AbstractThe role of NF‐xB in regulating FasL‐mediated cytotoxicity was investigated by using lactacystin. Lactacystin is a microbial metabolite known to inhibit only the protease activity of the proteasome, which is required for NF‐xB translocation. When activated by immobilized anti‐CD3 monoclonal antibody, hybridoma T cells (5D5) degraded IxBβ, translocated NF‐xB into the nucleus, transcribed immediate‐early genes and the Fas ligand (FasL) gene, and expressed FasL‐mediated cytotoxicity. Lactacystin strongly blocked IxBβ degradation and the translocation of NF‐xB (p50/RelA heterodimer), but had little effect on the expression of the transcription factors, Oct‐1 and AP‐1. Moreover, lactacystin did not inhibit the nuclear translocation of NF‐ATp whereas cyclosporin A inhibited the translocation of both NF‐xB and NF‐ATp. The expression ofc‐mycandnur77, two immediate‐early genes implicated in FasL gene activation, was blocked by lactacystin. Subsequently, the expression of FasL gene and FasL‐mediated cytotoxicity was inhibited. LLnL, a well‐known peptide aldehyde which inhibits the protease activities of the proteasome and cysteine proteases, also inhibited NF‐xB translocation and FasL‐mediated cytotoxicity. However, these events were not inhibited by the highly specific cysteine protease inhibitor E64. These observations provide further evidence that FasL cytotoxicity is regulated by the proteasome. Furthermore, lactacystin must be added early in order to efficiently inhibit the induction of FasL cytotoxicity, indicating that the early events are critical for FasL gene activation. Our study integrates the proteasome‐dependent IxB degradation and NF‐xB translocation into a T cell activation cascade which results in FasL gene activation and t
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