O'Connell M A, Kelleher D, Liskamp R M, Hall N, O'Neill L A, Long A
Department of Clinical Medicine, Trinity College, Dublin, Ireland.
Cytokine. 1997 Feb;9(2):83-92. doi: 10.1006/cyto.1996.0140.
The role of protein kinase C (PKC) in tumour necrosis factor (TNF)-mediated cytotoxicity was investigated, using the L929 cell line. The PKC activator phorbol-12-myristate 13-acetate (PMA) increased proliferation and inhibited TNF-induced cytotoxicity of L929 cells. A range of specific PKC inhibitors had no effect on TNF-mediated killing, suggesting that PKC does not play a direct role in this response. However, staurosporine enhanced cytotoxicity by TNF in the presence of actinomycin D, a protein synthesis inhibitor. In view of this finding, the authors investigated the role of specific PKC isozymes in both TNF-mediated cytotoxicity and staurosporine-induced sensitization to killing. PKC-alpha, beta, epsilon and zeta were detected in L929 cells. PKC-beta was only weakly detected in the cytoplasm, PKC alpha, epsilon and zeta were all found in resting cytoplasm and membrane. Stimulation with PMA caused a strong translocation of PKC-alpha but not zeta to the membrane. TNF had no effect on these isozymes but interestingly caused a translocation of PKC-epsilon, which also occurred in response to PMA. Staurosporine caused a translocation of PKC-zeta to the plasma membrane and a loss of PKC-epsilon from the cytosol. Although TNF induced PKC-epsilon translocation, this is unlikely to be involved in cytotoxicity as this effect was also induced by PMA which protected against TNF-mediated cytotoxicity. Staurosporine also induced translocation of PKC-zeta, an isozyme whose activity was previously found to be resistant to inhibition by staurosporine. These findings suggest the possibility that the mechanism by which staurosporine potentiates TNF action does not involve inhibition of PKC, but in contrast may involve modulation of PKC-zeta.
利用L929细胞系研究了蛋白激酶C（PKC）在肿瘤坏死因子（TNF）介导的细胞毒性中的作用。PKC激活剂佛波醇-12-肉豆蔻酸酯13-乙酸酯（PMA）可增加L929细胞的增殖并抑制TNF诱导的细胞毒性。一系列特异性PKC抑制剂对TNF介导的杀伤作用无影响，这表明PKC在该反应中不发挥直接作用。然而，在蛋白质合成抑制剂放线菌素D存在的情况下，星形孢菌素可增强TNF的细胞毒性。鉴于这一发现，作者研究了特异性PKC同工酶在TNF介导的细胞毒性和星形孢菌素诱导的杀伤敏感性中的作用。在L929细胞中检测到了PKC-α、β、ε和ζ。PKC-β仅在细胞质中微弱检测到，PKC-α、ε和ζ均存在于静息细胞质和细胞膜中。用PMA刺激导致PKC-α强烈转位至细胞膜，但PKC-ζ未转位。TNF对这些同工酶无影响，但有趣的是可导致PKC-ε转位，对PMA的反应也会出现这种转位。星形孢菌素导致PKC-ζ转位至质膜，PKC-ε从细胞质中丢失。尽管TNF诱导了PKC-ε转位，但这不太可能参与细胞毒性，因为PMA也诱导了这种效应，而PMA可保护细胞免受TNF介导的细胞毒性。星形孢菌素还诱导了PKC-ζ转位，该同工酶的活性先前被发现对星形孢菌素的抑制具有抗性。这些发现提示，星形孢菌素增强TNF作用的机制可能不涉及PKC的抑制，相反可能涉及PKC-ζ的调节。
