signaling cascade that involves signal transducer and activator of transcription (STAT)5 (22, 23), whereas stimulation of IGF-IR results in activation of protein kinase B/Akt signaling (24, 25).
The GH/IGF-I pathway was investigated in growth plates from wild-type, TR
1PV/+, TRßPV/+, and TRßPV/PV mice by in situ hybridization and immunohistochemistry. In 4-wk wild-type mice, GHR was expressed at low levels only in prehypertrophic chondrocytes at the junction between the PZ and HZ. GHR expression was markedly increased in TRßPV/PV mice and extended throughout the PZ, whereas increased expression was also observed in TRßPV/+ heterozygotes, but this was restricted to prehypertrophic chondrocytes. In contrast, GHR expression was absent from the growth plate in TR
1PV/+ mice, although low levels of expression were evident in immature chondrocytes populating the incompletely formed growth plate from the region of the developing secondary epiphysis (Fig. 8
). Low levels of IGF-I expression were also observed in these immature chondrocytes in TR
1PV/+ mice, but not in the growth plate itself. In contrast, there were no differences in levels of IGF-I expression in TRßPV/+ or TRßPV/PV mice compared with wild type, in which IGF-I mRNA was expressed in proliferating chondrocytes (Fig. 8
). The patterns of expression of the IGF-IR were similar to those observed for expression of GHR. In wild-type animals IGF-IR was restricted to prehypertrophic chondrocytes. Expression was increased in the same region in TRßPV/+ heterozygotes but was markedly increased throughout the growth plate in TRßPV/PV mice. In the TR
1PV/+, IGF-IR expression was not detected in the growth plate but was present in immature chondrocytes located in the secondary epiphysis (Fig. 8
).
To investigate whether the absence of GHR, IGF-I, and IGF-IR expression from growth plates in TR
1PV/+ mice was because of the immaturity of the TR
1PV/+ growth plate, we compared levels of expression in 2-, 3-, and 4-wk-old wild-type, TR
1PV/+, TRßPV/+, and TRßPV/PV mice (Fig. 9
and data not shown). Expression of all three mRNAs was absent from growth plates of TR
1PV/+ mice at all ages but was present at low levels in immature chondrocytes in the region of the secondary epiphysis, consistent with findings in Fig. 8
. In wild-type mice, levels of GHR and IGF-IR decreased with age between 2 and 4 wk and became localized to prehypertrophic chondrocytes (Figs. 8
and 9
and data not shown). In contrast, in TRßPV/+ and TRßPV/PV mice, expression of both GHR and IGF-IR remained persistently increased throughout the growth plate at all ages (Figs. 8
and 9
and data not shown). No changes in expression of IGF-I mRNA were observed in wild-type or mutant mice. These data indicate that altered patterns of expression of GHR and IGF-IR mRNAs in TR
1PV/+, TRßPV/+, and TRßPV/PV mice are not related to the maturity of the growth plate per se and suggest they result from altered skeletal T3 signaling as a consequence of the TR
1PV or TRßPV mutation.

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Fig. 9. In Situ Hybridizations (x200 Magnification) for IGF-IR Expression in Tibial Growth Plates from 2-, 3-, and 4-wk Wild-Type (TRß+/+), Heterozygote TRßPV/+, and TRßPV/PV Mice
The extent of the PZs and HZs are shown.
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To investigate whether changes in mRNA expression correlated with changes in functional activation of GHR and IGF-IR, we investigated the STAT5 and Akt downstream signaling pathways by immunohistochemistry. In TR
1PV/+ mice, basal expression of STAT5 and Akt was no different than that of wild type, whereas concentrations of phosphorylated STAT5 and phosphorylated Akt were markedly reduced (Fig. 10
). Thus, reduced levels of GHR and IGF-IR mRNAs in TR
1PV/+ mice correlated with reduced activation of downstream signaling pathways. In TRßPV/+ and TRßPV/PV mice, levels of basal STAT5 were reduced compared with wild type, whereas levels of basal Akt expression were unchanged. Concentrations of phosphorylated STAT5 and phosphorylated Akt, in contrast, were similar to wild type in TRßPV/+ mice but were elevated in TRßPV/PV mice, although the increase in phosphorylated STAT5 was small (Fig. 11
). Thus, increased expression of GHR and IGF-IR mRNAs in TRßPV mice correlated with increased activation of downstream signaling pathways. Taken together, these data demonstrate that expression and activity of the local growth plate GH/IGF-I signaling pathway is markedly reduced in TR
1PV mice but increased in TRßPV mice (Table 1
), indicating that skeletal thyroid status is a key determinant of the sensitivity of growth plate chondrocytes to the local actions of GH and IGF-I.

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Fig. 10. Immunohistochemistry (x200 Magnification) of STAT5 (Upper Panels), pSTAT5 (Second Row), Akt (Third Row) and pAkt (Fourth Row) ProteinExpression in Tibial Growth Plates from Wild-Type (TR 1+/+) and TR 1PV/+ Mice
The right-hand column labeled "Controls" shows parallel experiments in which primary antibody was omitted from the immunohistochemistry protocol and which show that staining of STAT and Akt proteins occurred only in the presence specific primary antibody. pSTAT5, Phosphorylated STAT5; pAkt, phosphorylated AKT.
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Fig. 11. Immunohistochemistry (x200 Magnification) of STAT5 (Upper Panels), pSTAT5 (Second Row), Akt (Third Row), and pAkt (Fourth Row) ProteinExpression in Tibial Growth Plates from Wild-Type (TRß+/+), TRßPV/+, and TRßPV/PV Mice
The right-hand column labeled "controls" shows parallel experiments in which primary antibody was omitted from the immunohistochemistry protocol and which show that staining of STAT and Akt proteins occurred only in the presence of specific primary antibody. pSTAT5, Phosphorylated STAT5; pAkt, phosphorylated AKT.
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DISCUSSION
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We have demonstrated that TR
1PV/+ mice exhibit a severe 25% reduction in postnatal linear growth, a 3- to 4-wk delay in endochondral ossification, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed intramembranous ossification. Reduced expression of the T3-target gene FGFR1 indicates that skeletal hypothyroidism is responsible for this phenotype. The findings contrast with TRßPV mice, in which skeletal thyrotoxicosis was documented by increased FGFR1 expression, accelerated early linear growth, increased trabecular bone mineralization, and advanced endochondral and intramembranous ossification that resulted in short stature and craniosynostosis (13). We previously identified that FGFR1 is a T3-target gene in bone (19), and the major role of FGFR1 in skeletal development is to regulate intramembranous ossification of the skull (26). Activating mutations of FGFR1 cause Pfeiffers craniosynostosis syndrome (27), whereas craniosynostosis also occurs in severe childhood thyrotoxicosis (9). The presence of delayed closure of the skull sutures in TR
1PV/+ mice, together with craniosynostosis in TRßPV/PV mice, suggests FGFR1 mediates important T3 effects that regulate intramembranous ossification.
Nevertheless, prominent phenotypes in TR
1PV/+ and TRßPV mice involve abnormalities of endochondral bone formation. Thus, we investigated GH and IGF-I signaling in the growth plate, because they are major regulators of endochondral ossification and growth (21, 28, 29). The effects of GH were originally proposed to be mediated by liver-derived IGF-I (30), but this was challenged when IGF-I expression was identified in many tissues (21). A "dual effector theory" for GH action was proposed (31) and extrapolated to the growth plate (32). In this model GH initiates differentiation of PZ chondrocytes directly and is proposed to induce local IGF-I production in proliferating chondrocytes. Local IGF-I then acts in an autocrine/paracrine manner to stimulate clonal expansion and chondrocyte proliferation, resulting in longitudinal growth. However, evidence from IGF-I knockout (IGF-I/), liver-specific IGF-I knockout (LID), acid-labile subunit knockout (ALSKO) (acid-labile subunit forms a ternary complex with IGF-I and IGF-I binding protein-3 to stabilize serum IGF-I and facilitate its endocrine actions), and LID+ALSKO double-knockout mice have demonstrated that a threshold concentration of circulating IGF-I is also necessary for bone growth. Nevertheless, tissue IGF-I also plays an essential role because IGF-I/ mice are much more growth retarded than LID+ALSKO double-knockout mice (33, 34, 35, 36, 37). Data from GHR knockout (GHR/), IGF-I/, and GHR/IGF-I/ double mutants indicate that GH and IGF-I act on the growth plate by both independent and overlapping pathways, with IGF-I being the major determinant of embryonic and postnatal growth, and its actions being modulated by GH in the postnatal period (29). It has further been suggested that, because only 17% of somatic growth can be attributed to processes that do not require an intact GH/IGF-I axis, GH and IGF-I pathways in the growth plate act as a point of convergence and participate in the actions of most growth-promoting molecules (29).
This concept is supported by studies showing that IGF-I is stimulated by T3 in osteoblastic cells (38, 39) and IGF-IR is T3 responsive in chondrocyte cultures (40). A recent study also showed that T3 treatment of hypophysectomized rats resulted in increased GHR expression in the growth plate (41), although a previous study showed that GHR expression in the growth plate was independent of thyroid status (42). In addition to effects on local growth plate GH/IGF-I signaling, T4 and T3 influence pituitary GH secretion (43). Abnormalities of the GH/IGF-I axis have been documented in various TR knockout mice: TR
1/ß/ mice (which lack TR
1 and TRß) have GH- and mild IGF-I deficiency (44), and GH replacement restores their growth but does not improve defective ossification (45); TR
0/0ß/ mice (lacking all products of the Thra and Thrb genes) have GH deficiency (17); TR
0/0 mice have normal GH production (17); TRß/ mice have mildly reduced GH production (46); and TR
2/ mice (which lack TR
2 but overexpress TR
1) have normal GH levels but are IGF-I deficient (47). We previously showed that TR
1PV/+ mice have normal pituitary GH production (15), whereas GH expression is reduced by 80% and circulating IGF-I is reduced by 40% in TRßPV/PV mutants (14, 48). These data from various TR mutant mice indicate that T3, acting mainly via TRß, regulates systemic GH/IGF-I signaling pathways in vivo. Nevertheless, the presence of delayed endochondral ossification in TR
1PV mice despite normal levels of GH, and the presence of accelerated ossification in TRßPV/PV mice in the face of low levels of GH and IGF-I, is discordant with the known actions of GH/IGF-I in the growth plate. These findings strongly suggest that the skeletal consequences of the PV mutation result from dysregulated local GH/IGF-I signaling in the growth plate.
Data in Figs. 811


support this by clearly showing that GHR and IGF-IR expression and signaling are reduced in TR
1PV mice (skeletal hypothyroidism) but increased in TRßPV mice (skeletal thyrotoxicosis). Nevertheless, the increase in GHR expression in TRßPV/PV growth plates (Fig. 8
) was accompanied by a disproportionately small rise in activated STAT5 (Fig. 11
). This finding reflects the impaired GH production observed in these mice (14) and demonstrates that the net effect of the TRßPV mutation results from systemic and local consequences on GH action. In contrast, IGF-I expression was unchanged in TRßPV/+ and TRßPV/PV mice compared with wild type and was undetectable in TR
1PV growth plates, suggesting that TR
and/or GHR activity are necessary for IGF-I expression but indicating that growth plate IGF-I expression is not responsive to increased T3 or GH action. Nevertheless, increased activation of Akt was observed in TRßPV/PV mice and was independent of changes in IGF-I expression, instead correlating with increased IGF-IR expression. These findings suggest that levels of IGF-IR, rather than IGF-I ligand, are limiting in the growth plate. An alternative possibility is that an unidentified T3-stimulated, IGF-I independent pathway could increase Akt activation in TRßPV/PV mice. Taken together, these data indicate that local GH/IGF-I actions mediate important effects of T3 on endochondral ossification.
Nevertheless, in TR
1PV/+ mice with skeletal hypothyroidism and reduced GHR and IGF-IR activity, growth plates were observed to be wider than in wild-type mice (Fig. 4
), whereas in TRßPV/PV mice, with skeletal thyrotoxicosis and increased GHR and IGF-IR signaling, growth plates were narrower (13). In contrast, growth plates were observed to be narrower in GHR/ and IGF-I/ mice compared with wild type (29, 36, 49), suggesting that T3 exerts important effects on linear growth that are independent of GH and IGF-I. Indeed, TR
and TRß are expressed in growth plate chondrocytes (50, 51, 52, 53). T3 inhibits clonal expansion and proliferation but promotes hypertrophic differentiation of primary chondrocytes in suspension culture (53), and additional studies have shown T3 regulates the spatial organization of chondrocyte columns and is required for terminal hypertrophic differentiation (54). In contrast, IGF-I stimulates chondrocyte proliferation and differentiation (28). Furthermore, growth retardation in hypothyroidism results from disrupted growth plate architecture, impaired vascular invasion of the growth plate, and inhibition of hypertrophic chondrocyte differentiation (18, 55). Again, differences are apparent as growth retardation in GH and IGF-I deficiency results from a combination of impaired chondrocyte proliferation and a reduction in the linear dimension of terminal hypertrophic chondrocytes (28, 29, 36, 49, 54). Together, these considerations indicate that regulation of growth and endochondral ossification by T3 involves both GH/IGF-I-independent and GH/IGF-I-dependent pathways.
In these studies, we showed that TR
1PV/+ mice display skeletal hypothyroidism despite the presence of biochemical euthyroidism. In contrast, TRßPV mice have severe RTH but a phenotype of skeletal thyrotoxicosis (13). This paradox results from differing effects of the PV mutations at the level of the hypothalamic-pituitary-thyroid axis and in bone (Fig. 12
). The hypothalamus and pituitary predominantly express TRß, and mutation or deletion of TRß results in impaired feedback regulation of TSH and the syndrome of RTH with thyrotoxic levels of T4 and T3 and elevated TSH concentrations (14, 44, 46, 56, 57, 58). In this situation, the pituitary displays tissue hypothyroidism. In contrast, mutation or deletion of TR
does not interfere significantly with feedback regulation of TSH, and minor changes in circulating T4 and T3 levels result from impaired hormone production in the thyroid gland (11, 15, 16, 17, 58, 59). In this situation the pituitary functions normally and systemic T4 and T3 levels lie within or close to the normal range. Together with data from other mutant mice (reviewed in Refs.12 , 60 , and 61), these considerations establish that TRß is the physiological mediator of negative feedback control of TSH secretion. In contrast, our previous studies suggest that TR
is the major functional TR in bone (13, 17, 19). In the current studies, demonstration of skeletal hypothyroidism and impaired ossification in TR
1PV/+ mice establishes that TR
acts directly in bone as a physiological regulator of skeletal development. In this context, it is apparent that the skeletal consequences of disrupted TRß function in TRßPV mice result from impaired inhibition of TSH and the resulting elevated T4 and T3 concentrations, which act via TR
in bone to induce skeletal thyrotoxicosis (13, 62). In contrast, the skeletal hypothyroidism in TR
1PV/+ mice results from locally impaired TR
function in bone.

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Fig. 12. Relationship between Pituitary and Skeletal Thyroid Status Revealed by Analysis of TR 1PV/+ and TRßPV/PV Mice
Bone is a TR -responsive tissue, whereas pituitary is TRß responsive. The TR 1PV mutation does not affect pituitary T3 responses because mutant TR 1PV concentrations are too low to interfere with TRß. In bone, however, T3 responses are severely impaired because of high concentrations of dominant-negative TR 1PV, resulting in skeletal hypothyroidism. In contrast, the TRßPV mutation disrupts pituitary T3 responses and causes RTH, resulting in elevated circulating thyroid hormone concentrations and reduced GH production. In bone, mutant TRßPV concentrations are too low to interfere with TR 1, and elevated T4 and T3 concentrations hyperstimulate TR 1, resulting in skeletal thyrotoxicosis. TRE, Thyroid response element.
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Our analysis of TR
1PV/+ and TRßPV mice has provided a new understanding of the complex relationship between central pituitary thyroid status and peripheral skeletal thyroid status that arises because the pituitary gland is a TRß target tissue, whereas bone is a TR
target organ. The model in Fig. 12
can be extended to understand the relationship between central and peripheral thyroid status in any T3-target tissue, depending on whether the peripheral tissue in question is TR
or TRß responsive. Thus, in the heart, a TR
-responsive organ, features of thyrotoxicosis are seen in mice with TRß mutation (63), whereas features of hypothyroidism are seen in TR
mutants (59). In contrast, in the liver, a TRß target tissue, a hypothyroid phenotype of impaired cholesterol clearance is seen in TRß mutant mice but not in TR
mutants (64).
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MATERIALS AND METHODS
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TR
1PV and TRßPV Mutant Mice
Animal studies were conducted in strict 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, heterozygous (TR
1PV/+ and TRßPV/+), and homozygous (TRßPV/PV) mutant mice were bred and genotyped as described elsewhere (14, 15). Both TR
1PV and TRßPV strains were generated from genomic clones isolated from a 129Sv mouse genomic library and transfected into TC-1 embryonic stem cells. Both mutant strains have a mixed C57BL/6J and NIH Black Swiss genetic background. Initial studies have revealed that TR
1PV/+ mice exhibit growth retardation with only minor alterations in circulating thyroid hormones (15). In contrast, gross RTH is present in homozygous TRßPV/PV mice, with milder thyroid dysfunction in heterozygous TRßPV/+ mutants (14). Detailed analysis of bone development in TRßPV mice has revealed accelerated growth, advanced bone age, and short stature resulting from skeletal thyrotoxicosis (13).
Skeletal Preparations
E17.5, P1, P14, P21, P28, and P49 male and female littermate mice were obtained. E17.5 and neonatal mice and limbs from P14, P21, P28, and P49 animals were fixed in 95% ethanol before staining with alizarin red and alcian blue 8GX as described previously (13). Skeletal preparations were photographed using a Leica MZ 75 binocular microscope (Leica AG, Heerbrugg, Switzerland), Leica KL 1500 LCD light source, Leica DFC 320 digital camera, Leica IM50 Digital Image Manager, and Leica Twain Module DFC 320 image acquisition software. Bone lengths from wild-type and TR
1PV/+ male and female littermates were determined digitally after linear calibration of pixel size using the image acquisition software. Skull dimensions and open fontanelle and suture areas were calculated using Image J v1.33u software (http://rsb.info.nih.gov/ij/). The assessment of ossification stage in E17.5 and neonatal mice, as determined by the amount of alizarin red staining relative to alcian blue, was more subjective (Fig. 2
). In these studies differences that were observed in all mutant mice examined compared with wild-type littermates were considered to be indicative of a difference in the degree of ossification.
Histology
Limbs were fixed for 4872 h in 10% neutral buffered formalin followed by decalcification in 10% formic acid and 10% neutral buffered formalin at 20 C. E17.5 and P1 limbs were decalcified for 24 h; P14, P21, and P28 limbs were decalcified for 5 d and P49 limbs were decalcified for 7 d. Paraffin-embedded 3-µm sections were taken from anatomically oriented bones (three to five parallel levels per bone depending on the age of the animal; 20 sections per level) and stained with hematoxylin and eosin (Pioneer Research Chemicals, Colchester, UK) or van Gieson and alcian blue 8GX, as described elsewhere (13, 18). Some limbs from E17.5 and P14 mice were also fixed for 4872 h in 10% neutral buffered formalin and frozen in paraffin without prior decalcification for determination of mineralization by von Kossa staining of 3-µm cryosections with neutral red counterstain (13).
In Situ Hybridization and Analysis of Growth Plate Dimensions
mRNA expression was analyzed in growth plate sections from P14, P21, P28, and P49 mice using collagen II, collagen X, FGFR1, IGF-I, IGF-IR, and GHR cRNA probes. A bacterial neomycin resistance gene cRNA probe (Boehringer Mannheim, Lewes, Sussex, UK) was used as a negative control for all hybridizations, and collagen II (nucleotides 29823689; GenBank accession no. L48440) and X (nucleotides 418858; GenBank accession no. AJ31848) probes were used to identify proliferative and hypertrophic zones in growth plate sections, as described in previous studies in which we optimized in situ hybridization methods (13, 18, 19). A rat FGFR1 (nucleotides 104603; GenBank accession no. S54008) partial cDNA was isolated by RT-PCR as described previously (18, 19) from osteoblastic ROS 17/2.8 cells (65). The rat IGF-I partial cDNA (nucleotides 61314; GenBank accession no. D00698) was a gift from Dr. Cécile Kedzia (Institut National de la Santé et de la Recherche Médicale, Paris, France). Mouse IGF-1R (nucleotides 10631690; GenBank accession no. XM_133508) and GHR (nucleotides 470711; GenBank accession no. NM_010284) partial cDNAs were isolated by RT-PCR as described elsewhere (18, 19) from chondrogenic ATDC5 cells (66) with the following primers: IGF-1R, forward 5'-GAAGACCACCATCAACAAT-3', reverse 5'-GAAGGACAAGGAGACCAAG-3'; GHR, forward 5'-GACCCCAGGATCTATTCAGC-3', reverse 5'-CAGGTTGCACTATTTCGTCAAC-3'. PCR products were subcloned into pGEM-T (Promega, Southampton, Hampshire, UK) and sequenced. FGFR1, IGF-I, IGF-1R, and GHR constructs were linearized with SpeI, BamHI, DraII, and SpeI, and digoxigenin-labeled cRNA probes were synthesized using T7, T3, T7, and T7 RNA polymerases, respectively (Boehringer Mannheim). In situ hybridizations using alkaline phosphatase-labeled probes were performed on 3-µm deparaffinized sections as described elsewhere (13, 18, 19). Studies were performed on at least three mice per genotype in duplicate, and repeat experiments were performed on three separate occasions.
Measurements at four separate positions across the width of growth plates were obtained, using a Leica DM LB2 microscope, Leica DFC 320 digital camera, Leica IM50 Digital Image Manager, and Leica Twain Module DFC 320 image acquisition software, to calculate mean values for the heights of the RZ, PZ, HZ, and total growth plate in sections from wild-type and TR
1PV/+ mice. Results from adjacent levels of sectioning were compared to ensure consistency of the data. Cortical bone width measurements were performed at four separate positions in the midshaft of the tibia and adjacent levels of sectioning were compared. All studies are performed with the observer blinded to the genotype. For histology and histomorphometry analyses, at least three animals per genotype were examined.
Immunohistochemistry
Activation of IGF-IR and GHR downstream signaling was examined by immunohistochemical analysis of protein kinase B (Akt) and signal transducer and activator of transcription-5 (STAT5) expression in wild-type, TR
1PV/+, TRßPV/+, and TRßPV/PV growth plates. Sections were deparaffinized and rehydrated in ethanol and PBS. Sodium citrate antigen retrieval was performed for 6 min in a microwave oven on medium setting. Endogenous peroxidase activity was quenched with 1% H2O2 in methanol for 15 min at room temperature. Sections were then blocked with 5% fetal calf serum (Sigma Chemical Co., St. Louis, MO) in PBS with 0.5% Tween 20 for 1 h at room temperature, before addition of primary antibody and incubation overnight at 4 C. Polyclonal antibodies used to detect expression of Akt (Santa Cruz Biotechnology. Inc., Santa Cruz, CA), phosphorylated Akt (Cell Signaling Technology, Inc., Beverly, MA), STAT5 (Santa Cruz), and phosphorylated STAT5 (Santa Cruz) were diluted 1:175, 1:50, 1:200, and 1:140, respectively. Sections were subsequently incubated with peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Inc., Hercules, CA) diluted 1:2000 to 1:1800 for 30 min at room temperature. Peroxidase activity was detected using 3,3'-diaminobenzidine containing 0.02% H2O2 (Sigma). Negative controls lacking primary antibody were performed in parallel in all experiments, as described elsewhere (53, 67). Studies were performed on at least three mice per genotype in duplicate, and repeat experiments were performed on three separate occasions.
Statistical Analysis
Data were expressed as mean ± SEM. Differences between groups were examined for statistical significance using Students t test, in which P values <0.05 were considered significant.
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FOOTNOTES
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This work was supported by a Medical Research Council (MRC) Ph.D./Studentship (to P.J.OS.), MRC Clinician Scientist Fellowship (to J.H.D.B.), and MRC Career Establishment Grant (G9803002) (to G.R.W.).
First Published Online July 28, 2005
Abbreviations: ALSKO, Acid-labile subunit knockout; E17.5, embryonic d 17.5; FGFR, fibroblast growth factor receptor; GHR, GH receptor; IGF-1R, IGF-I receptor; LID, liver-specific IGF-I knockout; HZ, hypertrophic zone; P1, postnatal d 1; PZ, proliferative zone; RTH, resistance to thyroid hormone; RZ, reserve zone; STAT, signal transducer and activator of transcription; TR, T3 receptor.
Received for publication June 8, 2005.
Accepted for publication July 20, 2005.
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