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
The aim of this study was to identify the di(n-butyl) phthalate (DBP)-induced differentially expressed genes (DEGs) using a novel annealing control primer system in the testes of Sprague-Dawley male rats. Animals (4 weeks of age) were administered orally either corn oil only (vehicle control) or DBP (250, 500, or 750 mg/kg/day) for 30 days. Total RNA was isolated from the rat testes and GeneFishing PCR was used to determine the differential gene expression levels. Using this technique, a total of 59 DEG mRNA fragments were observed in the testes treated with DBP 750 mg/kg/day compared to vehicle control. Of these 59 genes, 31 genes were significantly altered after exposing rats to high dose DBP (750 mg/kg/day), and their sequences cloned. Based on the Basic Local Alignment Search Tool (BLAST), 4 expressed sequence tags (EST), 27 cloned genes (Insl3, pgrp, H1SHR, etc.) and 3 genes (LDHA, lactate dehydrogenase A; Spag4, sperm associated antigen 4 and PBR, peripheral-type benzodiazepine receptor) were found to be involved in spermatogenesis and steroidogenesis. In addition, the expression patterns of the steroidogenesis-related genes such as scavenger receptor class B-1 (SR-B1), steroidogenic acute regulated protein (StAR), P450 side chain cleavage (P450scc), CYP17, and CYP19 were further analyzed by RT-PCR. Significant increases in the mRNA levels of steroidogenesis-related genes (PBR, SR-B1, StAR, P450scc, and CYP17) were observed in the high dose DBP-treated rats. However, DBP significantly decreased the CYP19 mRNA levels compared with controls. DBP (750 mg/kg/day) significantly increased the TR-alpha1 and PPARgamma expression in testes, whereas the AR and ERbeta protein levels were significantly reduced in the same group. These data indicate that the steroidogenesis- or spermatogenesis-related genes identified in this study may provide insights into the molecular mechanisms underlying environmental pollutants-mediated male infertility.
