Reactive oxygen species regulate Smac mimetic/TNFα-induced necroptotic signaling and cell death

Necroptosis represents a key programmed cell death pathway involved in various physiological and pathophysiological conditions. However, the role of reactive oxygen species (ROS) in necroptotic signaling has remained unclear. In the present study, we identify ROS as critical regulators of BV6/tumor necrosis factor-alpha (TNF alpha)-induced necroptotic signaling and cell death. We show that BV6/TNF alpha-induced cell death depends on ROS production, as several ROS scavengers such as butylated hydroxyanisole, N-acetylcysteine, alpha-tocopherol and ethyl pyruvate significantly rescue cell death. Before cell death, BV6/TNF alpha-stimulated ROS generation promotes stabilization of the receptor-interacting protein kinase 1 (RIP1)/RIP3 necrosome complex via a potential positive feedback loop, as on the one hand radical scavengers attenuate RIP1/RIP3 necrosome assembly and phosphorylation of mixed lineage kinase domain like (MLKL), but on the other hand silencing of RIP1 or RIP3 reduces ROS production. Although MLKL knockdown effectively decreases BV6/TNF alpha-induced cell death, it does not affect RIP1/RIP3 interaction and only partly reduces ROS generation. Moreover, the deubiquitinase cylindromatosis (CYLD) promotes BV6/TNF alpha-induced ROS generation and necrosome assembly even in the presence of BV6, as CYLD silencing attenuates these events. Genetic silencing of phosphoglycerate mutase 5 or dynamin-related protein 1 (Drp1) fails to protect against BV6/TNF alpha-induced cell death. By demonstrating that ROS are involved in regulating BV6/TNF alpha-induced necroptotic signaling, our study provides new insights into redox regulation of necroptosis.