Johnson S E, Baglioni C
Department of Biological Sciences, State University of New York, Albany 12222.
J Biol Chem. 1988 Apr 25;263(12):5686-92.
Human HeLa cells and murine L(S) cells are highly sensitive to the cytocidal activity of tumor necrosis factor (TNF) when simultaneously treated with the inhibitor of protein synthesis cycloheximide. This cytocidal activity of TNF was inhibited up to 90% in both cell lines after a 15-60-min pretreatment with 3-10 ng/ml of phorbol 12-myristate 13-acetate (PMA). This inhibition was long lasting for HeLa cells but transient for L(S) cells. The protection afforded by PMA was most effective when the cells were pretreated with this phorbol ester, but it decreased when PMA was added together with TNF or after TNF addition. This finding suggested that PMA interfered with one of the early steps in the mechanism of action of TNF. A pretreatment with the calcium ionophore A23187 also reduced the cytocidal activity of TNF in both HeLa and L(S) cells to about the same extent. Treatment of these cells with either PMA or A23187 significantly decreased the binding of 125I-TNF to cell surface receptors. This decrease paralleled the time course and dose-response of the inhibition of cytocidal activity. In addition, treatment of HeLa cells with 1-oleyl-2-acetyl-glycerol (OAG) also induced a rapid loss of TNF binding capacity. Since OAG, PMA, and A23187 are all activators of protein kinase C (Ca2+/phospholipid-dependent enzyme), these results suggest that this kinase is involved in modulation of TNF sensitivity. Furthermore, depletion or inhibition of protein kinase C antagonized PMA-induced effects on TNF cytotoxicity and binding to receptors. Internalization of bound TNF was not significantly affected by PMA treatment, and Scatchard analysis of binding data indicated that PMA decreased TNF receptor binding affinity rather than the number of TNF-binding sites. These findings suggest that protein kinase C may have a physiological role in mediating TNF sensitivity.
当用蛋白质合成抑制剂环己酰亚胺同时处理时，人宫颈癌细胞系HeLa细胞和小鼠L(S)细胞对肿瘤坏死因子（TNF）的杀细胞活性高度敏感。在用3-10 ng/ml佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）进行15-60分钟预处理后，这两种细胞系中TNF的这种杀细胞活性被抑制高达90%。这种抑制对HeLa细胞是持久的，但对L(S)细胞是短暂的。当细胞用这种佛波醇酯预处理时，PMA提供的保护最为有效，但当PMA与TNF一起添加或在TNF添加后添加时，保护作用会降低。这一发现表明PMA干扰了TNF作用机制中的早期步骤之一。用钙离子载体A23187预处理也在大致相同程度上降低了HeLa细胞和L(S)细胞中TNF的杀细胞活性。用PMA或A23187处理这些细胞显著降低了125I-TNF与细胞表面受体的结合。这种降低与杀细胞活性抑制的时间进程和剂量反应平行。此外，用1-油酰基-2-乙酰甘油（OAG）处理HeLa细胞也导致TNF结合能力迅速丧失。由于OAG、PMA和A23187都是蛋白激酶C（钙/磷脂依赖性酶）的激活剂，这些结果表明该激酶参与了TNF敏感性的调节。此外，蛋白激酶C的耗竭或抑制拮抗了PMA对TNF细胞毒性和与受体结合的诱导作用。PMA处理对结合的TNF的内化没有显著影响，结合数据的Scatchard分析表明PMA降低了TNF受体结合亲和力而不是TNF结合位点的数量。这些发现表明蛋白激酶C可能在介导TNF敏感性方面具有生理作用。
