Necrosis Through RIP

Abstract

If caspase activation is blocked, cells cannot undergo apoptosis but may still die through a process of necrotic cell death. Temkin et al. investigated the mechanism by which cell death occurred in response to activation of the death receptors in the presence of the nonspecific caspase inhibitor zVADfmk. Cell death caused by exposure of the cells to tumor necrosis factor-α (TNF-α) and zVADfmk was inhibited if receptor-interacting protein (RIP) abundance was diminished by geldanamycin or RNA interference (RNAi). In TNF-treated cells, RIP colocalized with mitochondria both in isolated mitochondria and by microscopic techniques. In cells exposed to both TNF-α and zVADfmk, the activity of mitochondrial adenine nucleotide translocase (ANT) was inhibited and adenosine triphosphate (ATP) concentrations dropped. Although reactive oxygen species were also produced, addition of antioxidants did not prevent death induced by TNF-α together with zVADfmk. ANT appeared to be a direct target of zVADfmk, because biotinylated zVADfmk was associated with ANT in the TNF-α-treated cells but not in cells in which RIP was decreased. Addition of zVADfmk to mitochondria isolated from TNF-α-treated cells inhibited the activity of ANT in two different assays. Cyclophilin D coimmunoprecipitated with ANT in control cells; however, TNF-α alone disrupted this interaction. Overexpression of cyclophilin D protected the cells from death induced by TNF-α+zVADfmk, and reduction of cyclophilin D by RNAi sensitized the cells to death induced by zVADfmk in the absence of TNF-α. Cyclophilin D and zVADfmk appeared to compete for the same interaction site on ANT. Thus, the authors propose a model whereby activation of a surface death receptor stimulates the translocation of the kinase RIP to the mitochondria, which results in displacement of cyclophilin D from ANT through an as-yet-undefined mechanism. In the presence of zVADfmk, the translocase activity of ANT is inhibited and ATP concentration falls, leading to necrotic cell death. These results have important implications for interpreting experiments using the inhibitor zVADfmk, because it not only inhibits apoptotic cell death through inhibition of caspases but also can promote necrotic cell death in the presence of death receptor signaling through inhibition of ANT. V. Temkin, Q. Huang, H. Liu, H. Osada, R. M. Pope, Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis. Mol. Cell. Biol. 26 , 2215-2225 (2006). [Abstract] [Full Text]