Caspase-3-dependent proteolytic cleavage of protein kinase Cδ is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl

In the present study, we characterized oxidative stress-dependent cellular events in dopaminergic cells after exposure to an organic form of manganese compound, methylcyclopentadienyl manganese tricarbonyl (MMT). In pheochromocytoma cells, MMT exposure resulted in rapid increase in generation of reactive oxygen species (ROS) within 5-15 min, followed by release of mitochondrial cytochrome C into cytoplasm and subsequent activation of cysteine proteases, caspase-9 (twofold to threefold) and caspase-3 (15- to 25-fold), but not caspase-8, in a time- and dose-dependent manner. Interestingly, we also found that MMT exposure induces a time- and dose-dependent proteolytic cleavage of native protein kinase Cδ (PKCδ, 72-74 kDa) to yield 41 kDa catalytically active and 38 kDa regulatory fragments. Pretreatment with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked MMT-induced proteolytic cleavage of PKCδ, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCδ activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCδ cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCδ. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCδ fragment. Attenuation of ROS generation, caspase-3 activation, and PKCδ activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCδ^K376R (dominant-negative mutant) prevented MMT-induced apoptosis in immortalized mesencephalic dopaminergic cells. For the first time, these data demonstrate that caspase-3-dependent proteolytic activation of PKCδ plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.