title: Molecular mechanisms of TNFalpha cytotoxicity: activation of NF-kappaB and nuclear translocation.
abstract: 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.
authors.affiliation: Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo,Spain.
authors.forename: E
authors.initials: E
authors.lastname: Claudio
authors.forename: F
authors.initials: F
authors.lastname: Segade
authors.forename: K
authors.initials: K
authors.lastname: Wrobel
authors.forename: S
authors.initials: S
authors.lastname: Ramos
authors.forename: R
authors.initials: R
authors.lastname: Bravo
authors.forename: P S
authors.initials: PS
authors.lastname: Lazo
mesh_terms: D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005354:Fibrosarcoma; D020672:I-kappa B Proteins; D008387:Marine Toxins; D051379:Mice; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D008387:Marine Toxins; D016328:NF-kappa B; D009281:Naphthalenes; C000606205:Nfkbia protein, mouse; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; C058819:calphostin C