RIP3 Expression and APAP-Induced Cell Death
RIP3 protein expression in livers of untreated C57Bl/6J mice and hepatocytes was low, but was significantly induced after APAP treatment. RIP3 morpholinos were only modestly effective in reducing the baseline levels of RIP3 but prevented APAP-induced transcriptional activation of RIP3. In contrast to control morpholinos, which did not affect RIP3 induction or APAP-induced liver injury, RIP3 morpholinos were highly effective in reducing APAP-induced cell death. These data suggest that the early transcriptional induction of RIP3 is critical for APAP-induced necrosis. The importance of RIP3 in APAP-induced liver injury was confirmed in RIP3-deficient mice and in cultured hepatocytes, suggesting that RIP3 expression in parenchymal cells is involved in cell death. The mode of cell death after APAP is generally considered to be necrosis due to characteristics such as cell swelling and massive cell contents release, the absence of active caspases, and the lack of a protective effect of caspase inhibitors in this model.[6, 7, 11, 12, 17] The current findings that RIP3 is critical for the injury combined with recent reports on the beneficial effect of the RIP1 inhibitor necrostatin[30, 32] indicate that early APAP-induced hepatocyte cell death fits many characteristics of programmed necrosis (necroptosis). Interestingly, in the absence of RIP3 the remaining cell death is still necrotic based on the TUNEL assay, which shows the typical features of necrotic DNA fragmentation. Although the most studied initiator of necroptosis is TNF-α and TNF receptor 1 signaling, TNF-α is unlikely the initiator in the context of APAP hepatotoxicity. First, there is very limited TNF-α protein formation in response to APAP overdose.[33, 34] Second, TNF-deficient or TNF receptor type 1-deficient mice are not protected against APAP hepatotoxicity[35, 36] and anti-TNF-α antibodies or soluble TNF-α receptor treatment did not affect APAP-induced liver injury. Third, treatment with a low dose of endotoxin, which produces large amounts of TNF-α, did not enhance APAP-induced liver injury. Together, these data suggest that TNF receptor 1 signaling is probably not responsible for the formation of the necrosome and initiation of programmed necrosis during APAP hepatotoxicity. Although TNF receptor signaling is the best-studied initiating event for necroptosis, it is well established that there are multiple ways to trigger this mode of cell death. Further studies are needed to identify potential activators of this pathway in APAP hepatotoxicity.
Downstream Events of RIP3 Activation
A critical question is what downstream events are triggered by RIP3 activation, which may lead to cell death. Mitochondrial dysfunction has emerged as the central and most critical event in the pathophysiology of APAP toxicity in experimental animals and humans. Inhibition of respiration, a selective mitochondrial oxidant stress, mitochondrial DNA damage, nuclear DNA fragmentation dependent on mitochondrial dysfunction, and eventual mitochondrial permeability transition (MPT) pore opening with collapse of the membrane potential and ATP depletion are all hallmarks of APAP-induced cell death. Our current data indicate that the absence of RIP3 or prevention of RIP3 induction can eliminate mitochondrial AIF release and attenuate nuclear DNA fragmentation and the oxidant stress which is almost exclusively located in the mitochondria. Recent insight into the development of the mitochondrial oxidant stress suggests the presence of an amplification loop. The initial metabolic stress, presumably protein adducts in mitochondria, induces a moderate mitochondrial oxidant stress, which triggers JNK phosphorylation through activation of MLK3 and apoptosis signal-regulating kinase 1 (ASK1). P-JNK translocates to the mitochondria and potentiates the oxidant stress, which eventually triggers the MPT and cell necrosis.[5, 40] The fact that RIP3-deficiency prevented mitochondrial dysfunction and oxidant stress suggests that RIP3 acted upstream of JNK activation. Consistent with this conclusion is the observation that the RIP1 inhibitor necrostatin reduced APAP-induced liver injury by inhibiting JNK activation. In addition, overexpression of ARC (apoptosis repressor with caspase recruitment domain), which interacts with JNK downstream of the necrosome, attenuated JNK activation, oxidant stress, and liver cell injury. Thus, the emerging evidence from our data in combination with recently published findings strongly suggests that RIP3 is involved in regulating the mitochondrial oxidant stress through JNK activation.
In addition to modulation of the APAP-induced oxidant stress by RIP3, we observed the mitochondrial translocation of Drp1, which is a protein involved in mitochondrial fission. Mitochondria are now recognized to be dynamic organelles, which undergo continuous cycles of fusion and fission to maintain function. Mitochondrial fission has been shown to occur during cell death, predominantly in models of apoptosis. It was recently shown that RIP3-mediated necrotic cell death can also occur through activation of mitochondrial fission. The molecular control of mitochondrial fission is mediated by Drp1, which polymerizes and constricts mitochondria to facilitate organelle division. Our data provide evidence that mitochondrial fission seems to be a feature of APAP-induced hepatocyte necrosis and this is controlled by RIP3-mediated Drp1 translocation to the mitochondria. The Drp1 inhibitor MDIVI prevented cell death, suggesting that Drp1 translocation and mitochondrial fission are critical events in APAP-induced cell death. Mitochondrial fission has been associated with production of reactive oxygen species (ROS). Thus, RIP3 can affect the critical mitochondrial oxidant stress by controlling JNK activation and mitochondrial fission during APAP hepatotoxicity.
Despite convincing evidence for the involvement of RIP3 in the signaling mechanisms of cell death early after APAP (6 hours in vivo and 24 hours in vitro), the protective effect of RIP3 elimination was lost at later timepoints (24 hours in vivo and 48 hours in vitro). Interestingly, the recently reported protection by the RIP1 inhibitor necrostatin was confirmed in our study; however, this protection was also lost at 48 hours in vitro, as was the reduced cell death with the Drp1 inhibitor MDIVI. Although it is beyond the scope of the present study to elucidate the mechanistic details of this effect, there are some similarities to our previous findings on the role of mitochondrial bax translocation in APAP hepatotoxicity. Bax deficiency was found to prevent the outer mitochondrial membrane permeability, DNA fragmentation, and cell necrosis at 6 hours. However, this effect was lost by 12 hours because of continued mitochondrial oxidant stress, which eventually became the dominant injury mechanism. Our data suggest that the initiation of cell death by RIP3-dominant programmed necrosis signaling is eventually overtaken by a secondary event. The fact that protein adduct formation is unaffected by RIP3 but the downstream mitochondrial dysfunction and oxidant stress is drastically reduced indicates that RIP3 is one factor that controls JNK activation and the mitochondrial oxidant stress. However, if this pathway is eliminated, the effect on mitochondria is delayed but not prevented. More studies are needed to elucidate the delicate network of mitogen-activated protein kinase (MAPK) regulation of the mitochondrial oxidant stress, which may be the ultimate deciding factor in cell death after APAP overdose.
In conclusion, our data identified a unique molecular mediator for early APAP-induced hepatocyte necrosis, namely RIP3. APAP overdose triggers the transcriptional activation of RIP3 and the newly expressed RIP3 is critical for cell necrosis by acting upstream of mitochondrial dysfunction and oxidant stress, which is controlled by JNK activation and mitochondrial translocation of P-JNK. We have also shown that mitochondrial dynamics are relevant to the mechanism of APAP-induced cell death and are controlled by activation of RIP3, which induces translocation of Drp1 from the cytosol to the mitochondria. The resulting mitochondrial fission and oxidant stress lead to activation of the mitochondrial permeability transition, release of AIF, nuclear DNA fragmentation, and hepatocyte necrosis. However, despite its prominent role in the cell death signaling pathway, elimination of RIP3 does not cause long-term protection, most likely due to alternate pathways of amplification of the mitochondrial oxidant stress. Controlling RIP3 induction or modulating function could be a promising new therapeutic approach to prevent the early APAP-induced liver injury, but requires complementary strategies to control the mitochondrial dysfunction for long-term protection.