Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate the biological activities of the thyroid hormone (T3). There are two TR genes, α and β, that are located on two different chromosomes. Alternative splicing of the primary transcripts gives rise to four major T3-binding TR isoforms-α1, β1, β2 and β3. These TRs regulate the expression of T3 target genes by binding to the thyroid hormone response elements (TREs) on the promoters. The expression of these TR isoforms is both tissue- and development-regulated (1). The regulation of the transcriptional activity of TRs is complex in that it depends on multiple factors including the types of TREs, the promoter context and many coregulatory proteins (coactivators and corepressors) (1).

Resistance to thyroid hormone (RTH) is a syndrome characterized by reduced sensitivity of tissues to the actions of thyroid hormone (2, 3). Refetoff et al. first described RTH in 1967 (4). But it was not until the tight linkage between the affected RTH family members and the thyroid hormone receptor β (TRβ) gene was discovered in 1988 (5) did it become possible to study this syndrome at the molecular level. The identification of a Pro453His mutation in the TRβ gene of one kindred (6) established that RTH is caused by mutations of the TRβ gene. To date, about 100 different mutations in the TRβ gene have been reported in more than 300 families (2, 3).

The hallmark of RTH is elevated thyroid hormone associated with nonsuppressible thyroid stimulating hormone (TSH). Other clinical signs are goiter, short stature, decreased weight, tachycardia, hearing loss, attention-deficit hyperactivity disorder, decreased IQ, and dyslexia (2, 3). The clinical manifestations vary between families with different mutations, between families with the same mutation, and also between members of the same family with identical mutations. Most patients are heterozygous with only one mutated TRβ gene, and the clinical symptoms are mild (2, 3). Only one patient homozygous for a mutant TRβ has been reported (7). This patient, who died at a young age, displayed an extraordinary and complex phenotype of extreme RTH with very high levels of thyroid hormones and TSH (7).

In the past decade, novel biochemical techniques and the availability of various genetically engineered mouse models have advanced the understanding of the physiological functions of TRs in vivo and the underlying molecular mechanisms for RTH. This article will focus on new developments in RTH.
 
2. GENERATION OF MOUSE MODELS OF HUMAN RTH: THE TRβPV MOUSE
 
Earlier work on the characterization of TRβ mutants and elucidation of their molecular actions in RTH mainly used in vitro biochemical methods. It soon became clear that these approaches have limitations when one tries to extrapolate to the physiological context. Such limitations led to the creation of two knock-in mouse models of RTH, one harboring a carboxyl-terminal 14 amino acid frame-shift mutation (TRβPV mouse) (8) and the other harboring a 337T mutation (TRβ337T mouse) (9) in the TRβ gene. The two mutations identified in RTH patients were targeted to the TRβ gene locus via homologous recombination. These two knock-in mice exhibit RTH phenotypes including dysregulation of the pituitary-thyroid axis and neurological dysfunction (8 -10). Consistent with phenotypes of RTH patients, TRβPV mice also exhibit growth retardation (8), abnormal regulation of serum cholesterol (11), hearing defects (12) and thyrotoxic skeletal phenotype (13). These phenotypes indicate that the TRβPV mouse is a valid model to study the molecular basis of human RTH.
  
2.1. Molecular mechanisms of the dominant negative activity of TRβ mutants in vivo
 
Genetic analyses indicate that almost all RTH patients are heterozygous for the mutant TRβ allele, a finding consistent with the autosomal dominant pattern of inheritance (2, 3). Early in vitro studies suggested four possible mechanisms to account for the dominant negative activity of TRβ mutants: (i) formation of inactive dimers between mutant and wild-type TRs (w-TRs), (ii) competition between mutant and w-TRs for binding to TREs, (iii) competition for limited amounts of auxiliary proteins, such as the retinoid X receptors (RXRs), and (iv) stable association of TRβ mutants with corepressors, resulting in repression of T3 target genes (14).

Using TRβPV mice, Zhang et al determined which of these possibilities operate in vivo (15). In the liver nuclear extracts of TRβ^PV/+ mice, PV forms not only TRE-bound homodimers, but also TRE-bound heterodimers with w-TRβ1, w-TRα1, or RXR. In TRβ^PV/PV mice, in addition to PV/PV homodimers, the lack of w-TRβ1 facilitates the formation of TRE-bound PV/TRα1 and PV/RXR heterodimers. Therefore, in vivo, PV competes with w-TRβ or w-TRα1 for binding to TRE and for heterodimerization with RXRs (15). Such competition leads to repression of the positively T3-regulated target genes-S14, malic enzyme, and type 1 deiodinase-in the liver of TRβPV mice. These studies demonstrated that one of the molecular mechanisms by which TRβ mutants exert their dominant negative activity in vivo is through competition (i) of inactive PV dimers with w-TRs for binding to TRE and (ii) of the mutant PV with w-TRs for binding to RXR to bind to TRE of T3-target genes.
 
2.2. The molecular basis of variable clinical manifestations in RTH

Although most heterozygous RTH patients are clinically euthyroid, some are hypothyroid and some may appear thyrotoxic (2, 3). Intriguingly, the same individual may present evidence of hypothyroidism in one tissue, while showing signs of thyrotoxicosis in other tissues (2, 3). Using TRβPV mice, Zhang et al. showed that differential expression of TR isoforms in tissues contributes to variable clinical manifestations in RTH (15). Since inactive PV/TRα1 and PV/TRβ1 heterodimers and PV/PV homodimers compete with w-TRs for binding to TRE, in tissues, such as liver and pituitary, in which the major TR isoform is TRβ1, the positive regulated genes are repressed owing to the more effective competition of PV with w-TRs for binding to TREs. The result is tissue hypothyroidism. In tissues, such as heart and bone, in which TRα1 is the major TR isoform, PV cannot compete effectively with the more abundantly expressed TRα1 for binding to TREs. Thus the positively regulated genes are activated by the elevated serum thyroid hormone in TRβPV mice, thereby resulting in a thyrotoxic phenotype (15).
Differential expression of coactivators such as the steroid hormone receptor coactivator-1 (SRC-1) also contributes to the variable clinical manifestations in RTH. Using mice from the cross of TRβPV mice and SRC-1-deficient mice, Kamiya et al. showed that lack of SRC-1 modulates the degree of resistance to thyroid hormone in a target-tissue-dependent manner and alters abnormal expression patterns of several T3 target genes in tissues (11). Thus, complex regulation of actions of TRβ mutants leads to varied manifestations of RTH phenotypes.

 2.3. Compensatory role of TRα1 in heterozygous patients with RTH
 
Most heterozygous RTH patients are clinically euthyroid (2, 3). One possible explanation is that TRα1 plays a compensatory role in maintaining the normal physiological functions of T3 in these patients. To test this hypothesis, Suzuki and Cheng crossed TRβPV mice with mice deficient in TRα1 (16) and compared the phenotypes of TRβPV mice with or without TRα1 (17). The lack of TRα1 worsened the dysregulation of the thyroid-pituitary axis in TRβPV mice and resulted in more severe impairment of postnatal growth. Furthermore, abnormal expression patterns of T3-target genes in TRβPV mice [e.g., the TSHβ, glycoprotein common α-subunit, α-myosin heavy chain, and β-myosin heavy chain genes] were altered by the lack of TRα1. These results show that the lack of TRα1 intensifies the manifestations of RTH in TRβPV mice. One can deduce, therefore, that TRα1 plays an important and previously unrecognized compensatory role in maintaining the physiological functions of T3 in heterozygous patients with RTH.

The TRβPV mouse provides a valuable model to uncover novel phenotypes due to mutations of the TRβ gene. Indeed, an unexpected, but remarkable discovery was that TRβ^PV/PV mice, but not TRβ^PV/+ mice, spontaneously develop follicular thyroid carcinoma with sequential capsular invasion, vascular invasion, anaplasia, and, eventually, metastasis (18). The molecular genetics of follicular thyroid carcinoma is not well understood. TRβ^PV/PV mice provide an unprecedented opportunity to study gene alterations that contribute to tumor progression and metastasis and to identify potential molecular targets for prevention and treatment. Using cDNA microarrays, Ying et al. have identified altered expression of ~100 genes involved in tumor induction and progression, cell proliferation, metastasis, and cell cycle regulation (19). Furthermore, these investigators further demonstrated that the repression of the signaling pathways of the peroxisome proliferator-activated receptor (PPAR) is associated with thyroid carcinogenesis (20). This finding is consistent with the frequent occurrence of the PAX8-PPAR fusion gene in human follicular thyroid carcinomas, its less frequent occurrence in adenomas, and its absence in papillary thyroid carcinomas (21, 22). The fusion of PAX8, a thyroid transcription factor, to the amino terminus of PPAR results in the loss of the transcriptional activity of PPAR. Moreover, PAX8-PPAR protein acts to inhibit thiazolidinedione-induced transactivation by PPAR in a dominant negative manner (21). These studies suggest that PPAR could potentially be tested as a molecular target for prevention or treatment of follicular thyroid carcinoma (20).
 

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