RESUMEN
As the genome of experimental animals has become easier to manipulate, a number of mouse models have been developed to understand in vivo thyroid hormone action. A major site of thyroid hormone action is the HPT axis. While several methods are available that provide a detailed understanding of the HPT axis in mice, many authors choose to include only cursory data about this axis, which can lead to erroneous conclusions about in vivo thyroid hormone action. A standard protocol is proposed to evaluate the HPT axis in mice.
Asunto(s)
Sistema Hipotálamo-Hipofisario/metabolismo , Transducción de Señal , Glándula Tiroides/metabolismo , Animales , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Hipotiroidismo/diagnóstico , Hipotiroidismo/genética , Hipotiroidismo/metabolismo , Ratones , Hormonas Tiroideas/sangre , Hormonas Tiroideas/metabolismo , Tirotropina/sangre , Tirotropina/metabolismoRESUMEN
AIMS: To understand how thyroid hormone (TH) regulates tissue-specific gene expression in patients with the syndrome of resistance to TH (RTHß), we used a mouse model that replicates the human RTHß, specifically the ∆337T mutation in the thyroid hormone receptor ß (THRß). MAIN METHODS: We investigated the expression of key TH target genes in the pituitary and liver of TRß∆337T and wild type THRß mice by qPCR before and after a T3 suppression test consisting of the administration of increasing concentrations of T3 to hypothyroid mice. KEY FINDINGS: Pituitary Tshb and Cga expression decreased and Gh expression increased in TRß∆337T mice after T3 suppression. The stimulation of positively regulated TH genes was heterogeneous in the liver. Levels of liver Me1 and Thsrp were elevated in TRß∆337T mice after T3 administration. Slc16a2 and Gpd2 did not respond to T3 stimulation in the liver of TRß∆337T mice whereas Dio1 response was lower than that observed in WT mice. Moreover, although Chdh and Upd1 genes were negatively regulated in the liver, the expression of these genes was elevated after T3 suppression. We did not observe significant changes in THRα expression in the liver and pituitary, while THRß levels were diminished in the pituitary and increased in the liver. SIGNIFICANCE: Using a model expressing a THRß unable to bind T3, we showed the expression pattern of liver negative and positive regulated genes by T3.
Asunto(s)
Regulación de la Expresión Génica , Triyodotironina/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Perfilación de la Expresión Génica , Hormona del Crecimiento/metabolismo , Humanos , Hipotiroidismo/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Hipófisis/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
Mice bearing the genomic mutation Δ337T on the thyroid hormone receptor ß (TRß) gene present the classical signs of resistance to thyroid hormone (TH), with high serum TH and TSH. This mutant TR is unable to bind TH, remains constitutively bound to co-repressors, and has a dominant negative effect on normal TRs. In this study, we show that homozygous (TRßΔ337T) mice for this mutation have reduced body weight, length, and body fat content, despite augmented relative food intake and relative increase in serum leptin. TRßΔ337T mice exhibited normal glycemia and were more tolerant to an i.p. glucose load accompanied by reduced insulin secretion. Higher insulin sensitivity was observed after single insulin injection, when the TRßΔ337T mice developed a profound hypoglycemia. Impaired hepatic glucose production was confirmed by the reduction in glucose generation after pyruvate administration. In addition, hepatic glycogen content was lower in homozygous TRßΔ337T mice than in wild type. Collectively, the data suggest that TRßΔ337T mice have deficient hepatic glucose production, by reduced gluconeogenesis and lower glycogen deposits. Analysis of liver gluconeogenic gene expression showed a reduction in the mRNA of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme, and of peroxisome proliferator-activated receptor-γ coactivator 1α, a key transcriptional factor essential to gluconeogenesis. Reduction in both gene expressions is consistent with resistance to TH action via TRß, reproducing a hypothyroid phenotype. In conclusion, mice carrying the Δ337T-dominant negative mutation on the TRß are leaner, exhibit impaired hepatic glucose production, and are more sensitive to hypoglycemic effects of insulin.
Asunto(s)
Adiposidad/genética , Glucosa/metabolismo , Crecimiento/genética , Homeostasis/genética , Hígado/metabolismo , Mutación/genética , Receptores beta de Hormona Tiroidea/genética , Animales , Ingestión de Alimentos , Glucógeno/metabolismo , Hipoglucemia/inducido químicamente , Hipoglucemia/metabolismo , Insulina/efectos adversos , Leptina/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos AnimalesRESUMEN
Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.
Asunto(s)
Células Fotorreceptoras Retinianas Conos/embriología , Células Fotorreceptoras Retinianas Conos/metabolismo , Opsinas de Bastones/metabolismo , Receptores beta de Hormona Tiroidea/metabolismo , Animales , Tipificación del Cuerpo , Femenino , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Fotorreceptoras Retinianas Conos/crecimiento & desarrollo , Opsinas de Bastones/genética , Receptores beta de Hormona Tiroidea/deficiencia , Receptores beta de Hormona Tiroidea/genética , Triyodotironina/metabolismoRESUMEN
The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors [NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)] in the absence of ligand to silence transcription. NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT. Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains. An analogous domain is not present in SMRT. This domain is specific for TR and interacts poorly with RAR. Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells. Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions. In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1. Additional regions distal to the CoRNR box sequence are needed for optimal binding. Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1. These preferences are likely to be important in corepressor action in vivo.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Línea Celular , Secuencia de Consenso , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Glutatión Transferasa/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Co-Represor 1 de Receptor Nuclear , Co-Represor 2 de Receptor Nuclear , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/metabolismo , Proteínas Recombinantes de Fusión , Proteínas Represoras/genética , Relación Estructura-Actividad , Factores de Transcripción/genética , TransfecciónRESUMEN
Growth factors such as epidermal growth factor (EGF) and insulin regulate development and metabolism via genes containing both POU homeodomain (Pit-1) and phorbol ester (AP-1) response elements. Although CREB binding protein (CBP) functions as a coactivator on these elements, the mechanism of transactivation was previously unclear. We now demonstrate that CBP is recruited to these elements only after it is phosphorylated at serine 436 by growth factor-dependent signaling pathways. In contrast, p300, a protein closely related to CBP that lacks this phosphorylation site, binds only weakly to the transcription complex and in a growth factor-independent manner. A small region of CBP (amino acids 312-440), which we term GF box, contains a potent transactivation domain and mediates this effect. Direct phosphorylation represents a novel mechanism controlling coactivator recruitment to the transcription complex.
Asunto(s)
Sustancias de Crecimiento/farmacología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Transcripción Genética , Activación Transcripcional/efectos de los fármacos , Secuencias de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteína de Unión a CREB , Línea Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Macromoleculares , Mutación/genética , Proteínas Nucleares/genética , Fosforilación/efectos de los fármacos , Prolactina/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Transactivadores/genética , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción Pit-1 , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Transfección , Células Tumorales CultivadasRESUMEN
Resistance to thyroid hormone (RTH) is due to mutations in the beta-isoform of the thyroid hormone receptor (TR-beta). RTH patients display inappropriate secretion of thyrotropin-releasing hormone (TRH) from the hypothalamus and thyrotropin (TSH) from the anterior pituitary, despite elevated levels of thyroid hormone thyroxine (T4) and triiodothyronine (T3). Thyrotropin-secreting tumors are presumed to represent clonal expansion of abnormal cells. Because the diagnosis of TSH-secreting tumors tends to be delayed and curative surgical resection remains under 50%, early diagnosis is paramount. Current diagnostic strategies suggest that RTH patients are distinguishable from patients with TSH-secreting pituitary tumors by the use of standard laboratory tests and imaging. Here, we present a woman in whom the standard evaluation for inappropriate TSH secretion was insufficient to distinguish these entities. The patient had a low-normal TRH stimulation test and an unmeasurable alpha-glycoprotein subunit level; however, a pituitary magnetic resonance imaging (MRI) revealed an adenoma. More testing using a T3 suppression test supported a RTH diagnosis and a R438H mutation was found in the TR-beta gene. To our knowledge, this represents the first report of an apparently incidental pituitary adenoma in the setting of documented resistance to thyroid hormone. As such, it raises the question of whether RTH predisposes to pituitary hyperplasia and adenoma development.
Asunto(s)
Adenoma/diagnóstico , Neoplasias Hipofisarias/diagnóstico , Síndrome de Resistencia a Hormonas Tiroideas/diagnóstico , Adenoma/complicaciones , Adulto , Femenino , Humanos , Imagen por Resonancia Magnética , Linaje , Neoplasias Hipofisarias/complicaciones , Mutación Puntual , Prolactina/sangre , Receptores de Hormona Tiroidea/genética , Síndrome de Resistencia a Hormonas Tiroideas/complicaciones , Síndrome de Resistencia a Hormonas Tiroideas/genética , Tirotropina/sangre , Tirotropina/metabolismo , Hormona Liberadora de Tirotropina , Triyodotironina/administración & dosificación , Triyodotironina/sangreRESUMEN
Thyroid hormone thyroxine (T(4)) and tri-iodothyronine (T(3)) production is regulated by feedback inhibition of thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) synthesis in the pituitary and hypothalamus when T(3) binds to thyroid hormone receptors (TRs) interacting with the promoters of the genes for the TSH subunit and TRH. All of the TR isoforms likely participate in the negative regulation of TSH production in vivo, but the identity of the specific TR isoforms that negatively regulate TRH production are less clear. To clarify the role of the TR-beta2 isoform in the regulation of TRH gene expression in the hypothalamic paraventricular nucleus, we examined preprothyrotropin-releasing hormone (prepro-TRH) expression in mice lacking the TR-beta2 isoform under basal conditions, after the induction of hypothyroidism with propylthiouracil, and in response to T(3) administration. Prepro-TRH expression was increased in hypothyroid wild-type mice and markedly suppressed after T(3) administration. In contrast, basal TRH expression was increased in TR-beta2-null mice to levels seen in hypothyroid wild-type mice and did not change significantly in response to induction of hypothyroidism or T(3) treatment. However, the suppression of TRH mRNA expression in response to leptin reduction during fasting was preserved in TR-beta2-null mice. Thus TR-beta2 is the key TR isoform responsible for T(3)-mediated negative-feedback regulation by hypophysiotropic TRH neurons.
Asunto(s)
Núcleo Hipotalámico Paraventricular/metabolismo , Receptores de Hormona Tiroidea/fisiología , Hormona Liberadora de Tirotropina/metabolismo , Animales , Ayuno , Leptina/metabolismo , Ratones , Ratones Noqueados , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Tirotropina/metabolismo , Hormona Liberadora de Tirotropina/genética , Tiroxina/metabolismoRESUMEN
Congenital hypothyroidism and the thyroid hormone (T(3)) resistance syndrome are associated with severe central nervous system (CNS) dysfunction. Because thyroid hormones are thought to act principally by binding to their nuclear receptors (TRs), it is unexplained why TR knock-out animals are reported to have normal CNS structure and function. To investigate this discrepancy further, a T(3) binding mutation was introduced into the mouse TR-beta locus by homologous recombination. Because of this T(3) binding defect, the mutant TR constitutively interacts with corepressor proteins and mimics the hypothyroid state, regardless of the circulating thyroid hormone concentrations. Severe abnormalities in cerebellar development and function and abnormal hippocampal gene expression and learning were found. These findings demonstrate the specific and deleterious action of unliganded TR in the brain and suggest the importance of corepressors bound to TR in the pathogenesis of hypothyroidism.
Asunto(s)
Hipotiroidismo/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Animales , Enfermedades Cerebelosas/etiología , Enfermedades Cerebelosas/metabolismo , Enfermedades Cerebelosas/fisiopatología , Hipotálamo/metabolismo , Hipotiroidismo/complicaciones , Hipotiroidismo/genética , Discapacidades para el Aprendizaje/etiología , Discapacidades para el Aprendizaje/metabolismo , Ratones , Ratones Noqueados , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Hormona Tiroidea/deficiencia , Receptores de Hormona Tiroidea/genética , Glándula Tiroides/metabolismoRESUMEN
Type 2 iodothyronine deiodinase (D(2)) catalyzes intracellular 3, 5, 3' triiodothyronine (T(3)) production from thyroxine (T(4)), and its messenger RNA mRNA is highly expressed in human, but not rodent, myocardium. The goal of this study was to identify the effects of D(2) expression in the mouse myocardium on cardiac function and gene expression. We prepared transgenic (TG) mice in which human D(2) expression was driven by the alpha-MHC promoter. Despite high myocardial D(2) activity, myocardial T(3) was, at most, minimally increased in TG myocardium. Although, plasma T(3) and T(4), growth rate as well as the heart weight was not affected by TG expression, there was a significant increase in heart rate of the isolated perfused hearts, from 284 +/-12 to 350 +/- 7 beats/min. This was accompanied by an increase in pacemaker channel (HCN2) but not alpha-MHC or SERCA II messenger RNA levels. Biochemical studies and (31)P-NMR spectroscopy showed significantly lower levels of phosphocreatine and creatine in TG hearts. These results suggest that even mild chronic myocardial thyrotoxicosis, such as may occur in human hyperthyroidism, can cause tachycardia and associated changes in high energy phosphate compounds independent of an increase in SERCA II and alpha-MHC.
Asunto(s)
Corazón/fisiopatología , Yoduro Peroxidasa/metabolismo , Proteínas Musculares , Tirotoxicosis/genética , Tirotoxicosis/fisiopatología , Nucleótidos de Adenina/metabolismo , Animales , ATPasas Transportadoras de Calcio/genética , Creatina Quinasa/metabolismo , Metabolismo Energético , Corazón/fisiología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Yoduro Peroxidasa/genética , Canales Iónicos/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Cadenas Pesadas de Miosina/genética , Tamaño de los Órganos , Canales de Potasio , Regiones Promotoras Genéticas , ARN Mensajero/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tiroxina/sangre , Tiroxina/metabolismo , Transcripción Genética , Triyodotironina/sangreRESUMEN
Transcription of pituitary alpha-glycoprotein hormone subunit (alpha-GSU) and thyrotropin beta subunit (TSH-beta) genes is stimulated by thyrotropin-releasing hormone (TRH). Since cAMP response element-binding protein (CREB)-binding protein (CBP) integrates a number of cell signaling pathways, we investigated whether CBP is important for TRH stimulation of the TSH subunit genes. Cotransfection of E1A in GH(3) cells completely blocked TRH stimulation of the TSH subunit genes, suggesting that CBP is a key factor for TRH signaling in the pituitary. CBP and Pit-1 acted synergistically in TRH stimulation of the TSH-beta promoter, and amino acids 1-450 of CBP were sufficient for the TRH effect. In contrast, on the human alpha-GSU promoter, CREB and P-Lim mediated TRH signaling. Intriguingly, CREB was phosphorylated upon TRH stimulation, leading to CBP recruitment to the alpha-GSU promoter. CBP also interacted with P-Lim in a TRH-dependent manner, suggesting that P-Lim is an important factor for non-cAMP response element-mediated TRH stimulation of this promoter. Distinct domains of CBP were required for TRH signaling by CREB and P-Lim on the alpha-GSU promoter, amino acids 450-700 and 1-450, respectively. Thus, the amino terminus of CBP plays a critical role in TRH signaling in the anterior pituitary via both Pit-1-dependent and -independent pathways, yielding differential regulation of pituitary gene products.
Asunto(s)
Proteínas Nucleares/fisiología , Hormona Liberadora de Tirotropina/farmacología , Tirotropina/genética , Transactivadores/fisiología , Proteína de Unión a CREB , AMP Cíclico/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas con Homeodominio LIM , Proteínas Nucleares/química , Fosforilación , Regiones Promotoras Genéticas , Subunidades de Proteína , Relación Estructura-Actividad , Transactivadores/química , Factor de Transcripción Pit-1 , Factores de Transcripción/fisiologíaRESUMEN
The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms have the capacity to silence gene expression in the absence of their ligands on target response elements. This active repression is mediated by the ability of the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to recruit a complex containing histone deacetylase activity. Interestingly, NCoR and SMRT share significant differences in the their two nuclear receptor-interacting domains (IDs), suggesting that they may recruit receptors with different affinities. In addition, the role of the receptor complex bound to a response element has not been fully evaluated in its ability to recruit separate corepressors. We demonstrate in this report that the proximal ID in NCoR and SMRT, which share only 23% homology, allows preferential recognition of nuclear receptors, such that TR prefers to recruit NCoR, and RAR prefers to recruit SMRT, to DNA response elements. However, mutations in the TR found in the syndromes of resistance to thyroid hormone can change the corepressor recruited by changing the complex (homodimer or heterodimer) formed on the TRE. These results demonstrate that the corepressor complex recruited can be both nuclear receptor- and receptor complex-specific.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Proteínas Represoras/metabolismo , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Glutatión Transferasa/genética , Mutación , Proteínas Nucleares/química , Co-Represor 1 de Receptor Nuclear , Co-Represor 2 de Receptor Nuclear , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/genética , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Elementos de Respuesta , Receptores X Retinoide , Síndrome de Resistencia a Hormonas Tiroideas/genética , Factores de Transcripción/metabolismoRESUMEN
Thyroid hormone deficiency has profound effects on the cardiovascular system, resulting in decreased cardiac contractility, adrenergic responsiveness, and vascular volume and increased peripheral vascular resistance. To determine the importance of direct cardiac effects in the genesis of hypothyroid cardiac dysfunction, the cardiac myocyte was specifically targeted with a mutant thyroid hormone receptor (TR)-beta (Delta337T-TR-beta(1)) driven by the alpha-myosin heavy chain (alpha-MHC) gene promoter. As a control in these experiments, a wild-type (Wt) TR-beta(1) was also targeted to the heart by using the same promoter. Transgenic mice expressing the mutant TR displayed an mRNA expression pattern consistent with cardiac hypothyroidism, even though their peripheral thyroid hormone levels were normal. When these animals were rendered hypothyroid or thyrotoxic, mRNA expression of MHC isoforms remained unchanged in the hearts of the Delta337T transgenic animals, in contrast to Wt controls or transgenic animals expressing Wt TR-beta(1), which exhibited the expected changes in steady-state MHC mRNA levels. Studies in Langendorff heart preparations from mutant TR-beta(1) transgenic animals revealed evidence of heart failure with a significant reduction in +dP/dT, -dP/dT, and force-frequency responses compared with values in Wt controls and transgenic mice overexpressing the Wt TR-beta(1). In contrast, in vivo measures of cardiac performance were similar between Wt and mutant animals, indicating that the diminished performance of the mutant transgenic heart in vitro was compensated for by other mechanisms in vivo. This is the first demonstration indicating that isolated cardiac hypothyroidism causes cardiac dysfunction in the absence of changes in the adrenergic or peripheral vascular system.
Asunto(s)
Corazón/fisiopatología , Mutación/fisiología , Miocardio/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Animales , Expresión Génica , Humanos , Ratones , Ratones Transgénicos/genética , Cadenas Pesadas de Miosina/genética , Especificidad por Sustrato , Transgenes/fisiologíaRESUMEN
Thyroid hormone receptors (TRs) mediate hormone action by binding to DNA response elements (TREs) and either activating or repressing gene expression in the presence of ligand, T(3). Coactivator recruitment to the AF-2 region of TR in the presence of T(3) is central to this process. The different TR isoforms, TR-beta1, TR-beta2, and TR-alpha1, share strong homology in their DNA- and ligand-binding domains but differ in their amino-terminal domains. Because TR-beta2 exhibits greater T(3)-independent activation on TREs than other TR isoforms, we wanted to determine whether coactivators bound to TR-beta2 in the absence of ligand. Our results show that TR-beta2, unlike TR-beta1 or TR-alpha1, is able to bind certain coactivators (CBP, SRC-1, and pCIP) in the absence of T(3) through a domain which maps to the amino-terminal half of its A/B domain. This interaction is specific for certain coactivators, as TR-beta2 does not interact with other co-factors (p120 or the CBP-associated factor (pCAF)) in the absence of T(3). The minimal TR-beta2 domain for coactivator binding is aa 21-50, although aa 1-50 are required for the full functional response. Thus, isoform-specific regulation by TRs may involve T(3)-independent coactivator recruitment to the transcription complex via the AF-1 domain.
Asunto(s)
Receptores de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/metabolismo , Transactivadores/metabolismo , Sitios de Unión , Línea Celular , Glutatión Transferasa/metabolismo , Humanos , Ligandos , Unión Proteica , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
p120 was originally isolated as a novel nuclear co-activator for thyroid hormone receptor. In this study, we characterized its interaction and transactivation of peroxisome proliferator-activated receptor-gamma (PPARgamma) and 9-cis-retinoic acid receptor (RXR) heterodimers. Transient transfection study revealed that p120 enhanced the transcriptional activation of PPARgamma/RXR induced by PPARgamma- or RXR-specific ligands. In the glutathione-S-transferase pull-down assay, while steroid receptor coactivator-1 showed apparent interactions with both RXR and PPARgamma, p120 bound only to RXR in a 9-cis-retinoic acid (RA)-dependent manner and also did not bind to PPARgamma even in the presence of thiazolidinediones. The yeast two-hybrid analysis showed no interaction of p120 with PPARgamma under any conditions, and electophoretic mobility shift assay showed apparent DNA-PPARgamma/RXR/p120 complex formation only in the presence of 9-cis-RA. Furthermore, the yeast three-hybrid assay clearly revealed a significant interaction between p120 and PPARgamma via RXR of PPARgamma/RXR heterodimer only in the presence of 9-cis-RA. These findings indicate that p120 acts as a specific co-activator for the RXR of PPARgamma/RXR heterodimer in a 9-cis-RA-dependent manner.
Asunto(s)
Proteínas Portadoras/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea , Factores de Transcripción/metabolismo , Tejido Adiposo/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Humanos , Riñón/citología , Riñón/metabolismo , Ratones , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor alfa de Ácido Retinoico , Factores de Transcripción/genética , Activación Transcripcional , Tretinoina/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Resistance to thyroid hormone (RTH) action is due to mutations in the beta-isoform of the thyroid hormone receptor (TR-beta). RTH patients display inappropriate central secretion of TRH from the hypothalamus and of TSH from the anterior pituitary despite elevated levels of thyroid hormone (T4 and T3). RTH mutations cluster in three hot spots in the C-terminal portion of the TR-beta. Most individuals with TR-beta mutations have generalized resistance to thyroid hormone, where most tissues in the body are hyporesponsive to thyroid hormone. The affected individuals are clinically euthyroid or even hypothyroid depending on the severity of the mutation. Whether TR-beta mutations cause a selective form of RTH that only leads to central thyroid hormone resistance is debated. Here, we describe an individual with striking peripheral sensitivity to graded T3 administration. The subject was enrolled in a protocol in which she received three escalating T3 doses over a 13-day period. Indexes of central and peripheral thyroid hormone action were measured at baseline and at each T3 dose. Although the patient's resting pulse rose only 11% in response to T3, her serum ferritin, alanine aminotransferase, aspartate transaminase, and lactate dehydrogenase rose 320%, 117%, 121%, and 30%, respectively. In addition, her serum cholesterol, creatinine phosphokinase, and deep tendon reflex relaxation time fell (25%, 36%, and 36%, respectively). Centrally, the patient was sufficiently resistant to T3 that her serum TSH was not suppressed with 200 microg T3, orally, daily for 4 days. The patient's C-terminal TR exons were sequenced revealing the mutation R383H in a region not otherwise known to harbor TR-beta mutations. Our clinical evaluation presented here represents the most thorough documentation to date of the central thyroid hormone resistance phenotype in an individual with an identified TR-beta mutation.
Asunto(s)
Mutación , Receptores de Hormona Tiroidea/genética , Síndrome de Resistencia a Hormonas Tiroideas/genética , Hormonas Tiroideas/farmacología , Peso Corporal , Femenino , Corazón/fisiopatología , Humanos , Hipotálamo/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Procesos Mentales , Persona de Mediana Edad , Linaje , Adenohipófisis/metabolismo , Prolactina/sangre , Síndrome de Resistencia a Hormonas Tiroideas/fisiopatología , Tirotropina/metabolismo , Hormona Liberadora de Tirotropina/metabolismo , Triyodotironina/administración & dosificaciónRESUMEN
Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Pit-1 contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for DNA binding and activation of the GH and PRL genes and regulation of the PRL, TSH-beta subunit (TSH-beta), and Pit-1 genes. Pit-1 is also necessary for retinoic acid induction of its own gene during development through a Pit-1-dependent enhancer. Combined pituitary hormone deficiency is caused by defective transactivation of target genes in the anterior pituitary. In the present report, we provide in vivo evidence that retinoic acid induction of the Pit-1 gene can be impaired by a Pit-1 gene mutation, suggesting a new molecular mechanism for combined pituitary hormone deficiency in man.
Asunto(s)
Proteínas de Unión al ADN/genética , Mutación , Hormonas Hipofisarias/deficiencia , Factores de Transcripción/genética , Tretinoina/metabolismo , Animales , Preescolar , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/metabolismo , Humanos , Hipotiroidismo/metabolismo , Masculino , Prolactina/genética , Prolactina/metabolismo , ARN Mensajero , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Transducción de Señal , Factor de Transcripción Pit-1 , Factores de Transcripción/metabolismoRESUMEN
The pituitary-specific transcription factor, Pit-1, is necessary to mediate protein kinase A (PKA) regulation of the GH, PRL, and TSH-beta subunit genes in the pituitary. Since these target genes lack classical cAMP DNA response elements (CREs), the mechanism of this regulation was previously unknown. We show that CREB binding protein (CBP), through two cysteine-histidine rich domains (C/H1 and C/H3), specifically and constitutively interacts with Pit-1 in pituitary cells. Pit-1 and CBP synergistically activate the PRL gene after PKA stimulation in a mechanism requiring both an intact Pit-1 amino-terminal and DNA-binding domain. A CBP construct containing the C/H3 domain [amino acids (aa) 1678-2441], but not one lacking the C/H3 domain (aa 1891-2441), is sufficient to mediate this response. Neither construct augments PKA regulation of CRE-containing promoters. Fusion of either CBP fragment to the GAL4 DNA-binding domain transferred complete PKA regulation to a heterologous promoter. These findings provide a mechanism for CREB-independent regulation of gene expression by cAMP.
Asunto(s)
AMP Cíclico/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Transactivadores/genética , Factores de Transcripción/genética , Animales , Western Blotting , Proteína de Unión a CREB , Células Cultivadas , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Glutatión Transferasa/fisiología , Humanos , Luciferasas/análisis , Proteínas Nucleares/fisiología , Hipófisis/fisiología , Pruebas de Precipitina , Prolactina/fisiología , Regiones Promotoras Genéticas/fisiología , Proteínas Recombinantes de Fusión , Hormona Liberadora de Tirotropina/fisiología , Transactivadores/fisiología , Factor de Transcripción Pit-1 , Factores de Transcripción/fisiología , TransfecciónRESUMEN
Patients with resistance to thyroid hormone (RTH) exhibit elevated thyroid hormone levels and inappropriate thyrotropin (thyroid-stimulating hormone, or TSH) production. The molecular basis of this disorder resides in the dominant inhibition of endogenous thyroid hormone receptors (TRs) by a mutant receptor. To determine the relative contributions of pituitary versus hypothalamic resistance to the dysregulated production of thyroid hormone in these patients, we developed a transgenic mouse model with pituitary-specific expression of a mutant TR (Delta337T). The equivalent mutation in humans is associated with severe generalized RTH. Transgenic mice developed profound pituitary resistance to thyroid hormone, as demonstrated by markedly elevated baseline and non-triodothyronine (T3)-suppressible serum TSH and pituitary TSH-beta mRNA. Serum thyroxine (T4) levels were only marginally elevated in transgenic mice and thyrotropin-releasing hormone (TRH) gene expression in the paraventricular hypothalamus was downregulated. After TRH administration, T4 concentrations increased markedly in transgenic, but not in wild-type mice. Transgenic mice rendered hypothyroid exhibited a TSH response that was only 30% of the response observed in wild-type animals. These findings indicate that pituitary expression of this mutant TR impairs both T3-mediated suppression and T3-independent activation of TSH production in vivo. The discordance between basal TSH and T4 levels and the reversal with TRH administration demonstrates that resistance at the level of both the thyrotroph and the hypothalamic TRH neurons are required to elevate thyroid hormone levels in patients with RTH.
Asunto(s)
Síndrome de Resistencia a Hormonas Tiroideas/genética , Tirotropina/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Mutación/genética , Hipófisis/metabolismo , Precursores de Proteínas/genética , ARN Mensajero/metabolismo , Receptores de Hormona Tiroidea/genética , Tirotropina/sangre , Hormona Liberadora de Tirotropina/genética , Tiroxina/sangre , Triyodotironina/farmacologíaRESUMEN
On positive thyroid hormone response elements (pTREs), thyroid hormone receptor (TR) binding to DNA in the absence of ligand (thyroid hormone, T3) decreases transcription (silencing). Silencing is due to a family of recently described nuclear corepressor proteins (NCoR and SMRT) which bind to the CoR box in the hinge region of TR. Ligand-dependent activation of TR is associated with displacement of corepressors and recruitment of coactivating proteins. Resistance to thyroid hormone (RTH) is due to mutations in the beta isoform of the thyroid hormone receptor (TR-beta). To date, three RTH mutations reportedly with near-normal T3 binding (A234T, R243Q, and R243W) have been described in or near the CoR box. To determine the mechanism of RTH caused by these mutants, the interaction of wild type (wt) and mutant TRs with the corepressor, NCoR, and the coactivator, SRC-1, was tested in gel-shift assays. As expected, NCoR bound wt TR in the absence of T3 and dissociated from TR with increasing T3 concentration. SRC-1 failed to bind wt TR in the absence of T3, but bound to TR with increasing avidity as T3 concentrations rose. At no T3 concentration did both NCoR and SRC-1 bind to wt TR, indicating that their binding to TR was mutually exclusive. Hinge mutants bound NCoR normally in the absence of T3; however, dissociation of NCoR and recruitment of SRC-1 was markedly impaired except at very high T3 concentrations. Importantly, hinge mutant TRs when complexed to DNA bound T3 poorly despite their near-normal T3 binding in solution. These binding studies correlated with functional assays showing defective transactivation of pTREs by hinge mutants except at high T3 concentrations. Thus, we describe a novel mechanism of RTH whereby TR hinge mutants selectively affect T3 binding when complexed to DNA, and prevent NCoR dissociation from TR. Our data also suggest that solution T3 binding by RTH mutants may not accurately reflect physiologically relevant T3 binding by TR when bound to DNA.