Claudio E, Segade F, Wrobel K, Ramos S, Bravo R, Lazo P S
Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo,Spain.
Exp Cell Res. 1996 Apr 10;224(1):63-71. doi: 10.1006/excr.1996.0111.
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.
鼠纤维肉瘤细胞系WEHI 164因其对肿瘤坏死因子（TNFα）敏感而闻名。我们研究了用TNFα刺激WEHI 164细胞时转录因子NF-κB的激活情况。在未受刺激的细胞中，NF-κB被其抑制剂IκB-α保留在细胞质中。细胞受到刺激后，IκB-α功能失活，NF-κB转位至细胞核。细胞毒性作用的程度和NF-κB的核转位程度表现出相同的TNFα依赖性。TNFα在WEHI 164细胞中诱导NF-κB快速短暂激活，随后进入第二个持久阶段，细胞核中NF-κB复合物的量维持在最大值的约50%。用TNFα处理后，IκB-α迅速降解。然而，新合成的IκB-α随后可在细胞质中检测到。NF-κB持续定位于细胞核是细胞毒性作用发生的必要步骤。因此，只要NF-κB以无活性形式重新定位于细胞质中，用TNFα处理长达6小时的WEHI 164细胞就可以被挽救。另一方面，只要NF-κB保留在细胞核中，短至15分钟的TNFα处理就会导致细胞毒性作用。NF-κB的激活受磷酸化和蛋白水解的控制。NF-κB的激活可被半胱氨酸蛋白酶抑制剂钙蛋白酶抑制剂I和丝氨酸蛋白酶抑制剂TPCK阻断。信号诱导的IκB-α磷酸化不会导致抑制剂与NF-κB解离。磷酸化似乎对IκB-α的抑制活性有正向和负向调节作用，因为蛋白激酶抑制剂有相反的作用。因此，用星形孢菌素处理细胞会诱导NF-κB部分激活，并与TNFα诱导的激活具有协同作用。另一方面，钙泊三醇C可阻断TNFα对NF-κB的激活，也可阻断其蛋白水解降解。
