RESUMEN
Thyroid hormone (TH) action is mediated by the products of two genes, TH receptor (THR)α (THRA) and THRß (THRB) that encode several closely related receptor isoforms with differing tissue distributions. The vast majority of THR isoform-specific effects are thought to be due to tissue-specific differences in THR isoform expression levels. We investigated the alternative hypothesis that intrinsic functional differences among THR isoforms mediate these tissue-specific effects. To achieve the same level of expression of each isoform, we created tagged THR isoforms and tested their DNA and functional properties in vitro. We found significant homodimerization and functional differences among the THR isoforms. THRA1 was unable to form homodimers on direct repeat separated by 4 bp DNA elements and was also defective in TH-dependent repression of Tshb and Rxrg in a thyrotroph cell line, TαT1.1. In contrast, THRB2 was both homodimer sufficient and fully functional on these negatively regulated genes. Using domain exchanges and individual amino acid switches between THRA1 and THRB2, we identified three amino acids in helix 10 of the THRB2 ligand-binding domain that are required for negative regulation and are absent in THRA1.
Asunto(s)
Regulación de la Expresión Génica , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/metabolismo , Aminoácidos/fisiología , Animales , Células Cultivadas , Ratones , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Isoformas de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Receptores de Hormona Tiroidea/genética , Hormonas Tiroideas/metabolismoRESUMEN
Thyroid hormone action is predominantly mediated by thyroid hormone receptors (THRs), which are encoded by the thyroid hormone receptor α (THRA) and thyroid hormone receptor ß (THRB) genes. Patients with mutations in THRB present with resistance to thyroid hormone ß (RTHß), which is a disorder characterized by elevated levels of thyroid hormone, normal or elevated levels of TSH and goitre. Mechanistic insights about the contributions of THRß to various processes, including colour vision, development of the cochlea and the cerebellum, and normal functioning of the adult liver and heart, have been obtained by either introducing human THRB mutations into mice or by deletion of the mouse Thrb gene. The introduction of the same mutations that mimic human THRß alterations into the mouse Thra and Thrb genes resulted in distinct phenotypes, which suggests that THRA and THRB might have non-overlapping functions in human physiology. These studies also suggested that THRA mutations might not be lethal. Seven patients with mutations in THRα have since been described. These patients have RTHα and presented with major abnormalities in growth and gastrointestinal function. The hypothalamic-pituitary-thyroid axis in these individuals is minimally affected, which suggests that the central T3 feedback loop is not impaired in patients with RTHα, in stark contrast to patients with RTHß.
Asunto(s)
Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Enfermedades de la Tiroides/genética , Enfermedades de la Tiroides/metabolismo , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Animales , Humanos , Mutación/fisiología , Enfermedades de la Tiroides/diagnósticoRESUMEN
Thyroid hormones have a profound influence on human development and disease. The hypothalamic-pituitary-thyroid axis involves finely tuned feedback mechanisms to maintain thyroid hormone (TH) levels. Despite the important role of TH-negative feedback in regulating this axis, the mechanism by which this occurs is not clearly defined. Previous in vivo studies suggest separate roles for the two thyroid hormone receptor isoforms, THRA and THRB, in this axis. We performed studies using a unique pituitary thyrotroph cell line (TαT1.1) to determine the relative roles of THRA and THRB in the regulation of Tshb. Using chromatin immunoprecipitation assays, we found that THRB, not THRA, bound to the Tshb promoter. By selectively depleting THRB, THRA, or both THRA and THRB in TαT1.1 cells, we found that simultaneous knockdown of both THRB and THRA abolished T(3)-mediated down-regulation of Tshb at concentrations as high as 100 nm T(3). In contrast, THRA knockdown alone had no effect on T(3)-negative regulation, whereas THRB knockdown alone abolished T(3)-mediated down-regulation of Tshb mRNA levels at 10 nm but not 100 nm T(3) concentrations. Interestingly, chromatin immunoprecipitation assays showed that THRA becomes enriched on the Tshb promoter after knockdown of THRB. Thus, a likely mechanism for the differential effects of THR isoforms on Tshb may be based on their differential DNA-binding affinity to the promoter.