zVAD-induced autophagic cell death requires c-Src-dependent ERK and JNK activation and reactive oxygen species generation.
The treatment of L929 fibrosarcoma cells with zVAD has been shown to induce necroptosis. However, whether autophagy is involved or not in this event remains controversial. In this study, we re-examined the role of autophagy in zVAD-induced cell death in L929 cells and further elucidated the signaling pathways triggered by caspase inhibition and contributing to autophagic death. First, we found that zVAD can stimulate LC3-II formation, autophagosome and autolysosome formation, and ROS accumulation. Antioxidants, beclin 1 or Atg5 silencing, and class III PtdIns3K inhibitors all effectively blocked ROS production and cell death, suggesting ROS accumulation downstream of autophagy contributes to cell necrosis. zVAD also stimulated PARP activation, and the PARP inhibitor DPQ can reduce zVAD-induced cell death, but did not affect ROS production, suggesting the increased ROS leads to PARP activation and cell death. Notably, our data also indicated the involvement of Src-dependent JNK and ERK in zVAD-induced ROS production and autophagic death. We found caspase 8 is associated with c-Src at the resting state, and upon zVAD treatment this association was decreased and accompanied by c-Src activation. In conclusion, we confirm the autophagic death in zVAD-treated L929 cells, and define a new molecular pathway in which Src-dependent ERK and JNK activation can link a signal from caspase inhibition to autophagy, which in turn induce ROS production and PARP activation, eventually leading to necroptosis. Thus, in addition to initiating proteolytic activity for cell apoptosis, inactivated caspase 8 also functions as a signaling molecule for autophagic death.
Department of Pharmacology, College of Medicine, National Taiwan University, Taiwan, Taiwan.
Szu-ying
SY
Chen
Ling-Ya
LY
Chiu
Ming-Chei
MC
Maa
Jang-Shiun
JS
Wang
Chung-Liang
CL
Chien
Wan-Wan
WW
Lin
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D060588:LIM Domain Proteins; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009842:Oligopeptides / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones; C120696:LDB3 protein, human; D060588:LIM Domain Proteins; D009842:Oligopeptides; D017382:Reactive Oxygen Species; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid; D011065:Poly(ADP-ribose) Polymerases; D016392:Proto-Oncogene Proteins pp60(c-src); D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D053181:Caspase 8