Rip3 Mediated Cellular ROS Accumulation Is Involved In TNF-α Induced Caspase-independent Cell Death

As a proinflammatory cytokine,TNF-αplays an important role in many cellular events.TNF-αelicits its biological effects by binding to the receptors,TNFRⅠand TNFRⅡ.But only TNFRⅠis a death receptor.By binding to the receptor TNFRⅠ, TNF-αinduces both apoptotic and necrotic cell death,depending on cell types and treatment condition.It means that TNF-αcan induce cell death through distinct signal transduction pathways.It has been extensively studied that the mechanisms of TNF-induced apoptotic cell death,which is mediated by TRADD,FADD and caspase-8.Caspases are required for apoptotic cell death.In some types of cells, TNF-αcan induced caspase-independent cell death,of which mechanism is still largely unknown.Further study elucidating the mechanism of TNF-induced cell death and difference and relationship among different signal pathways will facilitate our understanding of cell death control and be helpful for clinical treatment.In this study,we used two cell lines derived from NIH3T3 cells:NIH3T3(R) and NIH3T3(S).In NIH3T3(R) cell,TNF-αcan induce apoptosis which requires caspases; but in NIH3T3(S) cell,under a caspase-inhibited condition,TNF-αcan effectively induce cell death through an unknown signal pathway.We believe that these two cell lines would be good material for studying these two distinct mechanisms of the caspase-dependent or caspase-independent cell death induced by TNF-αat the same time,because which differentiated from the same ancestor in the long culture process. Large-scale gene expression profiles between these two cell lines were detected with cDNA microarray.As a result,one gene,namely Rip3,is selected for further study, which was detected only in NIH3T3(S) cell not in NIH3T3(R) cell in mRNA level, which was confirmed by RT-PCR.Rip3 is the member of the Rip family,which processes N-terminal Ser/Thr kinase domain,RHIM and a unique C-terminus.Rip1,belonging to the Rip family too,is an important NF-κB-activating molecule mediated by TNFR I or Trif.Rip3,via its RHIM,interacts with,and subsequently phosphorylates Rip1,resulting in diminished NF-κB activation.In addition,overexpression of Rip3 in some cell lines can induce apoptosis,which requires the C-terminus of Rip3.But additional studies are required to better understand the function of Rip3.This thesis reveals a novel role for Rip3,which is crucial in mediating ROS accumulation in TNF-induced caspase-independent cell death.And generation of cytocidal ROS requires an aerobic glycolytic pathway.This result will facilitate our understanding of the mechanism of TNF-induced cell death and improve the value in clinical application of TNF.