Boland M P, O'Neill L A
Department of Biochemistry, Trinity College, Dublin 2, Ireland.
J Biol Chem. 1998 Jun 19;273(25):15494-500. doi: 10.1074/jbc.273.25.15494.
The role of ceramide as a second messenger in tumor necrosis factor (TNF)-mediated signal transduction has been much debated. It is supported by recent reports describing an expanding number of potential targets for this lipid, but is opposed by those describing how ceramide is not necessary for many TNF-mediated cellular events. In this paper, we directly compare the effects of the cell-permeable ceramide analogue, N-acetylsphingosine (C2-ceramide), with TNF, on NFkappaB function, a transcription factor whose activation is central to many TNF-mediated effects. We describe how C2-ceramide failed to drive kappaB-linked chloramphenicol acetyltransferase gene expression in either HL60 promyelocytic or Jurkat T lymphoma cells. Furthermore, it had no effect on TNF-mediated transcription of this reporter gene. However, electrophoretic mobility shift analysis following cell stimulation with this ceramide analogue revealed a dose-responsive activation of NFkappaB, which was not apparent following cell treatment with the inactive dihydro form. Activated complexes from treated cells were shown to contain predominantly the p50 subunit, in contrast to complexes from TNF-treated cells, where both p50 and p65/RelA subunits were present. The specific activation of p50 homodimeric complexes by C2-ceramide, which are known to lack trans-activating activity, was strongly suggested from these data. Further investigations revealed that C2-ceramide had only a marginal effect on IkappaBalpha degradation but strongly promoted the processing of p105 to its p50 product as revealed by immunoblot analysis. The increase in p50 arising from the processing of its p105 precursor was further established from p105/p50 ratios obtained by scanning densitometric analysis of bands from immunoblots. TNF, on the other hand, stimulated both IkappaBalpha degradation and p105 processing, in accordance with previous findings. Furthermore, the effect of TNF on NFkappaB activation was rapid, whereas C2-ceramide required an optimal treatment time of 1 h. Interestingly, TNF was found to increase ceramide in cells but only after a 1-h contact time. Our data therefore suggest that ceramide promotes the activation of NFkappaB complexes that lack transactivating activity by enhanced processing of p105.
神经酰胺作为肿瘤坏死因子（TNF）介导的信号转导中的第二信使，其作用一直备受争议。近期报告称这种脂质的潜在靶点数量不断增加，这支持了该观点，但也有报告称神经酰胺对许多TNF介导的细胞事件并非必需，这又对此观点表示反对。在本文中，我们直接比较了细胞可渗透的神经酰胺类似物N - 乙酰鞘氨醇（C2 - 神经酰胺）与TNF对NFκB功能的影响，NFκB是一种转录因子，其激活对许多TNF介导的效应至关重要。我们描述了C2 - 神经酰胺在HL60早幼粒细胞或Jurkat T淋巴瘤细胞中均未能驱动κB连接的氯霉素乙酰转移酶基因表达。此外，它对该报告基因的TNF介导转录没有影响。然而，用这种神经酰胺类似物刺激细胞后进行的电泳迁移率变动分析显示NFκB呈剂量反应性激活，而用无活性的二氢形式处理细胞后则未出现这种情况。与TNF处理的细胞中的复合物不同，处理过的细胞中的活化复合物主要含有p50亚基，TNF处理的细胞中的复合物同时存在p50和p65/RelA亚基。这些数据强烈表明C2 - 神经酰胺特异性激活了已知缺乏反式激活活性的p50同二聚体复合物。进一步研究表明，C2 - 神经酰胺对IκBα降解的影响很小，但通过免疫印迹分析显示它强烈促进了p105向其p50产物的加工。通过对免疫印迹条带进行扫描光密度分析获得的p105/p50比值进一步证实了由p105前体加工产生的p50增加。另一方面，TNF如先前发现的那样，刺激了IκBα降解和p105加工。此外，TNF对NFκB激活的作用迅速，而C2 - 神经酰胺需要1小时的最佳处理时间。有趣的是，发现TNF仅在接触1小时后才会增加细胞中的神经酰胺。因此，我们的数据表明神经酰胺通过增强p105的加工促进了缺乏反式激活活性的NFκB复合物的激活。
