The stress-response molecule NR4A1 resists ROS-induced pancreatic ß-cells apoptosis via WT1.

Pancreatic ß-cells often face endoplasmic reticulum stress and/or ROS-associated oxidative stress under adverse conditions. Our previous work has verified that NR4A1 protects pancreatic ß-cells from ER-stress induced apoptosis. However, It remains unknown whether NR4A1 is able to protect pancreatic ß-cells against ROS-associated oxidative stress. In the present study, our data showed that NR4A1 protein expression rapidly increased in MIN6 cells upon H2O2 treatment, and overexpression of NR4A1 in MIN6 cells conferred resistance to cell apoptosis induced by H2O2. These results were further substantiated in isolated islets from mice infected with an adenovirus overexpressing NR4A1. 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used as a biomarker for oxidative stress or a marker for ROS damage. We found that the 8-OHdG level in the islets from NR4A1 knockout mice fed with high-fat diet was much higher than that in the islets from parental control mice; and higher apoptotic rate was observed in the islets from NR4A1 KO mice compared to control mice. Further investigation of underlying mechanisms of NR4A1's protective effects showed that NR4A1 overexpression in MIN6 cells reduced Caspase 3 activation caused by H2O2, and increased expression of WT1 and SOD1. There is a putative NR4A1 binding site (-1118bp to -1111bp) in WT1 promoter; our data demonstrated that NR4A1 protein physically associates with the WT1 promoter, and enhanced WT1 promoter transactivation and knockdown of WT1 in MIN6 cells induced apoptosis. These findings suggest that NR4A1 protects pancreatic ß-cells against H2O2 mediated apoptosis by up-regulating WT1 expression.