Kinase RIP3 Is An Essential Regulator In The TNF-Induced Necrosis

Cell death is a crucial process for development and tissue homeostasis in multicellular organisms by eliminating unwanted and harmful cells.Apoptosis,a form of programmed cell death,is the principal mechanism by which cells are physiologically removed.In contrast to apoptosis,necrosis was originally described as an accidental and passive form of cell death. However,recent evidences suggest necrosis is also well controlled and programmed,and plays a prominent role in pathological conditions including ischemia and neurodegeneration. Understanding the molecular mechanisms of programmed necrosis and exploring potent inhibitors could lead to powerful therapeutic strategies for these devastating diseases. Tumor necrosis factor(TNF)αis a pleiotropic factor involved in diverse cellular responses, including inflammation,apoptosis and necrosis.Previously,our lab has shown that Smac mimetic,a small molecule mimic of Smac/Diablo protein,induces TNFα-dependent apoptosis in a receptor interacting protein kinase 1(RIPK1)-dependent manner.Interestingly,when apoptosis was bloCked by a pan- caspase inhibitor,z-VAD,cell death phenotype was switched from apoptosis to necrosis in certain cell lines.This Smac mimetic induced necrosis is also TNFαand RIPK1-dependent.Using the high-throughput RNA interference(RNAi) screening assay,we identified RIP3,a serine-threonine kinase and homolog of RIPK1,as an essential regulator in the TNFα-induced necrosis.Stable expression of shRNA targeting RIP3 blocked necrotic cell death while the cell death phenotype was rescued by introducing a shRNA-resistant wild type form of RIP3,but not a kinase deficient RIP3.Upon necrotic stimuli,a protein complex containing both RIPKI and RIP3 was formed.Overexpression of a kinase-dead form of RIP3 blocked necrosis by sequestering endogenous RIPK1.We also noted that RIP3 was selectively expressed in certain cell lines and the endogenous expression of RIP3 correlates very well with the cellular response to TNFα-induced necrosis.Furthermore,ectopic expression of RIP3 rendered the resistant cells to TNFα-induced necrosis.RIP3 knockout mice were generated to further investigate the physiological functions of RIP3.Consistently,RIP3-/- MEF was resistant to TNFα-induced necrosis.RIP3 knockout mice are devoid from inflammation inflicted tissue damage in an acute pancreatitis model.These findings demonstrate that RIP3 is a key determinant for TNFα-induced necrotic cell death and therefore offers a new drug target for evading necrotic damage.In addition,we screened a chemical library of 200,000 compounds for the chemical inhibitor of the TNFα-induced necrosis.More than 20 compounds exerted profound inhibition on the TNFα-induced necrosis by interfering with necrosis at different stages of the signaling pathway and provide novel opportunities to develop successful therapies.Further insight into the specific targets of the compounds will uncover novel molecules in the regulation of necrosis.