TRADD mediates the tumor necrosis factor-induced apoptosis of L929 cells in the absence of RIP3.

Receptor-interacting protein kinase 3 (RIP3) is a critical initiator in mediating necroptosis induced by tumor necrosis factor alpha (TNFα) in L929 cells, so knockdown of RIP3 inhibits TNFα-induced L929 cell necroptosis. However, RIP3 knockdown was shown to switch TNFα-induced necroptosis to apoptosis in L929 cells in other studies. Therefore, whether RIP3 knockdown blocks the TNFα-induced death of L929 cells is controversial. In this study, TNFα activated caspase pathway and induced cell death in RIP3 knockdown L929 cells, and the RIP3-independent cell death had been blocked by Z-VAD-FMK (pan-caspase inhibitor) or caspase 8 knockdown, demonstrating that RIP3 knockdown switched TNFα-induced necroptosis to caspase-dependent apoptosis. Although both TNF receptor type 1-associated death domain protein (TRADD) and RIP1 have been reported to mediate TNFα-induced apoptosis, the knockdown of TRADD, but not RIP1, suppressed TNFα-induced activation of the caspase pathway and subsequent apoptosis in RIP3 knockdown L929 cells. In addition, TRADD bound and activated caspase 8 during the RIP3-independent apoptosis process, indicating that TRADD initiates RIP3-independent apoptosis by activating the caspase pathway. Collectively, we identified the target and mechanism underlying RIP3-independent apoptosis and elucidated the coordinated roles of RIP3 and TRADD in mediating the programmed cell death of L929 cells following TNFα stimulation.