Gao Weina, Fu Yuchang, Yu Cong, Wang Shunke, Zhang Yuchao, Zong Chen, Xu Tongfu, Liu Yong, Li Xia, Wang Xiangdong
Department of Cell Biology, Shandong University School of Medicine, Jinan, China; School of Medical Technology and Engineering, Henan University of Science and Technology, Luoyang, China.
Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
PLoS One. 2014 Mar 17;9(3):e91462. doi: 10.1371/journal.pone.0091462. eCollection 2014.
NR4A3/NOR-1 is a member of the NR4A orphan nuclear receptor subfamily, which contains early response genes that sense and respond to a variety of stimuli in the cellular environment. The role of NR4A3 in insulin expression in pancreatic beta cells remains unknown.
Dynamic changes in NR4A3 were examined in a pancreatic beta-cell line, MIN6, treated with thapsigargin (TG), palmitate (PA), tunicamycin (TM), and dithiothreitol (DTT), chemicals that produce cell stress and even apoptosis. We exploited virus infection techniques to induce expression of NR4A3 or three deletion mutants, and determined expression of insulin and insulin regulatory genes in MIN6 cells.
TG and PA, two endoplasmic reticulum (ER) stress inducers, were able to induce unfolded protein response (UPR) activation and elevation of NR4A3 expression in MIN6 cells, whereas TM and DTT, two other ER stress inducers, were able to induce UPR activation but not NR4A3 elevation. MIN6 cells over-expressing NR4A3 protein after adenoviral infection exhibited reduced transcription of the insulin genes Ins1 and Ins2, and reduced insulin protein secretion, which were negatively correlated with NR4A3 expression levels. Functional analysis of different deletion mutants of NR4A3 showed that deleting the activation domain AF1 or the DNA-binding domain abolished the down-regulation of insulin transcription by NR4A3 in MIN6 cells, indicating that this down-regulative role was closely related to the NR4A3 trans-activation activity. Over-expression of NR4A3 in MIN6 cells resulted in reduced mRNA transcription of the insulin positive-regulation genes, Pdx1 and NeuroD1.
Some ER stress inducers, such as TG or PA, are able to elevate NR4A3 expression in MIN6 cells, while others, such as TM or DTT, are not. Over-expression of NR4A3 in MIN6 cells results in down-regulation of insulin gene transcription and insulin secretion. NR4A3 reduces insulin gene expression by modulating the expression of Pdx1 and NeuroD1.
NR4A3/NOR-1是NR4A孤儿核受体亚家族的成员，该亚家族包含可感知并响应细胞环境中多种刺激的早期反应基因。NR4A3在胰腺β细胞胰岛素表达中的作用尚不清楚。
在经毒胡萝卜素（TG）、棕榈酸（PA）、衣霉素（TM）和二硫苏糖醇（DTT）处理的胰腺β细胞系MIN6中检测NR4A3的动态变化，这些化学物质会产生细胞应激甚至凋亡。我们利用病毒感染技术诱导NR4A3或三种缺失突变体的表达，并测定MIN6细胞中胰岛素和胰岛素调节基因的表达。
两种内质网（ER）应激诱导剂TG和PA能够诱导MIN6细胞中未折叠蛋白反应（UPR）激活和NR4A3表达升高，而另外两种ER应激诱导剂TM和DTT能够诱导UPR激活但不能使NR4A3升高。腺病毒感染后过表达NR4A3蛋白的MIN6细胞表现出胰岛素基因Ins1和Ins2的转录减少以及胰岛素蛋白分泌减少，这与NR4A3表达水平呈负相关。对NR4A3不同缺失突变体的功能分析表明，缺失激活域AF1或DNA结合域可消除NR4A3对MIN6细胞中胰岛素转录的下调作用，表明这种下调作用与NR4A3的反式激活活性密切相关。MIN6细胞中NR4A3的过表达导致胰岛素正调控基因Pdx1和NeuroD1的mRNA转录减少。
一些ER应激诱导剂，如TG或PA，能够使MIN6细胞中NR4A3表达升高，而其他一些，如TM或DTT，则不能。MIN6细胞中NR4A3的过表达导致胰岛素基因转录和胰岛素分泌下调。NR4A3通过调节Pdx1和NeuroD1的表达来降低胰岛素基因表达。
