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 susceptibility to tumor necrosis factor (TNF alpha). We have studied the activation of the transcription factor NF-kappa B when WEHI 164 cells are challenged with TNF alpha. NF-kappa B is retained in the cytoplasm of unchallenged cells by its inhibitor I kappa B-alpha. Upon cellular stimulation, I kappa B-alpha is functionally inactivated and NF-kappa B translocates to the nucleus. The extent of the cytotoxic effect and that of nuclear translocation of NF-kappa B show the same TNF alpha dependence. TNF alpha induces a rapid and transient activation of NF-kappa B in WEHI 164 cells which is followed by a second, long lasting phase 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 rapidly degraded, However, newly synthesized I kappa B-alpha can be demonstrated later in the cell cytosol. A persistent nuclear localization of NF-kappa B is an obligatory step for the cytotoxic effect to take place. 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 inactive 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 nucleus. The activation of NF-kappa B is controlled by both phosphorylation and proteolysis. The activation of NF-kappa B can be blocked by the cysteine protease inhibitor calpain inhibitor I and the serine protease inhibitor TPCK. Signal-induced phosphorylation of I kappa B-alpha does not lead to the dissociation of the inhibitor from NF-kappa B. Phosphorylation appears to regulate the inhibitory activity of I kappa B-alpha both positively and negatively, since inhibitors of protein kinases have opposite effects. Thus, treatment of cells with staurosporin induced a partial activation of NF-kappa B and was synergistic with TNF alpha-induced activation. Calphostin C, on the other hand, can block the activation of NF-kappa B by TNF alpha, also blocking its proteolytic degradation. (C) 1996 Academic Press, Inc.