Analysis of regulatory mechanism for signal transduction responding to oxidative stress

众所周知，PKC在受体介导的影响多种细胞反应的信号转导中起着至关重要的作用。我们已经证明了氧化二酰甘油(DAG-OHO)与佛波酯(PMA)一样有效地激活了大鼠脑内的PKC，佛波酯是一种人工的、强大的PKC激活剂。这一结果明显表明，DAG-OOH可能作为氧化应激中的生物信使，有效地改变依赖PKC的信号转导系统，其程度与PMA相似。此外，DAG-OOH还通过PKC Delta和MAP激酶的过度激活对培养的神经元造成损伤。在这些研究中，一直怀疑大鼠脑中存在两种类型的PKC亚型(PKC-X：比其他PKC亚型更容易受到DAG-OHO的影响，PKC dsv：PKC Delta剪接变异体，它只有DAG结合结构域，而不是激酶结构域)。PKC dsv被认为是DAG-OHO和PKC Delta诱导的神经细胞损伤的调节分子。在本研究中，通过细胞过度表达PKC dsV、PKC Delta和PKCα基因来研究PKC dsv的作用。2006年，针对这些基因的腺病毒表达载体制备完成。将这些表达载体导入PC12细胞。2007年，通过加入PMA等PKC激活剂，对PKC双链病毒转基因细胞进行了刺激。PMA刺激诱导的生长信号通过PKC双链病毒基因的导入而被抑制。这些结果提示，PKC dsv对DAG-OOH刺激引起的PKCβ过度激活具有调节作用。

It is well known that PKC plays a crucial role in receptor-mediated signal transduction affecting diverse range of cellular responses. We have demonstrated that oxidized-diacylglycerol (DAG-OOH) activates rat brain PKC as efficiently as phorbol ester (PMA), artificial and powerful activator for PKC. This result markedly suggested that DAG-OOH might act as a biological messenger in oxidative stress to efficiently alter the PKC-dependent signal transduction system to a similar extent to PMA. Furthermore, DAG-OOH injured the cultured neurons with the over-activation of PKC delta and MAP kinase. Among these studies, it has been suspected that two types of PKC isoforms (PKC-X: more susceptible to DAG-OOH than other PKC isoforms, PKC DSV: PKC delta splicing variant, which has only DAG binding domain but not kinase domain) exist in rat brain. PKC DSV is thought to act as a regulatory molecule for the DAG-OOH and PKC delta-induced neuronal cell injury. In the present study, the role of PKC DSV has been examined by means of the cells over-expressed the genes for PKC DSV, PKC delta and PKC alpha. In 2006, the adenovirus expression vectors for these genes were prepared. PC12 cells were transfected with these expression vectors. In 2007, PKC DSV-transfected cells were stimulated by the addition of PKC activator such as PMA. The growth signal induced by PMA irritation has been suppressed by the transfection of PKC DSV gene. These results suggested that PKC DSV regulate the over-activation of PKC delta provoked by DAG-OOH stimulation.