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Analysis of signal transduction during ceramide-mediated apoptotic pathway in hypoxic PC12 cell death

缺氧PC12细胞死亡过程中神经酰胺介导的凋亡途径信号转导分析

基本信息

批准号：
11557104
负责人：
SAKAI Noboru
金额：
$ 7.23万
依托单位：
GIFU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Hypoxia is one of the pervasive environmental stimuli which lead to neuronal cell death. During hypoxic incubation of PC12 cells, we have previously demonstrated that the apoptotic cell death dominates over the necrotic one and a sphingolipid, ceramide generated through the activation of neutral sphingomyeuinase (N-SMase) functions as one of the apoptotic mediators. Although it has been reported that reduced gluthatione (GSH) appeared to enhance hippocampal neuronal survival after transient forebrain ischemia in rats, the precise preventive mechanism of the action of GSH in hypoxic or ischemic injury remains poorly understood. We have demonstrated that GSH protects cells from hypoxic injury by direct inhibition of N-SMase activity and ceramide formation, resulting in inhibition of caspase-3 activation, These findings suggest that generation of reactive oxygen species (ROS) has been proposed as a crucial event for ischemic or hypoxic cell death signaling. Therefore, the aim of the next study is to understand the signal transduction pathway leading to apoptotic cell death in PC12 cells exposed to H2O2 (an important precursor of highly reactive free radicals). To be interested, compared with hypoxic cell death pathway, ceramide was not involved in H2O2-induced apoptotic cell death of PC12 cells. This difference indicates the existence of multiple apoptotic pathways in PC12 cells in response to oxygen-related stresses as shown in the previous study. Moreover. we further examined the changes of PLD activity during hypoxia-induced PC12 cell death, since its activity is reported to be inhibited by ceramide. Subsequently, PLD2 is activated at the early stage and thereafter down-regulated during hypoxic cell death pathway. In addition, the number of apoptotic cells significantly reduced in PC12 cells overexpressing PLD2. These results raise the possibility that PLD2 activation may play an anti-apoptotic role in hypoxia-induced cell death.

缺氧是导致神经细胞死亡的普遍环境刺激之一。在PC12细胞的低氧孵育过程中，我们已经证明了细胞的死亡主要是细胞的死亡，而神经鞘磷脂是中性鞘氨酸酶(N-sMase)激活产生的神经酰胺，是细胞凋亡的介质之一。尽管已有报道表明还原型谷胱甘肽(GSH)可提高大鼠短暂性前脑缺血后海马神经元的存活率，但GSH在缺氧或缺血损伤中的确切预防机制尚不清楚。我们已经证明，GSH通过直接抑制N-sMase活性和神经酰胺的形成来保护细胞免受缺氧性损伤，从而抑制caspase-3的激活。这些发现表明，ROS的产生是缺血或缺氧性细胞死亡信号的关键事件。因此，下一步研究的目的是了解过氧化氢(高活性自由基的重要前体)导致PC12细胞凋亡的信号转导途径。有趣的是，与缺氧细胞死亡途径相比，神经酰胺不参与过氧化氢诱导的PC12细胞的凋亡。这一差异表明PC12细胞存在多种凋亡途径，以响应氧相关的压力，如先前的研究所表明的那样。更有甚者。我们进一步检测了PLD活性在低氧诱导的PC12细胞死亡过程中的变化，因为据报道它的活性被神经酰胺抑制。随后，PLD2在早期被激活，随后在缺氧细胞死亡途径中下调。此外，在过表达PLD2的PC12细胞中，凋亡细胞数量显著减少。这些结果提示PLD2的激活可能在低氧诱导的细胞死亡中起到抗凋亡的作用。