Lipid rafts uncouple surface expression of transmembrane TNF-α from its cytotoxicity associated with ICAM-1 clustering in Raji cells

Since transmembrane tumor necrosis factor-alpha (tmTNF-α) has been reported to have a palmitoylated site at Cys−47, and therefore its functions may be linked to lipid raft membrane microdomains. The present study tested a hypothesis that lipid rafts may serve as a signaling platform to mediate the bioactivity of tmTNF-α. We found that destruction of lipid rafts with methyl-beta-cyclodextrin (MCD) in Raji cells almost completely blocked the cytotoxicity of tmTNF-α, as did an anti-TNF-α antibody. Although a proportion of tmTNF-α was colocated with lipid rafts, either the replacement of Cys at −47 by Ala, destructing its possible lipid rafts-attaching site or the displacement of its cytoplasmic domain by the C-terminal sequence (131-157) of caveolin-1, making all tmTNF-α target to lipid rafts, had no effect on tmTNF-α cytotoxicity. The data suggest that the cytotoxicity of tmTNF-α is not associated with its lipid rafts location. Unparallel to decreased cytotoxicity, moreover, MCD significantly increased tmTNF-α expression on the cell surface, and these increased tmTNF-α molecules were capable of binding to sTNFR1. To further explore the mechanism of lipid rafts-mediated cytotoxicity of tmTNF-α, we demonstrated that MCD led to a marked decrease in adhesion of Raji cells to T24 cells, which was due to dissociation of adhesion molecule ICAM-1 from lipid rafts. These results indicate that lipid rafts importantly participate in the cytotoxicity of tmTNF-α through ICAM-1 clustering and consequent enhancement of the cell-cell contact. The data suggest that lipid rafts are essential for the killing of tmTNF-α through the cell-cell contact mediated by ICAM-1. However, lipid rafts may limit exposure of tmTNF-α to the cell surface.