Abstract P049: Molecular Mechanisms of PARP1-Mediated Necrosis

Abstract

Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme associated with DNA replication, transcription, repair, and cell death. Although, PARP1 is most commonly associated with apoptosis, a specific PARP1-mediated caspase-independent necrosis pathway has recently been described. Two main models for this pathway have been proposed: one involving RIP1 and JNK kinases and mitochondrial permeability transition (MPT), the other involving activation of Bax and subsequent release of apoptosis inducing factor (AIF) from mitochondria. However, these models still remain untested. Consequently, the purpose of this study was to determine which (if any) of these molecules are truly involved in PARP1-mediated necrosis. Murine embryonic fibroblasts (MEFs) were treated with either N-Methyl-N’-Nitro-N-Nitrosoguanidine (MNNG) or β-Lapachone, 2 chemically distinct PARP1 activators. To inhibit RIP1, CypD, and AIF, MEFs were transfected with specific siRNAs for 48 hrs prior to treatment. RIP1 and CypD-null cells were also used. To block JNK activity, cells were incubated with the JNK-specific inhibitor SP600125. Inhibition of Bax was achieved using either Bax/Bak-null MEFs or Bcl2 overexpression. Necrosis, MPT, JNK activation, and AIF translocation were then analyzed using a combination of cell death assays, western blotting, and immunocytochemistry. Treatment of MEFs with MNNG and β-Lapachone induced translocation of AIF from the mitochondria to nucleus, and necrotic cell death. However, inhibition of RIP1, CypD, and Bax had no significant effect on MNNG or β-Lapachone- induced necrotic cell death. Moreover, neither PARP1 activator induced MPT. In contrast, the JNK inhibitor SP600125 significantly reduced MNNG- and β-Lapachone-induced JNK activation, AIF translocation, and necrosis. This suggests that JNK plays a key role in the pathway and that it is upstream of AIF. To our surprise, however, depletion of AIF did not influence PARP1-mediated cell death, thus indicating that AIF is more of a marker, rather than a key mediator, of this particular process. We conclude that JNK plays a significant signaling role in PARP1-mediated necrosis. However, RIP1, Bax, CypD, and AIF do not appear to be essential components of the PARP1 death pathway.