Dual functions of the steroid hormone receptor coactivator 3 in modulating resistance to thyroid hormone

Mutations of the thyroid hormone receptor beta (TRbeta) gene cause resistance to thyroid hormone (RTH). RTH is characterized by increased serum thyroid hormone associated with nonsuppressible thyroid-stimulating hormone (TSH) and impaired growth. It is unclear how the actions of TRbeta mutants are modulated in vivo to affect the manifestation of RTH. Using a mouse model of RTH that harbors a knockin mutation of the TRbeta gene (TRbetaPV mouse), we investigated the effect of the steroid hormone receptor coactivator 3 (SRC-3) on RTH. In TRbetaPV mice deficient in SRC-3, dysfunction of the pituitary-thyroid axis and hypercholesterolemia was lessened, but growth impairment of RTH was worsened. The lessened dysfunction of the pituitary-thyroid axis was attributed to a significant decrease in growth of the thyroid and pituitary. Serum insulin-like growth factor 1 (IGF-1) was further reduced in TRbetaPV mice deficient in SRC-3. This effect led to reduced signaling of the IGF-1/phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway that is known to mediate cell growth and proliferation. Thus, SRC-3 modulates RTH by at least two mechanisms, one via its role as a receptor coregulator and the other via its growth regulatory role through the IGF-1/PI3K/AKT/mTOR signaling.     

A novel thyroid hormone receptor-beta mutation,not anticipated to occur in resistance to thyroid hormone,causes variable phenotypes

Resistance to thyroid hormones (RTH) is a syndrome characterized by a variable tissue hyposensitivity to thyroid hormones and is linked to mutations in the thyroid hormone receptor-beta (TRbeta) gene. We report here for the first time in vivo the mutation R429W (CCG-->TCG) located in the exon 10. The artificial mutant obtained in vitro displayed a normal T(3)-binding affinity and transactivation function. Therefore, it was thought to produce little, if any, clinical effect and to escape to clinical detection. The present report is at least in part discordant with this prediction since the propositus and his grandmother had an authentic hyperthyroidism with high FT(4) plasma level in the presence of inappropriate TSH. On the other hand, spontaneous variations of clinical features and - interestingly - of plasma FT(4) concentrations with time in the propositus, and the phenotype observed in his mother who never complained with thyrotoxic symptoms, confirmed the in vitro binding and functional predictions. The most intriguing is the clinical course of the grandmother as she first presented with predominant pituitary RTH and a diffuse goiter and finally with a toxic multinodular goiter with normal T(3) and T(4) plasma concentrations and suppressed TSH. In conclusion, we report a novel mutation in the gene encoding the thyroid hormone receptor responsible for predominant pituitary RTH already described in vitro but not in vivo. The fluctuant phenotype of the propositus suggests that other factors modulate the degree of tissue resistance that is under genetic control. Toxic multinodular goiter, possibly due to chronic TSH stimulation during RTH, in addition to the phenotype variability, increases the difficulty to diagnose this thyroid disorder.     

Thyroid hormone receptor-beta mutations conferring hormone resistance and reduced corepressor release exhibit decreased stability in the N-terminal ligand-binding domain

Resistance to thyroid hormone (RTH) syndrome is associated with mutations in the human thyroid hormone receptor-beta (hTRbeta), many of which show marked reduction in hormone binding. Here, we investigated the structural consequences of two RTH mutants (A234T and R243Q), residing in the flexible N-terminal portion of the ligand binding domain (LBD), which exhibit modestly reduced hormone binding with impaired release of corepressor. X-ray crystallography analyses revealed that these two RTH mutants modulate the position of this flexible region by either altering the movement of helix 1 (A234T) or disrupting a salt bridge (R243Q). The subsequent increased flexibility and mobility in regions after the two sites of mutation coincided with a disorganized LBD. Consistent with this finding, the ability of these mutant N-terminal regions (234-260) to recruit the remaining LBD was decreased in a ligand-dependent helix assembly assay. Collectively, these data suggest that structural information imparted by the flexible segment in the N-terminal LBD is critical for overall stability of the LBD. Thus, these structural analyses provide mechanistic insight into the etiology of RTH disease in human TRbeta mutants that exhibit hormone binding with decreased ligand-dependent corepressor release.     

Differential expression of thyroid hormone receptor isoforms dictates the dominant negative activity of mutant Beta receptor

Mutations in the thyroid hormone receptor beta gene (TRbeta) cause resistance to thyroid hormone (RTH). Genetic analyses indicate that phenotypic manifestation of RTH is due to the dominant negative action of mutant TRbeta. However, the molecular mechanisms underlying the dominant negative action of mutants and how the same mutation results in marked variability of resistance in different tissues in vivo are not clear. Here we used a knock-in mouse (TRbetaPV mouse) that faithfully reproduces human RTH to address these questions. We demonstrated directly that TRbeta1 protein was approximately 3-fold higher than TRalpha1 in the liver of TRbeta(+/+) mice but was not detectable in the heart of wild-type and TRbetaPV mice. The abundance of PV in the liver of TRbeta(PV/PV) was more than TRbeta(PV/+) mice but not detectable in the heart. TRalpha1 in the liver was approximately 6-fold higher than that in the heart of wild-type and TRbetaPV mice. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed not only with TR isoforms for binding to thyroid hormone response elements (TRE) but also competed with TR for the retinoid X receptors in binding to TRE. These competitions led to the inhibition of the thyroid hormone (T(3))-positive regulated genes in the liver. In the heart, however, PV was significantly lower and thus could not effectively compete with TRalpha1 for binding to TRE, resulting in activation of the T(3)-target genes by higher levels of circulating thyroid hormones. These results indicate that in vivo, differential expression of TR isoforms in tissues dictates the dominant negative activity of mutant beta receptor, thereby resulting in variable phenotypic expression in RTH.     

Marked potentiation of the dominant negative action of a mutant thyroid hormone receptor beta in mice by the ablation of one wild-type beta allele

Mutations in the thyroid hormone receptor (TR) beta gene result in resistance to thyroid hormone (RTH), characterized by reduced sensitivity of tissues to thyroid hormone. To understand which physiological TR pathways are affected by mutant receptors, we crossed mice with a dominantly negative TRbeta mutation (TRbetaPV) with mice carrying a TRbeta null mutation (TRbeta(-/-)) to determine the consequences of the TRbetaPV mutation in the absence of wild-type TRbeta. TRbeta(PV/-) mice are distinct from TRbeta(+/-) mice that did not show abnormalities in thyroid function tests. TRbeta(PV/-) mice are also distinct from TRbeta(PV/+) and TRbeta(-/-) mice in that the latter shows mild dysfunction in the pituitary-thyroid axis, whereas the former exhibit very severe abnormalities, including extensive papillary hyperplasia of the thyroid epithelium, indistinguishable from that observed in TRbeta(PV/PV) mice. Similar to TRbeta(PV/PV) mice, TRbeta(PV/-) mice exhibited impairment in weight gain. Moreover, the abnormal regulation patterns of T3-target genes in the tissues of TRbeta(PV/-) and TRbeta(PV/PV) mice were strikingly similar. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed with TRalpha1 for binding to thyroid hormone response elements in TRbeta(PV/-) mice as effectively as in TRbeta(PV/PV) mice. Thus, the actions of mutant TRbeta are markedly potentiated by the ablation of the second TRbeta allele, suggesting that interference with wild-type TRalpha1-mediated gene regulation by mutant TRbeta leads to severe RTH.     

Rearrangements in thyroid hormone receptor charge clusters that stabilize bound 3,5',5-triiodo-L-thyronine and inhibit homodimer formation

In this study, we investigated how thyroid hormone (3,5',5-triiodo-l-thyronine, T3) inhibits binding of thyroid hormone receptor (TR) homodimers, but not TR-retinoid X receptor heterodimers, to thyroid hormone response elements. Specifically we asked why a small subset of TRbeta mutations that arise in resistance to thyroid hormone syndrome inhibit both T3 binding and formation of TRbeta homodimers on thyroid hormone response elements. We reasoned that these mutations may affect structural elements involved in the coupling of T3 binding to inhibition of TR DNA binding activity. Analysis of TR x-ray structures revealed that each of these resistance to thyroid hormone syndrome mutations affects a cluster of charged amino acids with potential for ionic bond formation between oppositely charged partners. Two clusters (1 and 2) are adjacent to the dimer surface at the junction of helices 10 and 11. Targeted mutagenesis of residues in Cluster 1 (Arg338, Lys342, Asp351, and Asp355) and Cluster 2 (Arg429, Arg383, and Glu311) confirmed that the clusters are required for stable T3 binding and for optimal TR homodimer formation on DNA but also revealed that different arrangements of charged residues are needed for these effects. We propose that the charge clusters are homodimer-specific extensions of the dimer surface and further that T3 binding promotes specific rearrangements of these surfaces that simultaneously block homodimer formation on DNA and stabilize the bound hormone. Our data yield insight into the way that T3 regulates TR DNA binding activity and also highlight hitherto unsuspected T3-dependent conformational changes in the receptor ligand binding domain.     

Cardiac glucose utilization in mice with mutated alpha- and beta-thyroid hormone receptors

Abnormal thyroid function is usually associated with altered cardiac function. Mutations in the thyroid hormone (TH)-binding region of the TH beta-receptor (TRbeta) that eliminate its TH-binding ability lead to the thyroid hormone resistance syndrome (RTH) in humans, which is characterized by high blood TH levels, goiter, hyperactivity, and tachycardia. Mice with "knock-in" mutations in the TH alpha-receptor (TRalpha) or TRbeta that remove their TH-binding ability have been developed, and those with the mutated TRbeta (TRbeta(PV/PV)) appear to provide a model for RTH. These two types of mutants show different effects on cerebral energy metabolism, e.g., negligible change in glucose utilization (CMR(Glc)) in TRbeta(PV/PV) mice and markedly reduced CMR(Glc), like that found in cretinous rats, in the mice (TRalpha(PV/+)) with the knock-in mutation of the TRalpha gene. Studies in knockout mice have indicated that the TRalpha may also influence heart rate. Because mutations in both receptor genes appear to affect some parameters of cardiac function and because cardiac functional activity and energy metabolism are linked, we measured heart glucose utilization (HMR(Glc)) in both the TRbeta(PV/PV) and TRalpha(PV/+) mutants. Compared with values in normal wild-type mice, HMR(Glc) was reduced (-77 to -95%) in TRalpha(PV/+) mutants and increased (87 to 340%) in TRbeta(PV/PV) mutants, the degree depending on the region of the heart. Thus the TRalpha(PV/+) and TRbeta(PV/PV) mutations lead, respectively, to opposite effects on energy metabolism in the heart that are consistent with the bradycardia seen in hypothyroidism and the tachycardia associated with hyperthyroidism and RTH.     

Compensatory role of thyroid hormone receptor (TR) alpha 1 in resistance to thyroid hormone: study in mice with a targeted mutation in the TR beta gene and deficient in TR alpha 1

Resistance to thyroid hormone (RTH) is caused by mutations of the thyroid hormone receptor beta (TR beta) gene. Almost all RTH patients are heterozygous with an autosomal dominant pattern of inheritance. That most are clinically euthyroid suggests a compensatory role of the TR alpha1 isoform in maintaining the normal functions of thyroid hormone (T3) in these patients. To understand the role of TR alpha1 in the manifestation of RTH, we compared the phenotypes of mice with a targeted dominantly negative mutant TR beta (TR betaPV) with or without TR alpha1. TR betaPV mice faithfully recapitulate RTH in humans in that these mice demonstrate abnormalities in the pituitary-thyroid axis and impairment in growth. Here we show that the dysregulation of the pituitary-thyroid axis was worsened by the lack of TR alpha1 in TR betaPV mice, and severe impairment of postnatal growth was manifested in TR betaPV mice deficient in TR alpha1. Furthermore, abnormal expression patterns of T3-target genes in TR betaPV mice were altered by the lack of TR alpha1. These results demonstrate that the lack of TR alpha1 exacerbates the manifestation of RTH in TR betaPV mice. Therefore, TR alpha1 could play a compensatory role in mediating the functions of T3 in heterozygous patients with RTH. This compensatory role may be especially crucial for postnatal growth.     

DNA binding affinity of hTRbeta1 mutants as heterodimers with traps from different tissues.

Patients with generalized resistance to thyroid hormone (GRTH) show various organ-specific features, for example mental retardation, growth abnormalities, liver damage, delayed bone age or cardiac disorders. Could this reflect aberrant mutant thyroid hormone receptor beta1 (TRbeta1) heterodimerization with specific TR auxiliary proteins (TRAPs) from different tissues, altering the mutant's ability to transactivate tissue-specific genes? To answer this question, we examined the heterodimerization of TRbeta1 mutants and TRAPs of several rat tissues (cerebrum, cerebellum, liver, heart, lung, spleen, and kidney), and in vitro translated RXRalpha, beta and gamma by electrophoretic gel mobility shift assay (EMSA). Mutant TRbeta1 proteins, synthesized in reticulocyte lysate, were incubated with 32P rat malic enzyme (rME) thyroid hormone response elements (TRE) and nuclear extracts of rat tissues. The TRbeta1 mutants used were Mf (G345R), and GH (R316H). Both have non-detectable T3 binding affinity. GH has weak dominant negative effect and Mf has strong dominant negative effect. Two major bands were observed in EMSA. Cerebrum, cerebellum, lung and liver extracts formed a slower migrating band than a TR homodimer, while kidney extracts formed a faster migrating band, and heart and spleen extracts had both bands. There were no qualitative differences in heterodimerization between TRbeta1wt, and TRbeta1 mutants, when using tissue extracts and DNA in excess ratio to TR. We found that RXRalpha, beta, and gamma were differentially expressed in each rat tissue and formed heterodimer complexes with wild type (WT) TRbeta1. Scatchard analysis of affinity and capacity of the binding of TR-TRAP heterodimers to response elements was performed by competing with 2.5-, 5-, 10-, 25-, and 250-fold excess non-radiolabeled rME-TRE. When using kidney extract, the DNA binding affinity of heterodimers was significantly decreased both in wild type and mutant TRs, suggesting that the DNA binding affinity of the faster migrating band was lower than that of the slower migrating band. Mutant GH, which causes 'pituitary RTH' and shows weak dominant negative effect, tended to form heterodimers with lower DNA binding affinity than TRbeta1wt with all extracts. Mutant Mf, which has strong dominant negative effect, tended to show higher DNA binding affinity than TRbeta1WT. When the data were pooled for all tissues, GH and Mf were found to form heterodimers with significantly lower, or higher, affinity for TREs than TRbeta1wt. These results indicate that: 1) differences of DNA binding affinity of mutant TR-TRAP heterodimers to response elements in DNA play a part in its reduced or strong dominant negative effect; and 2) differences in formation of heterodimers with TRAPs present in tissues do not appear to explain the apparent tissue-specific and mutant-specific variations seen in RTH.     

Finding the right balance between resistance and sensitivity: a review of the cardiac manifestations of the syndrome of resistance to thyroid hormone and the implications for treatment

ObjectiveTo review cardiac manifestations in the syndrome of resistance to thyroid hormone (RTH) and to question the general recommendation that the thyroidstimulating hormone (TSH) value be the guide to thyroid hormone replacement.MethodsThe syndrome of RTH is caused by mutations in the carboxyterminal portion of the β isoform of the thyroid hormone receptor, resulting in variable clinical manifestations. It is generally recommended that the replacement of thyroid hormone in patients with RTH be guided by the serum TSH concentration. The variable responsiveness of tissues to thyroid hormone, however, makes it difficult to balance the correct replacement dose. We present a case that brings into question the conventional wisdom about the replacement dose of thyroid hormone in this scenario, and we review the pertinent literature.ResultsA 54- year-old man with RTH was treated with levothyroxine and increasing doses of liothyronine sodium as part of an evaluation of RTH. On day 10 of theprotocol, he developed atrial fibrillation despite a normal level of TSH (1.1 mIU/L). Administration of liothyronine was discontinued, and cardioversion was planned; however, the patient’s heart rhythm converted spontaneously to normal sinus rhythm.ConclusionReplacement of thyroid hormone in patients with RTH should include careful monitoring of thyrotoxic cardiac side effects in addition to consideration of normalization of the TSH level. (Endocr Pract. 2012;18:252- 255)