Molecular mechanisms of TNFalpha cytotoxicity: activation of NF-kappaB and nuclear translocation.

The murine fibrosarcoma cell line WEHI 164 is well known for its susceptibility to tumor necrosis factor (TNFalpha). We have studied the activation of the transcription factor NF-kappaB when WEHI 164 cells are challenged with TNFalpha. NF-kappaB is retained in the cytoplasm of unchallenged cells by its inhibitor IkappaB-alpha. Upon cellular stimulation, IkappaB-alpha is functionally inactivated and NF-kappaB translocated to the nucleus. The extent of the cytotoxic effect and that of nuclear translocation of NF-kappaB show the same TNFalpha dependence. TNFalpha induces a rapid and transient activation of NF-kappaB in WEHI 164 cells which is followed by a second, long lasting phase in which the amount of NF-kappaB complex in the nucleus remains at about 50% of maximum. Upon TNFalpha treatment, IkappaB-alpha is rapidly degraded. However, newly synthesized IkappaB-alpha can be demonstrated later in the cell cytosol. A persistent nuclear localization of NF-kappaB is an obligatory step for the cytotoxic effect to take place. Thus, WEHI 164 cells treated with TNFalpha for up to 6 h can be rescued as long as NF-kappa relocalizes to the cytoplasm in its inactive form. On the other hand, TNFalpha treatments as short as 15 min cause the cytotoxic effect provided that NF-kappaB remains in the nucleus. The activation of NF-kappaB is controlled by both phosphorylation and proteolysis. The activation of NF-kappaB can be blocked by the cysteine protease inhibitor calpain inhibitor I and the serine protease inhibitor TPCK. Signal-induced phosphorylation of IkappaB-alpha does not lead to the dissociation of the inhibitor from NF-kappaB. Phosphorylation appears to regulate the inhibitory activity of IkappaB-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-kappaB and was synergistic with TNFalpha induced activation. Calphostin C, on the other hand, can block the activation of NF-kappaB by TNFalpha, also blocking its proteolytic degradation.