It is intriguing that some pan-caspase inhibitors such as zVAD-fmk (zVAD) are capable of inducing necrotic cell death in a selected group of cells. As earlier reports from our laboratory have ruled out the original notion that zVAD-induced necrosis in mouse fibrosarcoma L929 cells was autophagic cell death, the main objective of this study was thus to determine the underlying mechanism of this form of cell death. In this study, we provided clear evidence that zVAD-induced necroptosis in L929 cells and such cell death is dependent on autocrine production of tumor necrosis factor-a (TNFa) at the transcriptional level. More importantly, we identified that activating protein-1 (AP-1), but not nuclear factor j-B, is the transcription factor controlling zVADinduced TNFa transcription. Moreover, zVAD is able to activate AP-1 through activation of two upstream mitogen-activated kinases (MAPKs), c-Jun N-terminal kinase and extracellular signal-regulated kinase. Finally, we found that protein kinase C is the important upstream signaling molecule in mediating zVAD-induced activation of MAPKs and AP-1, and subsequent autocrine production of TNFa and cell death. Data from this study reveal the molecular mechanisms underlying zVAD-induced necroptosis, an important form of programmed necrotic cell death with increasing understanding of its biological significance in health and diseases.

Under oxidative stress, poly(ADP-ribose) polymerase-1 (PARP-1) is activated and contributes to necrotic cell death through ATP depletion. On the other hand, oxidative stress is known to stimulate autophagy, and autophagy may act as either a cell death or cell survival mechanism. This study aims to explore the regulatory role of PARP-1 in oxidative stress-mediated autophagy and necrotic cell death. Here, we first show that hydrogen peroxide (H 2 O 2 ) induces necrotic cell death in BaxÀ/À BakÀ/À mouse embryonic fibroblasts through a mechanism involving PARP-1 activation and ATP depletion. Next, we provide evidence that autophagy is activated in cells exposed to H 2 O 2 . More importantly, we identify a novel autophagy signaling mechanism linking PARP-1 to the serine/threonine protein kinase LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, leading to stimulation of autophagy. Finally, we demonstrate that autophagy plays a cytoprotective role in H 2 O 2 -induced necrotic cell death, as suppression of autophagy by knockdown of autophagy-related gene ATG5 or ATG7 greatly sensitizes H 2 O 2 -induced cell death. Taken together, these findings demonstrate a novel function of PARP-1: promotion of autophagy through the LKB1-AMPK-mTOR pathway to enhance cell survival in cells under oxidative stress. Oxidative stress refers to a status of cellular redox imbalance caused by an overabundance of reactive oxygen species (ROS) and/or a decline in antioxidant ability.1 Oxidative stress and ROS are known to be implicated in a number of physiological and pathological processes, leading to various biological consequences including cell death (either apoptotic or non-apoptotic/necrotic cell death).2 In comparison with ROS-mediated apoptosis, the molecular mechanisms underlying ROS-mediated non-apoptotic/necrotic cell death have not been well elucidated. Poly(ADP-ribose) polymerase-1 (PARP-1) is the founding member of the PARP family, a group of nuclear enzymes that play a critical role in DNA damage repair through poly(ADP-ribosyl)ation.3 It is known that poly(ADP-ribosyl)ation is an energetically expensive process, causing rapid depletion of cellular b-nicotinamide adenine dinucleotide (NAD þ ), failure in ATP production, and eventually necrotic cell death. 3,4 In cells under oxidative stress, PARP-1 activation has been well established as a key mechanism in necrotic cell death. [4][5][6]

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1À/À MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2À/À MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2À/À MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2À/À cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.