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Molecular Endocrinology, doi:10.1210/me.2007-0157
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Molecular Endocrinology 21 (8): 1893-1904
Copyright © 2007 by The Endocrine Society

Thyroid Status during Skeletal Development Determines Adult Bone Structure and Mineralization

J. H. Duncan Bassett, Kristina Nordström, Alan Boyde, Peter G. T. Howell, Shane Kelly, Björn Vennström and Graham R. Williams

Molecular Endocrinology Group (J.H.D.B., S.K., G.R.W.), Division of Medicine and Medical Research Council, Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom; Department of Cell and Molecular Biology (K.N., B.V.), Karolinska Institute, S-171 77 Stockholm, Sweden; Biophysics Oral Growth and Development (A.B., P.G.T.H.), Institute of Dentistry, Barts and The London School of Medicine, Queen Mary University of London, London E1 1BB, United Kingdom; and Department of Prosthetic Dentistry (P.G.T.H.), Eastman Dental Institute, University College London, London WC1X 8LD, United Kingdom

Address all correspondence and requests for reprints to: Graham Williams, Molecular Endocrinology Group, MRC Clinical Sciences Center, Clinical Research Building 5th Floor, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom. E-mail: graham.williams{at}ic.ac.uk or Björn Vennström, Department of Cell and Molecular Biology, Karolinska Institute, Box 285, S-171 77 Stockholm, Sweden. E-mail: Bjorn.Vennstrom{at}ki.se.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Childhood hypothyroidism delays ossification and bone mineralization, whereas adult thyrotoxicosis causes osteoporosis. To determine how effects of thyroid hormone (T3) during development manifest in adult bone, we characterized TR{alpha}1+/mß+/– mice, which express a mutant T3 receptor (TR) {alpha}1 with dominant-negative properties due to reduced ligand-binding affinity. Remarkably, adult TR{alpha}1+/mß+/– mice had osteosclerosis with increased bone mineralization even though juveniles had delayed ossification. This phenotype was partially normalized by transient T3 treatment of juveniles and fully reversed in compound TR{alpha}1+/mß–/– mutant mice due to 10-fold elevated hormone levels that allow the mutant TR{alpha}1 to bind T3. By contrast, deletion of TRß in TR{alpha}1+/+ß–/ – mice, which causes a 3-fold increase of hormone levels, led to osteoporosis in adults but advanced ossification in juveniles. T3-target gene analysis revealed skeletal hypothyroidism in TR{alpha}1m/+ß+/– mice, thyrotoxicosis in TR{alpha}1+/+ß–/– mice, and euthyroidism in TR{alpha}1+/ß–/– double mutants. Thus, TR{alpha}1 regulates both skeletal development and adult bone maintenance, with euthyroid status during development being essential to establish normal adult bone structure and mineralization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE INCIDENCE OF osteoporosis, which is characterized by low bone mass and bone fragility and fracture, rises as life expectancy increases. Already, osteoporotic fractures cost more than {euro}31.7 billion in Europe and $13.8 billion in the United States each year (1, 2). Adult bone strength and fracture risk are determined by peak bone mass attained during growth and the rate of age-related bone loss (3). Thyroid hormones are essential regulators of skeletal development and bone maintenance in adults (4, 5), but their importance in the pathogenesis of osteoporosis has not been recognized even though individuals with a history of exposure to excess thyroid hormone have an increased risk of fracture (6, 7, 8).

Euthyroid status is essential for normal skeletal development and adult bone maintenance (4, 9). Hypothyroidism in children causes growth arrest, delayed bone maturation, and epiphyseal dysgenesis, and T4 replacement results in rapid catch-up growth (10). By contrast, juvenile thyrotoxicosis accelerates growth and advances bone age but induces short stature due to premature fusion of the growth plates and may cause craniosynostosis (11). In adults, thyrotoxicosis accelerates bone loss causing osteoporosis (4, 12), and even minor disturbances of thyroid status increase fracture risk. Case control, thyroid registry, and population studies have revealed an increased fracture risk in patients with suppressed TSH (4, 6, 7, 8, 13, 14). A prospective study revealed a 3.6-fold increased risk of hip fracture and 4.5-fold risk of vertebral fracture in women over 65 with TSH below 0.1 mU/liter (15). These findings were supported in a population of postmenopausal women, in which low TSH was associated with low bone mineral density at the lumbar spine and femoral neck (16).

Thyroid hormone (T3) actions are mediated by thyroid hormone receptor (TR){alpha}1 and TRß nuclear receptors, which are expressed in chondrocytes and osteoblasts (5). Although thyroid hormone excess accelerates bone loss, it is unclear whether TRs are expressed in osteoclasts because conflicting studies suggest either that T3 induces osteoclastic bone resorption directly or that T3 acts indirectly via osteoblasts to induce the osteoclast response (17, 18, 19, 20). Even though previous exposure to excess thyroid hormone is an independent risk factor for fracture, the mechanism of T3 action in bone is uncertain, and it is unknown whether transient changes in thyroid status during development influence bone structure or mineralization in later life.

Studies of TR{alpha} and TRß knockout mice, and knock-in mice harboring a severe resistance to thyroid hormone mutation that abolishes T3 binding (PV), have suggested TR{alpha}1 is essential for skeletal development, but TRß is dispensable (21, 22, 23, 24, 25, 26, 27). We hypothesized that TR{alpha}1-dependent bone growth and mineralization are necessary to establish normal bone structure in adulthood. We reasoned that TR{alpha}1+/m mice (28), which harbor a milder TR{alpha}1R384C resistance to thyroid hormone mutation compared with TR{alpha}1PV, would allow us to address this possibility. TR{alpha}1R384C has a 10-fold reduced affinity for T3 and acts as a dominant-negative antagonist of ligand-bound wild-type TR{alpha}1 and TRß, but its dominant-negative actions can be overcome by increased concentrations of T3 in vitro and in vivo (28). TR{alpha}1+/m mice display a transient delay in development associated with hypothyroidism between postnatal d P10 and P35, but adult TR{alpha}1+/m mice have normal serum T3 and T4 levels. Importantly, developmental delay in TR{alpha}1+/m mice can be normalized by crossing them with TRß knockout mice (TRß–/–) because the resulting TR{alpha}1+/mß–/– double mutants have 10-fold elevated circulating hormone concentrations, which overcome the reduced T3 binding affinity of TR{alpha}1R384C (28). Furthermore, discrete neurological abnormalities occurring during postnatal development can be rescued permanently by treatment of TR{alpha}1+/m mice with thyroid hormones during the transient period of postnatal hypothyroidism (29). We hypothesized that TR{alpha}1+/m mice would be a unique model with which to investigate the relationship between skeletal development and adult bone structure. Thus, we compared skeletal development and adult bone structure in TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– littermate mice. In further studies the effect of T3 treatment during the period of transient hypothyroidism between P10 and P35 on adult bone structure was investigated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The thyroid status of mice in these studies has been described elsewhere (28, 29). TR{alpha}1+/+ß+/– control mice are euthyroid. In contrast, TR{alpha}1+/mß+/– mice are transiently hypothyroid between P10 and P35, but euthyroid thereafter. TR{alpha}1+/mß –/– double mutants have 10-fold elevated thyroid hormone concentrations from birth (data not shown), which overcome the reduced T3 binding affinity of the TR{alpha}1R384C mutant. TR{alpha}1+/+ß–/– mice have 3-fold elevated thyroid hormone levels (23, 28).

Deletion of TRß Rescues Delayed Skeletal Development in TR{alpha}1+/mß+/– Mice
Crossing TR{alpha}1+/+ß+/– females with TR{alpha}1+/mß–/– males resulted in TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß –/–, and TR{alpha}1+/+ß–/– littermates. In embryonic d 17.5 (E17.5) TR{alpha}1+/mß+/– and TR{alpha}1+/mß–/– mice, endochondral ossification was delayed in phalangeal bones compared with TR{alpha}1+/+ß+/– and TR{alpha}1+/+ß–/– mice (Fig. 1AGo). Delayed endochondral ossification in TR{alpha}1+/mß+/– mice was normalized by the TR{alpha}1+/mß–/– double mutation at P1 (Fig. 1BGo). Impaired intramembranous ossification of the skull was also present in TR{alpha}1+/mß+/– mice, with a 2.3-fold increased fontanelle area at P1 compared with TR{alpha}1+/+ß+/– mice (n = 4, P < 0.001). However, at P1 delayed intramembranous ossification of the skull was not rescued in double-mutant TR{alpha}1+/mß–/– mice, in which fontanelle area was increased 2.2-fold compared with TR{alpha}1+/+ß+/– mice (n = 3, P < 0.001). A marked delay of endochondral ossification was evident at P14 in TR{alpha}1+/mß+/– mice, which displayed 15% shortening of the tibia (Fig. 1CGo) and delayed formation of secondary ossification centers (Fig. 1DGo). Deletion of TRß partially normalized this because formation of the proximal secondary ossification center in TR{alpha}1+/mß–/– mice had begun by P14, and mice exhibited only 5% growth delay. TR{alpha}1+/+ß–/– mice were similar to TR{alpha}1+/+ß+/– mice at P14. Analysis of growth curves indicated that delayed growth in TR{alpha}1+/mß+/– mice was maximal at P14 but normalized by P70, whereas the 5% growth delay seen in P14 TR{alpha}1+/mß–/– mice had normalized by P28. TR{alpha}1+/+ß–/– mice were persistently shorter from P28 although this trend was not statistically significant (Fig. 1EGo). Thus, TR{alpha}1+/mß+/– mice displayed delayed ossification and growth retardation. This phenotype was ameliorated by deletion of TRß in TR{alpha}1+/mß–/– mice, in which the 10-fold elevated thyroid hormones enable mutant TR{alpha}1 to function normally (28).


Figure 1
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  		Fig. 1. Skeletal Development in TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– Mice

Whole-bone mounts of E17.5 (panel A) and P1 (panel B) littermates stained with alcian blue (cartilage) and alizarin red (bone). Low- and high-power views of the skull vault showing sutures and fontanelles demonstrate intramembranous ossification. Arrows show regions of delayed endochondral ossification in phalanges of TR{alpha}1+/mß+/– and TR{alpha}1+/mß–/– mice. C, Tibias from P14, P28, P70, and P112 mice. Arrows show delayed formation of secondary epiphyses in TR{alpha}1+/mß+/– mice. D, Midline longitudinal sections of P14 humeri stained with alcian blue and van Gieson. Arrows show delayed formation of secondary epiphyses in TR{alpha}1+/mß+/– mice and amelioration of the phenotype in TR{alpha}1+/mß–/– mice. E, Tibia and ulna growth; *, P < 0.05 vs. TR{alpha}1+/+ß+/–, n = 4 per group; ANOVA followed by Tukey’s multiple comparison post hoc test.

 
TR{alpha}1 Regulates Endochondral Ossification
In P28 TR{alpha}1+/mß+/– mice, growth plates were wider and secondary ossification centers were delayed (Fig. 2AGo). Similar features were seen at P14, P70, and P112. By contrast, growth plates were narrow in P14, P28, and P70 TR{alpha}1+/+ß–/– mice compared with TR{alpha}1+/+ß+/–, and secondary centers were advanced. By P112, TR{alpha}1+/+ß–/– growth plates were similar to TR{alpha}1+/+ß+/– mice. TR{alpha}1+/mß–/– and TR{alpha}1+/+ß+/– growth plates were indistinguishable at all ages. To investigate causes of delayed or advanced endochondral ossification, the reserve (RZ), proliferative (PZ), and hypertrophic (HZ) growth plate zones were defined by collagen II and X in situ hybridization (26, 27, 30). In P28 TR{alpha}1+/mß+/– mice, the wider growth plate consisted of an enlarged RZ containing chondrocyte progenitor cells and a slightly wider HZ that occupied a smaller percentage of the total width of the growth plate compared with TR{alpha}1+/+ß+/– mice (Fig. 2BGo). These features persisted until P112. Thus, delayed endochondral ossification in TR{alpha}1+/mß+/– mice resulted from impaired entry of progenitor cells into the PZ, together with impaired hypertrophic chondrocyte differentiation. By contrast, advanced endochondral ossification in P28 TR{alpha}1+/+ß–/– mice was accompanied by narrowing of the growth plate and RZ, but widening of the HZ. In P70 TR{alpha}1+/+ß–/– mice the RZ remained narrow and the HZ also narrowed compared with TR{alpha}1+/+ß+/– mice, indicating that advanced endochondral ossification in TR{alpha}1+/+ß –/– mice resulted from accelerated hypertrophic chondrocyte differentiation with faster transition of cells through the PZ. Growth plate measures in TR{alpha}1+/mß –/– and TR{alpha}1+/+ß+/– mice were similar at all ages. These data demonstrate the rate of growth plate chondrocyte maturation and progression of endochondral ossification are delayed in TR{alpha}1+/mß+/– mice, normalized in TR{alpha}1+/mß–/– mice, and accelerated in TR{alpha}1+/+ß–/– mice, indicating that chondrocyte differentiation is regulated by TR{alpha}1 and exquisitely sensitive to thyroid status.


Figure 2
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  		Fig. 2. Ossification in TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– Mice

A, Sections of proximal tibia from P28 littermates stained with alcian blue/van Gieson (magnification, x100). B, Upper graph shows the width of the total growth plate and RZ, PZ, and HZ zones in P28 mice. Graphs below show changes in the total growth plate, RZ, PZ, and HZ between ages P14 and P112. C, Change in tibia middiaphysis cortical bone width between P14 and P112. D, Longitudinal sections of tibia cortical bone from P14-P112 mice stained with alcian blue/van Gieson and transverse sections of middiaphysis femur from P112 mice (x100). #, P < 0.05 total growth plate width vs. TR{alpha}1+/+ß+/–; *, P < 0.05; **, P < 0.01 individual growth plate zone width or cortical bone width vs. TR{alpha}1+/+ß+/–; n = 4 per group, two-tailed Student’s t test.

 
TR{alpha}1 Is Required for Cortical Bone Deposition and Remodeling
Cortical bone was examined at P14, P28, P70, and P112. In P14 and P28 TR{alpha}1+/mß+/– mice, cortical bone width was reduced by 30–35% compared with TR{alpha}1+/+ß+/– mice but was similar thereafter. Cortical bone in TR{alpha}1+/mß–/–, TR{alpha}1+/+ß –/–, and TR{alpha}1+/+ß+/– mice was similar at all ages (Fig. 2Go, C and D). Analysis of transverse sections of cortical bone was consistent with studies performed on longitudinal sections and revealed no difference in cortical width at P112. However, in P112 TR{alpha}1+/mß+/– mice the femur lacked its normal oval transverse cross-section, and the ratio of the long medial-lateral to short anterior-posterior axis was reduced by 17% compared with TR{alpha}1+/+ß+/– mice (n = 4, P < 0.05). This abnormality was not present in TR{alpha}1+/mß–/– or TR{alpha}1+/+ß–/– mice. Thus, cortical bone deposition was grossly delayed in TR{alpha}1+/mß+/– mice, and a remodeling defect persisted into adulthood. These abnormalities were normalized by deletion of TRß in TR{alpha}1+/+ß–/– mice, indicating TR{alpha}1 is necessary for normal cortical bone deposition and remodeling.

TR{alpha}1 Regulates Trabecular Bone Remodeling during Postnatal Growth
Bone microarchitecture was analyzed by backscattered electron scanning electron microscopy (BSE-SEM). During endochondral ossification, trabecular bone is laid down beneath the growth plates, which advance proximally or distally resulting in linear growth. As growth continues, trabecular bone is continually remodeled by osteoclasts and osteoblasts, maintaining a roughly constant amount of trabecular bone (Fig. 3Go). In P28 TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß –/–, and TR{alpha}1+/+ß –/– mice the extent of trabecular bone in proximal tibia was similar (Fig. 3AGo). In P70 and P112 mice the amount of trabecular bone remained similar in TR{alpha}1+/+ß+/– and TR{alpha}1+/mß–/– mice but was markedly increased in TR{alpha}1+/mß+/– mice and progressively reduced in TR{alpha}1+/+ß–/– mice (Fig. 3Go, B and C). Thus, TR{alpha}1 regulates trabecular bone remodeling, which was impaired during growth in TR{alpha}1+/mß+/– mice, normalized in TR{alpha}1+/mß–/– mice but accelerated in TR{alpha}1+/mß–/– mice.


Figure 3
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  		Fig. 3. Bone Structure during Growth in TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– Mice

BSE-SEM views showing trabecular and cortical bone in midline sections of proximal tibias from P28 (panel A), P70 (panel B), and P112 (panel C) mice. Scale bars, 200 µm.

 
The Mutant TR{alpha}1 Causes Receptor-Mediated Skeletal Hypothyroidism, whereas Deletion of TRß Leads to Skeletal Thyrotoxicosis
We demonstrated previously that thyroid hormones positively regulate expression of fibroblast growth factor receptor-1 (FGFR1) and FGFR3 in chondrocytes and osteoblasts in vivo and in vitro (31, 32). To define skeletal thyroid status, we examined expression of these target genes in P14 mice (Fig. 4Go). Expression of both FGFR1 and FGFR3 was reduced in TR{alpha}1+/mß+/– growth plates, consistent with receptor-mediated skeletal hypothyroidism due to impaired TR{alpha}1 action. By contrast, increased expression in TR{alpha}1+/+ß –/– chondrocytes demonstrated skeletal thyrotoxicosis. In TR{alpha}1+/mß –/– mice FGFR1 and FGFR3 expression was similar to TR{alpha}1+/+ß+/– mice, indicating that additional deletion of TRß in TR{alpha}1+/mß –/– mice normalized skeletal thyroid status thereby rescuing the TR{alpha}1+/mß+/– phenotype. Expression of the T3-insensitive gene, FGFR2 (31), was similar in all four genotypes. Thus, postnatal skeletal development, as determined by endochondral ossification, cortical bone formation, and trabecular bone remodeling, correlates with tissue thyroid status in TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– mice.


Figure 4
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  		Fig. 4. FGFR Expression in TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– Growth Plates

A, Proximal tibia growth plates from P14 littermates stained with alcian blue/van Gieson. In situ hybridizations showing FGFR1 (B), FGFR2 (C), and FGFR3 (D) expression in tissue sections adjacent to those shown in panel A. Magnification, x200.

 
The Mutant TR{alpha}1 Causes Osteosclerosis, whereas Deletion of TRß Results in Osteoporosis
We determined the effect of the TR{alpha}1 mutation on the adult skeleton and investigated the importance of the transient period of hypothyroidism in TR{alpha}1+/mß+/– mice to adult bone structure and mineralization. TR{alpha}1+/mß+/– and TR{alpha}1+/+ß+/– mice were treated with T3 between P10 and P35, which results in a transient increase in circulating thyroid hormone levels in TR{alpha}1+/mß+/– mice and generates a period of thyrotoxicosis in TR{alpha}1+/+ß+/– mice (28, 29). T3-treated and untreated mice were also compared with TR{alpha}1+/+ß–/– mice, in which circulating T3 is increased 3-fold throughout life (23, 28). Bone microarchitecture was characterized by BSE-SEM in adult mice at P98 (Fig. 5Go). Remarkably, trabecular bone mass was substantially increased in adult TR{alpha}1+/mß+/– mice, whereas TR{alpha}1+/+ß–/– mice were severely osteoporotic. Trabecular bone in TR{alpha}1+/mß+/– mice was robust containing thicker trabeculae with increased connectivity and plate-like morphology, whereas TR{alpha}1+/+ß–/– trabecular bone was gracile, containing thinner trabeculae with reduced connectivity and rod-like morphology (Fig. 5BGo). Surprisingly, transient T3 treatment of juveniles induced loss of trabecular bone that was still evident 63 d later in adult TR{alpha}1+/+ß+/– and TR{alpha}1+/mß+/– mice. Trabecular bone volume fraction was increased 2.6-fold in TR{alpha}1+/mß+/– mice (n = 3–4, P < 0.01) but was similar in TR{alpha}1+/+TRß–/– and TR{alpha}1+/+ß+/– mice (Fig. 5DGo). Transient T3 treatment reduced the trabecular bone volume fraction by 63% in TR{alpha}1+/mß+/– mice (n = 3–4, P < 0.01) and by 12% in TR{alpha}1+/+ß+/– mice, although this value did not reach statistical significance. Thus, adult TR{alpha}1+/mß+/– mice with receptor-mediated skeletal hypothyroidism are osteosclerotic, and this phenotype is sensitive to T3 treatment during development. By contrast, adult TR{alpha}1+/+TRß–/– mice with skeletal thyrotoxicosis are osteoporotic.


Figure 5
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  		Fig. 5. Bone Structure in Untreated and T3-Treated TR{alpha}1+/+ß+/– and TR{alpha}1+/mß+/– Mice, and Untreated TR{alpha}1+/+ß–/– Adult Mice

A, Low-power BSE-SEM views of trabecular and cortical bone in midline longitudinal sections of distal femurs from P98 mice. B, Higher power views of distal femur metaphysis trabecular bone. C, Low-power views of trabecular and cortical bone from P98 proximal tibias. D, qBSE-SEM gray-scale views of polymethylmethacrylate-embedded proximal humerus from the same mice. Scale bars, 200 µm.

 
TR{alpha}1 Regulates Trabecular Bone Mineralization in Adults
Trabecular and cortical bone mineralization was determined in humerus and vertebra by quantitative BSE-SEM (qBSE-SEM) (Fig. 6Go). Trabecular bone mineralization was markedly increased in TR{alpha}1+/mß+/– mice, in which abnormal retention of highly mineralized calcified cartilage was evident (Fig. 6Go, B and D). By contrast, mineralization was reduced in TR{alpha}1+/+ß–/– mice. Transient T3 treatment of juveniles resulted in a remarkable reduction of trabecular bone mineralization in adult TR{alpha}1+/mß+/– mice, with a smaller loss evident in T3-treated TR{alpha}1+/+ß+/– mice. By contrast, cortical bone mineralization did not differ between TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, and TR{alpha}1+/+ß–/– mice and was unaffected by T3 treatment (Fig. 6Go, E and F). Thus, adult TR{alpha}1+/mß+/– mice have increased trabecular bone mineralization density, which is sensitive to T3 treatment during development, whereas trabecular mineralization density is reduced in TR{alpha}1+/+TRß–/–mice.


Figure 6
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  		Fig. 6. Trabecular and Cortical Bone Mineralization in Untreated and T3-Treated TR{alpha}1+/+ß+/– and TR{alpha}1+/mß+/– Mice and Untreated TR{alpha}1+/+ß–/– Adult Mice

qBSE-SEM images of longitudinal sections showing mineralization densities of trabecular bone from proximal humerus (panel A; magnification, x33) and vertebra (panel C; magnification, x100), and cortical bone from humerus (panel E; x100) of P98 mice. Mineralization densities were derived from halogenated standards, and gray-scale images were colored according to the palette shown in which low mineralization density is blue and high density is gray. In panel A the boxes represent the regions of trabecular bone from which mineralization densities shown in panel B were obtained. Panels B, D, and F show relative and cumulative frequency histograms of bone micromineralization densities. Arrows indicate the increased relative frequency of high micromineralization densities corresponding to retained calcified cartilage in TR{alpha}1+/mß+/– mice. ***, P < 0.001 micromineralization density vs. TR{alpha}1+/+ß+/–; n = 4 per group, Kolmogorov-Smirnov test. Scale bars, 200 µm.

 
Mutation of TR{alpha}1 Impairs Osteoclastic Bone Resorption, whereas Deletion of TRß Increases Osteoclast Numbers
Cortical and trabecular bone surfaces were analyzed in P98 adult mice (Fig. 7Go, A–C). Areas of bone resorption were identified by surfaces that contained osteoclastic resorption pits in contrast to smooth unresorbed surfaces that lacked evidence of osteoclastic activity. In TR{alpha}1+/mß+/– mice, the resorption surface was reduced by 30% in trabecular bone compared with TR{alpha}1+/+ß+/– mice (n = 3, P < 0.05). The 40% reduction in endosteal bone resorption surface observed in TR{alpha}1+/mß+/– mice did not reach statistical significance. Transient T3 treatment of juvenile TR{alpha}1+/+ß+/– mice resulted in a 53% (n = 3, P < 0.05) and 18% increase (n = 3, P < 0.05) in adult endosteal and trabecular resorption areas, respectively. T3 treatment of TR{alpha}1+/mß+/– mice increased endosteal and trabecular resorption surfaces by 300% (n = 3, P < 0.001) and 71% (n = 3, P < 0.01), respectively. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts surfaces were quantified in P112 adult mice (Fig. 7Go, D–E). In TR{alpha}1+/mß+/– mice osteoclast surfaces were reduced by 36% in trabecular bone compared with TR{alpha}1+/+ß+/– mice (n = 4, P < 0.01). The 29% reduction at the endosteal surface in TR{alpha}1+/mß+/– mice did not reach statistical significance. By contrast, in TR{alpha}1+/+ß–/– mice osteoclast surfaces were increased 76% in trabecular bone (n = 4, P < 0.001). The 44% increase at the endosteal surface in TR{alpha}1+/+ß–/– mice also did not reach statistical significance. Deletion of TRß in TR{alpha}1+/mß–/– mice normalized osteoclast numbers. Thus, adult TR{alpha}1+/mß+/– mice have evidence of reduced osteoclastic bone resorption, a phenotype that is normalized by additional deletion of TRß and sensitive to T3 treatment during development. By contrast, adult TR{alpha}1+/+TRß–/– mice have increased numbers of osteoclasts.


Figure 7
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  		Fig. 7. Cortical and Trabecular Bone Surfaces and Osteoclast Numbers in TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– Mice

BSE SEM views of mid-diaphysis endosteal cortical bone surface (A) and metaphyseal trabecular bone (B) from distal femurs of P98 mice. Arrows illustrate the junction between osteoclastic resorption surfaces and unresorbed bone surfaces. Scale bars, 200 µm. C, Graphs demonstrate the amount of endosteal and trabecular bone occupied by resorption surfaces. *, P < 0.05 vs. TR{alpha}1+/+ß+/–; n = 3 per group, two-tailed Student’s t test. D, Sections of proximal humerus from P112 mice stained with TRAP to reveal osteoclasts lining trabecular bone surfaces (magnification, x400). E, Number of TRAP-positive osteoclasts per unit bone surface per mm of endosteal or trabecular bone surface. **, P < 0.01; ***, P < 0.001 vs. TR{alpha}1+/+ß+/–; n = 4 per group, two-tailed Student’s t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In these studies adult TR{alpha}1+/mß+/– mice, which harbor a mutant TR{alpha}1 that inhibits the action of ligand-bound wild-type TR{alpha}1 and TRß, have osteosclerosis and increased bone mineralization even though juveniles have delayed endochondral and intramembranous ossification. This phenotype was normalized in TR{alpha}1+/mß–/– double mutants, in which deletion of TRß leads to a persistent 10-fold elevation of thyroid hormones that enables the mutant TR{alpha}1 to function normally. Surprisingly, the adult phenotype in TR{alpha}1+/mß+/– mice was also ameliorated by transient T3 treatment during development. By contrast, deletion of TRß alone, which persistently elevates hormone levels 3-fold in TR{alpha}1+/+ß–/– mice, resulted in advanced endochondral ossification in juveniles but osteoporosis in adults. Thus, TR{alpha}1 mediates T3 action in bone, and euthyroid status during development is essential to establish normal adult bone structure and mineralization. The current data extend our recent model describing the relationship between central and skeletal thyroid status (33) and provide a unifying hypothesis linking thyroid hormone actions during skeletal development to their effects in adult bone. Receptor-mediated skeletal hypothyroidism in TR{alpha}1+/m mice results in adult osteosclerosis, whereas systemic hyperthyroidism in TRß-deficient mice causes osteoporosis. Thus, thyroid hormones exert anabolic actions during skeletal growth but catabolic actions in adults, and these effects are mediated principally via TR{alpha}1.

TR{alpha}1 Regulates Skeletal Development and Maintenance of Adult Bone Structure and Mineralization
Reduced T3 target gene expression and delayed ossification in TR{alpha}1+/mß+/– mice demonstrate a phenotype of skeletal hypothyroidism consistent with abnormalities identified in human juvenile hypothyroidism, hypothyroidism in rats, and other TR{alpha} mutant mice (10, 21, 27, 30). Thus, actions of TR{alpha}1R384C recapitulate the effects of tissue hypothyroidism mediated by unliganded apo-TR{alpha}1, which acts as a repressor to regulate the timing of postnatal development (34). Adult TR{alpha}1+/mß+/– mice also display extensive trabecular bone of increased density with retained mineralized cartilage and middiaphysis cortical bone of abnormal cross-section, suggesting a persistent bone remodeling defect also seen in hypothyroidism (12). By contrast, increased activation of skeletal TR{alpha}1 in TR{alpha}1+/+ß–/– mice causes advanced endochondral ossification and persistent short stature consistent with juvenile thyrotoxicosis (11). Furthermore, adult TR{alpha}1+/+ß–/– mice display accelerated bone remodeling and osteoporosis, consistent with increased activation of skeletal TR{alpha}1 (33) and typical of thyrotoxicosis (4). Thus, TR{alpha}1 regulates skeletal growth and development and, together with the rescue of impaired bone remodeling in TR{alpha}1+/mß–/– mice, these data establish that TR{alpha}1 also regulates adult bone mass and mineralization. The presence of reduced bone resorption surfaces and osteoclast numbers in TR{alpha}1+/mß+/– mice, the normalization of these parameters in TR{alpha}1+/mß–/– mice or after T3 treatment, and the increased osteoclast numbers in TR{alpha}1+/+ß–/– mice all suggest that TR{alpha}1 controls adult bone mass by regulating osteoclastic bone resorption.

Thyroid Hormones and TR{alpha}1, Rather than TSH, Mediate Skeletal Responses to Altered Thyroid Status
A recent study surprisingly suggested that osteoporosis in thyrotoxicosis results from TSH deficiency rather than thyroid hormone excess. Abe et al. (35) proposed TSH as a direct negative regulator of bone remodeling. Our data, however, are incompatible with this model. First, adult TR{alpha}1+/+ß–/– mice display osteoporosis with reduced bone mass and mineralization accompanied by increased bone remodeling despite a 3.5-fold rise in TSH and elevated thyroid hormone levels (23, 28). Second, adult TR{alpha}1+/mß+/– mice have osteosclerosis with increased bone mass and mineralization accompanied by reduced bone remodeling despite normal thyroid hormone levels. The additional deletion of TRß in TR{alpha}1+/mß–/– double mutant mice causes 10-fold elevation of thyroid hormones and a 2-fold increase in pituitary TSH mRNA (28), but nevertheless results in a marked loss of bone (Fig. 3CGo) and an increased number of osteoclasts (Fig. 7Go, D and E) compared with single mutant TR{alpha}1+/mß+/– mice. We conclude that thyroid hormones and TR{alpha}1, rather than TSH, mediate skeletal responses to abnormalities of the hypothalamic-pituitary-thyroid axis in vivo.

Euthyroid Status during Development Is Essential to Establish a Normal Adult Skeleton
TR{alpha}1+/mß+/– mice have skeletal hypothyroidism mediated by a mutant receptor. During development this is exacerbated by a transient period of hypothyroidism, and we explored its importance to the adult skeleton by treating juveniles with T3. Importantly, T3 treatment increases hormone levels to an extent that allows the mutant TR{alpha}1R384C to bind ligand and activate target genes normally, thus overcoming the aporeceptor activity (28, 29). The T3 treatment reduced both trabecular bone mineralization and volume in adult TR{alpha}1+/mß+/– mice, whereas in TR{alpha}1+/+ß+/– mice T3 only affected mineralization to a lesser degree. The exaggerated response in TR{alpha}1+/mß+/– mice indicates an increased sensitivity to T3 analogous to the period of rapid catch-up growth in hypothyroid children after thyroid hormone replacement (10). Thus, the effects of T3 treatment in TR{alpha}1+/mß+/– and TR{alpha}1+/+ß+/– mice indicate that a period of transient hypothyroidism or hyperthyroidism in juveniles results in persistent skeletal abnormalities in adults, demonstrating that euthyroid status during development is required to establish normal adult bone structure and mineralization. These findings provide the first insight into previously unexplained observations that a prior history of thyrotoxicosis is an independent risk factor for future fracture (6, 7, 8, 15). TR{alpha}1+/mß+/– mice represent a unique resource to investigate mechanisms that determine how transient changes in thyroid status can permanently influence adult bone mass, and these studies identify TR{alpha}1 as a new potential drug target for the prevention and treatment of osteoporosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
TR{alpha}1+/m and TRß–/– Mice
The knock-in mouse strain carrying the dominant-negative R384C mutation in TR{alpha}1 (TR{alpha}1+/m), the TRß–/– strain, genotyping procedures, and animal husbandry have been described previously (28). Animal care procedures were conducted in accordance with the guidelines set by the European Community Council Directives (86/609/EEC). Necessary permissions were obtained from the appropriate local ethical committees. TR{alpha}1+/+ß+/– females were crossed with TR{alpha}1+/mß–/– males to obtain TR{alpha}1+/+ß+/–, TR{alpha}1+/mß+/–, TR{alpha}1+/mß–/–, and TR{alpha}1+/+ß–/– littermates for experiments. In some studies TR{alpha}1+/+ß+/– and TR{alpha}1+/mß+/– juvenile mice were treated with daily sc injections of vehicle or T3 (20–30 ng T3/gram body weight) starting at P10 and ending at P35.

Skeletal Preparations
E17.5 and P1 mice, and limbs from P14, P28, P70, P98, and P112 animals were stained with alizarin red and alcian blue as described previously (26, 27). Preparations were photographed using a Leica MZ75 binocular microscope (Leica AG, Heerbrugg, Switzerland), Leica KL1500 light source, Leica DFC320 digital camera, and Leica IM50 Digital Image Manager. Bone lengths 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 version 1.33u software (http://rsb.info.nih.gov/ij/).

Histology
Limbs were fixed for 48–72 h in 10% neutral buffered formalin and decalcified in 10% formic acid and 10% formaldehyde. E17.5 and P1 limbs were decalcified for 24 h, P14 and P28 limbs were decalcified for 5 d, and P70, P98, and P112 limbs were decalcified for 7 d. Decalcified bones were embedded in paraffin and 3-µm sections were cut onto 3-aminopropyltriethoxysilane-coated slides (Sigma Chemical Co., St. Louis, MO), deparaffinized in xylene, and rehydrated. Sections were stained with hematoxylin and eosin or van Gieson and alcian blue. Osteoclast numbers were determined after staining sections from limbs decalcified in 10% EDTA, pH 7.4, for 14 d for TRAP. Staining using a Sigma TRAP kit (386A-1KT) was performed according to manufacturer’s instructions except that fast garnet dye was diluted 10-fold, and the incubation time was 10 min. TRAP-positive cells were counted using ImageJ. Osteoclast numbers were normalized to the amount of bone surface present in each section, and data were expressed as number of TRAP-positive cells per mm of endosteal or trabecular bone surface.

In Situ Hybridization
mRNA expression was analyzed in growth plate sections using collagen II, collagen X, FGFR1, FGFR2, and FGFR3 cRNA probes as described elsewhere(26, 27, 31, 32). A bacterial neomycin resistance gene cRNA probe (Boehringer Mannheim, Lewes, Sussex, UK) was used as a negative control for all hybridizations, and collagen II and X 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 (30). In situ hybridizations were performed on at least three mice per genotype in duplicate, and repeat experiments were performed on three occasions.

Analysis of Growth Plate and Cortical Bone Dimensions
Measurements of at least four separate positions across the width of growth plates were obtained using a Leica DM LB2 microscope and the Leica DFC320 digital camera to calculate mean values for the heights of the RZ, PZ, HZ, and total growth plate in sections of proximal tibia. Results from two different levels of sectioning were compared to ensure consistency of the data. Cortical bone measurements were performed in longitudinal and transverse sections: at least 10 separate positions in the midshaft of long bones were analyzed, and adjacent levels of sectioning were compared to ensure consistency of the data.

BSE-SEM
Analysis of bone microarchitecture was determined by BSE-SEM. Freshly dissected bones were fixed and stored in 70% ethanol. Bones were opened longitudinally along anatomical curvatures, and half the cortical bone and medullary trabecular elements were removed with a fine tungsten carbide milling tool in a dental workshop hand piece. Samples were cleaned of cell remnants by maceration with an alkaline bacterial pronase, which also removes unmineralized cartilage matrix. Samples were coated with carbon and imaged using backscattered electrons, generally at 30 kV beam potential, and tomographic approaches when necessary. The images provide the best and the most detailed view of bone surfaces, from which surface activity states (forming, resting, resorbing, resorbed) can be investigated (36, 37). The distribution of mineralization densities within calcified tissues was examined by BSE-SEM quantified by digital image analysis at the cubic micron volume resolution scale (38, 39). Fixed bones were embedded in polymethylmethacrylate. Block faces were cut through the specimens, which were then polished, coated with carbon, and analyzed using backscattered electrons in a Zeiss DSM962 digital scanning electron microscope, equipped with an annular solid state BSE detector (KE Electronics, Toft, Cambridgeshire, UK), operated at 20 kV and 0.5 nA and a 17-mm working distance (11 mm, sample to detector distance). The mineralization densities of calcified tissues were determined by comparison with halogenated dimethacrylate standards: C22H25O10Br [mean BSE coefficient according to the procedure of Lloyd (40) = 0.1159] to C22H25O10I (mean BSE coefficient = 0.1519). Increasing gradations of micromineralization density were represented in eight equal intervals by a pseudocolor scheme for presentation of digital images (38, 39).

Statistics
Normally distributed data were analyzed by Student’s t test, or ANOVA followed by Tukey’s multiple comparison post hoc test. P values < 0.05 were considered significant. Frequency distributions of bone micromineralization densities obtained by qBSE were compared using the Kolmogorov-Smirnov test, in which P values for the D statistic in 1024 pixel data sets were D = > 6.01, P < 0.05; D = > 7.20, P < 0.01; and D = > 8.62, P < 0.001.


   ACKNOWLEDGMENTS
 
We thank Maureen Arora for providing valuable technical assistance for SEM studies.


   FOOTNOTES
 
This work was supported by a Medical Research Council (MRC) Clinician Scientist Fellowship (to J.H.D.B.), a grant from the Horserace Betting Levy Board (to A.B.), grants from the Swedish Cancer Society, the Swedish Science Council, and the Wallenberg Foundations (to B.V.), and a MRC Career Establishment Grant and Arthritis Research Campaign Project Grant (to G.R.W.). The automated digital SEM facility was funded by the MRC.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 8, 2007

Abbreviations: BSE-SEM, Back scattered electron-scanning electron microscopy; E17.5, embryonic d 17.5; FGFR, fibroblast growth factor receptor; HZ, hypertrophic zone; P10, postnatal d 10; PZ, proliferative zone; qBSE-SEM, quantitative BSE-SEM; RZ, reserve zone; TR, thyroid hormone receptor; TRAP, tartrate-resistant acid phosphatase.

Received for publication March 27, 2007. Accepted for publication May 3, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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Nuclear Receptors:   TRα  |  TRβ
Ligands:   Thyroid hormone



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