RESUMO
OBJECTIVE: The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of TH receptor alpha (TRα) mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. MATERIALS AND METHODS: It was examined the involvement of PI3K pathway in mediating T3 effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI =100 nM) T3 doses during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey's test was used at 5% significance level. RESULTS: T3 increased TRα mRNA expression in P (1.91±0.13, p<0.001), SI (2.14±0.44, p<0.001) compared to C group (1±0.08). This increase was completely abrogated by LY294002 in P (0.53±0.03, p<0.001) and SI (0.31±0.03, p<0.001). To examine whether TRα is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). The presence of CHX completely abrogated levels TRα mRNA in P (1.15±0.05, p>0.001) and SI (0.99±0.15, p>0.001), induced by T3. CONCLUSION: These results demonstrate that the activation of the PI3K signaling pathway has a role in T3-mediated indirect TRα gene expression in 3T3-L1 adipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , RNA Mensageiro/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/farmacologia , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Diferenciação Celular , Cromonas/farmacologia , Expressão Gênica/genética , Genes erbA/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Receptores alfa dos Hormônios Tireóideos/genética , Fatores de TempoRESUMO
Objective The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of TH receptor alpha (TRα) mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. Materials and methods: It was examined the involvement of PI3K pathway in mediating T3 effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI =100 nM) T3 doses during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey’s test was used at 5% significance level. Results T3 increased TRα mRNA expression in P (1.91±0.13, p<0.001), SI (2.14±0.44, p<0.001) compared to C group (1±0.08). This increase was completely abrogated by LY294002 in P (0.53±0.03, p<0.001) and SI (0.31±0.03, p<0.001). To examine whether TRα is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). The presence of CHX completely abrogated levels TRα mRNA in P (1.15±0.05, p>0.001) and SI (0.99±0.15, p>0.001), induced by T3. Conclusion These results demonstrate that the activation of the PI3K signaling pathway has a role in T3-mediated indirect TRα gene expression in 3T3-L1 adipocytes. .
Objetivo O objetivo do presente estudo foi analisar os efeitos do hormônio tireoidiano (HT), triiodotironina (T3), na modulação da expressão de mRNA do receptor alfa (TRα) de HT e o envolvimento da via de sinalização da via fosfatidilinositol 3-quinase (PI3K) em adipócitos, 3T3-L1. Materiais e métodos: Foi examinado o envolvimento da via PI3K nos efeitos do T3 nos tratamentos de adipócitos, 3T3-L1, nas doses fisiológica (P=10nM) ou suprafisiológica (SI =100 nM) durante uma hora (tempo curto), na ausência ou na presença do inibidor da PI3K (LY294002). A ausência de qualquer tratamento foi considerada o grupo controle (C). RT-qPCR foi utilizado para analisar a expressão do mRNA. Para as análises dos dados, utilizou-se ANOVA complementada com o teste de Tukey a 5% de significância. Resultados O T3 aumentou a expressão de mRNA de TRα em P (1,91±0,13, p<0,001) e SI (2,14±0,44, p<0,001) em comparação com o grupo C (1±0,08). Esse aumento foi completamente abolido por LY294002 em P (0,53±0,03, p<0,001) e SI (0,31±0,03, p<0,001). Para examinar se a expressão de TRα foi diretamente induzida pelo T3, utilizou-se o inibidor de tradução, ciclohexamida (CHX). A presença de CHX reduziu os níveis de mRNA de TRα em P (1,15±0,05, p>0,001) e SI (0,99±0,15, p>0,001), induzidos pelo T3. Conclusão Esses resultados demonstram que a ativação da via de sinalização de PI3K tem um papel importante na expressão do gene TRα mediada indiretamente pelo T3, em adipócitos 3T3-L1. .
Assuntos
Animais , Camundongos , Adipócitos/efeitos dos fármacos , /metabolismo , RNA Mensageiro/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/farmacologia , Adipócitos/metabolismo , Diferenciação Celular , Cromonas/farmacologia , Expressão Gênica/genética , Genes erbA/efeitos dos fármacos , Morfolinas/farmacologia , Fatores de Tempo , Receptores alfa dos Hormônios Tireóideos/genéticaRESUMO
OBJECTIVE: To examine the effect of different doses of triiodothyronine (T3) on mRNA levels of thyroid hormone receptors, TRα and TRß, at different times. MATERIALS AND METHODS: 3T3-L1 adipocytes were incubated with T3 (physiological dose: F; supraphysiological doses: SI or SII), or without T3 (control, C) for 0.5, 1, 6, or 24h. TRα and TRß mRNA was detected using real-time polymerase chain reaction. RESULTS: F increased TRß mRNA levels at 0.5h. After 1h, TRα levels increased with F and SI and TRß levels decreased with SII compared with C, F, and SI. After 6h, both genes were suppressed at all concentrations. In 24h, TRα and TRß levels were similar to those of C group. CONCLUSIONS: T3 action with F began at 1h for TRα and at 0.5h for TRß. These results suggest the importance of knowing the times and doses that activate T3 receptors in adipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Modulação Antigênica/imunologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/administração & dosagem , Adipócitos/metabolismo , Animais , Linhagem Celular , Esquema de Medicação , RNA Mensageiro/análise , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Tri-Iodotironina/farmacologiaRESUMO
OBJECTIVE: To examine the effect of different doses of triiodothyronine (T3) on mRNA levels of thyroid hormone receptors, TRα and TRβ, at different times. MATERIALS AND METHODS: 3T3-L1 adipocytes were incubated with T3 (physiological dose: F; supraphysiological doses: SI or SII), or without T3 (control, C) for 0.5, 1, 6, or 24h. TRα and TRβ mRNA was detected using real-time polymerase chain reaction. RESULTS: F increased TRβ mRNA levels at 0.5h. After 1h, TRα levels increased with F and SI and TRβ levels decreased with SII compared with C, F, and SI. After 6h, both genes were suppressed at all concentrations. In 24h, TRα and TRβ levels were similar to those of C group. CONCLUSIONS: T3 action with F began at 1h for TRα and at 0.5h for TRβ. These results suggest the importance of knowing the times and doses that activate T3 receptors in adipocytes.
OBJETIVO: Examinar o efeito de diferentes doses de triiodotironina (T3) sobre a expressão gênica dos receptores TRα e TRβ em diferentes tempos. MATERIAIS E MÉTODOS: Adipócitos, 3T3-L1, foram incubados com T3 nas doses fisiológica (F, 10nM) e suprafisiológicas (SI, 100nM ou SII, 1000nM) ou veículo (controle, C) durante 0,5, 1, 6 ou 24h. mRNA dos TRs foram detectados utilizando PCR em tempo real. RESULTADOS: Níveis de TRβ aumentaram em F em 0,5h. Após 1h, níveis de TRα aumentaram em F e SI comparado ao C, enquanto TRβ diminuiu no SII comparado com C, F, e SI. Após 6h, ambos os genes foram suprimidos em todas concentrações. Em 24h, níveis de TRα e TRβ retornaram aos do C. CONCLUSÕES: Ação do T3 em F iniciou-se em 1h para TRα e 0,5h para TRβ. Esses resultados são importantes para determinar tempo inicial e dose de T3 em que os receptores de HT são ativados em adipócitos.
Assuntos
Animais , Adipócitos/efeitos dos fármacos , Modulação Antigênica/imunologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/administração & dosagem , Adipócitos/metabolismo , Linhagem Celular , Esquema de Medicação , RNA Mensageiro/análise , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Tri-Iodotironina/farmacologiaRESUMO
As previously reported, the activity of liver glutathione S-transferases, an important family of enzymes for detoxification processes, is regulated by thyroid hormone levels. Here, we specifically studied glutathione S-transferase α (Gsta) gene expression in livers of mice. First, in wild-type (WT) mice, hypothyroidism was induced by 5 weeks of a diet containing 5-propyl-2-thiouracil plus water containing metimazole, whereas hyperthyroidism was induced by daily injections of 50 µg (100 g body weight)(-1) of 3,3, 5-triiodo-L-thyronine (L-T(3)) for 15 days. Importantly, hypothyroidism induced liver Gsta mRNA (>500%) and protein levels (70%; P < 0.01), indicating an important role of baseline thyroid hormone levels to repress this gene; however, surprisingly, no differences were seen in hyperthyroid mice. To further investigate Gsta repression by T(3), we used animals expressing a naturally occurring mutation of the gene for thyroid hormone receptor (TR)-ß (Δ337T), which prevents T(3) binding and causes a general resistance to thyroid hormone. At baseline, homozygous animals showed increased Gsta levels (mRNA 3.5 times, protein 1.3 times) similar to those found in hypothyroid animals. After a T(3) suppression test, we found a blunted response of liver Gsta after the lower doses of T(3) in homozygous animals, as expected. However, after the highest dose of T(3), we observed a decrease in Gsta expression (80%), similar to normal animals, explained by a higher expression of TR-α1 (60%; P < 0.01) and a lower expression of Src1 (steroid coactivator receptor) in the mutant animals (50% decrease). In summary, a decrease in Gsta expression caused by T(3) was observed only in the hypothyroid state. In addition, an essential role of TR-ß1 is to mediate Gsta suppression in response to T(3) and, in the absence of a functional TR-ß, there is a compensatory action of TR-α1 that depends on low levels of Src1.
Assuntos
Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hipotireoidismo/sangue , Hipotireoidismo/genética , Fígado/enzimologia , Tireotropina/sangue , Tri-Iodotironina/sangue , Animais , Feminino , Glutationa Transferase/biossíntese , Hipertireoidismo/sangue , Hipertireoidismo/genética , Hipotireoidismo/enzimologia , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação Puntual , RNA Mensageiro/genética , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/metabolismo , Tireotropina/genética , Tri-Iodotironina/farmacologiaRESUMO
Thyroid hormones (THs) exert a broad range of actions on development, growth, and cell differentiation by both genomic and nongenomic mechanisms. THs regulate lymphocyte function, but the participation of nongenomic actions is still unknown. Here the contribution of both genomic and nongenomic effects on TH-induced division of T cells was studied by using free and noncell permeable THs coupled to agarose (TH-ag). THs-ag led to cell division, but to a lesser extent than free hormones. THs induced nongenomically the rapid translocation of protein kinase C (PKC) ζ isoform to cell membranes, extracellular-signal-regulated kinases (ERK1/2) phosphorylation and nuclear factor-κB (NF-κB) activation. The signaling cascade include sphingomyelinases acting up-stream the activation of PKCζ isoform, while ERK and NF-κB are activated downstream this PKC isoenzyme. Both free and THs-ag increased the protein and mRNA levels of TH nuclear receptor TRα1, while only free hormones incremented the inducible NOS gene and protein levels as well as a calcium independent NOS activity. Both effects were blunted by PKCζ inhibition. These results indicate that THs, by triggering a nongenomic signaling cascade that involves Smases-mediated activation of PKCζ, lead to ERK 1/2 and NF-κB activation and to the genomic increase of TRs and the inducible nitric oxide synthase protein and mRNA levels, improving T lymphocyte proliferation. These finding not only contribute to the understanding of the mechanisms involved in TH modulation of lymphocyte physiology, but would also point out for the first time the interplay between genomic and nongenomic TH actions in T cells.
Assuntos
Proliferação de Células , Óxido Nítrico Sintase Tipo II/metabolismo , Linfócitos T/enzimologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , RNA Mensageiro/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Linfócitos T/efeitos dos fármacos , Receptores alfa dos Hormônios Tireóideos/genética , Fatores de Tempo , Regulação para CimaRESUMO
Understanding the molecular basis of the binding modes of natural and synthetic ligands to nuclear receptors is fundamental to our comprehension of the activation mechanism of this important class of hormone regulated transcription factors and to the development of new ligands. Thyroid hormone receptors (TR) are particularly important targets for pharmaceuticals development because TRs are associated with the regulation of metabolic rates, body weight, and circulating levels of cholesterol and triglycerides in humans. While several high-affinity ligands are known, structural information is only partially available. In this work we obtain structural models of several TR-ligand complexes with unknown structure by docking high affinity ligands to the receptors' ligand binding domain with subsequent relaxation by molecular dynamics simulations. The binding modes of these ligands are discussed providing novel insights into the development of TR ligands. The experimental binding free energies are reasonably well-reproduced from the proposed models using a simple linear interaction energy free-energy calculation scheme.
Assuntos
Simulação de Dinâmica Molecular , Receptores dos Hormônios Tireóideos/química , Receptores dos Hormônios Tireóideos/metabolismo , Ligantes , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Receptores alfa dos Hormônios Tireóideos/química , Receptores alfa dos Hormônios Tireóideos/metabolismoRESUMO
The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics simulations of the TRalpha and TRbeta LBDs in the absence and in the presence of the natural ligand Triac. The simulations show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary structure elements, while the structure remains essentially compact, resembling a molten globule state. This differs from most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TRalpha and TRbeta subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H11, and the interaction of the region between H1 and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.
Assuntos
Receptores alfa dos Hormônios Tireóideos/química , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/química , Receptores beta dos Hormônios Tireóideos/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Desnaturação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
Nuclear receptors are important targets for pharmaceuticals, but similarities between family members cause difficulties in obtaining highly selective compounds. Synthetic ligands that are selective for thyroid hormone (TH) receptor beta (TRbeta) vs. TRalpha reduce cholesterol and fat without effects on heart rate; thus, it is important to understand TRbeta-selective binding. Binding of 3 selective ligands (GC-1, KB141, and GC-24) is characterized at the atomic level; preferential binding depends on a nonconserved residue (Asn-331beta) in the TRbeta ligand-binding cavity (LBC), and GC-24 gains extra selectivity from insertion of a bulky side group into an extension of the LBC that only opens up with this ligand. Here we report that the natural TH 3,5,3'-triodothyroacetic acid (Triac) exhibits a previously unrecognized mechanism of TRbeta selectivity. TR x-ray structures reveal better fit of ligand with the TRalpha LBC. The TRbeta LBC, however, expands relative to TRalpha in the presence of Triac (549 A(3) vs. 461 A(3)), and molecular dynamics simulations reveal that water occupies the extra space. Increased solvation compensates for weaker interactions of ligand with TRbeta and permits greater flexibility of the Triac carboxylate group in TRbeta than in TRalpha. We propose that this effect results in lower entropic restraint and decreases free energy of interactions between Triac and TRbeta, explaining subtype-selective binding. Similar effects could potentially be exploited in nuclear receptor drug design.