Abstract
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)-α-induced activation of NF-κB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-α-induced activation of NF-κB 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-α for NF-κB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-κB 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-κB activation triggered by TNF-α and caspase-8. In addition, expression of an NF-κB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-α and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-κB. 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-α-induced activation of NF-κB via FLASH.
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Acknowledgements
We thank Dr P Chaudhary (University of Texas, USA) for caspase-8 (D73A) mutant expression plasmid. J Jun was partially supported by the Brain Korea 21 project. This work was supported by National Research Laboratory program (to YK Jung), 21 C Frontier on Functional Genomics and Brain of the Korean Ministry of Science and Technology.
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Jun, JI., Chung, CW., Lee, HJ. et al. Role of FLASH in caspase-8-mediated activation of NF-κB: dominant-negative function of FLASH mutant in NF-κB signaling pathway. Oncogene 24, 688–696 (2005). https://doi.org/10.1038/sj.onc.1208186
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DOI: https://doi.org/10.1038/sj.onc.1208186
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