PARP1-mediated necrosis is dependent on parallel JNK and Ca2+/calpain pathways

Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme that can trigger caspase-independent necrosis. Two main mechanisms for this have been proposed: one involving RIP1 and JNK kinases and mitochondrial permeability transition (MPT), the other involving calpain-mediated activation of Bax and mitochondrial release of apoptosis-inducing factor (AIF). However, whether these two mechanisms represent distinct pathways for PARP1-induced necrosis, or whether they are simply different components of the same pathway has yet to be tested. Mouse embryonic fibroblasts (MEFs) were treated with either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or beta-Lapachone, resulting in PARP1-dependent necrosis. This was associated with increases in calpain activity, JNK activation and AIF translocation. JNK inhibition significantly reduced MNNG-and beta-Lapachone-induced JNK activation, AIF translocation, and necrosis, but not calpain activation. In contrast, inhibition of calpain either by Ca2+ chelation or knockdown attenuated necrosis, but did not affect JNK activation or AIF translocation. To our surprise, genetic and/or pharmacological inhibition of RIP1, AIF, Bax and the MPT pore failed to abrogate MNNG-and beta-Lapachone-induced necrosis. In conclusion, although JNK and calpain both contribute to PARP1-induced necrosis, they do so via parallel mechanisms.