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Molecular Endocrinology, doi:10.1210/me.2007-0033
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Molecular Endocrinology 21 (5): 1095-1107
Copyright © 2007 by The Endocrine Society

Thyroid Hormone Excess Rather Than Thyrotropin Deficiency Induces Osteoporosis in Hyperthyroidism

J. H. Duncan Bassett, Patrick J. O’Shea, Srividya Sriskantharajah, Bénédicte Rabier, Alan Boyde, Peter G. T. Howell, Roy E. Weiss, Jean-Paul Roux, Luc Malaval, Phillipe Clement-Lacroix, Jacques Samarut, Olivier Chassande and Graham R. Williams

Molecular Endocrinology Group (J.H.D.B., P.J.O., S.S., G.R.W.), Division of Medicine and Medical Research Council (MRC) Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom; Institut Nationale de la Santé et de la Recherche Médicale (INSERM) Unité (U) 577 (B.R., O.C.,), Bordeaux F-33000, France; Université Victor Segalen (B.R., O.C.), Bordeaux F-33076, France; Biophysics Oral Growth and Development (A.B., P.G.T.H.), Institute of Dentistry, Bart’s and The London School of Medicine, Queen Mary University of London, London E1 1BB, United Kingdom; Division of Prosthetic Dentistry (P.G.T.H.), Eastman Dental Institute, University College London, London WC1X 8LD, United Kingdom; Thyroid Study Unit (R.E.W.), Department of Medicine, University of Chicago, Chicago, Illinois 60645; INSERM, U831 (J.-P. R.), Lyon, France; Institut Fédératif de Recherche (IFR) 62, Lyon, France; Université Claude Bernard-Lyon I, Faculté de Médecine Laënnec, Lyon, France; INSERM, U890 (L.M.), Saint-Etienne, France; IFR 62, Lyon, France; Université Jean Monnet, Faculté de Médecine, 42023 Saint-Etienne Cedex 2, France; ProSkelia a Galapagos Company (P.C.-L.), 93230 Romainville, France; Laboratoire de Biologie Moléculaire et Cellulaire de l’Ecole Normale Supérieure de Lyon (J.S.), Unité Mixte de Recherche 5665 Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique 913, Lyon, France

Address all correspondence and requests for reprints to: Graham 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}imperial.ac.uk; or Olivier Chassande, Institut National de la Santé et de la Recherche Médicale Unité 577, Université Victor Segalen Bordeaux 2, Bâtiment 4A, zone Nord, 146, rue Lèo Saignat, 33076 Bordeaux cedex, France. E-mail: Olivier.Chassande{at}bordeaux.inserm.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyrotoxicosis is an important but under recognized cause of osteoporosis. Recently, TSH deficiency, rather than thyroid hormone excess, has been suggested as the underlying cause. To investigate the molecular mechanism of osteoporosis in thyroid disease, we characterized the skeleton in mice lacking either thyroid hormone receptor {alpha} or ß (TR{alpha}0/0, TRß–/–). Remarkably, in the presence of normal circulating thyroid hormone and TSH concentrations, adult TR{alpha}0/0 mice had osteosclerosis accompanied by reduced osteoclastic bone resorption, whereas juveniles had delayed endochondral ossification with reduced bone mineral deposition. By contrast, adult TRß–/– mice with elevated TSH and thyroid hormone levels were osteoporotic with evidence of increased bone resorption, whereas juveniles had advanced ossification with increased bone mineral deposition. Analysis of T3 target gene expression revealed skeletal hypothyroidism in TR{alpha}0/0 mice, but skeletal thyrotoxicosis in TRß–/– mice. These studies demonstrate that bone loss in thyrotoxicosis is independent of circulating TSH levels and mediated predominantly by TR{alpha}, thus identifying TR{alpha} as a novel drug target in the prevention and treatment of osteoporosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OSTEOPOROSIS IS CHARACTERIZED by low bone mass, deterioration of bone microarchitecture, increased bone fragility, and susceptibility to fracture. Annually, osteoporotic fractures cost {euro}31 billion in Europe and $13 billion in the United States, and their prevalence will increase substantially as the population ages (1, 2). The risk of osteoporotic fracture is determined by the acquisition of peak bone mass during growth and the rate of age-related bone loss thereafter (3). Accrual of bone mineral is modified by endocrine status, and the risk of osteoporosis is influenced by fetal programming of vitamin D levels (4) and the set points of the gonadal steroid (5), cortociosteroid (6), and GH/IGF-I (7) axes. Even though thyroid hormones (T4, T3) are essential for skeletal development and the maintenance of adult bone (8), their role in the pathogenesis of osteoporosis has been largely overlooked.

Thyrotoxicosis increases bone turnover, accelerates bone loss, and causes osteoporosis. Moreover, case control, thyroid registry, and population studies indicate that even minor or transient disturbances of thyroid status also increase fracture risk (8, 9, 10, 11). Furthermore, in prospective studies of postmenopausal women (12, 13), suppressed TSH from any cause is associated with an increased risk of hip (3.6-fold) and vertebral (4.5-fold) fracture. Because more than 3% of 50-yr-old women are treated with T4 and overreplacement is frequent (14), it is clear that thyroid dysfunction is an underestimated contributor to the burden of osteoporosis. During development, thyrotoxicosis accelerates growth and advances bone age but induces short stature due to premature fusion of the growth plates and may result in craniosynostosis due to early closure of the skull sutures (15). In contrast, juvenile hypothyroidism causes growth arrest with delayed bone maturation whereas T4 replacement results in rapid catch-up growth (16). Thus, euthyroidism during skeletal growth is likely to be a key determinant of peak bone mass in adulthood.

The THRA and THRB genes encode three receptors (TR{alpha}1, ß1, and ß2), which regulate gene expression in response to T3 (17). TR{alpha}1 and ß1 are expressed widely, whereas ß2 is restricted to the central nervous system where it regulates the hypothalamic-pituitary-thyroid (HPT) axis (18, 19). TR{alpha}1, {alpha}2, and ß1 are expressed in chondrocytes and osteoblasts, whereas reports of TR expression in osteoclasts are inconsistent (17). It remains unclear whether osteoclasts respond directly to T3 or to T3 actions in osteoblasts that subsequently regulate osteoclast activity (20, 21, 22, 23). Several TR-knockout mice have been generated (18, 19, 24, 25, 26, 27, 28), but analyses of the developing skeleton have yielded conflicting phenotypes that are difficult to reconcile because of the retention or overexpression of some TR isoforms (17). Importantly, descriptions of these mice lack longitudinal data, do not include analysis of the adult skeleton, and have not investigated the effects of altered thyroid status. A recent study has also suggested that TSH acts directly in the skeleton to inhibit bone turnover (29). Osteoblasts and osteoclasts were found to express TSH receptors in vitro, and hypothyroid juvenile TSH receptor knockout mice treated with thyroid extract displayed high bone turnover. These findings were interpreted to indicate that thyrotoxic bone loss results from TSH deficiency rather than T3 excess (29).

To clarify the roles of TR{alpha}, TRß, and TSH in bone we studied TR{alpha}0/0 and TRß–/– mice, which lack all TR{alpha} or TRß isoforms. TR{alpha}0/0 mice have normal levels of thyroid hormones and TSH, enabling the role of TR{alpha} to be determined independently of thyroid status. By contrast, thyroid hormones and TSH are both elevated in TRß–/– mice due to disruption of the HPT axis. This dissociation of their normal reciprocal relationship enabled us to clarify in vivo the relative importance of thyroid hormones and TSH to bone physiology. Thus, we characterized skeletal development, postnatal growth, adult bone structure, and the effects of exogenous thyroid hormones in these mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TR{alpha}0/0 Mice Are Euthyroid but TRß–/– Mice Have Elevated Thyroid Hormones
In TR{alpha}0/0 mice, T4 levels were equivalent to wild type (WT) at postnatal d 21 (P21) and P154 but reduced slightly at P56. In TRß–/– mice, T4 was elevated at all ages. TSH levels at P168 did not differ between wild-type (WT) and TR{alpha}0/0 mice but were elevated 10-fold in TRß–/– mice (Table 1Go). The findings are consistent with previous data (27, 30) indicating TR{alpha}0/0 mice are predominantly euthyroid whereas TRß–/– mice have resistance to thyroid hormone. Osteocalcin levels were reduced in P21 and P56 TR{alpha}0/0 mice and P56 TRß–/– mice, and IGF-I was reduced in P21 TR{alpha}0/0 and TRß–/– mice (supplemental Table 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).


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  		Table 1. Free T4 and TSH Levels

 
TR{alpha}0/0 Mice Have Transient Growth Retardation and Delayed Ossification but TRß–/– Mice Have Advanced Ossification and Persistent Short Stature
Ossification was similar in embryonic d 17.5 (E17.5) (data not shown) and P1 WT and TR{alpha}0/0 littermates (Fig. 1AGo). TR{alpha}0/0 limbs were shorter with delayed ossification at P21, but growth was similar to WT by P56 although delayed ossification persisted (Fig. 1BGo). By contrast, endochondral ossification was accelerated in E17.5 and P1 TRß–/– mice, and a 64% reduction in skull fontanelle area (P < 0.05, n = 3–5) revealed advanced intramembranous ossification (Fig. 1CGo). TRß–/– limbs still displayed advanced ossification at P21 but were now shorter than WT, whereas ossification was similar by P56 (Fig. 1DGo). A comprehensive assessment of longitudinal growth was performed (Fig. 1EGo). TR{alpha}0/0 tibias were similar to WT before P21, up to 7% shorter between P21 and P56, but not different after P70. Similarly, TR{alpha}0/0 tails were up to 17% shorter between P21 and P56, but no different after P70. TRß–/– tibias were also similar to WT before P21 and up to 5% shorter between P21 and P56; however, adult TRß–/– mice remained short. TRß–/– tails were shorter after P21 and progressively diverged from WT. Body weight measurements were consistent with these findings (supplemental Table 2 published as supplemental data on The Endocrine Society’s Journals Online web site).


Figure 1
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  		Fig. 1. Growth and Skeletal Development of TR{alpha}0/0 and TRß–/– Mice

Whole-bone mounts from P1 TR{alpha}0/0 (A) or TRß–/– (C) and WT littermates stained with alcian blue 8GX (cartilage) and alizarin red (bone). Arrows indicate regions of increased bone formation in TRß–/– mice. Tibias from P21 and P56 WT, TR{alpha}+/0, and TR{alpha}0/0 mice are shown in panel B; arrows indicate delayed formation of secondary epiphyses and wider growth plates in TR{alpha}0/0 mice. Tibias from P21 and P56 WT, TRß+/–, and TRß–/– mice are shown in panel D; arrow indicates advanced formation of the secondary epiphysis in P21 TRß–/– mice. Growth of tibias and tails of TR{alpha}0/0 or TRß–/– mice and WT littermates are shown in graphs (panels E and F); *, P < 0.05; **, P < 0.01; ***, P < 0.001 tibia or tail length in mutant vs. WT; n = 3–60 per group; two-tailed Student’s t test.

 
During Growth TR{alpha}0/0 Mice Have Impaired Chondrocyte Differentiation with Reduced Bone Mineralization but TRß–/– Mice Have Accelerated Differentiation with Increased Mineralization
In P21 TR{alpha}0/0 mice, growth plates were wider and secondary ossification centers were delayed (Fig. 2AGo). Similar features were seen at P56 and, to a lesser extent, at P154 (data not shown). By contrast, P21 TRß–/– growth plates were narrow and secondary ossification centers were advanced (Fig. 2BGo). By P56, TRß–/– growth plates were indistinguishable from those of WT (data not shown). Reserve (RZ), proliferative (PZ), and hypertrophic (HZ) growth plate zones were defined by collagen II and X in situ hybridization (31, 32, 33). In P21 TR{alpha}0/0 mice the wider growth plate resulted from an enlarged RZ but a narrower HZ (Fig. 2CGo), and these features persisted at P56 (data not shown). Thus, delayed ossification in TR{alpha}0/0 mice resulted from impaired recruitment of RZ progenitor cells, together with impaired chondrocyte differentiation. By contrast, advanced ossification in TRß–/– mice was accompanied by narrowing of the RZ, PZ, and HZ (Fig. 2DGo), suggesting accelerated chondrocyte differentiation with faster transition of cells through the PZ. Delayed ossification in TR{alpha}0/0 mice was accompanied by reduced calcified bone (Fig. 2EGo), whereas advanced ossification in TRß–/– mice resulted in increased calcified bone (Fig. 2FGo). Midfemur cortical bone was also examined. In P21 TR{alpha}0/0 mice, cortical width was reduced by 30% but was similar to WT thereafter (Fig. 2Go, C and E). By contrast, cortical width was increased by 11% in P21 TRß–/– mice, similar at P56 but reduced thereafter (Fig. 2Go, D and F). Microcomputerized tomography of P21 TR{alpha}0/0 and TRß–/– tibias and histomorphometry studies were consistent with these findings (supplemental Table 3 published as supplemental data on The Endocrine Society’s Journals Online web site).


Figure 2
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  		Fig. 2. Ossification in TR{alpha}0/0 and TRß–/– Mice

Sections of proximal tibia from P21 TR{alpha}0/0 (A) or TRß–/– (B) and WT littermates stained with alcian blue/van Gieson (magnifications x50, x100, x200). Bar graphs (C) and (D) show widths of individual growth plate zones from P21 WT and TR{alpha}0/0 or TRß–/– mice. Line graphs show midfemur diaphysis cortical bone widths in P21–P154 WT and TR{alpha}0/0 or TRß–/– mice. Panels E and F show von Kossa staining of proximal tibia from P21 WT and TR{alpha}0/0 or TRß–/– mice (upper panels, x50) and alcian blue/van Gieson staining of middiaphysis cortical bone from P21 and P56 femurs (lower two panels, x100). E, Epiphysis; IE, immature epiphysis; GP, growth plate; PS, primary spongiosum. *, P < 0.05; **, P < 0.01; ***, P < 0.001 growth plate zone width or cortical bone thickness in mutant vs. WT; n = 3–4 per group; two-tailed Student’s t test.

 
TR{alpha}0/0 Mice Have Skeletal Hypothyroidism but TRß–/– Mice Have Skeletal Thyrotoxicosis
Skeletal thyroid status was determined by characterizing expression of the T3 target gene, fibroblast growth factor receptor-1 (FGFR1) (34). FGFR1 mRNA was reduced in P21 TR{alpha}0/0 growth plates compared with WT littermates, but increased in TRß–/– chondrocytes (Fig. 3Go, A and B). Similar findings were present at P14 and P28 (data not shown), indicating changes were independent of growth plate maturity. Expression of FGFR2, a gene unresponsive to T3 (34), was indistinguishable in WT and TR{alpha}0/0 mice, whereas FGFR3, an additional T3 target gene (35), was also elevated in TRß–/– chondrocytes (data not shown). Thus, delayed ossification in TR{alpha}0/0 mice and advanced ossification in TRß–/– mice correlate with tissue thyroid status.


Figure 3
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  		Fig. 3. T3-Responsive Signaling Pathways in Growth Plates from TR{alpha}0/0 and TRß–/– Mice

In situ hybridization of FGFR1, GHR, IGF-I, and IGF-1R expression in growth plates from P21 TR{alpha}0/0 (A) or TRß–/– (B) and WT mice. C and D, Immunohistochemical analysis of STAT5, pSTAT5, AKT, and pAKT expression in growth plates from P21 WT and TR{alpha}0/0 or TRß–/– mice. Magnification, x200.

 
T3, Acting via TR{alpha}, Positively Regulates Local GH and IGF-I Signaling
Postnatal growth is regulated by paracrine actions of GH and IGF-I in chondrocytes, and this key pathway is sensitive to T3 (33). To investigate mechanisms of abnormal ossification in TR{alpha}0/0 and TRß–/– mice, we determined GH receptor (GHR), IGF-I and IGF-I receptor (IGF-1R) expression in the growth plate. IGF-I expression was indistinguishable in WT and TR{alpha}0/0 littermates and in WT and TRß–/– littermates, whereas GHR and IGF-1R levels were reduced in P21 TR{alpha}0/0 mice but increased in TRß–/– mice (Fig. 3Go, A and B). Similar results were obtained in P14 and P28 mice (data not shown). To determine the consequences of these changes, we examined the signal transducer and activator of transcription (STAT)5 and protein kinase B (AKT) pathways that lie downstream of GHR and IGF-1R (Fig. 3Go, C and D). In TR{alpha}0/0 chondrocytes, even though STAT5 expression was increased compared with WT, activated phospho-STAT5 (pSTAT5) was reduced. Similarly, activated pAKT was reduced in TR{alpha}0/0 chondrocytes. These findings are consistent with reduced GHR and IGF-1R expression in TR{alpha}0/0 mice. pSTAT5 and pAKT levels in TRß–/– mice were indistinguishable from WT. Thus, skeletal hypothyroidism in TR{alpha}0/0 mice correlates with reduced GH/IGF-I growth plate signaling, whereas skeletal thyrotoxicosis in TRß–/– mice correlates with increased expression of GHR and IGF-1R in growth plate chondrocytes.

Adult TR{alpha}0/0 Mice Have Osteosclerosis but TRß–/– Mice Are Osteoporotic
Bone microarchitecture was characterized in untreated P154 mice and mice rendered thyrotoxic between P70 and P154 (Table 1Go) using backscattered electron scanning electron microscopy (BSE-SEM) (Fig. 4Go). Unexpectedly, trabecular bone mass was substantially increased in TR{alpha}0/0 mice, but reduced in TRß–/– mice. In TR{alpha}0/0 mice, trabecular bone was robust and contained thicker trabeculae with increased connectivity and plate-like morphology. By contrast, trabecular bone in TRß–/– mice was gracile and contained thinner trabeculae with reduced connectivity and rod-like morphology. T4 treatment resulted in trabecular thinning in WT and TR{alpha}0/0 mice but had no further effect in TRß–/– mice (Fig. 4Go, A–C). Bone volume fraction (BVF) was quantified in embedded specimens (Fig. 4DGo). Trabecular BVF was increased 1.8-fold in TR{alpha}0/0 mice but similar to WT in TRß–/– mice (WT, 9 ± 2; TR{alpha}0/0, 16 ± 2*; TRß–/–, 8 ± 0; mean (%) ± SEM; n = 4–5, ANOVA and Tukey’s multiple comparison test; *, P < 0.05). T4 reduced trabecular BVF in TR{alpha}0/0 mice but had no effect in WT or TRß–/– mice (WT, 9 ± 2 vs. WT + T4 7 ± 1; TR{alpha}0/0 16 ± 2 vs. TR{alpha}0/0 + T4 9 ± 2*; TRß–/– 8 ± 0 vs. TRß–/– + T4 8 ± 1; mean (%) ±SEM, n = 2–7; ANOVA and Tukey’s multiple comparison test, *P < 0.05). Bone length in WT, TR{alpha}0/0, or TRß–/– P154 mice was unaffected by T4, and dimensions of growth plate regions were also unaffected (data not shown), indicating that T4 treatment of adults did not affect the continued slow growth of bone in mature mice. By contrast, T4 resulted in a 15%, 26%, and 29% loss of cortical bone width in WT + T4, TR{alpha}0/0 + T4, and TRß–/– + T4 tibias (WT, 0.19 ± 0.01 vs. WT + T4 0.16 ± 0.01; TR{alpha}0/0 0.20 ± 0.01 vs. TR{alpha}0/0 + T4 0.15 ± 0.01**; TRß–/– 0.16 ± 0.01 vs. TRß–/– + T4 0.12 ± 0.01*; mean (mm) ± SEM, n =3–4; two-tailed Student’s t test; *, P < 0.05; **, P < 0.01) (Fig. 4EGo). Remarkably, the juvenile TR{alpha}0/0 phenotype of delayed ossification and reduced calcified bone deposition was associated with an adult phenotype of osteosclerosis, whereas advanced ossification and increased calcified bone in juvenile TRß–/– mice preceded an osteoporotic phenotype in adults.


Figure 4
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  		Fig. 4. BSE-SEM Analysis of Bone Structure in Adult TR{alpha}0/0 and TRß–/– Mice

3D BSE-SEM views (A, x12 magnification; B, x75) of distal femurs from untreated P154 WT, TR{alpha}0/0, and TRß–/– mice and mice treated with T4 from P70 until P154. BSE-SEM views (x12) of proximal tibias from the same animals are shown (C). qBSE-SEM gray scale views (x12) of polymethylmethacrylate-embedded proximal tibias from the same mice are shown (D). Decalcified sections of middiaphysis cortical bone from tibias of the same mice stained with alcian blue/van Gieson are shown (E). Scale bars represent 200 µm in all panels.

 
TR{alpha}0/0 Mice Have Normal Bone Mineralization but TRß–/– Mice Have Reduced Mineralization
Bone mineralization was determined in P154 femurs by quantitative BSE-SEM (qBSE-SEM) (Fig. 5Go). The distributions of mineralization densities in trabecular and cortical bone were indistinguishable in WT and TR{alpha}0/0 mice (Kolmogorov-Smirnov test; trabecular bone D statistic 3.3, P > 0.05; cortical bone D statistic 3.1, P > 0.05), although abnormal retention of highly mineralized calcified cartilage was evident in TR{alpha}0/0 trabeculae (Fig. 5CGo). By contrast, mineralization in TRß–/– mice was markedly reduced (trabecular D statistic 14.5, P < 0.001; cortical D statistic 18.2, P < 0.001). T4 treatment did not affect trabecular or cortical bone mineralization (WT trabecular, 4.6; P > 0.05; cortical, 5.6; P > 0.05; TR{alpha}0/0 trabecular, 4.78; P > 0.05; cortical, 4.88; P > 0.05; TRß–/– trabecular, 1.57; P > 0.05; cortical, 3.63; P > 0.05). Thus, adult TR{alpha}0/0 mice display osteosclerosis with increased trabecular bone of robust architecture, but mineralization that was similar to WT. By contrast, adult TRß–/– mice were osteoporotic with reduced trabecular bone of gracile architecture, decreased cortical width, and reduced mineralization. T4 induced loss of trabecular and cortical bone in TR{alpha}0/0 mice and loss of cortical bone in TRß–/– mice but did not alter mineralization.


Figure 5
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  		Fig. 5. qBSE-SEM of Bone Mineralization Densities in Adult TR{alpha}0/0 and TRß–/– Mice

qBSE-SEM images (A, x33 magnification; B, x100) showing mineralization densities of trabecular bone in proximal femurs from untreated P154 WT, TR{alpha}0/0, and TRß–/– mice and animals treated with T4 from P70 until P154. Mineralization densities were derived from halogenated standards, and gray scale images were pseudocolored according to the palette shown in which low mineralization density is blue and high density is gray. C, Graphs show relative and cumulative frequency histograms of trabecular bone micromineralization densities. Arrow indicates the increased relative frequency of high micromineralization densities corresponding to retained calcified cartilage in TR{alpha}0/0 mice. D, qBSE-SEM images of middiaphyseal cortical bone (x100) from the same mice. E, Relative and cumulative frequency histograms of cortical bone micromineralization densities. Scale bars represent 200 µm in all panels. ***, P < 0.001 micromineralization density in mutant vs. WT; n = 3–5 per group, Kolmogorov-Smirnov test.

 
TR{alpha}0/0 Mice Have Reduced Osteoclastic Bone Resorption but TRß–/– Mice Have Increased Resorption
Cortical and trabecular bone surfaces were analyzed by higher resolution BSE-SEM (Fig. 6Go, A and B). Areas of resorption were identified by surfaces that contained osteoclastic resorption pits and contrasted sharply with smooth unresorbed surfaces that lacked evidence of osteoclastic activity. In P154 TR{alpha}0/0 mice, the resorption surface was reduced by 21% (P < 0.01) in cortical bone and 9% (P < 0.05) in trabecular bone, whereas in TRß–/– mice it was increased by 22% (P < 0.05) and 10% (P < 0.05) in cortical and trabecular bone. Quantitation of osteoclasts per unit bone surface (Fig. 6Go, C and D) revealed a 13% reduction (P < 0.05) in TR{alpha}0/0 mice and 20% increase (P < 0.05) in TRß–/– mice. Thus, increased bone mass in TR{alpha}0/0 mice was associated with reduced numbers of osteoclasts and reduced cortical and trabecular bone resorption, whereas osteoporosis in TRß–/– mice was accompanied by increased osteoclast numbers and increased resorption.


Figure 6
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  		Fig. 6. BSE-SEM Analysis of Bone Resorption in Adult TR{alpha}0/0 and TRß–/– Mice

BSE-SEM views of (A) middiaphysis endosteal cortical bone surface and (B) submetaphyseal trabecular bone from distal femurs of P154 WT, TR{alpha}0/0, and TRß–/– mice. Arrows indicate junctions between osteoclast resorption surfaces and smooth unresorbed bone surfaces, which are clearly distinguished in 3D SEM images. Decalcified sections of distal femur metaphysis from P98 WT, TR{alpha}0/0, and TRß–/– mice stained with TRAP to reveal osteoclasts lining trabecular bone surfaces are shown (C). D, Graphs demonstrate the amount of endosteal (left) and trabecular (middle) bone occupied by osteoclast resorption surfaces, and the numbers of TRAP-positive osteoclasts per unit bone surface (right), in WT, TR{alpha}0/0, and TRß–/– mice. All scale bars represent 100 µm. *, P < 0.05; **, P < 0.01 mean percentage area of bone surface occupied by resorption surfaces (n = 2–5 per group); or number of osteoclasts per millimeter bone surface (n = 5 per group), in mutant vs. WT, two-tailed Student’s t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In these studies euthyroid TR{alpha}0/0 adult mice unexpectedly displayed a marked increase in trabecular bone mass even though juveniles had growth retardation with delayed endochondral ossification and reduced bone mineral deposition. By contrast, adult TRß–/– mice, with elevated TSH and thyroid hormone levels, displayed an opposite phenotype of osteoporosis despite advanced ossification and increased bone mineral in juveniles. These data indicate that thyroid hormones, acting via TR{alpha}, promote anabolic effects during postnatal bone growth but have catabolic actions on the adult skeleton.

T3 Acts via Skeletal TR{alpha} to Regulate Bone Mass in Adults
Adult TR{alpha}0/0 mice exhibited an osteosclerotic phenotype characterized by a marked increase in trabecular bone volume of normal mineralization density. Furthermore, a remodeling defect was revealed by the presence of retained and highly mineralized cartilage in trabecular bone that also displayed a robust and plate-like morphology. These features are similar to the reduced bone turnover and impaired bone remodeling seen in adult hypothyroidism (36) and correlate with target gene studies indicating skeletal hypothyroidism in TR{alpha}0/0 mice. By contrast, adult TRß–/– mice were osteoporotic with reduced trabecular and cortical bone of reduced mineralization density, indicating a phenotype of increased bone remodeling. These features are typical of the rapid bone turnover and osteoporosis observed in thyrotoxicosis (37) and correlate with the increased skeletal T3 target gene expression observed in TRß–/– mice. Thus, TR{alpha} regulates the maintenance of bone mass in adults.

Regulation of Adult Bone Mass by T3 Is Mediated Principally by Osteoclasts
Increased trabecular bone in adult TR{alpha}0/0 mice correlated with reduced bone resorption surfaces and reduced osteoclast numbers, whereas the osteoporotic phenotype in TRß–/– mice was associated with increased resorption surfaces and increased numbers of osteoclasts. Moreover, levels of osteocalcin, a systemic marker of osteoblastic bone formation, did not differ between TR{alpha}0/0 and TRß–/– mice. These data indicate that altered osteoclastic bone resorption was primarily responsible for the divergent phenotypes in TR{alpha}0/0 and TRß–/– mice. Although the data further support the conclusion that TR{alpha} is functionally predominant in adult bone, severe thyrotoxicosis also induced bone loss in TR{alpha}0/0 mice, indicating that TRß can partially compensate for lack of TR{alpha} in the presence of thyroid hormone excess.

GH/IGF-I Signaling Lies Downstream of TR{alpha} in Growth Plate Chondrocytes
Delayed ossification and growth retardation were observed in juvenile TR{alpha}0/0 mice. These features are similar to the characteristic skeletal defects of hypothyroidism in children (16) and correlate with reduced T3 target gene expression in the growth plate, supporting the conclusion that the developing TR{alpha}0/0 skeleton is hypothyroid. By contrast, advanced ossification in juvenile TRß–/– mice was analogous to the accelerated growth and advanced bone age seen in childhood thyrotoxicosis (15) and correlated with increased T3 target gene expression in the growth plate, thus demonstrating that the developing TRß–/– skeleton is thyrotoxic. In TR{alpha}0/0 mice the growth plate abnormalities resulted from impaired recruitment of RZ progenitor cells, together with impaired chondrocyte differentiation, whereas in TRß–/– mice chondrocyte differentiation was accelerated with more rapid entry of cells into the HZ.

GH and IGF-I are major regulators of postnatal growth (38), whereas IGF-I and its receptor are regulated by T3 in skeletal cells (39). Furthermore, the GH dual effector theory proposes that GH directly initiates proliferating chondrocyte differentiation in the growth plate and also induces local IGF-I production, which acts locally to stimulate clonal expansion of RZ cells and maintain the supply of chondrocytes for longitudinal growth (40). In our studies, circulating levels of IGF-I were similar in TR{alpha}0/0 and TRß–/– mice, indicating their divergent phenotypes are independent of the systemic GH/IGF-I axis. Nevertheless, reduced expression of GHR and IGF-1R and reduced STAT and AKT activation in TR{alpha}0/0 growth plates, together with increased expression of GHR and IGF-1R in TRß–/– mice, indicates the paracrine GH/IGF-I pathway lies downstream of TR{alpha} in the growth plate. Thus, GH/IGF-I signaling is a local downstream mediator of T3 responses in developing bone.

Relationship between Central and Skeletal Thyroid Status
The divergent phenotypes observed in TR{alpha}0/0 and TRß–/– mice can be understood by considering how physiological control of the HPT axis by TRß (18, 19) relates to T3 action in bone mediated by TR{alpha}. After analysis of skeletal development in mice with dominant-negative mutations in Thra or Thrb (32, 33), we proposed recently that delayed ossification in TR{alpha} mutant mice results from direct interference with T3 action in bone, whereas advanced skeletal development in TRß mutant mice is due to elevated thyroid hormone levels and the supraphysiological stimulation of skeletal TR{alpha} resulting from disruption of the HPT axis (41). In the current studies TRß–/– mice express TR{alpha} and also have elevated thyroid hormones, whereas TR{alpha}0/0 mice are euthyroid but lack TR{alpha} in bone. Thus, the phenotypes of skeletal hypothyroidism in TR{alpha}0/0 mice and skeletal thyrotoxicosis in TRß–/– mice are consistent with this model, which is further supported by the much higher levels of expression of TR{alpha} than TRß in bone (32, 42).

Thyroid Hormone Excess Rather Than TSH Deficiency Induces Bone Loss in Thyrotoxicosis
A recent study, however, challenges our interpretation of the role of the HPT axis in skeletal physiology. Abe et al. (29) reported osteoporosis in juvenile TSH receptor knockout (TSHR–/–) mice and, surprisingly, proposed TSH as a major and direct inhibitor of bone remodeling. In the current study, however, we show that adult TR{alpha}0/0 mice have osteosclerosis despite normal levels of TSH, whereas TRß–/– mice have osteoporosis despite elevated levels of thyroid hormones and TSH. Furthermore, TR{alpha}1–/–ß–/– double-knockout mice from a different genetic background display mildly impaired bone mineralization despite grossly elevated TSH levels (28). The current studies, therefore, demonstrate that bone loss in hyperthyroidism results from T3 excess rather than TSH deficiency.

The reason for the striking difference between our studies and those of Abe et al. is unclear. Importantly, TSHR–/– mice have congenital hypothyroidism and require thyroid hormone replacement for survival (43). Juvenile hypothyroidism results in severe growth retardation and delayed bone maturation (44). Thyroid hormone replacement results in a period of increased growth velocity termed catch-up growth, indicating the hypothyroid skeleton becomes exquisitely sensitive to thyroid hormone (16, 44). In mice, the peak physiological rise in circulating T4 and T3 occurs at 2 wk of age, and this coincides with normal maximum growth velocity (33, 45). In the study of Abe et al. TSHR–/– mice only received hormone replacement with thyroid extract from weaning at 3 wk of age (29) and were, therefore, grossly hypothyroid during this critical stage of skeletal development. Thus, a possible explanation is that high bone remodeling described in TSHR–/– mice reflects the skeletal manifestations of catch-up growth and accelerated bone development in response to delayed thyroid hormone replacement. Nevertheless, the milder phenotype observed in TSHR+/– heterozygotes and effects of TSH in skeletal cells in vitro (29) means a role for TSH in bone cannot be excluded completely by our findings.

In summary, our studies demonstrate that T3 acts via TR{alpha} to regulate skeletal development, the accrual of peak bone mass during growth, and the maintenance of bone mass, microarchitecture, and mineralization in adults. We conclude that bone loss in thyrotoxicosis is independent of circulating TSH concentrations and mediated principally by TR{alpha}. These studies identify TR{alpha} as a novel drug target in the prevention and treatment of osteoporosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
TR{alpha}0/0 and TRß–/– Mice
Studies were approved by the Université Victor Segalen Animal Care and Use Committee. TR{alpha}0/0 and TRß–/– mice were derived in the same genetic background. Studies were performed on littermates from heterozygote crosses to overcome confounding influences of a maternal homozygote genotype (19, 27). Some P70 WT, TR{alpha}0/0, and TRß–/– mice were treated with T4 in their drinking water (5 µg/ml) until P154.

Biochemical Measurements
Free T4 was measured using an Access RIA (Beckman Coulter, Marseilles, France), and TSH was measured by RIA (46). Osteocalcin was measured with an ELISA (Biomedical Technologies, Inc., Stoughton, MA) or an immunoradiometric assay (Immutopics, San Clemente, CA). IGF-I was determined by RIA (Nichols Institute Diagnostics, Paris, France). All blood samples were taken after an overnight fast.

Skeletal Preparations
E17.5 and P1 mice and limbs from P14, P21, P28, and P56 animals were stained with alcian blue/alizarin red (32, 33). Preparations were photographed using a Leica MZ75 binocular microscope (Leica AG, Heerbrugg, Switzerland), Leica KL1500 light source, Leica DFC320 digital camera, Leica IM50 Digital Image Manager, and Leica Twain Module DFC320 image acquisition software. Bone lengths from wild-type, heterozygous, and homozygous male and female littermates were determined digitally after linear calibration. Skull dimensions and open fontanelle and suture areas were calculated using ImageJ 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, P21, and P28 limbs were decalcified for 5 d. Limbs from older mice were decalcified for 7 d. Decalcified bones were embedded in paraffin and sections stained with van Gieson and alcian blue. Some limbs from P21 mice were 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 of 3-µm cryosections with neutral red counterstain. Growth plate and cortical bone dimensions were measured in at least four separate positions to calculate mean values for the heights of the RZ, PZ, HZ, and total growth plate. Results from at least two different levels of sectioning were compared to ensure consistency of data. Cortical bone measurements were performed in at least 10 separate positions in the midshaft of long bones, and adjacent levels of sectioning were compared. Osteoclast numbers in long bone metaphyses were determined after staining sections for tartrate-resistant acid phosphatase (TRAP) in limbs decalcified in 10% EDTA, pH 7.4, for 14 d. 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 (Cell-counter macro). Osteoclast numbers were normalized for the amount of bone surface quantitated using Image J, and data were expressed as number of TRAP-positive cells per millimeter of bone surface in each section.

In Situ Hybridization
mRNA expression was analyzed using collagen II, collagen X, FGFR1, FGFR2, FGFR3, IGF-I, IGF-1R, and GHR probes (32, 33, 34, 35). A neomycin probe (Boehringer Mannheim, Lewes, Sussex, UK) was used as a negative control, and collagen II and X probes were used to identify proliferative and hypertrophic zones in growth plate sections. In situ hybridizations were performed on at least three mice per genotype in duplicate, and repeated three times. For comparison of WT and TR{alpha}0/0 littermates, studies were performed in parallel, and development times were optimized separately for each probe. In situ hybridizations were also optimized in separate experiments for comparison of WT and TRß–/– littermates.

Immunohistochemistry
IGF-1R and GHR signaling was examined by immunohistochemical analysis of AKT, pAKT, STAT5, and pSTAT5. Negative controls lacking primary antibody were performed in all experiments (33). Studies were performed in duplicate on at least three mice per genotype and repeated three times. For comparison between WT and TR{alpha}0/0 littermates, and between WT and TRß–/– littermates, immunohistochemistry was performed in parallel and optimized separately for each antibody.

Microcomputerized Tomography
Three dimensional (3D)-architecture of the tibia was evaluated in P21 TR{alpha}0/0 and TRß–/– mice (µCT20; Scanco Medical AG, Bassersdorf, Switzerland). The proximal metaphysis was scanned at 250-µm increments beginning at 500 µm and extending 1.5mm below the growth plate. Morphometric parameters were computed using triangulation algorithms. Trabecular bone volume and total tissue volume were determined from the binarized bone volume. Bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th/µm) and trabecular number (Tb.N, 1/mm) were derived from these parameters.

Histomorphometry
Undecalcified bones from P21 and P56 mice were fixed, dehydrated in ethanol, and embedded in methyl-methacrylate. Sections (7 µm) were obtained using a Polycut E microtome (Leica) and stained with von Kossa/van Gieson. Histomorphometry of a standardized area of the metaphysis was performed with a Leica Quantimet Q570 image processor coupled to a Leitz DM/RBE microscope at x40 magnification. Standard analysis terms and abbreviations adhere to the American Society for Bone and Mineral Research histomorphometry nomenclature: BV/TV (%) = cancellous bone volume/ tissue volume, corresponding to the amount of cancellous bone within the cancellous space; Tb.Th (µm) = mean thickness of the trabeculae; Tb.N (mm) = mean number of trabeculae. These parameters reflect the spatial distribution of trabeculae and are derived from surface to volume ratio (BS/BV) measures according to stereological formulas.

BSE-SEM
Freshly dissected bones were fixed and stored in 70% ethanol. Bones were opened longitudinally by removing half the cortical bone and medullary trabecular elements with a fine tungsten carbide milling tool in a dental workshop handpiece, using standard orientations and following anatomical curvatures. Samples were cleaned of cell remnants using alkaline bacterial pronase, coated with carbon, and imaged using back-scattered 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 (47). 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. Fixed bones were embedded in polymethylmethacrylate. Block faces were cut through the specimens, which were polished, coated with carbon, and analyzed using back-scattered electrons in a Zeiss DSM962 digital scanning electron microscope, with an annular solid state BSE detector (KE Electronics, Toft, Cambridgeshire, UK), operated at 20 kV and 0.5 nA. The mineralization densities of calcified tissues were determined by comparison with halogenated dimethacrylate standards, C22H25O10Br [mean BSE coefficient according to the procedure of Lloyd (48) = 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 (49, 50).

Statistics
Normally distributed data were analyzed using Student’s t test, or ANOVA followed by Tukey’s multiple comparison post hoc test. P < 0.05 was considered significant. Distributions of qBSE mineralization densities were analyzed by the Kolmogorov-Smirnov test, in which significant 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 technical assistance.


   FOOTNOTES
 
This work was supported by a Medical Research Council (MRC) Clinician Scientist Award (to J.H.D.B.), MRC Studentship and Marie-Curie Outgoing International Fellowship (to P.J.O’S.), MRC Career Establishment (to G.R.W.), Horserace Betting Levy Board (to A.B.), and grants from the French Ministry of Research (to O.C.). The SEM was funded by the MRC.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 27, 2007

Abbreviations: AKT, Protein kinase B; BSE-SEM, back-scattered electron scanning electron microscopy; BVF, bone volume fraction; 3D, three-dimensional; E17.5, embryonic d 17.5; FGFR1, fibroblast growth factor receptor-1; GHR, GH receptor; HPT, hypothalamic-pituitary-thyroid; HZ, hypertrophic zone; IGF-1R, IGF-I receptor; P21, postnatal d 21; pAKT, phosphor-AKT; pSTAT, phospho-STAT; PZ, proliferative zone; qBSE-SEM, quantitative BSE-SEM; RZ, reserve zone; STAT, signal transducer and activator of transcription; TR, thyroid hormone receptor; TRAP, tartrate-resistant acid phosphatase; WT, wild type.

Received for publication January 18, 2007. Accepted for publication February 20, 2007.


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