TNF receptor-associated factor-2 binding site is involved in TNFR75-dependent enhancement of TNFR55-induced cell death

Shanghai Research Center of Biotechnology, Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences, Shanghai 200233, China

TNF recepter-55 is the main mediator of TNF-induced apoptosis. TNF receptor-75-dependent induction or enhancement of cytotoxicity has been explained by intracellular signaling, ``ligand passing", or induction of endogenous TNF. To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFa-induced cytotoxicity, HEp-2 cells were transfected with bicistronic expression vector of hTR75 and its deletion mutants genes. hTNFa-induced cytotoxicity was determined by crystal violet colorimetric method. The expression of hTR75 and its deletion mutants in HEp-2 cells was demonstrated by RT-PCR and indirect ELISA. We found that the overexpressed hTR75 could significantly increase the susceptibility of HEp-2 cells to hTNFa which especially required TRAF2 binding site. hTR75 could not only mediate partial hTNFa-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity.

Key words: hTNFa, hTNF receptors, TRAF2, cytotoxicity, signal transduction.