RESUMO
Thyroid hormone receptor (TR) α and ß mediate thyroid hormone action at target tissues. TR isoforms have specific roles in development and in adult tissues. The mechanisms underlying TR isoform-specific action, however, are not well understood. We demonstrate that posttranslational modification of TR by conjugation of small SUMO to TRα and TRß plays an important role in triiodothyronine (T3) action and TR isoform specificity. TRα was sumoylated at lysines 283 and 389, and TRß at lysines 50, 146, and 443. Sumoylation of TRß was ligand-dependent, and sumoylation of TRα was ligand-independent. TRα-SUMO conjugation utilized the E3 ligase PIASxß and TRß-SUMO conjugation utilized predominantly PIAS1. SUMO1 and SUMO3 conjugation to TR was important for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The functional role of SUMO1 and SUMO3 in T3 induction in transient expression assays was closely matched to the pattern of TR and cofactor recruitment to thyroid hormone response elements (TREs) as determined by ChIP assays. SUMO1 was required for the T3-induced recruitment of the co-activator CREB-binding protein (CBP) and release of nuclear receptor co-repressor (NCoR) on a TRE but had no significant effect on TR DNA binding. SUMO1 was required for T3-mediated recruitment of NCoR and release of CBP from the TSHß-negative TRE. SUMO3 was required for T3-stimulated TR binding to the TSHß-negative TRE and recruitment of NCoR. These findings demonstrate that conjugation of SUMO to TR has a TR-isoform preference and is important for T3-dependent gene induction and repression.
Assuntos
Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/farmacologia , Ubiquitinas/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Células Hep G2 , Humanos , Masculino , Camundongos , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Elementos de Resposta/fisiologia , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Tireotropina Subunidade beta/genética , Tireotropina Subunidade beta/metabolismo , Ubiquitinas/genéticaRESUMO
Thyroid hormone regulates the balance between lipolysis and lipogenesis. We previously reported that male mice with a dominant-negative P398H mutation introduced into the TRalpha gene have visceral obesity, hyperleptinemia, and reduced catecholamine-stimulated lipolysis in white adipose tissue. Based on our observation of hepatic steatosis in the TRalpha P398H male mice, we used in vitro and in vivo models to investigate the influence of the TRalpha P398H mutant on peroxisome proliferator-activated receptor-alpha (PPARalpha) signaling. Wild-type TRalpha and the P398H mutant significantly reduced PPARalpha-mediated transcription in transient transfection assays. T(3) reversed the inhibition of PPARalpha action by wild-type TRalpha but not the P398H mutant. Chromatin immunoprecipitation assays demonstrated that the P398H mutant reduces PPARalpha binding to peroxisome proliferator receptor elements. In gel shift assays, the P398H mutant directly bound the peroxisome proliferator-activated receptor response element and inhibited PPARalpha binding, which was not reversed by addition of retinoid X receptor. The TRalpha R384C and PV dominant-negative mutants are not associated in vivo with a metabolic phenotype and had reduced (PV) or absent (R384C) PPARalpha inhibition compared with P398H. The metabolic phenotype of the P398H mutant mice is due, in part, to unique properties of the P398H mutant receptor interfering with PPARalpha signaling. The P398H mutant is a potential probe to characterize the physiological role of thyroid hormone receptor/PPARalpha interactions.
Assuntos
Ácidos Graxos/metabolismo , Proteínas Mutantes/fisiologia , PPAR alfa/antagonistas & inibidores , Receptores alfa dos Hormônios Tireóideos/fisiologia , Animais , Fígado Gorduroso/genética , Glicogênio/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredução , Elementos Reguladores de Transcrição , Transdução de Sinais , Receptores alfa dos Hormônios Tireóideos/genética , Ativação TranscricionalRESUMO
Thyroid hormone negatively regulates the amyloid-beta precursor protein (APP) gene in thyroid hormone receptor (TR)-transfected neuroblastoma cells. A negative thyroid hormone response element (nTRE) that mediates this regulation has been identified in the first exon of the APP gene. We demonstrate in an in vivo system that expression of APP mRNA, APP protein, and APP secretase cleavage products in mouse brain is influenced by thyroid status. Adult female mice were made hyperthyroid or hypothyroid for 3 weeks and compared to euthyroid mice. APP gene product expression was increased in hypothyroid mouse brain and reduced in hyperthyroid mouse brain, when compared to euthyroid controls. We observed similar effects of thyroid hormone on endogenous APP gene expression in human neuroblastoma cells. The incidence of hypothyroidism increases with age, and localized hypothyroidism of central nervous system has been reported in some patients with Alzheimer's disease (AD). Reduced action of thyroid hormone on the APP gene may contribute to AD pathology by increasing APP expression and the levels of processed APP products. These findings may be an underlying mechanism contributing to the association of hypothyroidism with AD in the elderly, as well as identifying a potential therapeutic target. Pharmacologic supplementation of thyroid hormone, or its analogs, may reduce APP gene expression and beta amyloid peptide accumulation.
Assuntos
Precursor de Proteína beta-Amiloide/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Tiroxina/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Hipertireoidismo/metabolismo , Hipotireoidismo/metabolismo , Camundongos , Neuroblastoma/metabolismo , Tretinoína/farmacologiaRESUMO
Lactating breast tissue and some breast cancers express the sodium/iodide symporter (NIS) and concentrate iodide. We recently demonstrated that all-trans retinoic acid (tRA) induces both NIS gene expression and iodide accumulation in vitro in well-differentiated human breast cancer cells (MCF-7). In the present study, we investigated the in vivo efficacy and specificity of tRA-stimulated iodide accumulation in mouse breast cancer models. Immunodeficient mice with MCF-7 xenograft tumors were treated with systemic tRA for 5 days. Iodide accumulation in the xenograft tumors was markedly increased, approximately 15-fold greater than levels without treatment, and the effects were tRA dose dependent. Iodide accumulation in other organs was not significantly influenced by tRA treatment. Significant induction of NIS mRNA and protein in the xenograft tumors was observed after tRA treatment. Iodide accumulation and NIS mRNA expression were also selectively induced in breast cancer tissues in transgenic mice expressing the oncogene, polyoma virus middle T antigen. These data demonstrate selective induction of functional NIS in breast cancer by tRA. Treatment with short-term systemic retinoic acid, followed by radioiodide administration, is a potential tool in the diagnosis and treatment of some differentiated breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Radioisótopos do Iodo/farmacocinética , Simportadores/genética , Tretinoína/farmacologia , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Cintilografia , Simportadores/efeitos dos fármacos , Simportadores/efeitos da radiação , Distribuição Tecidual , Transplante HeterólogoRESUMO
Thyroid hormone plays an important role in bone development and metabolism. We used a polymerase chain reaction (PCR)-based mRNA differential display (DD) analysis to obtain a profile of thyroid hormone-responsive genes in osteoblast-like cells (ROS 17/2.8). ROS 17/2.8 cells were treated with 10(-8) M triiodothyronine (T(3)) for 2 and 24 hours. Total RNA was isolated, reverse-transcribed, and amplified using a total of 72 combinations (2 hours) and 240 combinations (24 hours) of 5' and 3' primers. At the 2-hour time point, 1 true-positive novel clone was identified and shown to be the mitochondrial gene, subunit 6 of ATP synthase (ATPase-6). At the 24-hour time point, 3 differentially expressed (DE) mRNAs were confirmed as true-positives including; nonmuscle alkali myosin light chain (NM aMLC), ATPase-6, and one novel clone. T(3)-induction of ATPase-6 mRNA in ROS 17/2.8 cells was seen at 2 and 4 hours, but was maximal at 24 hours (2.1-fold). T(3) induction of ATPase-6 mRNA was increased to fourfold in ROS 17/2.8 cells cultured at a low density. NM aMLC mRNA was modestly upregulated by T(3) in ROS 17/2.8 cells by 1.4-fold, and induction was augmented at low cell density to 1.7-fold. T(3) action on NM aMLC and on the mitochondrial gene ATPase 6, represent novel targets and potential mediators of thyroid hormone action on bone. Cell type, and the extent of cell differentiation, influences T(3) regulation of genes in osteoblast-derived cells.
Assuntos
Perfilação da Expressão Gênica , Osteoblastos/fisiologia , Tri-Iodotironina/farmacologia , Adenosina Trifosfatases/genética , Animais , Expressão Gênica/efeitos dos fármacos , Mitocôndrias/genética , Cadeias Leves de Miosina/genética , Osteoblastos/citologia , Osteossarcoma , Ratos , Células Tumorais CultivadasRESUMO
Cold-induced adaptive (or nonshivering) thermogenesis in small mammals is produced primarily in brown adipose tissue (BAT). BAT has been identified in humans and becomes more active after cold exposure. Heat production from BAT requires sympathetic nervous system stimulation, T(3), and uncoupling protein 1 (UCP1) expression. Our previous studies with a thyroid hormone receptor-beta (TR beta) isoform-selective agonist demonstrated that after TR beta stimulation alone, adaptive thermogenesis was markedly impaired, although UCP-1 expression in BAT was normal. We used mice with a dominant-negative TR beta PV mutation (frameshift mutation in resistance to thyroid hormone patient PV) to determine the role of TR beta in adaptive thermogenesis and UCP1 expression. Wild-type and PV mutant mice were made hypothyroid and replaced with T(3) (7 ng/g x d) for 10 d to produce similar serum thyroid hormone concentration in the wild-type and mutant mice. The thermogenic response of interscapular BAT, as determined by heat production during iv infusions of norepinephrine, was reduced in PV beta heterozygous and homozygous mutant mice. The level of UCP1, the key thermogenic protein in BAT, was progressively reduced in PV beta(+/-) and PV beta(-/-) mutant mice. Brown adipocytes isolated from PV mutant mice had some reduction in cAMP and glycerol production in response to adrenergic stimulation. Defective adaptive thermogenesis in TR beta PV mutant mice is due to reduced UCP1 expression and reduced adrenergic responsiveness. TR beta mediates T(3) regulation of UCP1 in BAT and is required for adaptive thermogenesis.
Assuntos
Adaptação Fisiológica/fisiologia , Tecido Adiposo Marrom/metabolismo , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Termogênese , Receptores beta dos Hormônios Tireóideos/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/fisiologia , Animais , Catecolaminas/farmacologia , Células Cultivadas , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Hipotireoidismo/metabolismo , Hipotireoidismo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Especificidade por Substrato , Termogênese/efeitos dos fármacos , Termogênese/genética , Termogênese/fisiologia , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/farmacologia , Proteína Desacopladora 1RESUMO
Thyroid hormone has profound effects on metabolic homeostasis, regulating both lipogenesis and lipolysis, primarily by modulating adrenergic activity. We generated mice with a point mutation in the thyroid hormone receptor alpha (TRalpha) gene producing a dominant-negative TRalpha mutant receptor with a proline to histidine substitution (P398H). The heterozygous P398H mutant mice had a 3.4-fold (p < 0.02) increase in serum thyrotropin (TSH) levels. Serum triiodothyronine (T3) and thyroxine (T4) concentrations were slightly elevated compared with wild-type mice. The P398H mice had a 4.4-fold increase in body fat (as a fraction of total body weight) (p < 0.001) and a 5-fold increase in serum leptin levels (p < 0.005) compared with wild-type mice. A 3-fold increase in serum fasting insulin levels (p < 0.002) and a 55% increase in fasting glucose levels (p < 0.01) were observed in P398H compared with wild-type mice. There was a marked reduction in norepinephrine-induced lipolysis, as reflected in reduced glycerol release from white adipose tissue isolated from P398H mice. Heart rate and cold-induced adaptive thermogenesis, mediated by thyroid hormone-catecholamine interaction, were also reduced in P398H mice. In conclusion, the TRalpha P398H mutation is associated with visceral adiposity and insulin resistance primarily due to a marked reduction in catecholamine-stimulated lipolysis. The observed phenotype in the TRalpha P398H mouse is likely due to interference with TRalpha action as well as influence on other metabolic signaling pathways. The physiologic significance of these findings will ultimately depend on understanding the full range of actions of this mutation.