RESUMO
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.
Assuntos
Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , UbiquitinaçãoRESUMO
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.