RESUMO
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.
Assuntos
Regulação da Expressão Gênica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores dos Hormônios Tireóideos/química , Receptores dos Hormônios Tireóideos/metabolismo , Aminoácidos/fisiologia , Animais , Células Cultivadas , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Isoformas de Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Receptores dos Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismoRESUMO
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.
Assuntos
Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Doenças da Glândula Tireoide/genética , Doenças da Glândula Tireoide/metabolismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Animais , Humanos , Mutação/fisiologia , Doenças da Glândula Tireoide/diagnósticoRESUMO
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.
Assuntos
DNA/metabolismo , Receptores dos Hormônios Tireóideos/fisiologia , Tireotrofos/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Subunidade alfa de Hormônios Glicoproteicos/genética , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Tireotropina Subunidade beta/genética , Tireotropina Subunidade beta/metabolismo , Transcrição Gênica , Tri-Iodotironina/fisiologiaRESUMO
Cerebellar development on the postnatal period is mainly characterized by cellular proliferation in the external granular layer (EGL) followed by migration of granular cells in the molecular layer through the Bergmann glia (BG) fibers in order to form the granular layer in the adult. All these events are drastically affected by thyroid hormones (TH), which actions are mainly mediated by alpha (TRalpha) and beta (TRbeta) nuclear receptor isoforms. Here, we analyzed the effects of a natural human mutation (337T) in the TRbeta locus, which impairs T3 binding to its receptor, on the mouse cerebellum ontogenesis. We report that target inactivation of TRbeta-TH binding leads to a smaller cerebellum area characterized by impaired lamination and foliation. Further, TRbeta mutant mice presented severe deficits in proliferation of granular precursors, arborization of Purkinje cells and organization of BG fibers. Together, our data suggest that the action of TH via TRbeta regulates important events of cerebellar ontogenesis contributing to a better understanding of some neuroendocrine disorders. Further, our data correlate TRbeta with cerebellar foliation, and provide, for the first time, evidence of a receptor-mediated mechanism underlying TH actions on this event.
Assuntos
Cerebelo/anormalidades , Cerebelo/metabolismo , Receptores beta dos Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/patologia , Diferenciação Celular/genética , Movimento Celular/genética , Células Cultivadas , Cerebelo/fisiopatologia , Dendritos/metabolismo , Dendritos/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Transgênicos , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/fisiopatologia , Neurogênese/genética , Ligação Proteica/genética , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Doenças da Glândula Tireoide/complicações , Receptores alfa dos Hormônios Tireóideos/genéticaRESUMO
Thyroid hormone (TH) regulates many cardiac genes via nuclear thyroid receptors, and hyperthyroidism is frequently associated with atrial fibrillation. Electrical activity propagation in myocardium depends on the transfer of current at gap junctions, and connexins (Cxs) 40 and 43 are the predominant junction proteins. In mice, Cx40, the main Cx involved in atrial conduction, is restricted to the atria and fibers of the conduction system, which also express Cx43. We studied cardiac expression of Cx40 and Cx43 in conjunction with electrocardiogram studies in mice overexpressing the dominant negative mutant thyroid hormone receptor-beta Delta337T exclusively in cardiomyocytes [myosin heavy chain (MHC-mutant)]. These mice develop the cardiac hypothyroid phenotype in the presence of normal serum TH. Expression was also examined in wild-type mice rendered hypothyroid or hyperthyroid by pharmacological treatment. Atrial Cx40 mRNA and protein levels were decreased (85 and 55%, respectively; P < 0.001) in MHC-mt mice. Atrial and ventricular Cx43 mRNA levels were not significantly changed. Hypothyroid and hyperthyroid animals showed a 25% decrease and 40% increase, respectively, in Cx40 mRNA abundance. However, MHC-mt mice presented very low Cx40 mRNA expression regardless of whether they were made hypothyroid or hyperthyroid. Atrial depolarization velocity, as represented by P wave duration in electrocardiograms of unanesthetized mice, was extremely reduced in MHC-mt mice, and to a lesser extent also in hypothyroid mice (90 and 30% increase in P wave duration). In contrast, this measure was increased in hyperthyroid mice (19% decrease in P wave duration). Therefore, this study reveals for the first time that Cx40 mRNA is up-regulated by TH acting in cardiac atria via the TH receptor and that this may be one of the mechanisms contributing to atrial conduction alterations in thyroid dysfunctions.
Assuntos
Conexinas/genética , Átrios do Coração/metabolismo , RNA Mensageiro/genética , Receptores dos Hormônios Tireóideos/fisiologia , Hormônios Tireóideos/farmacologia , Animais , Conexina 43/genética , Primers do DNA , Eletrocardiografia , Átrios do Coração/efeitos dos fármacos , Camundongos , Camundongos Mutantes , Cadeias Pesadas de Miosina/genética , Proteína C/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tri-Iodotironina/sangue , Proteína alfa-5 de Junções ComunicantesRESUMO
PURPOSE: The expression of S- and M-opsins in the murine retina is altered in different transgenic mouse models with mutations in the thyroid hormone receptor (TR)-beta gene, demonstrating an important role of thyroid hormone (TH) in retinal development. METHODS: The spatial expression of S- and M-opsin was compared in congenital hypothyroidism and in two different TR mutant mouse models. One mouse model contains a ligand-binding mutation that abolishes TH binding and results in constitutive binding to nuclear corepressors. The second model contains a mutation that blocks binding of coactivators to the AF-2 domain without affecting TH binding. RESULTS: Hypothyroid newborn mice showed an increase in S-opsin expression that was completely independent of the genotype. Concerning M-opsin expression, hypothyroidism caused a significant decrease (P < 0.01) only in wild-type animals. When TRbeta1 and -beta2 were T3-binding defective, the pattern of opsin expression was similar to TRbeta ablation, showing increased S-opsin expression in the dorsal retina and no expression of M-opsin in the entire retina. In an unexpected finding, immunostaining for both opsins was detected when both subtypes of TRbeta were mutated in the helix 12 AF-2 domain. CONCLUSIONS: The results show, for the first time, that the expression of S- and M-opsin is dependent on normal thyroid hormone levels during development.