Abstract
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.
Highlights
Based on its morphological and biochemical features, programmed cell death has been classified into several distinct forms, including apoptosis, necroptosis and autophagic cell death[1,2]
Caspase 8 activity was increased in Receptor-interacting protein kinase 3 (RIP3) knockdown L929 cells but did not exhibit a significant change in the negative control L929 cells following TNFα stimulation (Fig. 1C), further confirming that RIP3 knockdown promotes the activation of the caspase pathway
Because caspase 8 is a key initiator of apoptosis induced by TNF receptor 1 (TNFR1) ligation, we determined the role of caspase 8 in the RIP3-independent cell death process
Summary
Based on its morphological and biochemical features, programmed cell death has been classified into several distinct forms, including apoptosis, necroptosis and autophagic cell death[1,2]. By recruiting the adaptor protein Fas-associated death domain (FADD) and pro-caspase 8, Complex II initiates apoptosis by activating the caspase pathway[16,18,19,20]. In cells expressing high levels of receptor-interacting protein 3 (RIP3), RIP1 binds RIP3 to form a “necrosome” and triggers necrotic cell death by activating the RIP1/RIP3 signaling pathway[8,17,21]. The RIP3 knockdown and negative control L929 cells were treated with or without TNFα for 12 h and harvested to measure the activity of caspase 8. Ectopic expression of RIP3 in HeLa or 3T3 cells promotes the activation of the necroptotic signaling pathway, resulting in a shift from TNFα-induced apoptosis to necroptosis[26,27]. The exact target and detailed mechanisms involved in initiating the RIP3-independent cell death are unclear
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