MOLECULAR MECHANISM FOR ALTERATION IN SIGNAL TRANSDUCTION BY OXIDATIVE STRESS -Isoform specific activation of C kinase by oxidized DAG and cell injury-

蛋白激酶C （PKC）在受体介导的信号转导中起着至关重要的作用，影响多种细胞反应，如细胞增殖、分化和肿瘤促进。PKC是一种独特的酶，由受体介导的磷酸肌醇水解产生的1,2-二酰基甘油（DAG）激活。另一方面，活性氧如超氧化物、过氧化氢和羟基自由基已被认为在许多病理事件中发挥重要作用，如炎症、自身免疫性疾病、缺血再灌注损伤、动脉粥样硬化和心血管疾病以及致癌。这表明PKC的作用与活性氧诱导的功能障碍和细胞损伤之间存在密切的关系，然而，这种可能性尚未得到彻底的研究。在本研究中，我们观察到1-硬脂酰-2-亚油基甘油过氧化氢（SLG- ooh）和SLG氢氧化物（SLG- oh）在磷脂酰丝氨酸和钙离子存在下比未氧化的SLG更有效地刺激了大鼠胸膜PKC的活性。然后，氧化DAG对PKC在大鼠脑中表达最多的7种具有代表性的PKC异构体进行了激活作用，发现α和δ异构体被氧化DAG强烈激活。此外，我们试图证明DAG-OOH对过度表达α和δ亚型的培养神经元的神经退行性作用。培养细胞为18日龄大鼠胎儿大脑皮层神经细胞（PN细胞）。我们在PN细胞中使用腺病毒载体系统，该系统允许PKC α和δ基因的高水平表达。这些培养的神经元经溶链素O处理，使天然DAG和DAG- ooh渗透到细胞中。观察处理后的细胞：1)相对比显微镜，2)细胞活力，3)电镜，4)磷酸化tau蛋白免疫印迹分析，5)免疫组织化学定位磷酸化tau蛋白。暴露于DAG-OOH 6h内，过表达PKC δ异构体的PN细胞表现出神经性变薄和特征性的束状，而过表达α异构体的PN细胞未表现出这种变化。MEK抑制剂PD98059可预防DAG-OOH引起的神经退行性变。常规电镜观察病变，发现病变内微管（MT）解体。免疫印迹分析显示，在dag - oh处理的细胞中，tau蛋白在Thr 181、Ser 202和Thr 205上被部分磷酸化。tau蛋白的磷酸化可能导致MT的解体，并在细胞体和头部病变中积累。这些发现表明，DAG-OOH对PKC δ的明显激活可能是引起神经变性的具体原因。少

Protein kinase C (PKC) plays a crucial role a receptor-mediated signal transduction affecting diverse range of cellular responses such as cell proliferation, differentiation, and tumor promotion. PKC is a unique enzyme, which is activated by 1,2-diacylglycerol (DAG) produced from receptor-mediated hydrolysis of inositol phosphate. On the other hand, active oxygen species such as superoxide, hydrogen peroxide, and hydroxy radical have been suggested to play important roles in many pathological events such as inflammation, autoimmune diseases, ischemia-reperfusion injury, atherosclerosis and cardiovascular diseases, and carcinogenesis. This suggests a close relationship between the action of PKC and active oxygen species-induced dysfunction and cellular damage, however, this possibility has not been thoroughly examined. In the present study, we observed that 1-stearoyl-2-linoleoylglycerol hydroperoxide (SLG-OOH) and SLG Hydroxide (SLG-OH) stimulated the PKC activity isolated from rat bra … More in more efficiently than that of unoxidized SLG in the presences of phosphatidylserin and calcium ion. Then, the PKC activation effect of oxidized DAG has also been tried on 7 repsentative PKC isoforms, which were most pronouncedly expressed in rat brains, and alpha and delta isoforms were found to be intensely activated by the oxidized DAGs. Furthermore, we attempts to prove the neurodegenerative effects of DAG-OOH on cultured neurons overexpressing alpha and delta isoforms. As the cultured cells, neuronal cells established from 18-day rat fetus cerebral cortex (PN cells) were employed for the experiments. We used an adenovirus vector system in PN cells, which allows expression of PKC alpha and delta gene at a high level. These cultured neurons treated by streptolysin O to let the penetration of natural DAG and DAG-OOH into cells. Those treated cells were observed by 1) phase contrast microscopy, 2) cell viability, 3) electron microscopy, 4) immunoblot analysis of phoshorylated tau, 5) immunohistochemistry to localize phosphorylated tau. Within 6h of exposure to DAG-OOH, PN cells overexpressing PKC delta isoform, exhibited neuritic thinning and characteristic beading, while PN cells overexpressing alpha isoform did not shows such changes. The MEK inhibitor PD98059 prevented its neurodegeneration caused by DAG-OOH. Those lesions were observed routine EM which revealed the microtubule (MT) disassembly in the lesions. Immunoblot analysis showed that tau was partially phosphorylated on Thr 181, Ser 202 and Thr 205 in those DAG-OOH-treated cells. Phosphorylation of tau may have caused in MT disassembly and accumulated in cell body and beading lesions. Those findings indicate that the pronounced activation of PKC delta by DAG-OOH may specifically be responsible to elicit the neurodegeneration. Less