MOLECULAR MECHANISMS OF TNF-ALPHA CYTOTOXICITY - ACTIVATION OF NF-KAPPA-B AND NUCLEAR TRANSLOCATION

The murine fibrosarcoma cell line WEHI 164 is well known for its susce ptibility to tumor necrosis factor (TNF alpha). We have studied the ac tivation of the transcription factor NF-kappa B when WEHI 164 cells ar e challenged with TNF alpha. NF-kappa B is retained in the cytoplasm o f unchallenged cells by its inhibitor I kappa B-alpha. Upon cellular s timulation, I kappa B-alpha is functionally inactivated and NF-kappa B translocates to the nucleus. The extent of the cytotoxic effect and t hat of nuclear translocation of NF-kappa B show the same TNF alpha dep endence. TNF alpha induces a rapid and transient activation of NF-kapp a B in WEHI 164 cells which is followed by a second, long lasting phas e in which the amount of NF-kappa B complex in the nucleus remains at about 50% of maximum. Upon TNF alpha treatment, I kappa B-alpha is rap idly degraded, However, newly synthesized I kappa B-alpha can be demon strated later in the cell cytosol. A persistent nuclear localization o f NF-kappa B is an obligatory step for the cytotoxic effect to take pl ace. Thus, WEHI 164 cells treated with TNF alpha for up to 6 h can be rescued as long as NF-kappa B relocalizes to the cytoplasm in its inac tive form. On the other hand, TNF alpha treatments as short as 15 min cause the cytotoxic effect provided that NF-kappa B remains in the nuc leus. The activation of NF-kappa B is controlled by both phosphorylati on and proteolysis. The activation of NF-kappa B can be blocked by the cysteine protease inhibitor calpain inhibitor I and the serine protea se inhibitor TPCK. Signal-induced phosphorylation of I kappa B-alpha d oes not lead to the dissociation of the inhibitor from NF-kappa B. Pho sphorylation appears to regulate the inhibitory activity of I kappa B- alpha both positively and negatively, since inhibitors of protein kina ses have opposite effects. Thus, treatment of cells with staurosporin induced a partial activation of NF-kappa B and was synergistic with TN F alpha-induced activation. Calphostin C, on the other hand, can block the activation of NF-kappa B by TNF alpha, also blocking its proteoly tic degradation. (C) 1996 Academic Press, Inc.