Bott C M, Doshi J B, Li L L, McMurtry S A, Sanders J L, Fox D A
Rackham Arthritis Research Unit, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109.
J Immunol. 1994 Jul 1;153(1):1-9.
Activation of protein kinase C (PKC) has emerged as a major common signal-transducing mechanism for T cell activation and regulation of expression of T cell surface glycoproteins. Surface expression of the CD6 Ag is known to increase with T cell activation and CD6 itself may be involved in regulation of T cell activation. Therefore, we performed experiments to determine whether activation of PKC by phorbol ester induced an increase in CD6 expression and to investigate the mechanisms of such an effect. CD6 surface expression was up-regulated substantially in response to PMA on both mature and immature T cells, but only negligibly on B cell lines. This increase was blocked by PKC inhibitors. The PMA-induced increase in CD6 surface expression was accompanied by an increase in total CD6 protein, as detected by using Western blot analysis of whole cell lysates. After PMA stimulation, Northern blot analysis showed that steady state levels of CD6 mRNA increased in response to PMA treatment. This increase was blocked by cycloheximide, demonstrating that it was dependent on new protein synthesis. Nuclear run-on analysis showed that the rate of CD6 mRNA transcription increased by approximately two to threefold after PMA stimulation of Jurkat cells. Experiments in which we used actinomycin D showed that PMA had no significant effect on the t1/2 of CD6 mRNA. The data suggest that the effect of PMA on CD6 expression is mediated primarily by an increase in CD6 mRNA transcription after PKC activation. mAbs were used to determine whether augmented CD6 expression could be induced by perturbation of specific T cell surface molecules. Up-regulation of CD6 expression occurred when thymocytes were cultured with anti-CD2 Abs, but not with Abs to other functional T cell surface structures, and not when mature T cells were cultured with the anti-CD2 mAbs. Up-regulation of CD6 expression by activation of PKC, triggered in thymocytes by ligation of CD2, could allow CD6 to provide additional regulatory signals required for events in later stages of T cell activation and differentiation.
蛋白激酶C（PKC）的激活已成为T细胞激活以及T细胞表面糖蛋白表达调控的主要共同信号转导机制。已知CD6抗原的表面表达会随着T细胞激活而增加，并且CD6本身可能参与T细胞激活的调控。因此，我们进行了实验，以确定佛波酯对PKC的激活是否会诱导CD6表达增加，并研究这种效应的机制。在成熟和未成熟T细胞上，佛波醇酯（PMA）均可显著上调CD6的表面表达，但在B细胞系上的上调作用可忽略不计。这种增加被PKC抑制剂所阻断。通过对全细胞裂解物进行蛋白质印迹分析检测到，PMA诱导的CD6表面表达增加伴随着总CD6蛋白的增加。PMA刺激后，Northern印迹分析显示，CD6 mRNA的稳态水平因PMA处理而增加。这种增加被放线菌酮阻断，表明其依赖于新的蛋白质合成。核转录分析显示，PMA刺激Jurkat细胞后，CD6 mRNA的转录速率增加了约两到三倍。我们使用放线菌素D的实验表明，PMA对CD6 mRNA的半衰期没有显著影响。数据表明，PMA对CD6表达的影响主要是通过PKC激活后CD6 mRNA转录增加来介导的。使用单克隆抗体（mAbs）来确定是否可以通过干扰特定的T细胞表面分子来诱导CD6表达增加。当胸腺细胞与抗CD2抗体一起培养时，会出现CD6表达上调，但与针对其他功能性T细胞表面结构的抗体一起培养时则不会，并且成熟T细胞与抗CD2单克隆抗体一起培养时也不会。通过CD2的连接在胸腺细胞中触发PKC激活从而上调CD6表达，这可能使CD6提供T细胞激活和分化后期事件所需的额外调节信号。
