Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced Apoptosis. However, little is known about the mechanism of how ASK1 executes Apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced Apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum Caspase Inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced Apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and Caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced Caspase-3 activation and Apoptosis, suggesting that Caspase-8 is dispensable for ASK1-induced Apoptosis, whereas ASK1 failed to activate Caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of Caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute Apoptosis mainly by the mitochondria-dependent Caspase activation.