Acetaminophen knocks on death's door and receptor interacting protein 1 kinase answers
Authors: Maria Eugenia Guicciardi, Gregory J. Gores, Hartmut Jaeschke
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. Supported by National Institutes of Health grants DK63947 (to G.J.G.) and DK102142 (to H.J.). See Article on Page 1847 In the current issue of Hepatology, the study by Dara et al.1 provides fundamental insights regarding the mechanisms of cell death during N‐acetyl‐p‐aminophenol (acetaminophen; APAP)–induced liver injury. Hepatotoxicity with liver failure from intentional and unintentional overdoses of APAP continues to be a public health problem. Therapy is limited to early use of N‐acetylcysteine, which is ineffective later in the course of liver injury. Thus, mechanistic insights concerning the mechanisms of APAP hepatotoxicity remain highly germane. What Are the Mechanisms of APAP Hepatotoxicity, and Have Therapeutic Targets Been Identified? For many years, cell death was believed to occur mainly by two distinct mechanisms: (1) apoptosis, a tightly regulated cell death mediated by proteolytic caspase cascades, and (2) necrosis, an unregulated cell death characterized by massive organelle and cell disruption. Recent studies have identified an additional form of regulated cell death with necrotic morphology termed "necroptosis." APAP induces hepatocyte necrosis; hence, the question of whether this is necroptosis is pertinent. Necroptosis is mediated by two kinases of the receptor interacting protein (RIP) family, RIP1 and RIP3. Association of RIP1 and RIP3 into a complex termed the "necrosome" triggers a necroptotic signal mediated, at least in part, by the activation of mixed lineage kinase domain‐like protein (MLKL), which results in cellular membrane disruption.2 To date, necroptosis has been described mainly following stimulation of death receptors, in particular tumor necrosis factor receptor 1. Necroptosis occurs in settings in which apoptosis is impaired, specifically in the absence of functional caspase 8, an apical caspase that initiates proapoptotic signaling by death receptors. Although necroptosis can be provoked in vivo by genetic deletion of caspase 8, no physiologic or pathophysiologic context has been described where caspase 8 is sufficiently inhibited to permit necroptosis by death receptor signaling. This does not preclude another, yet to be identified, process for RIP‐mediated necrosis. Early observations that RIP3 messenger RNA (mRNA) is almost completely absent in the liver suggested that necroptosis may be unlikely to occur in hepatocytes as RIP3 is requisite for necroptosis. However, several recent studies have reported RIP3 mRNA up‐regulation and protein expression in mouse models of liver injury.3 In particular, two of these studies reported induction of RIP3 in the liver of APAP‐treated mice and confirmed RIP3 expression in human liver tissues from patients with severe APAP toxicity.5 Importantly, an increase in RIP3 protein expression was shown in response to APAP in isolated hepatocytes, an effect that was not detected in RIP3 knockout hepatocytes. Moreover, RIP3 genetic ablation or knockdown by means of antisense morpholinos protected the mice from APAP‐induced liver injury, although this was limited to the early injury phase.5 In support of a role of RIP3 in APAP hepatotoxicity, a recently developed, highly selective RIP3 inhibitor protected against APAP‐induced cell death in isolated human hepatocytes in vitro and in an in vivo mouse model.7 Interestingly, experiments in human hepatocytes demonstrated that the RIP3 inhibitor was more effective than necrostatin‐1 (nec‐1), a selective inhibitor of RIP1 kinase activity. Furthermore, only knockdown of RIP3, but not RIP1, in part reversed APAP‐induced cell death in human hepatocytes.7 Despite these findings, there are reasons to question whether necroptosis contributes to APAP toxicity. First, induction of necroptosis generally requires the engagement of death receptors or toll‐like receptors, none of which are involved in APAP toxicity. Second, the presence of RIP3 in the liver remains controversial, mainly due to the lack of specificity of numerous antibodies commercially available and commonly used to identify RIP3. This lack of specificity was confirmed by Dara et al., who showed that nine commercially available antibodies, including three of the antibodies used in previous studies in the liver, failed to recognize RIP3 by western blot and immunohistochemistry in the presence of appropriate positive and negative controls.1 This obviously is in strong contrast to the reported presence of RIP3 mRNA and protein in wild‐type mouse liver and hepatocytes but not in RIP3 knockout mice.5 On the contrary, only a monoclonal antibody generated at Genentech and not commercially available proved to be highly specific in the same conditions.1 Using this antibody, the authors demonstrated absence of RIP3 in hepatocytes, although high levels of RIP3 were detected in nonparenchymal cells. More importantly, no changes in RIP3 expression in hepatocytes were identified following APAP treatment, and no protection from APAP toxicity, even in the early phase, was reported in RIP3‐deficient mice. This is, again, in strong contrast to the reported induction of RIP3 mRNA and protein expression in liver and hepatocytes after APAP5 and the protection in mouse liver and mouse and human hepatocytes by RIP3 deficiency, knockdown, and pharmacological inhibition.5 The reasons for these discrepancies are unclear at this time; however, it should be noted that the RIP3‐deficient mice used by Dara et al.1 were on a different background (C57Bl/6N) compared to those used in a previous study (C57Bl/6J).5 Nevertheless, the fact that gene knockdown experiments in vivo and in vitro, as well as the use of an RIP3 inhibitor, consistently showed initial protection against APAP hepatotoxicity5 would argue against the hypothesis that susceptibility differences of mouse substrains could fully account for these different results. The absence of RIP3 in nonparenchymal cells (i.e., liver sinusoidal endothelial cells) could also indirectly provide some protective effect against liver injury. Interestingly, Dara et al.1 also observed no protection in MLKL‐deficient mice (C57Bl/6J background). Therefore, because necroptosis is, by definition, mediated by RIP3 and MLKL, necroptosis does not significantly contribute to APAP‐induced liver injury. However, the possibility remains that RIP3 promotes APAP‐induced cell death through MLKL‐independent mechanisms. Ramachandran et al. reported RIP3‐dependent dynamin‐related protein 1 translocation to mitochondria, which promotes mitochondrial fission and oxidant stress.5 The dynamin‐related protein 1 inhibitor mitochondrial division inhibitor 1 was shown to protect against APAP hepatotoxicity, an effect that was confirmed by Dara et al.1 Does RIP1 Mediate APAP Hepatotoxicity? The study by Dara and coworkers also confirmed that RIP1 is required for APAP hepatotoxicity, in line with previous findings mostly based on the hepatoprotective effect of nec‐1. This raises two interesting questions. How is RIP1 activated by APAP? And how does it induce cell death? The answer to these questions will likely require further investigation. Ubiquitination is the main posttranslational modification by which RIP1 is sorted into different signaling pathways, including necrotic cascades. The authors report the presence of slowly migrating RIP1 bands by western blot shortly after APAP treatment, which may be suggestive of RIP1 ubiquitination; but the nature of this posttranslational modification was not investigated, nor was the identity of a potential E3 ligase responsible for APAP‐induced RIP1 ubiquitination. Therefore, the mechanism by which RIP1 is activated by APAP remains to be determined. RIP1 is known to induce caspase‐dependent apoptosis and RIP3‐dependent necroptosis (Fig. 1), the outcome being determined by caspase 8 and/or RIP3 availability. Very little is known about its ability to cause necrosis independently of RIP3 as many previous studies employing nec‐1 or RIP1 kinase–deficient mice/cells in various experimental models of necrosis did not thoroughly examined the contribution of RIP3. In this regard, Dara and coworkers may be among the first to provide solid evidence of RIP1‐mediated necrosis in the absence of RIP3. The authors described RIP1 translocation to the mitochondria following APAP treatment upstream of c‐Jun N‐terminal kinase (JNK) activation and speculate that RIP1 may promote cell death, at least in part by inducing JNK activation (Fig. 1). However, RIP1 activation of JNK may be indirect because, despite RIP1 and active JNK colocalization on the outer mitochondrial membrane, the authors were unable to demonstrate direct interaction between JNK and RIP1. The study by Dara et al. is also notable as the use of RIP1 antisense oligonucleotides in vivo allowed evaluation of the effect of RIP1 knockdown in the whole adult animal, a challenging task given the perinatal mortality of RIP1‐deficient mice. The use of nec‐1 or RIP1 kinase–deficient mice (which are viable and have no overt phenotype) only provides insight on the requirement of the kinase activity of RIP1; however, RIP1 has an equally important function as a scaffold protein facilitating the assembly of intracellular signaling complexes, which can only be assessed by complete elimination of the protein, as achieved by use of the oligonucleotide antisense. Although considerable work remains to clarify these mechanisms, RIP1 may ultimately be a viable target to ameliorate APAP hepatotoxicity. However, potential differences in signaling mechanisms between mouse and human hepatocytes need to be considered.8 Although nec‐1 and RIP1 knockdown experiments consistently showed protection in the mouse model,1 these interventions did not protect in human hepatocytes.7 These findings would be consistent with the observation that APAP‐induced cell death is more dependent on the JNK pathway in mouse hepatocytes than in human hepatocytes.8 Thus, the study by Dara et al.1 clearly argues against the classical necroptosis pathway as a mechanism of APAP‐induced liver cell death, but it also raises a number of important questions about potential alternative roles of RIP1 and RIP3 in APAP hepatotoxicity in the mouse model and, more importantly, in the human system.Figure 1: RIP1‐regulated cell death pathways. Following binding of death receptors (tumor necrosis factor receptor 1, Fas, or tumor necrosis factor–related apoptosis‐inducing ligand receptors) in cells with functional caspase 8 and in the presence of inhibitor of apoptosis protein antagonism, which prevents RIP1 ubiquitination, RIP1 associates with the adaptor molecules FADD and TRADD and with caspase 8, triggering caspase 8–mediated apoptosis (left). When caspase 8 activity is inhibited and in the presence of RIP3, binding of death receptors or toll‐like receptors induces the formation of a signaling complex comprising RIP1, RIP3, and MLKL (named the necrosome), resulting in RIP1‐/RIP3‐dependent necroptosis (center). APAP treatment triggers an increase of cytosolic RIP1 and its translocation to mitochondria by a yet to be identified mechanism. RIP1 activation precedes and contributes to JNK activation, which results in RIP1‐dependent, RIP3‐independent necrotic hepatocyte death. How RIP1 regulates JNK activation is currently unknown (right). Abbreviations: FADD, Fas‐associated protein with death domain; TRADD, tumor necrosis factor receptor–associated protein with death domain.