Identification The Role And Mechanism For PI3K In Mediating Necroptosis Induced By Tumor Necrosis Factor

Background: Necroptosis is a new kind of regulated cell death with necrotic morphological features,including cellular vacuoles,organelles swelling and cell membrane rupture.Just like apoptosis,necroptosis also plays a critical role in some physiological and pathological process,such as embryo development,ischemia-reperfusion injury and inflammation.Therefore,identification of the new protein in mediating necroptosis would provide novel target for the medicine design and clinical treatment of disease mediated by necroptosis.Necroptosis can be initiated by many kinds of necroptotic stimulus,including ligands of death receptors,virus infection,and the activator of Toll like receptors,but TNF?-induced necroptosis has been widely and intensively studied and has become a classic model for the study of necroptosis.TNF? first binds to the receptor TNFR1 on the cell membrane,which promotes the conformational change of the intracellular domain of TNFR1,thereby recruiting and binding to various proteins such as RIP1 and TRADD to form a TNFR1 signaling complex known as Complex I.As an E3 ligase,cIAP1 mediates RIP1 ubiquitination,which facilitates RIP1 to binds TAB1 and TAK1 to form new protein complex to activate NF-?B signaling pathway,leading to cell survival or proliferation.In cells destined to die,RIP1 was deubiqtuitinated by CYLD,and then dissociates from TNFR1 and recruit other proteins to form a secondary protein complex known as Complex II.By recruiting the adaptor protein FADD and pro-caspase 8,Complex II initiates apoptosis by activating the caspase pathway.In contrast,when caspase 8 is blocked by the caspase inhibitor Z-VAD or in cells expressing high levels of RIP3,RIP1 binds RIP3 through the RHIM domain to form a “necrosome” and then triggers necroptosis.In addition,the oligomerization of RIP1 and RIP3 was also mediated by their RIP homology motifs(RHIMs)or the oxidation of cysteine disulfide bond induced by reactive oxygen species.Activated RIP3 recruits and phosphorylates its downstream target protein MLKL.Phosphorylated MLKL forms oligomer and translocates to membrane,leading to necroptosis by disrupting plasma membrane structure.Therefore,RIP1,RIP3 and MLKL are critical proteins in initiating necroptosis induced by TNF? alone or plus with other compounds.Phosphatidylinositol-3-kinase(PI3K)is an intracellular phosphatidylinositol kinase,which has both phosphatidylinositol kinase activity and serine/threonine kinase activity.PI3 K can be classified into 3 types according to its structure and substrate specificity.Among them,type I PI3 K is the most widely studied subtype,which is composed of the regulatory subunit p85 and the catalytic subunit p110.PI3 K can activate AKT through the activity of phosphatidyl inositol kinase,which promotes the survival and proliferation of cells and inhibits apoptosis.It also directly activates SGK and PKC and other protein kinases through serine/threonine protein kinase activity to promote cell migration and survival.However,recent studies have found that the PI3 K inhibitor LY294002 can block the programmed necrosis of L929 cells induced by TNF?.Therefore,PI3 K may be an important target protein for regulating necroptosis,but the exact mechanism is not clear.Objective: To identify the exact role of PI3 K in mediating necroptosis induced by TNF?,and then explore the detail mechanism for PI3 K in initiating necroptosis.Our results will reveal new mechanism of necroptosis,and provide a new target and idea for the drug design and clinical treatment of necroptosis-related diseases.Methods:In this study,necroptosis was induced in L929 cells treated with TNF? with or without Z-VAD,and then determined by microscopy and flow cytometry.The lentivirus-mediated RNA interference technique was used to construct the p110? knockdown,RIP1 knockdown and RIP1 and p110? double knockdown L929 cell line.Western blot and RT-PCR was used to determine the knockdown efficiency.In addition,Western blot was also used to detect the phosphorylation and oligomerization level of RIP1,RIP3 and MLKL in L929 cells treated with or without TNF? plus Z-VAD.Duolink assay kit was used to detect the interaction between different proteins or the same proteins.Results:In the first part of the study,we found that TNF?-induced necroptosis was inhibited by PI3 K inhibitors,LY294002 and A66,indicating that PI3 K may be the target protein in initiating necroptosis.Moreover,knockdown of PI3 K catalytic subunit,p110?,blocked L929 cell necroptosis induced by TNF? alone or plus Z-VAD,further confirming the critical role of PI3 K in mediating TNF?-induced necroptosis.In addition,we found that the inhibitors of AKT also protected L929 cells from TNF?-induced necroptosis,suggesting that AKT was also essential for TNF?-induced necroptosis.As AKT is the downstream target protein of PI3 K,PI3K may initiate necroptosis through activated AKT.However,AKT inhibitors have no protective effect against necroptosis of L929 cells induced by TNF? and Z-VAD,suggesting that PI3 K may mediate necroptosis induced by TNF? and Z-VAD through other mechanisms.Therefore,in the second part of the study,we further identified the specific mechanism of PI3K-mediated necroptosis induced by TNF? and Z-VAD.Our results demonstrates that phosphorylation and oligomerization of RIP1,RIP3 and MLKL induced by TNF? plus Z-VAD during the process of necroptosis was reversed by p110? knockdown,indicating that PI3 K may mediated necroptosis induced by TNF? plus Z-VAD through initiating the activation of RIP1/RIP3/MLKL signaling pathway.In addition,p110? knockdown also suppressed the interaction between RIP1 and RIP3 and the subsequent necrosome formation following TNF? plus Z-VAD stimulation,which may contribute to the inhibitory effect of p110? knockdown against RIP1/RIP3/MLKL signaling pathway activation.We also found that the phosphorylation level of RIP1 and RIP3 was decreased in p110? knockdown L929 cells,Which was essential for the interaction between RIP1 and RIP3.So p110? knockdown may suppress necrosome formation through inhibiting RIP1 and RIP3 autophosphorylation.Finally,we found that p110? bound RIP3,but not RIP1,to form new protein complex in response to TNF? plus Z-VAD stimulation,so it can not exclude the possibility that p110? activated RIP3 through direct phosphorylation to initiate necroptosis.Conclusion:In this study,PI3 K was identified as a new target protein in mediating necroptosis induced by TNF? or TNF? plus Z-VAD.As TNF?-induced necroptosis was suppressed by both PI3 K inhibitors and AKT inhibitors,so PI3 K may mediate TNF?-induced necroptosis through activating its downstream target protein AKT.In addition,PI3 K was essential for the necrosome formation and the subsequent activation of RIP1/RIP3/MLKL signaling pathway during the process of necroptosis induced by TNF? plus Z-VAD,which may be another important mechanism for PI3 K in mediating necroptosis.