ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4134–4145 doi:10.1242/jcs.128009 RESEARCH ARTICLE PARP1-mediated necrosis is dependent on parallel JNK and Ca2+/calpain pathways Diana L. Douglas1 and Christopher P. Baines1,2,3,* ABSTRACT receptor interacting proteins (RIP, or RIPK) 1 and 3 (Cho et al., 2009; He et al., 2009; Zhang et al., 2009; Li et al., 2012). This Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme that complex in turn has been proposed to facilitate necrotic killing can trigger caspase-independent necrosis. Two main mechanisms through a variety of mechanisms, including activation of NADPH for this have been proposed: one involving RIP1 and JNK kinases oxidases (Kim et al., 2007), induction of mitochondrial reactive and mitochondrial permeability transition (MPT), the other involving oxygen species (Irrinki et al., 2011; Vanlangenakker et al., 2011) calpain-mediated activation of Bax and mitochondrial release of and activation of the pseudokinase mixed lineage kinase like apoptosis-inducing factor (AIF). However, whether these two (MLKL) with subsequent activation of the mitochondrial-associated mechanisms represent distinct pathways for PARP1-induced phosphatase PGAM5 (Sun et al., 2012; Wang et al., 2012). necrosis, or whether they are simply different components of the In addition to the necroptosis pathway, another necrotic same pathway has yet to be tested. Mouse embryonic fibroblasts program involving the DNA repair enzyme poly(ADP-ribose) (MEFs) were treated with either N-methyl-N9-nitro-N-nitrosoguanidine polymerase-1 (PARP1) has also emerged. Genotoxic stresses, (MNNG) or b-Lapachone, resulting in PARP1-dependent necrosis. such as oxidants and alkylating agents, have long been This was associated with increases in calpain activity, JNK activation known to cause necrotic cell death that is associated with an and AIF translocation. JNK inhibition significantly reduced MNNG- overstimulation of PARP1 (Hassa, 2009). Moreover, ischemia/ and b-Lapachone-induced JNK activation, AIF translocation, and reperfusion-induced myocardial and cerebral necrosis is markedly necrosis, but not calpain activation. In contrast, inhibition of calpain attenuated by genetic inhibition of PARP1 (Zingarelli et al., 1998; either by Ca2+ chelation or knockdown attenuated necrosis, but did Li et al., 2010). It was originally thought that necrosis induced by not affect JNK activation or AIF translocation. To our surprise, genetic PARP1 hyperactivation was simply due to metabolic catastrophe, + and/or pharmacological inhibition of RIP1, AIF, Bax and the MPT pore where the overactive PARP1 used up the supply of NAD in the failed to abrogate MNNG- and b-Lapachone-induced necrosis. In cell, and subsequently ATP (van Wijk and Hageman, 2005). conclusion, although JNK and calpain both contribute to PARP1- However, it is now appreciated that PARP1-induced necrosis induced necrosis, they do so via parallel mechanisms. follows more specific molecular pathways (Xu et al., 2006; Moubarak et al., 2007; Artus et al., 2010; Dong et al., 2010; Chiu KEY WORDS: Cell death, Necrosis, PARP1, JNK, Calpain, AIF, et al., 2011; Park et al., 2011). Mitochondria Despite this, the signaling networks responsible for PARP1- mediated cell death have yet to be clearly defined. Moubarak and INTRODUCTION colleagues (Moubarak et al., 2007) have reported that PARP1- Apoptosis is regarded as a ‘programmed’ form of cell death due induced necrosis is dependent on activation of the Ca2+-activated to the fact that there are specific, genetically determined protease calpain, which in turn induces Bax translocation to the pathways that mediate this process. In contrast, until recently it mitochondrion where it elicits the release and nuclear was believed that necrosis was a random, uncontrolled process translocation of apoptosis-inducing factor (AIF). In contrast, Xu that leads to the ‘accidental’ death of the cell. However, recent et al. (Xu et al., 2006) have suggested that PARP1 activation evidence has indicated that distinct molecular pathways also activates the pro-necrotic kinase RIP1. RIP1 in turn activates mediate necrosis (Vanlangenakker et al., 2012). In particular, a JNK1, which induces necrosis through a mechanism that can be great number of recent studies have focused on death-receptor- blocked by the mitochondrial permeability transition (MPT) pore induced mechanisms of programmed necrosis, or necroptosis. inhibitor cyclosporine-A. However, whether these two Activation of tumor necrosis factor (TNF) receptors under mechanisms represent distinct pathways for PARP1-induced conditions of caspase-8 inhibition leads to the formation of the necrosis, or whether they are simply different components of necrosome, a complex consisting of the serine/threonine kinases the same pathway has yet to be tested. Moreover, the majority of studies have relied on only a single pharmacological PARP1 activator, and direct comparisons with another distinct activator are lacking. 1Dalton Cardiovascular Research Center, University of Missouri-Columbia, Consequently the purpose of the present study was to Columbia, Missouri, 65211, USA. 2Department of Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri, 65211, USA. 3Department of Medical systematically investigate the roles and functional interrelationships Pharmacology and Physiology, University of Missouri-Columbia, Columbia, of RIP1, JNK, calpain, Bax and MPT in PARP1-mediated necrosis Missouri, 65211, USA. induced by two distinct PARP1 activators, N-methyl-N9-nitro-N- *Author for correspondence ([email protected]) nitrosoguanidine (MNNG) and b-Lapachone. Although we were able to confirm independent roles for JNK and calpain in PARP1 necrosis, Received 25 January 2014; Accepted 8 July 2014 RIP1, Bax, AIF, and MPT appear to be dispensable. Journal of Cell Science 4134 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 4134–4145 doi:10.1242/jcs.128009 RESULTS interfering (si)RNA reduced RIP1 protein levels by ,80% b-Lapachone and MNNG induce PARP1-dependent, caspase- (Fig. 2A). In control siRNA-transfected cells b-Lapachone and independent necrosis MNNG induced necrotic death in a dose-dependent manner We first verified that b-Lapachone and MNNG were indeed (Fig. 2B,C). Depletion of RIP1, however, failed to attenuate the acting through activation of PARP1. Treatment of wild-type cell death effect of either agent (Fig. 2B,C). Pharmacological mouse embryonic fibroblasts (MEFs) with both b-Lapachone inhibition of RIP1 with necrostatin-1 similarly failed to attenuate and MNNG induced substantial cellular necrosis (Fig. 1A). b-Lapachone- and MNNG-induced necrotic death (supplementary Importantly, the cytotoxic effect of both compounds was material Fig. S1A,B). We repeated these experiments using ablated in PARP1-deficient MEFs (Fig. 1A). We observed no Ripk12/2 MEFs which are completely devoid of RIP1 (Fig. 2D). evidence of caspase-3 cleavage in the treated cells in contrast to However, even the complete absence of RIP1 did not affect the treatment with staurosporine, a canonical apoptosis inducer ability of b-Lapachone and MNNG to evoke necrosis (Fig. 2E,F). (Fig. 1B), and b-Lapachone- and MNNG-induced cell death With regards to JNK signaling, we first examined whether was insensitive to the pan-caspase inhibitor benzyloxycarbonyl- PARP1 activation elicited activation of JNK. Treatment of MEFs Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-FMK) (Fig. 1C). with either b-Lapachone or MNNG caused a dose-dependent These data confirmed that both b-Lapachone and MNNG induced increase in JNK phosphorylation (Fig. 3A), indicative of activation. PARP1-dependent necrosis, rather than apoptosis, in MEFs. Moreover, this was significantly attenuated by SP600125, an inhibitor of JNK signaling (Fig. 3A). Co-incubation with SP600125 b-Lapachone- and MNNG-induced necrosis is dependent on was also able to significantly inhibit b-Lapachone- and MNNG- JNK but not RIP1 induced cell death (Fig. 3B,C). Thus, unlike RIP1, JNK activation As a PARP1–RIP1–JNK pathway has been proposed (Xu et al., appears to be a crucial step in PARP1-mediated necrosis. We then 2006; Chiu et al., 2011), we next examined the role of the pro- tested which JNK isoform is involved by knocking down either necrotic signaling kinases RIP1 and JNK in PARP1-mediated JNK1 or JNK2 in the MEFs (Fig. 3D). Interestingly, silencing of necrosis. Transfection of MEFs with a RIP1-specific small JNK1 did not greatly affect either b-Lapachone- or MNNG-induced necrosis with only a small reduction observed at the highest concentration of the agents (Fig. 3E,F). In contrast JNK2 knockdown considerably attenuated cell death in response to both compounds suggesting that it is this isoform that plays a causative role in PARP1-mediated necrotic death. b-Lapachone- and MNNG-induced necrosis is dependent on Ca2+ and calpain In addition to JNK, previous studies have implicated the mobilization of Ca2+ and activation of the Ca2+-dependent protease calpain as key proximal signals in b-Lapachone- and MNNG-induced necrosis (Tagliarino et al., 2003; Moubarak et al., 2007; Dong et al., 2010). Consistent with this, co-treatment with the Ca2+ chelating agent BAPTA-AM significantly attenuated the degree of necrotic cell death in response to b-Lapachone and MNNG (Fig. 4A,B). Calpain activity was also dose-dependently increased by both b-Lapachone and MNNG (Fig. 4C,D). To genetically inhibit the m- and m-calpains we transfected the MEFs with siRNA