Caspase inhibition sensitizes inhibitor of NF-κB kinase β-deficient fibroblasts to caspase-independent cell death via the generation of reactive oxygen species

Cells lacking functional NF-kappa B die after ligation of some tumor necrosis factor (TNF) receptor family members through failure to express NF-kappa B-dependent anti-apoptotic genes. NF-kappa B activation requires the I kappa B kinase (IKK) complex containing two catalytic subunits named IKK alpha and IKK beta that regulate distinct NF-kappa B pathways. IKK beta is critical for classical signaling that induces pro- inflammatory and anti-apoptotic gene profiles, whereas IKK alpha regulates the non-canonical pathway involved in lymphoid organogenesis and B- cell development. To determine whether IKK alpha and IKK alpha differentially function in rescuing cells from death induced by activators of the classical and non-canonical pathways, we analyzed death after ligation of the TNF and lymphotoxin-receptors, respectively. Using murine embryonic fibroblasts (MEFs) lacking each of the IKKs, the caspase inhibitor benzyloxycarbonyl- Val-Ala-Asp-fluoromethyl ketone, and dominant negative Fas-associated death domain protein, we found that deletion of these kinases sensitized MEFs to distinct cell death pathways. MEFs lacking IKK alpha were sensitized to death in response to both cytokines that was entirely caspase-dependent, demonstrating that IKK beta functions in this process. Surprisingly, death of IKK beta(-/-) MEFs was not blocked by caspase inhibition, demonstrating that IKK beta negatively regulates caspase-independent cell death (CICD). CICD was strongly activated by both TNF and lymphotoxin-beta receptor ligation in IKK beta(-/-) MEFs and was accompanied by loss of mitochondrial membrane potential and the generation of reactive oxygen species. CICD was inhibited by the anti- oxidant butylated hydroxyanosole and overexpression of Bcl-2, neither of which blocked caspase-dependent apoptosis. Our findings, therefore, demonstrate that both IKK alpha and IKK beta regulate cytokineinduced apoptosis, and IKK beta additionally represses reactive oxygen species- and mitochondrial- dependent CICD.