RESUMEN
Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic ß-cells. ER stress or ROS causes c-Jun N-terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi-stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual-luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in ß-cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p-JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome-dependent manner. We found that NR4A1 increased the expression of cbl-b (an E3 ligase); knocking down cbl-b expression increased MKK4 and p-JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl-b promoter by physical association. We further confirmed that cbl-b expression in ß-cells was reduced in NR4A1-knockout mice compared with WT mice. NR4A1 down-regulates JNK activation by ER stress or ROS in ß-cells via enhancing cbl-b expression.
Asunto(s)
Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , UbiquitinaciónRESUMEN
Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor with diverse functions. It has been reported that NR4A1, as a transcriptional activator, is implicated in glucose and lipid metabolism. The aim of this study was to investigate the regulatory role of NR4A1 in adipogenesis and explore the underlying mechanisms. Quantitative real-time PCR and Western blotting were used to analyse the expression of genes involved in synthesis and mobilization of fats in vivo and in vitro. Dual-luciferase reporter assay was conducted to study the regulatory mechanisms of NR4A1. Our data from in vivo study confirmed that NR4A1 knockout (KO) mice fed with high-fat diet were more prone to obesity, and gene expression levels of PPARγ and FAS were increased in KO mice compared to controls; our data from in vitro study showed that NR4A1 overexpression in 3T3-L1 pre-adipocytes inhibited adipogenesis. Moreover, NR4A1 enhanced GATA binding protein 2 (GATA2) expression, which in turn inhibited peroxisome proliferator-activated receptor γ (PPARγ); NR4A1 inhibited sterol regulatory element binding transcription factor 1 (SREBP1) and its downstream gene fatty acid synthase (FAS) by up-regulating p53. NR4A1 inhibits the differentiation and lipid accumulation of adipocytes by enhancing the expression of GATA2 and p53.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis/genética , Factor de Transcripción GATA2/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Obesidad/genética , Proteína p53 Supresora de Tumor/genética , Células 3T3-L1 , Adipocitos/citología , Animales , Secuencia de Bases , Diferenciación Celular/genética , Dieta Alta en Grasa/efectos adversos , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Factor de Transcripción GATA2/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Metabolismo de los Lípidos/genética , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/deficiencia , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , PPAR gamma/genética , PPAR gamma/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Under adverse conditions, such as sustained or chronic hyperglycemia or hyperlipidemia, ROS (reactive oxygen species) or/and ER-stress (endoplasmic reticulum stress) will be induced in pancreatic ß cells. ROS or ER-stress damages ß-cells even leads to apoptosis. Previously we found ROS or ER-stress resulted in JNK activation in ß cells and overexpressing NR4A1 in MIN6 cells reduced JNK activation via modulating cbl-b expression and subsequent degrading the upstream JNK kinase (MKK4). To search other possible mechanisms, we found the mRNA level and protein level of MKP7 (a phosphatase for phospho-JNK) were dramatic reduced in pancreatic ß cells in the islets from NR4A1 KO mice compared with that from wild type mice. To confirm what we found in animals, we applied pancreatic ß cells (MIN6 cells) and found that the expression of MKP7 was increased in NR4A1-overexpression MIN6 cells. We further found that knocking down the expression of MKP7 increased the p-JNK level in pancreatic ß cells upon treatment with TG or H2O2. After that, we figured out that NR4A1 did enhance the transactivation of the MKP7 promoter by physical association with two putative binding sites. In sum, NR4A1 attenuates JNK phosphorylation incurred by ER-stress or ROS partially via enhancing MKP7 expression, potentially decreases pancreatic ß cell apoptosis induced by ROS or ER-stress. Our finding provides a clue for diabetes prevention.