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

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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.