A Thyrotoxic Skeletal Phenotype of Advanced Bone Formation in Mice with Resistance to Thyroid Hormone

Patrick J. O’Shea, Clare B. Harvey, Hideyo Suzuki, Masahiro Kaneshige, Kumiko Kaneshige, Sheue-Yann Cheng and Graham R. Williams

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

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone (T3) regulates bone turnover and mineralization in adults and is essential for skeletal development during childhood. Hyperthyroidism is an established risk factor for osteoporosis. Nevertheless, T3 actions in bone remain poorly understood. Patients with resistance to thyroid hormone, due to mutations of the T3-receptor ß (TRß) gene, display variable phenotypic abnormalities, particularly in the skeleton. To investigate the actions of T3 during bone development, we characterized the skeleton in TRßPV mutant mice. TRßPV mice harbor a targeted resistance to thyroid hormone mutation in TRß and recapitulate the human condition. A severe phenotype, which includes shortened body length, was evident in homozygous TRßPV/PV animals. Accelerated growth in utero was associated with advanced endochondral and intramembranous ossification. Advanced bone formation resulted in postnatal growth retardation, premature quiescence of the growth plates, and shortened bone length, together with increased bone mineralization and craniosynostosis. In situ hybridization demonstrated increased expression of fibroblast growth factor receptor-1, a T3-regulated gene in bone, in TRßPV/PV perichondrium, growth plate chondrocytes, and osteoblasts. Thus, the skeleton in TRßPV/PV mice is thyrotoxic and displays phenotypic features typical of juvenile hyperthyroidism.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
T3 IS ESSENTIAL for skeletal development (1). Childhood hypothyroidism results in growth arrest, delayed bone age, and stippled epiphyses. T4 replacement induces rapid catch-up growth, although maximum height is rarely achieved, and the resulting deficit correlates with the estimated duration of untreated hypothyroidism (2). Untreated childhood thyrotoxicosis causes accelerated growth and advanced bone age with premature closure of growth plates and skull sutures that results in short stature and craniosynostosis (3). In adults, thyrotoxicosis is an established risk factor for osteoporosis.

T3 stimulates bone resorption in vivo and in vitro (4, 5), but these effects require osteoblasts, which respond to T3 directly and are thought to communicate with osteoclasts via paracrine pathways (6). In the growth plate in vivo and in growth plate chondrocytes in vitro, T3 inhibits cell proliferation and stimulates hypertrophic chondrocyte differentiation (7, 8). Endochondral ossification, the process that results in skeletal development, linear growth, and bone formation, is regulated by thyroid hormones in vivo and involves the coordinated control of chondrocyte proliferation, differentiation, and apoptosis (8, 9). Closure of the skull sutures is also stimulated by T3 in vivo (10). Osteoblasts (11, 12, 13) and growth plate chondrocytes (7, 8, 14) express T3 receptors (TRs), and these cells respond directly to T3 in vitro. Nevertheless, although several specific T3-target genes have been identified in bone (1, 15, 16, 17), the mechanisms of T3-induced gene regulation in bone have not been defined, and it is not known which TRs mediate the skeletal actions of T3.

T3 actions are mediated by TRs, which act as ligand-inducible transcription factors that are expressed as multiple isoforms, each with unique developmental and tissue-specific patterns of expression (18, 19). The TR{alpha}1, TRß1, TRß2, and TRß3 isoforms are functional T3-responsive receptors but the {alpha}2, {Delta}{alpha}1, {Delta}{alpha}2, and {Delta}ß3 splice variants act as repressors in vitro, although their physiological significance is unknown (18, 20). It is unclear which TRs are coexpressed in individual cells or whether alterations in their relative concentrations influence cellular T3 responses in vivo. This complexity has resulted in difficulty establishing thyroid status and characterizing T3 action in individual tissues.

Resistance to thyroid hormone (RTH) is an autosomal dominant condition caused by mutation of the TRß gene (21). The mutant receptor acts as a dominant-negative antagonist that interferes with transcriptional control of T3-target genes. Nevertheless, a single severe case caused by a homozygous mutation of TRß has been reported (22), and a family has been identified with RTH due to homozygous deletion of the TRß coding region (23). RTH is characterized by reduced tissue sensitivity to T3 that is manifest by elevated circulating T3 and T4 concentrations with inappropriately normal or elevated serum TSH levels. The clinical features are variable and include goiter, tachycardia, hearing loss, attention deficit hyperactivity disorder, reduced IQ, and short stature (21). The complex phenotype is thought to result from hypothyroidism in tissues such as pituitary and brain, with thyrotoxic features evident in others such as heart. Phenotypic differences occur between families with different mutations, between families harboring the same mutation, and also between members of the same family with identical mutations. Furthermore, RTH has been characterized in families in which TRß mutations have not been identified (24), indicating that modifier genes influence target organ responses to thyroid hormones.

Phenotype variability in RTH is especially seen in the skeleton. Features include stippled epiphyses with scattered calcification in growth plate cartilage, high bone turnover osteoporosis and fracture, reduced bone mineral density, craniosynostosis, and various developmental defects of the facial bones or vertebrae (25). Growth retardation and short stature are frequently noted and have been attributed to skeletal hypothyroidism. However, objective measurements including growth curves are available in only a minority of patients; short stature has been estimated to occur in up to 26%, with evidence of variably delayed bone age in up to 47% (21, 25). In contrast, bone age has been shown, additionally, to be advanced by more than 2 SD in two families, and lesser degrees of advanced bone age have also been documented. These observations have been interpreted to indicate that an intact TRß gene is required for normal bone development and growth (25).

To investigate further, we analyzed skeletal development in TRßPV mutant mice (26). The PV mutation was derived from a patient with severe RTH (27). PV is a C-insertion in codon 448 leading to a frame shift of the carboxy-terminal 14 amino acids of TRß1, which produces a receptor that fails to bind T3, has no transactivation activity, and interferes with the actions of wild-type TR in vitro (28, 29). Heterozygous TRßPV/+ mice recapitulate human RTH with circulating T3 and T4 concentrations elevated 2- and 2.5-fold relative to wild type in association with mild goiter and a 2.1-fold increase in TSH. Homozygous TRßPV/PV mutants have severe disruption of the pituitary thyroid axis with T3, T4, and TSH levels elevated 9-, 15-, and 412-fold, respectively (26). In these studies, we demonstrate, unexpectedly, that shortened bone and body length in TRßPV/PV mice are associated with advanced endochondral and intramembranous bone formation.

Animal studies were conducted in strict accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the National Cancer Institute Animal Care and Use Committee.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Growth curves were plotted for TRß+/+, heterozygous TRßPV/+, and homozygous TRßPV/PV mice. No difference was observed between body weights of male or female TRßPV/+ mice compared with TRß+/+, but male and female TRßPV/PV mice were 20% smaller (Fig. 1AGo). Similar data were obtained from body length measurements, indicating proportionate reductions of both parameters in homozygous mutants. Measurement of long bones confirmed a reduction in their length in TRßPV/PV mice in the early postnatal period, with no difference observed between wild-type and heterozygous animals. A different pattern was observed at embryonic d 17.5 (E17.5), when bones of homozygous TRßPV/PV mice were longer than heterozygous and wild-type littermates (Fig. 1BGo). At birth, however, bone lengths were identical in all three genotypes.



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Figure 1. Growth Curves of Wild-Type and TRßPV Mice

A, Graphs showing mean weights (g) ± SEM of male (left) and female (right) wild-type (+/+), heterozygote (PV/+), and homozygote TRßPV mutant (PV/PV) mice at weekly intervals up to 10 wk of age. Data analyzed by Students’ t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; PV/PV vs. +/+. Males (+/+, n = 6–12 each time point; PV/+, n = 13–16; PV/PV, n = 5). Females (+/+, n = 10; PV/+, n = 14; PV/PV, n = 3). B, Graphs showing mean lengths of tibia + ulna combined (mm) ± SEM of +/+, PV/+, and PV/PV mice from E17.5 to 4 wk (upper). Lower graph shows increased magnification of the upper graph between ages E17.5 and 4 d postnatal. Data analyzed by Students’ t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; PV/PV vs. +/+. Total numbers of animals (both sexes); E17.5 (+/+, n = 7; PV/+, n = 14; PV/PV, n = 7); neonate (+/+, n = 9; PV/+, n = 15; PV/PV, n = 9); 2 wk (+/+, n = 4; PV/+, n = 4; PV/PV, n = 3); 3 wk (+/+, n = 3; PV/+, n = 16; PV/PV, n = 3); 4 wk (+/+, n = 4; PV/+, n = 4; PV/PV, n = 2).

 
Circulating total T4 concentrations were determined in E17.5, neonatal, and in 2-wk-, and 4-wk-old mice. T4 values in E17.5 animals of each genotype were below the level of detection using a highly sensitive T4 RIA. These findings are in keeping with a recent study in which serum T4 levels were undetectable in neonatal wild-type mice and increased with age to a peak on d 15 (30). In our studies, total T4 levels were barely detectable in neonatal wild-type and heterozygous TRßPV/+ mice, but were elevated 4-fold in neonatal TRßPV/PV animals (Fig. 2Go). T4 concentrations were elevated 2-fold in both 2- and 4-wk-old TRßPV/+ mice but were elevated 7-fold and 11-fold, respectively, in 2- and 4-wk-old homozygous TRßPV/PV mutants. These data demonstrate that circulating T4 concentrations increase in an age-dependent manner and that T4 levels become elevated in hetererozygous TRßPV/+ mice only after birth, but are already raised in homozygous TRßPV/PV animals at the time of birth. The undetectable and very low circulating T4 levels in E17.5 and neonatal animals, however, may not reflect thyroid status in individual tissues (30) and do not exclude the presence of significant thyroid hormone levels in fetal bone. It is not possible, therefore, to determine the precise timing of the onset of tissue hyperthyroidism in the skeleton of TRßPV/PV animals.



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Figure 2. Comparison of Serum Total T4 Concentrations in Neonatal (0 day), 2-wk- and 4-wk-Old TRßPV Mice

Data are expressed as mean ± SEM (n = 1–9; *, P < 0.05; **, P < 0.01; ***, P < 0.001 as compared with wild-type (+/+) mice for each age.

 
RNA was extracted from the femur and tibia of 7-wk-old wild-type mice and analyzed by real-time RT-PCR to determine the relative levels of expression of TR{alpha}1 and TRß1 mRNAs. TR{alpha}1 was expressed at a 12-fold higher concentration than TRß1 (Fig. 3Go), indicating that TR{alpha}1 is expressed predominantly in bone.



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Figure 3. Comparison of the Relative Levels of Expression of TRß1 and TR{alpha}1 mRNA Isoforms in Bone

Total RNAs were extracted from tibia and femur of 7-wk-old male wild-type mice. Real-time RT-PCR using specific primers for TR{alpha}1 and TRß1 was performed. Relative quantification of the TR{alpha}1 mRNA was determined by designating expression of TRß1 mRNA to 1. Differences in total RNA input were normalized by signals obtained with specific primers for glyceraldehyde-3-phosphate dehydrogenase. Data are expressed as mean ± SEM (n = 3; *, P < 0.0001).

 
The observed accelerated growth of homozygous TRßPV/PV mutant mice in utero, which was followed by a slowing of the growth rate in the postnatal period (Fig. 1Go), was supported by studies of skeletal preparations from E17.5 and neonatal mice, in which Alizarin Red-stained ossified bone pink and Alcian Blue 8GX-stained cartilage blue (Fig. 4Go). In the thorax at E17.5 there was increased Alizarin Red staining in TRßPV/+ heterozygous ribs and vertebral arches compared with wild type, with a further increase seen in TRßPV/PV mice (Fig. 4Go, arrows). In the upper and lower limbs a similar graded pattern was identified, with increased Alizarin Red staining of ossified bone in TRßPV/PV mice compared with heterozygous and wild-type littermates. Furthermore, correlating with limb measurements in Fig. 1BGo, the upper and lower limbs at E17.5 from TRßPV/PV mice were larger. These data demonstrate advanced bone formation in TRßPV/PV mutant mice by E17.5 and confirm that accelerated growth of homozygous mutants occurs in utero. Examination of the skull in neonatal mice revealed further differences between TRßPV/PV animals and heterozygous and wild-type littermates. The neonatal TRßPV/PV skull displayed an exaggerated curvature of the cranium together with increased areas of Alizarin Red staining and reduced regions of Alcian Blue in the craniofacial bones (Fig. 4Go, arrowheads). These features suggested the possibility of advanced bone formation in the skull, which develops via intramembranous ossification of the cranial bones and mandible and by endochondral ossification of the facial bones.



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Figure 4. Skeletal Preparations from E17.5 and Neonatal Wild-Type (+/+), Heterozygote (PV/+), and Homozygote TRßPV Mutant (PV/PV) Mice Stained with Alizarin Red (Bone) and Alcian Blue (Cartilage)

Rib cages are shown in the upper panels; upper limbs in the middle left; lower limbs in the middle right; and skulls in the bottom panels (all x8 magnification). Arrows indicate ossified vertebral arches (pink) in mutant mice compared with wild type. Arrowheads indicate differences in Alcian Blue and Alizarin Red staining in the maxilla between the genotypes.

 
Thus, cranial development was examined in E17.5 and neonatal mice (Fig. 5Go). No evidence of altered skull development was seen in TRßPV/+ mice compared with wild type (not shown), but homozygous TRßPV/PV skulls were abnormal. The fontanelles were smaller and cranial sutures narrower in skulls from E17.5 and neonatal TRßPV/PV mice. Larger areas of ossified bone had formed in the TRßPV/PV skull relative to wild type by E17.5, as seen clearly in the occipital and parietal bones (Fig. 5Go). In neonates, there was deeper alizarin red staining and reduced skull translucency in TRßPV/PV mice relative to wild type. In high-power views of the coronal suture there was interdigitation of bony outgrowths from the frontal and parietal bones across the suture line, which was almost closed at the anterior fontanelle in TRßPV/PV mice (Fig. 5Go). These features were less advanced in wild-type animals, in which the anterior fontanelle remained broader. Taken together with the altered shape of the skull (Fig. 4Go), these data demonstrate advanced intramembranous ossification of the skull with craniosynostosis in TRßPV/PV mice that is evident by E17.5 and more pronounced at birth.



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Figure 5. Skull Preparations from E17.5 and Neonatal Wild-Type (+/+) and Homozygote TRßPV mutant (PV/PV) Mice Stained with Alizarin Red (Bone) and Alcian Blue (Cartilage)

Pairs of skulls were placed side by side, illuminated together by the same light source, and photographed individually using identical exposure settings. Magnification, x16 in top four panels, x25 in third set of panels, and x63 in lower panels. Anatomy of the skull sutures, bones, and fontanelles is shown in the adjacent diagram.

 
Analysis of limbs from 3-wk-old animals revealed differences between TRßPV/PV and wild-type mice (Fig. 6Go). In the forelimb, there was increased Alizarin Red staining of ossified bone in TRßPV/PV homozygous mutants compared with heterozygous and wild-type mice. Furthermore, growth plate cartilage in long bones from wild-type and heterozygous mice stained positive with Alcian Blue. In contrast, TRßPV/PV growth plates did not exhibit Alcian Blue staining. Identical findings were observed in the lower limbs of TRßPV/PV mice (Fig. 4Go). In contrast to findings at E17.5 (Figs. 1Go and 4Go), homozygous TRßPV/PV mutant limbs from 3-wk-old animals were shorter than wild type (Fig. 6Go), confirming short body length in these animals (Fig. 1Go). These data demonstrate advanced endochondral bone formation in TRßPV/PV mice at 3 wk of age. Reduced Alcian Blue staining of growth plate cartilage in 3-wk-old TRßPV/PV mice correlated with reduced growth rates of these animals.



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Figure 6. Skeletal Preparations from Upper and Lower Limbs of 3-wk-Old Wild-Type (+/+), Heterozygote (PV/+), and Homozygote TRßPV Mutant (PV/PV) Mice Stained with Alizarin Red (Bone) and Alcian Blue (Cartilage)

Upper panels show forelimbs (magnification, x8), middle panel shows tibias (x8), and lower panels show hindpaws (x16).

 
These findings were investigated further in histological and in situ hybridization studies. Undecalcified and decalcified limbs from 2-, 3-, and 4 wk-old mice were examined. Increased calcified bone was evident in 2-wk-old limbs from TRßPV/PV homozygous mutants compared with TRßPV/+ and TRß+/+ mice, as indicated by increased von Kossa staining in trabecular bone of the tibial epiphysis and metaphysis (Fig. 7Go). By 4 wk, increased von Kossa staining persisted in TRßPV/PV homozygous mice relative to wild type and became evident in TRßPV/+ heterozygotes, although the difference relative to wild type was less marked than in 2-wk-old limbs. These data indicate that trabecular bone mineralization is advanced in homozygous mutants by 2 wk of age, with increased mineralization present in both heterozygous and homozygous mutants at 4 wk. Advanced endochondral ossification was demonstrated further in TRßPV/PV homozygous mice by histological analysis of the fibula in 3-wk-old animals (Fig. 7Go, middle panels). By 3 wk of age the growth plate was fully organized and the secondary center of ossification that forms the epiphysis of the fibula was present in TRßPV/PV mice (arrow), whereas the growth plates were immature, and secondary ossification centers had not formed in 3-wk-old heterozygous and wild-type mice.



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Figure 7. Undecalcified Sections of the Upper Tibial Growth Plate Region from 2- and 4-wk-Old Wild-Type (+/+), Heterozygote (PV/+), and Homozygote TRßPV Mutant (PV/PV) Mice (Top and Bottom Panels, Respectively), Stained with von Kossa (Calcified Bone in Black) and Neutral Red Counterstain (Magnification, x40)

Middle panels show decalcified growth plate sections from fibulas of 3-wk-old animals (x200) stained with Alcian Blue (growth plate cartilage) and van Gieson (bone osteoid in red). The arrow indicates presence of the secondary center of ossification that forms the epiphysis of the fibula in PV/PV mice. Arrowheads show the tibial epiphysis and asterisks indicate the metaphysis.

 
Detailed studies of the growth plates were performed in 2-, 3-, and 4-wk-old animals to investigate mechanisms underlying advanced ossification in TRßPV mice. The period between 2 and 4 wk of age was chosen for these studies because it represents the time at which the growth curves of homozygous mutant mice diverge from those of wild-type and heterozygous animals (Fig. 1Go), indicating the critical postnatal period in which there is a slowing of the growth rate in TRßPV/PV mice. Histological studies enabled the reserve zone (RZ), proliferative zone (PZ), and hypertrophic zone (HZ) of the growth plate to be identified anatomically (Fig. 8AGo). In situ hybridization was performed to determine expression of collagen II, a marker of proliferating chondrocytes (31), to define the extent of the PZ (Fig. 8BGo) and facilitate accurate measurement of the PZ, HZ, and total growth plate widths in 2-, 3-, and 4-wk-old animals of each genotype (Fig. 8CGo).



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Figure 8. Analysis of Growth Plate Dimensions in Wild-Type and TRßPV Mice

A, Decalcified sections of the upper tibial growth plate from 3-wk-old wild-type (+/+), heterozygote (PV/+), and homozygote TRßPV mutant (PV/PV) mice stained with hematoxylin and eosin (magnification, x100). The RZ, PZ, and HZ of the growth plate are indicated. B, In situ hybridization for collagen II expression in the upper tibial growth plate (x200). (Legend continued on next page.)

The regions of the growth plate are indicated as in panel A and are shown relative to the total growth plate width (GP). C, Widths of the PZ and HZ (upper graph) and the ratios of the widths of PZ to HZ (lower graph) in tibial growth plates from 2-, 3-, and 4-wk-old +/+, PV/+, and PV/PV mice. Mean growth plate measurements (µm) ± SEM were obtained from three to four animals of each genotype at each age (except 4 wk PV/PV mice, n = 2); four separate measurements were obtained across each growth plate. Data were analyzed by Student’s t test for 3-wk- vs. 2-wk-old and 4-wk- vs. 3-wk-old animals: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Total growth plate width or PZ:HZ ratio: {dagger}{dagger}{dagger}, P < 0.001 PZ width; ###, P < 0.001 HZ width; NS, not significant.

 
Between 2 and 4 wk of age there was progressive narrowing of the growth plate in wild-type and heterozygous TRßPV/+ mice that was due to narrowing of both the PZ and HZ regions. This effect predominated in the PZ, as indicated by reduction of the PZ:HZ ratio over this time. Growth plate measurements, however, were considerably different in TRßPV/PV mice. At 2 and 3 wk, the TRßPV/PV growth plate was narrower than in heterozygous (P < 0.001) or wild-type (P < 0.001) littermates. This finding was associated with a reduction in width of both the PZ and HZ. In contrast, by 4 wk there was no further narrowing of the TRßPV/PV growth plate. Thus, the TRßPV/PV growth plate became quiescent (32) between 3 and 4 wk of age and remained broader than in wild-type (P < 0.001) and heterozygous (P < 0.001) mice, in which growth plate narrowing continued. These data suggest that premature quiescence of the growth plates accounts for shortened bone length in TRßPV/PV mice.

To investigate thyroid status in growth plate chondrocytes, in situ hybridization experiments were performed to determine expression of fibroblast growth factor receptor 1 (FGFR1) and collagen X mRNAs. In recent work (33), we showed that FGFR1 expression and functional activity is enhanced by T3 and that FGFR1 mRNA expression is considerably reduced in the growth plate and osteoblasts of TR{alpha}-null (TR{alpha}0/0) mice, which are growth retarded due to impaired endochondral ossification (34). FGFR1 mRNA is expressed predominantly in prehypertrophic and hypertrophic chondrocytes in the normal epiphyseal growth plate (35). First, we identified hypertrophic chondrocytes in wild-type, TRßPV/+, and TRßPV/PV mice by in situ hybridization. Collagen X is a specific marker of hypertrophic chondrocyte differentiation (36), and expression was restricted to HZ chondrocytes in wild-type and mutant mice [Fig. 9AGo (i)]. Together with studies of collagen II expression that determined the extent of the PZ (Fig. 8BGo), these findings enabled identification of the growth plate regions in which FGFR1 was expressed.




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Figure 9. In Situ Hybridizations of Growth Plate and Diaphyseal Cortical Bone from Wild-Type (+/+), Heterozygote (PV/+), and Homozygote TRßPV Mutant (PV/PV) Mice of Various Ages.

A (i), Collagen X expression (magnification, x200) is shown within the epiphyseal growth plate of 3-wk-old mice, in which the regions containing reserve zone (rz), prolifertive zone (pz), and hypertrophic zone (hz) chondrocytes are shown; (ii) growth plate sections from 3-wk-old mice hybridized to a neomycin cRNA probe are shown as negative controls (x100). B, FGFR1 mRNA expression is shown in the epiphyseal growth plates of (i) E17.5 (arrowheads show perichondrial region, x200); (ii) neonatal (arrowheads show perichondrial region, x100); (iii) 2-wk-old (arrowheads show prehypertrophic chondrocytes at the distal region of the proliferative zone, x200); (iv) 3-wk-old (arrowheads show prehypertrophic chondrocytes, x200); and (vi) 4-wk-old (arrowheads show prehypertrophic chondrocytes, x100) mice as well as in (v) endosteal osteoblasts (arrowheads) lining cortical bone within the tibial diaphysis of 3-wk-old animals (x600).

 
FGFR1 mRNA was restricted to perichondrial cells surrounding developing limbs in E17.5 and neonatal mice and was increased in both TRßPV/+ and TRßPV/PV mice compared with wild type [Fig. 9BGo, (i) and (ii)]. A greater increase in FGFR1 expression was evident in TRßPV/PV animals compared with heterozygous mutant littermates, although FGFR1 mRNA expression was not entirely restricted to the perichondrium in E17.5 TRßPV/+ mice and was seen in chondrocytes within the immature growth plate of these animals. In 2-, 3-, and 4-wk old mice, there was increased expression of FGFR1 mRNA in the TRßPV/PV growth plate compared with wild-type and heterozygous animals. Furthermore, FGFR1 mRNA expression extended throughout the growth plate in TRßPV/PV mice and was not restricted to prehypertrophic and hypertrophic chondrocytes [Fig. 9BGo, (iii), (iv), and (vi)]. FGFR1 expression was also increased and more extensive in growth plates from 2-wk-old TRßPV/+ mice compared with wild type [Fig. 9BGo (iii)]. Increased FGFR1 expression in heterozygous mutants was less pronounced in growth plates from 3-wk-old animals compared with observations in 2-wk-old animals and was not seen in 4-wk-old TRßPV/+ mice, when increased expression was retained in homozygous TRßPV/PV mice [Fig. 9BGo (vi)].

In addition to the findings in growth plate chondrocytes, FGFR1 mRNA expression was increased in osteoblasts lining diaphyseal cortical bone in 3-wk-old TRßPV/PV mice compared with wild-type and TRßPV/+ animals [Fig. 9BGo (v)]. Similar findings were observed in mice of all ages and in osteoblasts lining metaphyseal and epiphyseal trabecular bone (not shown). These data contrast with findings in TR{alpha}0/0 knockout mice (33) and provide evidence of increased T3 signaling in the TRßPV/PV skeleton during development between E17.5 and 4 wk of age.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several lines of evidence demonstrate that shortened bone length in TRßPV/PV mice results from advanced bone formation. Accelerated growth occurs in utero (Fig. 1Go) in association with premature ossification of the ribs, vertebrae, limbs, and facial bones (Fig. 4Go). In the postnatal period, the growth rate slows below that of heterozygous and wild-type littermates. Shortened body length becomes evident and persists after the wild-type and TRßPV/PV growth curves cross over at the neonatal stage (Fig. 1Go). At 2 wk, increased bone mineralization is evident in TRßPV/PV mice (Fig. 7Go), indicating premature skeletal maturation as the growth rate begins to slow down. This finding is strengthened by analysis of 3-wk-old animals in which there is early quiescence of the growth plates in the upper and lower limbs (Fig. 6Go) along with premature formation of the secondary center of ossification in the TRßPV/PV fibula (Fig. 7Go). Advanced endochondral ossification occurs in association with advanced intramembranous ossification in the skull (Fig. 5Go). These features of accelerated growth, advanced bone age (early formation of secondary ossification centers), shortened body and bone length, and craniosynostosis are those of juvenile thyrotoxicosis and indicate that the skeleton of TRßPV/PV mice, which exhibit severe RTH, unexpectedly displays a hyperthyroid phenotype. This is supported by data showing overexpression of FGFR1 mRNA in the perichondrial region, growth plate, and osteoblasts of TRßPV/PV mice (Fig. 9Go) and the finding of elevated T4 concentrations in TRßPV/PV mice from birth (Fig. 2Go). We recently showed that FGFR1 mRNA expression is stimulated by T3 in osteoblasts and found that FGFR1 expression is reduced in growth plate chondrocytes and osteoblasts of TR{alpha}0/0 mice (33), which display a hypothyroid phenotype of delayed endochondral ossification (34).

Having identified a thyrotoxic skeletal phenotype in TRßPV/PV mice, it is important to consider the mechanism of skeletal hyperthyroidism, to ask why the phenotype is more severe in homozygous mutants, and to understand how accelerated bone formation leads to ultimately reduced body length. Genetic studies indicate that the TR{alpha} gene is essential for skeletal development. We showed that TR{alpha}0/0 mice exhibit delayed endochondral ossification, impaired mineralization, and growth retardation (34) whereas TRß-/- mice display a normal skeletal phenotype (37). Thus, TR{alpha} is the major functional TR in bone. In recent studies in TRßPV mice, we showed that the abundance of mutant PV protein in a particular tissue determines its phenotype (38). The PV protein competes with wild-type TR and 9-cis-retinoic acid receptor proteins in vivo for DNA binding. This competition is more effective in homozygous mutants compared with heterozygous animals because of increased mutant receptor abundance in TRßPV/PV mice. Thus, in liver, there is a high level of TRß expression relative to TR{alpha}. Consequently, there is equal expression of mutant and wild-type TRß proteins in TRßPV/+ mice but high levels of mutant and no wild-type TRß in TRßPV/PV liver (38). In the heart the situation is different because levels of TRß are low compared with TR{alpha} and in both TRßPV/+ and TRßPV/PV mice the low levels of mutant receptor are unable to interfere with the action of TR{alpha} (38). Thus, in TRßPV/+ and TRßPV/PV mice, the liver displays a hypothyroid phenotype with reduced expression of T3 target genes. A similar picture is seen in pituitary where expression of the negatively regulated TSH gene is increased. In contrast, the heart is thyrotoxic with increased expression of the positively regulated target gene {alpha}-myosin heavy chain and reduced expression of the negatively regulated gene ß-myosin heavy chain. Effects in both hypothyroid and thyrotoxic tissues are more marked in TRßPV/PV compared with TRßPV/+ mice, reflecting increased expression of mutant receptor and higher circulating hormone concentrations in homozygous mice (38).

In accordance with these studies, we propose that the hyperthyroid TRßPV/PV skeleton results from increased TR{alpha} activity that is stimulated by thyrotoxic circulating hormone levels. The phenotype is less severe in heterozygous animals because peripheral hormone concentrations are less markedly elevated. Furthermore, T4 concentrations only become elevated in TRßPV/+ mice by 2 wk of age, whereas in TRßPV/PV animals T4 concentrations were clearly higher in neonates (Fig. 2Go). These temporal differences also probably contribute to the severity of the phenotype in homozygous mutants. Analysis of TR mRNA expression in wild-type mice demonstrated a 12-fold higher expression of TR{alpha}1 mRNA relative to ß1 in bone (Fig. 3Go). It is important to note here that relative levels of expression of TR mRNAs may not correlate with concentrations of expressed receptor protein, although this seems very unlikely given the magnitude of the difference in mRNA expression. Thus, we predict that TRß is expressed in bone at low levels relative to TR{alpha} and that low levels of expressed TRßPV mutant cannot compete efficiently with TR{alpha} in TRßPV/PV mice. This results in a thyrotoxic phenotype that is dictated by the abundance of mutant TR in bone, as in other tissues, and the circulating hormone concentrations (38). The present studies, therefore, support recent data from knockout mice (34, 37), which indicate that TR{alpha} is the major functional TR in bone.

An alternative explanation is that TR{alpha} and TRß proteins may not be coexpressed in bone cells. This would allow TR{alpha} to act unopposed by mutant TRß in the TRßPV/PV skeleton and respond to thyroid hormone excess to cause a thyrotoxic phenotype. This possibility is unlikely, however, because we showed that TR{alpha} and TRß mRNAs are both expressed in reserve and proliferating chondrocytes and in osteoblasts (7, 8, 12). Nevertheless, colocalization of TR{alpha} and TRß proteins in individual cells has not been demonstrated formally. A further conceivable alternative is that there may be a compensatory increased expression of TR{alpha} in bone in TRßPV/PV mice. Such an increase could mediate exaggerated TR{alpha} responses to elevated hormone concentrations to produce a thyrotoxic skeletal phenotype. However, we show that TR{alpha} is predominantly expressed in bone in wild-type mice (Fig. 3Go), and such a compensatory change in TR expression was not identified in other T3-target tissues in TRßPV/PV mice (38).

We investigated how accelerated bone formation results in shortened bone length by analysis of 2-, 3-, and 4-wk-old growth plates (Fig. 8Go), the time at which the TRßPV/PV growth curve diverges (Fig. 1Go). These studies revealed accelerated narrowing of the proliferating and hypertrophic zones in the TRßPV/PV growth plate, followed by growth plate quiescence at 3–4 wk of age. In contrast, growth plate narrowing continued between 3 and 4 wk in wild-type and heterozygous animals (Fig. 8Go). These findings indicate that growth plate maturation and quiescence are advanced in TRßPV/PV mice. No sexual dimorphism was evident, suggesting that accelerated maturation was independent of sex steroids. Similar features are seen in human juvenile thyrotoxicosis, in which accelerated growth occurs in boys and girls and is manifest by premature epiphyseal fusion and short stature. In the mouse, growth plate quiescence coincides with cessation of growth; fusion occurs only in later life and is independent of sex steroids (32).

An interesting feature is that the quiescent TRßPV/PV growth plate remained broader than in wild-type and heterozygous animals at 4 wk because growth plate narrowing and linear growth continued in wild-type and heterozygous mice. These observations suggest that complex mechanisms are responsible for the premature induction of quiescence in the thyrotoxic TRßPV/PV growth plate. In previous studies, we showed that T3 inhibits chondrocyte proliferation and simultaneously accelerates hypertrophic differentiation in vitro (7). Furthermore, hypothyroidism disrupts endochondral ossification and impairs chondrocyte differentiation in vivo (9). Similar abnormalities occur in TR{alpha}0/0 mice (34). These data suggest that accelerated bone formation in TRßPV/PV mice results from T3-induced acceleration of hypertrophic chondrocyte differentiation. However, the rate of growth plate chondrocyte differentiation is coupled to cell proliferation in vivo, and thyroid status regulates the set point of a key feedback loop involving Indian hedgehog and PTH-related peptide (8), which controls the pace of chondrocyte proliferation and differentiation during development (39, 40, 41). Thus, we propose that T3 promotes entry of reserve zone progenitor cells into the PZ and shortens the transit time of chondrocytes through the PZ either by shortening the cell cycle or by reducing the number of cell divisions before hypertrophic differentiation. Both mechanisms would result in the observed narrowing of the PZ in TRßPV/PV mice.

In addition, narrowing of the HZ is present at 2 and 3 wk in the TRßPV/PV growth plate. Thus, T3 accelerates differentiation of proliferating chondrocytes but must also shorten the transit time of differentiated chondrocytes through the HZ. This could occur via two mechanisms. We propose that acceleration of differentiation either produces hypertrophic chondrocytes of reduced diameter or that T3 stimulates accelerated apoptosis of hypertrophic chondrocytes, a mechanism that would be consistent with advanced bone formation and mineralization. Either process would result in narrowing of the HZ in TRßPV/PV mice. By 4 wk, the TRßPV/PV growth plate enters quiescence whereas growth plate narrowing and linear growth in wild-type and heterozygous mice continue. This suggests, that although bone formation and mineralization are advanced in TRßPV/PV mice, growth plate quiescence is induced before normal maturation is completed. The mechanism for this observation remains obscure and may involve other factors, but it is consistent with the persistence of reduced bone length in the presence of advanced bone formation. One factor that is likely to be important is GH, which is normally regulated by T3. GH acts directly on growth plate chondrocytes and also indirectly via a local paracrine pathway involving IGF-I (42). In previous studies we showed in 8-wk-old TRßPV/PV mice that pituitary GH mRNA expression, which correlates well with mean serum GH concentrations, is reduced to 20% of the levels in wild-type and heterozygous mice (26). Reduced GH levels may, therefore, contribute to the severity of the phenotype in homozygous mutant animals. Nevertheless, in TR{alpha}0/0 mice, which display growth retardation and delayed ossification, we have shown that pituitary GH mRNA expression is not altered (34), indicating that thyroid hormones exert important effects on skeletal growth and maturation that are independent of GH. Thus, the relationship between T3 and GH signaling in the skeleton is complex and requires further investigation (42).

What are the implications of these data for human RTH, in which growth retardation is considered to result from skeletal hypothyroidism (25)? The skeletal phenotype in RTH is variable and has not been well characterized. The description of delayed bone age is consistent with tissue hypothyroidism, but other features such as advanced bone age, reduced bone mineral density, high bone turnover, osteoporosis, and craniosynostosis are consistent with hyperthyroidism. Features such as short stature and epiphyseal dysgenesis may be nonspecific, merely reflecting abnormal ossification. The heterogeneity of skeletal abnormalities in RTH probably reflects incomplete or absent characterization of the phenotype in many cases (21, 25). Heterogeneity may also result from the fact that human RTH is caused by heterozygous mutations, from specific effects of different RTH mutations in the skeleton or from background variability of modifier genes that influence T3 action in bone. Nevertheless, our studies demonstrate a clear thyrotoxic skeletal phenotype in TRßPV/PV mice with severe RTH. The more subtle differences in heterozygous mice indicate a lesser degree of skeletal hyperthyroidism that may manifest in older animals as high bone turnover osteoporosis. Our studies predict that RTH patients are likely to be at particular risk of osteoporotic fracture, especially if treated with excessive doses of thyroid hormones. Thus, skeletal abnormalities in human RTH require definitive characterization both in children and during adulthood that will provide new contributions to our understanding of T3 action in bone.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRßPV Mutant Mice
Wild-type (TRß+/+), heterozygous (TRßPV/+), and homozygous (TRßPV/PV) mutant mice were bred and genotyped as described (26). Animal studies were conducted in strict accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the National Cancer Institute Animal Care and Use Committee. Growth curves were constructed during the first 10 postnatal weeks. E17.5, neonatal, and 2-, 3-, and 4-wk-old male and female littermate mice were obtained for skeletal analyses.

Hormone Assays
The serum total T4 (TT4) concentration was determined in E17.5, neonatal, and 2-wk- and 4-wk-old mice using a Gamma Coat T4 assay RIA kit (DiaSorin, Inc., Stillwater, MN) according to the manufacturer’s instructions.

Skeletal Preparations
E17.5 and neonatal mice and limbs from 2-, 3-, and 4-wk-old animals were fixed for 48–72 h in 80% ethanol at room temperature (RT) and for 48–72 h in 95% ethanol at 4 C. Samples were transferred to acetone for 48–72 h at 4 C and stained for 48–72 h at 37 C in Alizarin Red (Sigma, Poole, Dorset, UK) and Alcian Blue 8GX (Sigma) containing 5% Alcian Blue (0.3% in 70% ethanol), 5% Alizarin Red (0.1% in 95% ethanol), 5% glacial acetic acid, and 85% ethanol (70% in H2O). Samples were destained for 48–72 h in 1% KOH at 4 C and subsequently in 20%, 40%, 60%, and 80% glycerol/1% KOH for further periods of 48–72 h before storage in 100% glycerol at 4 C. Skeletal preparations were photographed using an MZF L111 binocular microscope (Leica Corp. AG, Heerbrugg, Switzerland) and Intralux 6000–1 light source (Volpi AG, Schlieren, Switzerland). Tibia and ulna lengths from TRß+/+, TRßPV/+, and TRßPV/PV male and female littermates were determined by measurement of dissected bones using Mitutoyo Absolute Digital Calipers (Mitutoyo Ltd., Andover, Hampshire, UK) and a Wild M3Z microscope (Leica Corp.).

Histology
Limbs were fixed for 48–72 h in 10% neutral buffered formalin followed by decalcification in 10% formic acid and 10% neutral buffered formalin at RT. E17.5 and neonatal limbs were decalcified for 24 h, and 3-wk-old limbs were decalcified for 5 d. Decalcified bones were embedded in paraffin and 3-µm sections were cut onto 3-aminopropyltriethoxysilane (APES)-coated slides (Sigma), deparaffinized in xylene, and rehydrated. Sections were stained with hematoxylin and eosin (Pioneer Research Chemicals, Colchester, UK), or van Gieson and Alcian Blue 8GX (8).

Limbs from 2- and 4 wk-old mice were also fixed for 48–72 h in 10% neutral buffered formalin and frozen in paraffin without prior decalcification for determination of mineralization by von Kossa staining. Cryosections (3 µm) were cut onto APES-coated slides and deparaffinized. Undecalcified sections were washed in several changes of distilled water before being placed into 1.5% silver nitrate solution, in the dark, for 10–20 min. Samples were washed at least 10 times in distilled water before exposure to 0.5% hydroquinone for 5 min at RT. Samples were washed in distilled water, exposed to 2.3% sodium thiosulfate for 5 min, and counterstained with neutral red.

Quantitative Real-Time RT-PCR
Total RNAs were extracted from tibia and femur with TRIzol (Invitrogen, Carlsbad, CA) using an SPEX CertiPrep 6750 Freezer/Mill (SPEX CertiPrep, Inc., Metuchen, NJ) according to the manufacturer’s instructions. Real-time RT-PCR of TR isoforms was performed employing a Roche Light Cycler PCR instrument and Light Cycler-RNA Amplification Kit SYBR Green I (Roche, Mannheim, Germany) with specific primers as follows:

TR{alpha}1: 5'-GTGACTGACCTCCGCATGAT-3'(sense) and

5'-ATCCTCAAAGACCTCCAGGAA-3'(antisense),

TRß1: 5'-GCAGACTTCCCCACACCTT-3'(sense) and

5'-ACAGGTGATGCAGCGATAGT-3'(antisense),

Glyceraldehyde-3-phosphate dehydrogenase: 5'-ACATCATCCCTGCATCCACT-3'(sense) and

5'-GTCCTCAGTGTAGCCCAAG-3'(antisense).

Total RNA (200 ng) was incubated at 55 C for 30 min and 95 C for 30 sec, followed by 45 PCR cycles, consisting of 95 C for 15 sec, 58 C for 30 sec, and 72 C for 30 sec.

In Situ Hybridization
mRNA expression was analyzed in growth plate sections from 3-wk-old mice using collagen II, collagen X, and FGFR1 cRNA probes. A bacterial neomycin resistance gene cRNA probe (Roche Molecular Biochemicals, Lewes, Sussex, UK) was used as a negative control for all hybridizations, as described in studies in which we optimized in situ hybridization methods (8). Rat collagen II (nucleotides 2982–3689, GenBank accession no. L48440), collagen X (nucleotides 418–858, GenBank accession no. AJ131848), and FGFR1 (nucleotides 104–603, GenBank accession no. S54008) partial cDNAs were isolated by RT-PCR as described (8) using RNA from chondrogenic FTC5:3 cells (43) and osteoblastic ROS 17/2.8 cells (12). PCR products were subcloned into the pGEM-T vector (Promega Corp., Southampton, Hampshire, UK) and sequenced. Collagen II, collagen X, and FGFR1 constructs were linearized with NcoI, SpeI, and SpeI, and digoxigenin-labeled antisense cRNA probes were synthesized using SP6, T7, and T7 RNA polymerases (Roche Molecular Biochemicals).

Deparaffinized sections (3 µm) were cut onto APES-coated slides, and mRNA was exposed by digestion with 20 µg/ml proteinase K in TE buffer (10 mM Tris-Cl; and 1 mM EDTA, pH 7–8) for 12 min. Sections were acetylated in 0.1 M triethanolamine and 0.3 M acetic anhydride for 10 min before washing, dehydrating, and drying. Hybridization solution (1x Denhardt’s solution, 50% deionized formamide, 20% dextran sulfate, and 2 µg probe) was heated to 80 C for 90 sec, added to sections, and incubated in humidified chambers at 50 C overnight. Slides were washed in 50% formamide in 0.15 M NaCl, 5 mM NaH2PO4, 5 mM Tris/HCl, and 2.5 mM EDTA (pH 6.8) at 50 C for 30 min followed by six washes over 1 h at RT with constant agitation in 0.5 M NaCl, 0.1 M Tris/Cl, 0.2 M EDTA (pH 7.4). Sections were blocked in 3% BSA in 100 mM Tris/Cl and 150 mM NaCl (pH 7.5), for 1 h before the addition of alkaline phosphatase-conjugated antidigoxygenin Fab fragments (Roche Molecular Biochemicals) diluted 1:500 in blocking solution for 1 h at RT. Slides were washed in PBS and developed in nitroblue tetrazolium chlorine/5-bromo-4-chloro-3-indolyl-phosphate-4-toluidine for 20–30 min to detect alkaline phosphatase. Studies were performed on at least three mice per genotype in duplicate, and repeat experiments were performed on three separate occasions.

Growth Plate Measurements
In situ hybridization using the collagen II probe identified proliferating chondrocytes, and the collagen X probe enabled hypertrophic chondrocytes to be visualized. The height of the tibial growth plate and the PZ and HZ was determined by obtaining measurements, using a BH2 microscope (Olympus Corp. Optical Co. Ltd., London, UK) and AX0067 20.4-mm 10/100 eyepiece micrometer (Olympus Corp.), at four separate positions across the width of the growth plate to calculate mean PZ, HZ, and total growth plate heights for each genotype. These studies were performed independently by two observers (P.J.O. and G.R.W.), who were blinded to the genotype of the growth plate. The intraassay and interobserver coefficients of variation for these measurements were 2.87 ± 1.04% and 9.77 ± 3.8%, respectively.

Statistical Analysis
Data are expressed as mean ± SEM. Differences between groups were examined for statistical significance using Student’s t test or ANOVA with Fisher’s protected least significant difference post hoc test for multiple pair-wise comparisons as appropriate.


    ACKNOWLEDGMENTS
 
We thank Dr. J. H. D. Bassett and members of the Williams laboratory for helpful discussion during preparation of the manuscript.


    FOOTNOTES
 
This work was supported by a Medical Research Council (MRC) Ph.D. Studentship (to P.J.O.) and MRC Career Establishment Grant (G9803002) (to G.R.W.).

Abbreviations: APES, 3-Aminopropyltriethoxysilane; E17.5, embryonic d 17.5; FGFR, fibroblast growth factor receptor; HZ, hypertrophic zone; PZ, proliferative zone; RZ, reserve zone; RT, room temperature; RTH, resistance to thyroid hormone; TR, T3 receptor.

Received for publication August 27, 2002. Accepted for publication March 27, 2003.


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