Mechanisms of action of thyroid hormones in the skeleton

Biochimica et Biophysica Acta 1830 (2013) 3979–3986

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Review

Mechanisms of action of thyroid hormones in the skeleton☆ Anna Wojcicka a, b, J.H. Duncan Bassett b, Graham R. Williams b,⁎ a b

The Medical Centre of Postgraduate Education, Department of Biochemistry and Molecular Biology, ul.Marymoncka 99/103, 01–813 Warsaw, Poland Molecular Endocrinology Group, Department of Medicine and MRC Clinical Sciences Centre, Imperial College London, London W12 0NN, UK

a r t i c l e

i n f o

Article history: Received 25 January 2012 Received in revised form 19 April 2012 Accepted 18 May 2012 Available online 25 May 2012 Keywords: Thyroid hormone Thyroid hormone receptor Deiodinase Endochondral ossification Bone turnover Osteoporosis

a b s t r a c t Background: Thyroid hormones regulate skeletal development, acquisition of peak bone mass and adult bone maintenance. Abnormal thyroid status during childhood disrupts bone maturation and linear growth, while in adulthood it results in altered bone remodeling and an increased risk of fracture Scope of Review: This review considers the cellular effects and molecular mechanisms of thyroid hormone action in the skeleton. Human clinical and population data are discussed in relation to the skeletal phenotypes of a series of genetically modified mouse models of disrupted thyroid hormone signaling. Major Conclusions: Euthyroid status is essential for normal bone development and maintenance. Major thyroid hormone actions in skeletal cells are mediated by thyroid hormone receptor α (TRα) and result in anabolic responses during growth and development but catabolic effects in adulthood. These homeostatic responses to thyroid hormone are locally regulated in individual skeletal cell types by the relative activities of the type 2 and 3 iodothyronine deiodinases, which control the supply of the active thyroid hormone 3,5,3’-L-triiodothyronine (T3) to its receptor. General Significance: Population studies indicate that both thyroid hormone deficiency and excess are associated with an increased risk of fracture. Understanding the cellular and molecular basis of T3 action in skeletal cells will lead to the identification of new targets to regulate bone turnover and mineralization in the prevention and treatment of osteoporosis. This article is part of a Special Issue entitled Thyroid hormone signaling. © 2012 Elsevier B.V. All rights reserved.

1. Thyroid hormone action The synthesis and release of the pro-hormone 3,5,3′,5′-Ltetraiodothyronine (thyroxine, T4) and the biologically active thyroid hormone 3,5,3′-L-triiodothyronine (T3) are regulated by a classical negative feedback loop involving the paraventricular nucleus of the hypothalamus and the anterior pituitary thyrotropes. The hypothalamus secretes TRH (thyrotropin-releasing hormone) into the portal circulation to stimulate production and secretion of TSH (thyrotropin, thyroid-stimulating hormone) by the pituitary. TSH subsequently acts via the TSH receptor (TSHR) on thyroid follicular cells to stimulate synthesis and release of T4 and T3. The circulating thyroid hormones are predominantly bound to carrier proteins including thyroxine binding globulin, transthyretin (previously known as thyroxine binding pre-albumin) and albumin, with only approximately 0.2% of the total T3 and 0.02% of total T4 available as free unbound hormones (fT3, fT4) in plasma. Circulating fT3 and fT4 ultimately act in the hypothalamus and anterior pituitary to inhibit synthesis and secretion of TRH and TSH. Thus, systemic thyroid status is

☆ This article is part of a Special Issue entitled Thyroid hormone signaling. ⁎ Corresponding author at: Molecular Endocrinology Group, 7th Floor Commonwealth Building, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. Tel.: + 44 208 383 1383. E-mail address: [email protected] (G.R. Williams). 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2012.05.005

maintained within a normal reference range by the hypothalamicpituitary-thyroid (HPT) axis negative feedback loop (Fig. 1A). This negative feedback loop maintains a physiological inverse relationship between TSH and circulating T3 and T4 levels that defines the HPT axis set-point [1,2]. Systemic fT4, fT3 and TSH concentrations vary significantly among individuals, indicating each person has a unique HPT axis set point [3]. Twin studies suggest the HPT axis set point is predominantly genetically determined with heritability for fT3, fT4 and TSH of 65% [4]. Consistent with this, genome wide association studies (GWAS) have identified quantitative trait loci for fT4 (14q13 and 18q21), fT3 (7q36, 8q22 and 18q21) and TSH (2q36, 4q32) [5]. Circulating T4 is derived from thyroid gland secretion, whereas the majority of the pool of systemic T3 is generated by deiodination of T4 in peripheral tissues. Thyroid hormone metabolism is mediated by three iodothyronine deiodinases. The type 1 and type 2 enzymes (D1 and D2) catalyze deiodination of the pro-hormone T4 to the active hormone T3 by removal of an iodine atom from the outer ring of T4. Conversely, the type 3 enzyme (D3) irreversibly inactivates both T4 and T3 by removal of an inner ring iodine atom [6]. Circulating thyroid hormones are bound to carrier proteins including thyroxine-binding globulin (TBG), transthyretin and albumin. Uptake into peripheral tissues is mediated by several specific membrane transporter proteins including the monocarboxylate transporters 8 and 10 (MCT8 and MCT10) and the organic anion transporter protein 1c1 (OATP1C1).

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Fig. 1. Hypothalamic-pituitary-thyroid axis and thyroid hormone action. (A) The thyroid gland secretes the pro‐hormone T4 and a small amount of the active hormone T3. Their circulating concentrations are regulated by a classical negative feedback loop. TRH is synthesized and secreted in the hypothalamus and acts on pituitary thyrotropes to stimulate synthesis and secretion of TSH. TSH acts on thyroid follicular cell to stimulate growth of the gland and thyroid hormone secretion. T4 and T3 subsequently inhibit secretion of TRH and TSH acting via TRβ2 to complete a negative feedback loop. (B) Thyroid hormones enter target cells via specific membrane transporters including MCT8, MCT10 and OATP1C1. The intracellular concentration of T3 is determined by the relative activities of the deiodinases, D2 and D3. T3 enters the nucleus and bind to TRs, which act as hormone inducible transcription factors to regulate expression of T3‐target genes.

Thyroid hormone action is mediated primarily via the nuclear thyroid hormone receptors (TRα and TRβ), which act as ligand-inducible transcription factors that mediate diverse cellular responses including proliferation, differentiation and apoptosis. The 3 functional TR proteins, TRα1, TRβ1 and TRβ2 are encoded by THRA and THRB. TRα1 and TRβ1 are expressed in virtually all tissues, but their abundance and roles differ, depending on the developmental stage of the organism and on the particular tissue type. By contrast, expression of TRβ2 is restricted to the hypothalamus, pituitary and sensory organs where it regulates the HPT axis and timing of the onset of hearing and color vision [7]. TRs bind to specific thyroid hormone response element sequences (TREs) located in promoter regions of T3-target genes and regulate their expression in a ligand-dependent manner (Fig. 1B).

hedgehog (Ihh), bone morphogenetic proteins, fibroblast growth factors, vascular endothelial growth factors) that act in a paracrine and autocrine manner [11]. By contrast, the skull vault is formed by intramembranous ossification, in which condensations of mesenchyme differentiate into osteoblasts, which secrete and mineralize osteoid to form bone directly without an intermediate cartilage model [12] (Fig. 2B). During these processes of skeletal development and linear growth, bone mass accumulates and mineralization increases until peak bone mass [13] is achieved in early adulthood. Throughout adult life there is a gradual loss of bone mass, which in women is accelerated at the menopause.

2. Skeletal development

Structural integrity and strength of the adult skeleton is maintained by a continual process of regeneration and repair. The bone remodeling cycle has a duration of 150–200 days and is characterized by sequential periods of activation, bone resorption, reversal, bone formation and quiescence (Fig. 3). The cycle is mediated by osteoclasts and osteoblast-derived cells located in basic multicellular units (BMU) [14]. In the adult human skeleton up to 2 million BMUs are active and separated both spatially and temporally, thus demonstrating the importance and scale of continuous bone turnover and renewal. Local activation of bone remodeling is initiated by changes in mechanical load, structural damage or in response to systemic or paracrine factors. Activation of bone lining cells results in the recruitment of osteoclast progenitor cells and their differentiation to multinucleated bone-resorbing osteoclasts. Mature osteoclasts adhere to activated bone surfaces and resorb bone by creating a localized microenvironment into which they secrete acid and proteases resulting in demineralization of bone and degradation of matrix proteins. Subsequently reversal cells, which are alkaline phosphatase-expressing precursor cells of uncertain phenotype probably arising from the osteoblast lineage, engulf and remove demineralized and undigested matrix fragments from the resorbed bone surface [15,16]. The bone

The skeleton forms by two distinct mechanisms, endochondral and intramembranous ossification (Fig. 2). Long bones form via endochondral ossification, during which mesenchymal stem cells differentiate into chondrocytes that proliferate and secrete cartilage matrix to form a scaffold or anlage [8,9] (Fig. 2A). Chondrocytes undergo hypertrophic differentiation commencing at the centre of the anlage and this process is followed by cartilage mineralization, chondrocyte apoptosis and vascular invasion. This calcified cartilage forms a template for bone formation by invading osteoblasts that lay down and mineralize bone matrix (osteoid). Chondrocytes at both ends of the anlage organize to form epiphyseal growth plates and secondary ossification centers. The ordered process of growth plate chondrocyte proliferation, hypertrophic differentiation, apoptosis and subsequent new bone formation mediates linear growth until adulthood [10]. Progression of endochondral ossification and the rate of linear growth are tightly regulated by multiple systemic hormones (including thyroid hormones, growth hormone, insulin-like growth factor 1, glucocorticoids and sex steroids) and various cytokines and growth factors (including parathyroid hormone-related peptide (PTHrP), Indian

3. Adult bone remodeling

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Fig. 2. Endochondral and intramembranous ossification. (A) Long bones form by endochondral ossification. Chondrocytes first synthesize a cartilage model. Central chondrocytes undergo hypertrophic differentiation and then apoptose inducing vascular invasion and formation of a primary ossification centre. Linear growth occurs at the epiphyseal growth plates located at the proximal and distal ends of the bone. (B) The skull vault forms by intramembranous ossification. Mesenchyme condensations differentiate into osteoblasts, which synthesize and mineralize osteoid to form new without the requirement for a cartilage intermediate.

formation phase is subsequently initiated by local paracrine signals derived from the degraded bone matrix, osteoclasts and reversal cells resulting in increased recruitment and activity of bone-forming osteoblasts. Once the resorption cavity is repaired, bone formation ceases and at termination the bone surface returns to a resting quiescent state covered with bone-lining cells and the remodeling cycle is completed [16]. This orchestrated process of bone remodeling, in which osteoclastic bone resorption is coupled to osteoblastic bone formation, continually optimizes bone structure and strength in response to its changing environment and is also a key component of calcium and phosphate homeostasis [17].

4. Thyroid hormones and skeletal development in humans The detrimental effects of thyroid hormone excess on the skeleton were first described in 1891 [18]. In children, hypothyroidism causes cessation of linear growth resulting in a marked delay of bone age and epiphyseal dysgenesis. A recent report described the first heterozygous mutation of THRA resulting in expression of a dominantnegative TRα protein [19]. The 6 year old female proband had circulating free and total T4 levels within or just below the normal range, free and total T3 levels within or just above the normal range and normal levels of TSH. Despite this, she displayed a phenotype of

Fig. 3. The bone remodeling cycle. Schematic representation of a basic multicellular unit illustrating phases of the bone remodeling cycle. Bone remodeling is initiated by mechanical load, structural damage or systemic or paracrine factors. Hemopoietic stem cells of the monocyte/macrophage lineage differentiate to mature osteoclasts and resorb bone. During the reversal phase osteoblastic cells are recruited to the site of resorption, differentiate and synthesize and mineralize new bone matrix to repair the defect.

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developmental retardation consistent with classical features of hypothyroidism that included short stature, delayed intramembranous and endochondral ossification and skeletal dysplasia, thus demonstrating a critical role for TRα in human skeletal development [19]. Childhood thyrotoxicosis, by contrast, accelerates linear growth and advances bone maturation resulting in premature fusion of the growth plates and persistent short stature. In severe and early cases craniosynostosis results from premature fusion of the sutures of the skull and may be associated with cognitive deficits [20–22]. Similarly, congenital hyperthyroidism resulting from activating mutations of the TSH receptor gene (TSHR) leads to advanced bone age and shortening of the metacarpals and metatarsals although early thyroidectomy prevents the long-term sequelae of thyroid hormone excess [23]. Dominant-negative mutations of THRB impair negative feedback of TRH and TSH secretion from the hypothalamus and pituitary resulting in the syndrome of resistance to thyroid hormone (RTH), which is characterized by elevated T4 and T3 concentrations together with an inappropriately normal or elevated TSH. RTH patients display variable skeletal phenotypes that are difficult to interpret due to confounding effects of treatment interventions and the wide variety of TRβ mutations with heterogeneous functional properties [24,25]. 5. Thyroid hormones and adult bone maintenance in humans Initiation and duration of the bone remodeling cycle is regulated by thyroid hormone. Hypothyroidism has been shown to be associated with a 2- to 3-fold increased risk of fracture in large population studies [26,27]. Classical histomorphometry studies have demonstrated that hypothyroidism results in reduced bone turnover with prolongation of the bone remodeling cycle [28]. Conversely, thyrotoxicosis leads to increased bone turnover, shortening of the bone remodeling cycle and uncoupling of bone resorption and formation resulting in a net loss of about 10% of mineralized bone per cycle [29]. Thus, thyrotoxicosis results in osteoporosis and an increased risk of fragility fracture [30,31]. Population studies have further demonstrated that thyroid hormone replacement, sub-clinical hyperthyroidism and even variation of thyroid status within the normal reference range are inversely correlated with BMD and may be associated with an increased risk of fracture [32–39]. 6. Thyroid hormone action in bone Mouse chondrocytes, osteoblasts and osteoclasts express the thyroid hormone transporter MCT8 but not OATP1c1 but no data have been published in human cells or regarding skeletal expression of MCT10 [40,41]. Skeletal cells do not express the type 1 deiodinase, whereas the inactivating enzyme D3 is expressed in chondrocytes, osteoblasts and osteoclasts with the highest levels of activity detected in the growth plate prior to weaning [41]. By contrast, expression of the activating enzyme D2 is restricted during development to the fetal skeleton between embryonic days E14.5 and E18.5 [42] and to the perichondrium adjacent to the developing growth plate [43]. In adults, D2 activity is restricted to mature osteoblasts only [41]. Chondrocytes, osteoblasts and osteoclasts express TRα1 and TRβ1 mRNAs but studies of their relative expression using RNA extracted from whole bone and primary bone cell cultures demonstrate that expression of TRα1 is at least 10-fold higher than expression of TRβ1 [44–46]. These data are consistent with the recent finding of a phenotype suggestive of reduced skeletal thyroid hormone signaling in a child with a dominant-negative mutation of TRα1 [19]. 7. Effects of T3 in skeletal cells in vitro Skeletal cell responses to T3 have been studied in primary cultures and well-characterized immortalized cells including mouse chondrogenic

embryonal carcinoma cells (ATDC5) and various osteoblastic cell lines (e.g. MCT3T3-E1, UMR106 and ROS17/2.8). 7.1. Chondrocytes TRα1 and TRβ1 are expressed in reserve and proliferating chondrocytes in the growth plate, suggesting these cells are direct targets for T3 action [47] (Fig. 4). Accordingly, T3 stimulates clonal expansion of resting chondrocyte progenitor cells (reserve cells) in the reserve zone that may serve as chondrocyte stem cells, but inhibits subsequent chondrocyte proliferation while stimulating hypertrophic differentiation and eventual apoptosis [47–51]. The pace of chondrocyte proliferation and differentiation is tightly controlled by several paracrine factors including Indian hedgehog (Ihh) and PTHrP, IGF1, Wnt, bone morphogenetic proteins (BMPs) and FGFs [52,53]. Ihh, PTHrP and the BMP receptor-1A participate in a negative feedback loop that promotes growth plate chondrocyte proliferation and inhibits differentiation thereby controlling the rate of linear growth. The set-point of this feedback loop is sensitive to changes in thyroid status in vivo [54] and regulated by local thyroid hormone metabolism and T3 availability [43]. Furthermore, T3 stimulates expression of genes specifically involved in cartilage matrix synthesis, mineralization and degradation, and the regulation of progression of hypertrophic differentiation; including cyclin-dependent kinase inhibitors [55], BMP4, Wnt4 and FGFR3 [56–58]. Thus, thyroid hormone stimulates maturation of chondrocytes and the progression of endochondral ossification and is essential for linear growth (Fig. 4). 7.2. Osteoblasts In vitro studies of T3 action in osteoblasts have reported contradictory effects, but an overall consensus suggests T3 stimulates osteoblast activity [59]. T3 stimulates osteoblast differentiation and bone matrix synthesis, modification and mineralization. Thus, T3 promotes type I collagen synthesis and its posttranslational modification via regulation of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 and lysyloxidase [60]. Furthermore, T3 induces expression and activity of alkaline phosphatase [61,62], which is essential for matrix mineralization, and the synthesis and secretion of the bone matrix proteins osteopontin and osteocalcin [62,63]. T3 also stimulates matrix remodeling by increasing expression of matrix metalloproteinases‐9 and ‐13 (MMP-9 or gelatinase B and MMP-13 or collagenase-3) [64]. In addition, T3 regulates key pathways involved in osteoblast proliferation and differentiation. Thus, T3 induces IGF-I transcription via a TRE recently identified in intron 1 [45] while also stimulating expression of its regulatory binding proteins IGF1BP-2 and IGF1BP-4 [65]. Furthermore, T3 induces FGFR1 expression and activity leading to activation of MAPKsignaling and promotion of osteoblast differentiation [66]. Taken together, these observations demonstrate T3 stimulates osteoblast differentiation and function by complex direct and indirect mechanisms involving numerous paracrine and autocrine factors. Moreover, actions of T3 in osteoblasts may also indirectly influence bone resorption by regulating expression of osteoprotegerin (OPG), the decoy receptor that inhibits receptor activator of nuclear factor-kB ligand (RANKL) mediated activation of osteoclastogenesis [67], although other studies suggest effects of T3 on osteoclastogenesis may be independent of RANKL signaling [68,69] (Fig. 5). 7.3. Osteoclasts Thyroid hormone excess results in increased osteoclast numbers and activity in vivo leading to bone loss. Although osteoclasts express TRα1 and TRβ1 mRNAs it is not clear whether functional receptors are expressed because currently available TR antibodies lack sufficient sensitivity to detect endogenous proteins by immunohistochemistry or Western blotting. Thus, it remains unclear whether T3

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Fig. 4. The role of thyroid hormone in regulation of growth plate chondrocytes. Expression of TRα and β isoforms in the various growth plate is shown to the left of a histological section of mouse growth plate stained with Alcian blue (cartilage matrix, blue) and van Gieson (bone matrix, red). In the growth plate chondrocyte progenitor cells in the reserve zone undergo clonal expansion to form columns of proliferating cells that secrete a matrix rich in type II collagen. Pre‐hypertrophic chondrocytes differentiate to hypertrophic chondrocytes, which enlarge, secrete and mineralize a matrix that is rich in type X collagen before finally undergoing apoptosis. The remaining cartilage forms a scaffold for trabecular bone formation, a process that occurs in the primary spongiosum and requires vascular invasion. Growth plate chondrocyte proliferation is maintained by a long negative feedback loop. Pre‐hypertrophic chondrocytes secrete Indian hedgehog (Ihh), which acts to induce parathyroid hormone‐related peptide (PTHrP) synthesis in periarticular cells. PTHrP completes the loop by acting at the PTHR1 receptor to stimulate chondrocyte proliferation and inhibit further hypertrophic differentiation. In a short positive feedback loop bone morphogenic proteins (BMP) are synthesized by hypertrophic chondrocytes and induce Ihh expression, which in turn stimulates chondrocyte proliferation and further BMPs synthesis. The canonical Wnt signaling pathway stimulates chondrocyte terminal differentiation and collagen X matrix synthesis. Fibroblast growth factors (FGF) act via FGF receptors (FGFR) to inhibit chondrocyte proliferation, cartilage matrix synthesis and chondrocyte hypertrophy while also stimulating hypertrophic chondrocyte apoptosis and angiogenesis. Vascular invasion is mediated by the actions of vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs) and aggrecanase derived from chondrocytes and osteoblasts. The regulatory effects of T3 on these pathways are summarized on the right side of the figure. The growth hormone/insulin‐like growth factor 1 (GH/IGF1), Ihh/PTHrP, Wnt/β‐catenin and FGF/FGFR signaling pathways are all T3 targets. Expression of heparan sulphate proteoglycans (HSPGs), which are essential for FGF and Ihh signaling, is also regulated by T3. Furthermore, T3 inhibits chondrocyte proliferation by increasing expression of the cyclin‐dependent kinase inhibitors P21cip‐1 P27kip‐1. T3 also stimulates cartilage matrix synthesis, collagen X expression, alkaline phosphatase (ALP activity, and matrix metalloproteinase‐13 (MMP‐13) and aggrecanase‐2 expression.

acts directly in cells of the osteoclast lineage or whether responses to T3 are indirect and mediated by osteoblasts, bone marrow stromal cells or other cell types. Previous studies were performed in mixed cultures containing osteoclasts lineage cells and bone marrow stroma [70–74]. Nevertheless and consistent with an indirect action of T3 on

osteoclast function, treatment of immortalized osteoblasts or osteoblastic stromal cells (cells derived from bone marrow stroma with the capability of differentiation to the osteoblast lineage) results in increased expression of RANKL and interleukin 6 (IL-6), IL-8 and prostaglandin E2 (PGE2) [70,71].

Fig. 5. Responses to thyroid hormones in skeletal cells. Representation of the major pathways by which T3 regulates chondrocyte, osteoblast and osteoclast proliferation and differentiation. Abbreviations: FGFs (fibroblast growth factors), IGFs (insulin‐like growth factors), IL‐6, 8 (interleukins 6 and 8), M‐CSF (macrophage colony stimulating factor), OPG (osteoprotegerin), RANK (receptor activator of nuclear factor‐kB), RANKL (RANK ligand), Wnt (a mammalian homolog of the Drosophila morphogen wingless).

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8. Skeletal phenotypes in mutant mice with disrupted thyroid hormone signaling Manipulation of thyroid status in rodents recapitulates the skeletal effects of hypothyroidism and thyrotoxicosis observed in humans during both development and adulthood. More recent studies in knockout and mutant mice have been used to investigate the molecular and cellular mechanisms of thyroid hormone actions in bone and cartilage [75].

8.1. TRα mutants TRα 0/0 mice lack all TRα isoforms and are systemically euthyroid. During development TRα 0/0 mice display features of skeletal hypothyroidism with transient growth retardation and reduced bone mineralization due to delayed endochondral ossification and impaired growth plate chondrocyte differentiation [76]. Mice harboring heterozygous dominant-negative mutations in the Thra gene (TRα1 R384C/+, TRα1 PV/+) have mild and transient systemic hypothyroidism during development. Both mutants exhibit a more severe phenotype of skeletal hypothyroidism than TRα 0/0 mice, but TRα1 PV/+ mice are most severely affected, displaying persistent short stature and grossly delayed intramembranous and endochondral ossification [77,78]. These findings demonstrate an important and detrimental role for unliganded TRα1 in the skeletal consequences of hypothyroidism as demonstrated previously in other tissues including heart, intestine and cerebellum [79]. Moreover, the phenotype of TRα1PV/+ mice is more severe than observed in TRα1R384C/+ mutants because the truncated receptor fails to bind T3 and acts as a potent dominant-negative antagonist whereas activity of the TRα1R384C/+ mutant protein can be overcome by increased T3 concentrations [80]. Furthermore, the developmental skeletal phenotype of TRα1PV/+ mice is remarkably similar to the clinical features of

the child with a heterozygous TRα1E403X mutation, which also results in expression of a truncated TRα1 protein that fails to bind T3 and acts as a potent dominant-negative antagonist [19]. Adult TRα0/0 mice have osteosclerosis with increased trabecular bone volume associated with reduced osteoclast numbers and bone resorption [76] (Fig. 6). TRα1R384C/+ mutant mice also display a more severe phenotype with greatly increased trabecular bone mass and increased mineralization again associated with impaired osteoclastic bone resorption [77]. The adult skeletal phenotype of TRα1PV/+ mice has not been reported yet and no adult patients with THRA mutations have been identified. In summary, deletion or mutation of TRα results in delayed endochondral ossification with reduced bone mass and mineral deposition during growth, but an adult phenotype characterized by increased bone mass and mineralization.

8.2. TRβ mutants TRβ −/− mice lack all TRβ isoforms and have RTH with elevated T4, T3 and TSH levels. During development TRβ −/− mice display features of skeletal hyperthyroidism with persistent short stature, advanced endochondral and intramembranous ossification, and increased bone mineralization due to accelerated growth plate chondrocyte differentiation [76]. Mice harboring a dominant-negative mutation in Thrb (TRβ PV/PV, TRβ PV/+) have severe RTH and display a much more severe phenotype, particularly in homozygotes [46,78]. These findings are consistent with supra-physiological activation of the normal and predominant TRα in bone by elevated circulating T4 and T3 levels in TRβ mutants [81]. Adult TRβ −/− mice have osteoporosis with reduced trabecular bone volume and mineralization associated with increased osteoclast numbers and bone resorption [76] (Fig. 6). The adult skeletal phenotypes of TRβ PV/PV and TRβ PV/+ mice have not yet been reported and

Fig. 6. Effects of TRα and TRβ mutations on the HPT axis and the skeleton. Backscattered electron scanning electron microscopy images of trabecular bone micro‐ architecture in wild type (WT), TRα0/0 and TRβ−/− mice. TRα0/0 mice are euthyroid, but have impaired T3 action in skeletal cells and display features of tissue hypothyroidism including increased trabecular bone mass. TRβ−/− mice have resistance to thyroid hormone due to disruption of the HPT axis, resulting in elevated T4, T3 and TSH. Elevated thyroid hormones cause supraphysiological activation of TRα in bone resulting in tissue hyperthyroidism and osteoporosis. The white arrow in the WT image shows a normal trabecula within the trabecular bone network. The white arrow in the TRα0/0 image shows a thickened trabecula to highlight increased trabecular bone mass. The black arrow in the TRβ−/− image shows loss of a trabecula to highlight reduced trabecular bone mass and osteoporosis.

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interpretation of the skeletal consequences of RTH in adult patients with THRB mutations are confounded by often multiple treatment interventions and the functional diversity of the causative mutations [25]. In summary, deletion or mutation of TRβ results in advanced endochondral ossification with increased bone mass and mineral deposition during growth, but an adult phenotype of osteoporosis. 8.3. Genetic mouse models of hypothyroidism Pax8 −/− mice lack the thyroid specific transcription factor Pax8 required for thyroid follicular cell formation and hyt/hyt mice have a loss-of-function mutation in the TSH receptor (TSHR P556L). Both mutants have severe congenital hypothyroidism with no detectable circulating T4 and T3 and 2000-fold elevated TSH levels, and accordingly both display a phenotype of severe and persistent growth retardation with grossly impaired endochondral ossification [82]. Similarly, double knockout TRα0/0TRβ−/− mice that lack thyroid hormone receptors also display a skeletal phenotype characterized by growth retardation and delayed endochondral ossification [83], although these defects are less severe than seen in Pax8 −/− and hyt/hyt mice [82], suggesting a detrimental role for unoccupied TRs in the skeletal consequences of hypothyroidism. To investigate the roles of unoccupied TRα and TRβ during development, Pax8 −/− mice were crossed with TRα 0/0 and TRβ −/− mice [84]. The skeletal phenotype of Pax8 −/−TRα 0/0 mice was less severe than in Pax8 −/− mice, but was similar to that observed in TRα0/0TRβ−/− double knockout mice. By contrast, the skeletal phenotype of Pax8 −/−TRα 0/0 mice was similar to that seen in mice with deletion of Pax8 alone. Taken together, these observations demonstrate a role for unoccupied TRα but not TRβ in mediating effects of hypothyroidism on the skeleton [84]. Nevertheless, a further study showed that crossing Pax8−/− with TRα1−/− mice did not ameliorate the Pax8 −/− phenotype [85], thus questioning this conclusion. The restricted skeletal expression of the thyroid hormone activating enzyme, D2, in mature osteoblasts [41] enabled investigation of the effects of osteoblast-specific T3 deficiency to be studied in D2 knockout mice [86]. Growth and skeletal development of D2 knockout mice were normal indicating an insignificant role for D2 during endochondral and intramembranous ossification in vivo. Despite this, adult knockout mice had brittle bone with increased mineralization due to reduced osteoblastic bone formation and an extended period of secondary mineralization [86]. These findings suggest a possible mechanism to account for the increased risk of fracture observed in large population studies of patients with hypothyroidism [26,31], and demonstrate an essential for thyroid hormones in the optimization of adult bone strength and mineralization [86]. In summary, studies of a series of genetically modified mice have demonstrated that thyroid hormones exert anabolic actions during skeletal development and growth, but mediate catabolic responses in adult bone resulting in increased bone turnover and bone loss. The major effects of T3 in bone are mediated by TRα and the skeletal effects of mutation or deletion of TRβ are indirect and a consequence of disruption of the HPT negative feedback axis. Furthermore, local control of T3 availability in osteoblasts by the regulated activity of D2 is essential to maintain normal bone strength and mineralization. The physiological role of D3 in the skeleton has not yet been elucidated. References [1] J.H. Bassett, G.R. Williams, Critical role of the hypothalamic–pituitary–thyroid axis in bone, Bone 43 (2008) 418–426. [2] S. Andersen, N.H. Bruun, K.M. Pedersen, P. Laurberg, Biologic variation is important for interpretation of thyroid function tests, Thyroid 13 (2003) 1069–1078. [3] A.I. Gogakos, J.H. Duncan Bassett, G.R. Williams, Thyroid and bone, Arch. Biochem. Biophys. 503 (2010) 129–136. [4] V. Panicker, S.G. Wilson, T.D. Spector, S.J. Brown, M. Falchi, J.B. Richards, G.L. Surdulescu, E.M. Lim, S.J. Fletcher, J.P. Walsh, Heritability of serum TSH, free T4 and free T3 concentrations: a study of a large UK twin cohort, Clin. Endocrinol. (Oxf) 68 (2008) 652–659.

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