Role of FLASH in caspase-8-mediated activation of NF-kappaB: dominant-negative function of FLASH mutant in NF-kappaB signaling pathway.

Caspase-8 is the most receptor-proximal, upstream caspase in the caspase cascade and plays a key role in cell death triggered by various death receptors. Here, we addressed the role of endogenous caspase-8 in tumor necrosis factor (TNF)-alpha-induced activation of NF-kappaB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-alpha-induced activation of NF-kappaB in NIH3T3, HeLa, and HEK293 cells as determined with luciferase reporter gene and cell fractionation assays. Reconstitution of caspase-8-deficient C33A cells with processing-defective (P/D) mutant of caspase-8 sensitized the cells to TNF-alpha for NF-kappaB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-kappaB and to bind FLICE-associated huge protein (FLASH) in vitro and in vivo. Instead, caspase-8 (D73A) mutant bound to caspase-8 and blocked NF-kappaB activation triggered by TNF-alpha and caspase-8. In addition, expression of an NF-kappaB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-alpha and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-kappaB. Further, immunoprecipitation assays showed that caspase-8 formed triple complex with TRAF2 and FLASH. Taken together, these results suggest that endogenous caspase-8 mediates TNF-alpha-induced activation of NF-kappaB via FLASH.