TRADD Mediates Tumor Necrosis Factor Alpha-induced L929 Cell Necroptosis In The Absence Of RIP1 And Apoptosis In The Absence Of RIP3

Necroptosis,also named programmed necrosis,is a new kind of programmed cell death,which has been identified to be a tightly controlled,active process with a well-established regulatory mechanism.Moreover,increasing amounts of evidence indicate that necroptosis is immunogenic and is critically involved in vital physiological and pathological processes.A broad range of extracellular stimuli,including H2O2,genotoxic stressors,anti-cancer drugs and tumor necrosis factor α(TNFα),can induce necroptosis.In particular,TNFα-induced necroptosis has been widely and intensively studied and documented.TNFα is a pleiotropic inflammatory cytokine and plays an important role in multiple cellular functions,including cell proliferation,differentiation,apoptosis and necroptosis.Upon ligation,TNF receptor 1(TNFR1)recruits several adaptor/effector proteins bearing death domains(DDs)to form a TNFR1 signaling complex known as Complex I,which contains TNF receptor-associated death domain protein(TRADD)and receptor-interacting protein 1(RIP1).Complex I serves as a platform for the recruitment of downstream kinases and effector proteins to initiate NFκB and mitogen-associated protein kinase(MAPK)pathway activation.In cells destined to die,RIP1 dissociates from TNFR1 on cell membrane and translocates to cytoplasm,binds receptor-interacting protein 3(RIP3)to form a “necrosome” and then triggers necrotic cell death by activating the RIP1/RIP3 signaling pathway.RIP1 and RIP3 areserine/threonine protein kinases that activate through direct or indirect mutual phosphorylation in the “necrosome” and then recruit and activate the RIP3 substrate mixed linage kinase domain-like protein(MLKL)through phosphorylation.MLKL phosphorylation promotes its tetramerization and subsequent translocation to the plasma membrane,where the MLKL tetramer causes necroptosis by binding phosphatidylinositol phosphate phospholipids,leading to plasma membrane rupture.As the first identified target protein in mediating necroptosis,RIP1 is a serine/threonine kinase and activates through phosphorylation induced by RIP3 or other unidentified protein kinase,so its allosteric inhibitor,Necrostatin-1,efficiently blocks necroptosis in various cellular model or animal disease model via suppressing RIP1 activation.In addition,repression of RIP1 by gene silencing or depletion also blocks TNFα-induced necroptosis in many kinds of necroptotic cellular models,including mouse embryo fibroblast(MEF),HT-29 and 3T3 cells upon ectopically expressed RIP3,further confirming that RIP1 is essential for initiating necroptosis induced by TNFα.However,recently published studies have demonstrated that RIP1 depletion does not inhibit TNFα-induced necroptosis in L929 cells,as well as MEF cells with elevated RIP3.Therefore,TNFα induces a new kind of cell death which bypasses the need of RIP1 in L929 cells,however,the exact type,target protein and detail mechanism in mediating the RIP1-independent necroptosis remains largely unknown.In this study,we explored the target and mechanism in mediating the RIP1-independent necroptosis by using L929 cells,the well-established model of necroptosis.Through infecting L929 cells with lentivirus encoding RIP1 sh RNA,we established RIP1-knockdown L929 cells,whereas downregulation of RIP1 did not inhibit cell death induced by TNFα.In addition,Z-VAD-FMK,the inhibitor of pan-caspase,andcaspase 8 knockdown had no protective effect against the RIP1-independent cell death induced by TNFα,suggesting that TNFα induced RIP1-independent cell death through necroptotic pathway,but not apoptotic pathway.As the phosphorylation and oligomerization of RIP3 and its substrate protein MLKL increased during the necroptotic process in RIP1-knockdown L929 cells,and depletion of RIP3 or MLKL almost fully inhibited the RIP1-independent necroptosis,activation of RIP3/MLKL pathway is critical in mediating TNFα-induced necroptosis in the absence of RIP1.We also found that the activation of RIP3/MLKL pathway and cell death induced by TNFα in RIP1-knockdown L929 cells had been blocked by simultaneously knockdown of TRADD,indicating that TRADD is a critical target protein in mediating RIP1-independent necroptosis.Additionally,TRADD had been found to bind RIP3 and mediated the interaction between RIP3 and TNFR1,so TRADD bridged the necroptotic signal transduction through facilitating the formation of protein complex containing TNFR1 and RIP3.We also found that RIP3 knockdown resulted in a shift from TNFα-induced necroptosis to apoptosis in L929 cells since Z-VAD-FMK,the inhibitor of caspase-dependent apoptosis,significantly inhibited TNFα-induced cell death in RIP3-knockdown L929 cells.Furthermore,activation of caspase pathway had been detected during the process of RIP3-independent cell death,and knockdown of caspase 8 had an inhibitory role against TNFα-induced cell death in RIP3-knockdown L929 cells,further confirming cell died through apoptotic pathway in the absence of RIP3.As TRADD knockdown also blocked RIP3-independent apoptosis,TRADD is critical for initiating RIP3-independent apoptosis.We also found that TRADD bound caspase 8 and facilitated the activation of caspase 8 and the subsequent executor caspase,so TRADD initiated RIP3-independent apoptosis via activating caspase pathway.In conclusion,our study identified TRADD as the target protein in initiatingRIP1-indpendent necroptosis through facilitating the transduction of necroptotic signal from TNFR1 to RIP3,and the subsequent activation of RIP3/MLKL pathway.In addition,TRADD is also critical for mediating apoptosis in RIP3-knockdown L929 cells via activating caspase pathway.Therefore,our study systemically elucidates the compensatory and coordinative roles of RIP1,RIP3 and TRADD in the mediation of programmed cell death in L929 cells upon TNFα stimulation.