**2. Role of thyroid hormones in bone growth and metabolism**

Thyroid hormones are critical for the skeletal development and the bone maintenance. The thyroid hormone, 3,5,3'-triiodothyronine (T3), is responsible for major actions of thyroid hormones. T3 binds to nuclear receptors that regulate gene transcription via interaction with thyroid hormone response elements of specific genes (Sap et al., 1986; Weinberger et al., 1986; Thompson et al., 1987). Recently, non-genomic actions of T3 and T4 have been described (Cheng et al., 2010). Local tissue availability of T3 seems to be regulated by type 2 and 3 deiodinase (St Germain et al, 2009). The nuclear thyroid hormone receptors (TRs) are derived from the THRA and THRB genes coding for the TRα1 and β1-2 T3-binding isoforms, truncated isoforms ∆α1, ∆α2 and ∆β3, and a TRa2 non-T3-binding isoform of unknown function (Lazar et Chin et al, 1990; Lazar, 1993; Chassande et al, 1997; Williams, 2000; Cheng et al, 2010). Expression of TRα1 and TRβ1 was described in growth plate chondrocytes, osteoblasts, and stromal cells of bone marrow (Williams et al, 1994; Abu et al, 1997; Ballock et al, 1999; Bradley et al, 1992; Bassett et Williams, 2003; Siddiqi et al, 2002). Expression of TRα in the skeleton is higher than that of TRβ (Bookout et al, 2006; O'Shea et al, 2003).

Thyroid Disorders and Bone Mineral Homeostasis 253

partially reverted skeletal development and maturation defects in hypothyroid rats (Freitas

Thyroid hormones regulate bone development also indirectly through the growth hormone (GH) and insulin-like growth factor-I (IGF-I) axis. Previously, it was demonstrated that T4 enhanced the growth promoting effect of GH/IGF-I (Thorngren et Hansson, 1974) and could stimulate longitudinal bone growth in hypophysectomized rats (Thorngren et Hansson, 1973; Ray et al, 1954). T3 was shown to interact with the thyroid hormone receptor - thyroid hormone responsive element complex (TR-TRE) in the GH promoter to regulate GH gene transcription (Glass et al, 1987; Koenig et al, 1987). However, GH without T3 did not promote the maturation (Ballock et Reddi, 1994) and organization (Lewinson et al, 1989) of growth plate chondrocytes and GH replacement could not rescue the impaired ossification in TRα and TRβ-null mice (Kindblom et al, 2001). In TRα1−/−β−/− mice, GH substitution reversed the growth phenotype, but not the defective ossification (Kindblom et al, 2001). On the other hand, inactivation of GH or IGF-I receptors in mice was associated with delayed

Overall, the literature data indicate importance of both TRs and GH-IGF-I axis in skeletal development. Other factors, such as the Indian Hedgehog (Ihh), parathyroid hormonerelated peptide (PTHrP), fibroblast growth factor receptor and Wnt--catenin signaling pathways, are implicated in this process (Barnard et al, 2005; O'Shea et al, 2005; Stevens et al, 2000, 2003; Wang et al, 2007). Further studies are warranted to clarify the exact

Literature evidence points to the critical importance of thyroid hormones in bone remodeling and maintenance. Adult euthyroid mice invalidated for TRα have reduced osteoclastic bone resorption and increased trabecular bone volume and mineralization (Bassett et al, 2007a, 2007b), indicating a critical role of TRα in T3 action in bone cells. On the other hand, increased osteoclastic bone resorption and severe osteoporosis were demonstrated in adult TRβ mutant mice, suggestive of thyroid hormone excess in TRαexpressing skeletal cells (Bassett et al, 2007a, 2007b; Gauthier et al, 2001; O'Shea et al, 2006).

The bone architecture and strength are maintained by a balanced process of remodeling, which involves recruitment of osteoclast and osteoblasts. T3 can induce differentiation and inhibits proliferation of osteoblastic cells. T3 was shown to promote production of IL-6, IL-8, IGF-I and its binding proteins IGFBP2-4 in bone marrow stromal cells and osteoblasts (Milne et al, 2001; Siddiqi et al, 1998), and to increase the expression of several bone-related genes, including osteocalcin, collagen type I, gelatinase B and collagenase 3 (Gouveia et al 2001; Milne et al 1998; Pereira 1999; Varga et al 1997; Williams et al 1994). T3 is implicated in chondrogenesis, angiogenesis, bone matrix formation and mineralization (Himeno et al, 2002; Makihira et al, 2003; Pereira et al, 1999). In primary cultures of growth plate chondrocytes, T3 inhibits chondrocyte clonal expansion and cell proliferation, induces hypertrophic chondrocyte differentiation and promotes cartilage matrix mineralization (Robson et al, 2000).

mechanisms underlying the physiological regulation of bone development.

et al, 2005). This finding is suggestive of TRβ1 involvement in bone growth.

ossification (Liu et al, 1993; Sjogren et al, 2000).

**2.2. Thyroid hormone and bone remodeling** 

### **2.1. Thyroid hormone and bone development**

Studies on animal models have brought valuable insights into role of TRs in bone development and growth. Mice lacking TRβ or TRα1 did not display abnormalities in skeletal development (Forrest et al, 1996; Wikstrom et al, 1998). On the other hand, genetic disruption of both receptors (TRα1 and TRβ) led to delayed ossification and disorders in development of epiphyseal growth plates (EGPs; Gothe et al, 1999). Pax-8−/− mice, expressing all TR isoforms, but lacking the follicular cells producing T4 and T3 in the thyroid gland, displayed more severe abnormalities in bone development than mice KO for all TRs (TRα0/0, TRβ−/−) (Flamant et al, 2002). The authors concluded that the unliganded TRs (aporeceptors) on thyroid hormone responsive genes have repressor effects during bone development. In support of this, Pax-8−/−TRα0/0, but not Pax-8−/−TRβ−/−, compound mutants presented a partial rescue of the bone phenotype (O'Shea et Williams, 2002; Flamant et al, 2002). Another study was realized employing mice invalidated for TRα. These animals were euthyroid, but displayed a growth delay with abnormal bone development and ossification (Bassett et al 2007a, 2007b; Gauthier et al, 1999, 2001; O'Shea 2003, 2005). Mice lacking all TRα isoforms presented a less severe impairment of bone development than TRα-/- mice, pointing to the role of non-T3 binding TRα isoforms (∆α1 and ∆α2) (Gauthier et al, 2001). On the other hand, mice with nonfunctional TRβ displayed augmentation in circulating thyroid hormone levels associated with dysregulation of hypothalamo-pituitary-thyroid axis. These animals had skeletal signs of hyperthyroidism, increased bone mineral deposition and acceleration of growth-plate maturation, resulting in a short adult body size (Bassett et al, 2007a; O'Shea et al, 2003). These findings suggested an increased skeletal response to T3 via TRα, which was consistent with the hypothesis that elevated circulating thyroid hormone levels in TRβ mutant mice result in an increased skeletal response to T3 via TRα (O'Shea et al, 2006). Recently, GC-1, thyroid hormone analogue targeting preferentially TRβ1 over TRα1, has partially reverted skeletal development and maturation defects in hypothyroid rats (Freitas et al, 2005). This finding is suggestive of TRβ1 involvement in bone growth.

Thyroid hormones regulate bone development also indirectly through the growth hormone (GH) and insulin-like growth factor-I (IGF-I) axis. Previously, it was demonstrated that T4 enhanced the growth promoting effect of GH/IGF-I (Thorngren et Hansson, 1974) and could stimulate longitudinal bone growth in hypophysectomized rats (Thorngren et Hansson, 1973; Ray et al, 1954). T3 was shown to interact with the thyroid hormone receptor - thyroid hormone responsive element complex (TR-TRE) in the GH promoter to regulate GH gene transcription (Glass et al, 1987; Koenig et al, 1987). However, GH without T3 did not promote the maturation (Ballock et Reddi, 1994) and organization (Lewinson et al, 1989) of growth plate chondrocytes and GH replacement could not rescue the impaired ossification in TRα and TRβ-null mice (Kindblom et al, 2001). In TRα1−/−β−/− mice, GH substitution reversed the growth phenotype, but not the defective ossification (Kindblom et al, 2001). On the other hand, inactivation of GH or IGF-I receptors in mice was associated with delayed ossification (Liu et al, 1993; Sjogren et al, 2000).

Overall, the literature data indicate importance of both TRs and GH-IGF-I axis in skeletal development. Other factors, such as the Indian Hedgehog (Ihh), parathyroid hormonerelated peptide (PTHrP), fibroblast growth factor receptor and Wnt--catenin signaling pathways, are implicated in this process (Barnard et al, 2005; O'Shea et al, 2005; Stevens et al, 2000, 2003; Wang et al, 2007). Further studies are warranted to clarify the exact mechanisms underlying the physiological regulation of bone development.
