1. ¹Department of Life Science, Gwangju Institute of Science and Technology, 1-Oryong-dong, Buk-gu, Gwangju 500-712, Korea
2. ²Korea Research Institute of Bioscience and Biotechnology, 21C Frontier Center, Daejeon 305-600, Korea
3. ³Department of Biology, Yonsei University, Sudaemoon-gu, Seoul 120-749, Korea
4. ⁴Department of Life Science, PaiChai University, Daejeon 302-735, Korea

Correspondence: Y-K Jung, E-mail: ykjung@kjist.ac.kr

Received 18 December 2003; Revised 26 August 2004; Accepted 31 August 2004; Published online 13 December 2004.

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)--induced activation of NF-B. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF--induced activation of NF-B 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- for NF-B activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-B 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-B activation triggered by TNF- and caspase-8. In addition, expression of an NF-B-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF- and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-B. 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--induced activation of NF-B via FLASH.