Aims: Previously we reported that ATF4-mediated endoplasmic reticulum (ER) stress led to endothelial inflammation and retinal vascular leakage under diabetic condition. In this study, we aimed to investigate if ATF4 regulated the inflammatory responses through modulating NLRP3 inflammasome activation in diabetic retinopathy.
Methods: Streptozotocin (STZ) was applied to induced diabetes in mice. Mice were sacrificed at day 15 and day 30 after onset of diabetes, and retinas were collected for analyses. To mimic the diabetic condition in vitro, human retinal microvascular endothelial cells (HRMECs) were exposed to palmitate (50 μM), accompanied with adenoviruses overexpressing human ATF4 or shRNA of ATF4.
Results: ATF4 level was closely correlated with the activation of NLRP3 inflammasome in STZ-diabetic retinas and palmitate-treated HRMECs, followed by increases of inflammatory factors (ICAM-1, VCAM-1 and TNF-α) and loss of endothelial junction proteins (ZO-1, occludin-1 and VE-cadherin). Moreover, diabetes-induced ATF4 and NRLP3 were colocalized in the ganglion cell layer, inner plexiform layer and outer plexiform layer. Overexpression of ATF4 augmented the palmitate-elicited activation of NLRP3 inflammasome, whereas knocking-down of ATF4 abolished the activation. By analyzing the promoter region of NLRP3 gene, three potential binding sites of ATF4 were identified: -1418bp∼-1406bp, -1332bp∼-1320bp, -65∼-53bp. Overexpression of ATF4 potentiated the palmitate-induced nuclear translocation of ATF4, followed by enhanced activation of NLRP3 inflammasome. In addition, treatment with ISRIB, an inhibitor of ER stress, significantly abolished the palmitate-induced elF2α phosphorylation but not NLRP3 activation when ATF4 is overexpressed.
Conclusion: We have demonstrated that ATF4 is a directly regulator of NLRP3 inflammasome, independent of ER stress response, thus providing a novel insight into the pathogenesis of ATF4-elicited diabetic retinopathy.
T. Li: None. X. He: None. R. Peng: None. R. Gao: None. G. Shi: None. Y. Chen: None.
Research and Development Plans in Key Areas of Guangdong Province (2019B020227003); National Natural Science Foundation of China (81770826); National Key Research and Development Program of China (2017YFA0105803)