JNK mediates APPA-induced necrosis, apoptosis and other forms of cell death
In vivo and in vitro studies report that depletion of mitochondrial GSH results in necrosis [85, 86]. Depletion of mitochondrial GSH by DEM results in a significant increase in necrosis [4, 13]. Notably, DEM depletes cytoplasmic and mitochondrial GSH, leading to 100% necrosis instead of apoptosis despite exposure to TNF-α [13]. Administration of low concentration of DEM or APAP only depletes cytoplasmic GSH in hepatocytes, therefore, they become sensitive to TNF-α-induced apoptosis [13, 87]. Moreover, using GSH-EE to restore depleted GSH can reverse sensitivity to apoptosis, induced by TNF-α [87]. Furthermore, antioxidants can restore the levels of GSH and reduce levels of p-JNK [88, 89]. Previous studies report that JNK regulates apoptosis and necrosis [4, 45, 81].
Apoptosis:
JNK promotes cytokine receptor apoptosis signaling pathway. Previous studies report that JNK activation promoted the of release of Fas-L in an autocrine or paracrine manner to increase toxicity in adjacent liver cells [4, 90] and induce the cytokine receptor death signaling pathway. JNK is activated during APAP-induced oxidative stress in hepatocytes and non-parenchymal cells such as sinusoidal endothelial cells. On the other hand, inflammatory cells release pro-inflammatory cytokines such as TNF-α and INF-γ, thus promoting inflammatory reactions and aggravating liver damage under inflammatory conditions [91, 92]. The pro-apoptotic factors, p53 and c-Myc, are phosphorylated by activated JNK in cells exposed to stress factors [4]. Therefore, sustained activation of JNK mediates TNF-α [93] or FasL-induced apoptosis. Although endotoxin-induced production of TNF-α does not increase APAP-induced liver damage [94], Astaxanthin (ASX) reduces apoptosis by inhibiting TNF-α-mediated JNK signaling pathway [95]. Furthermore, TNFα-induced apoptosis following pretreatment with APAP, is inhibited by SNAP (producing NO). Furthermore, SNAP inhibits 50% of caspase3 activity, which is increased by combined treatment with APAP and TNF-α [13]. In addition, silencing TNF-α or TNFR1 exhibits protective effects against APAP-induced liver damage [96]. A previous study reported that JNK inhibitors partially block TNF-α-induced apoptosis in cells treated with APAP, without affecting GSH consumption [13]. Activation of TNF receptor induces activation of caspase 8, which in turn truncates Bid into tBid then tBid is translocated to the mitochondria. Bax is also translocated to the mitochondria where it forms MPT pores with Bak and Bad. These changes induce release of cytochrome C and AIF from mitochondria and translocation to the nucleus. As a result, apoptotic bodies are formed, caspase9 and caspase3 are activated in presence of sufficient ATP. Notably, the TNF superfamily activates death receptors including TNFα, (Fas ligand)FasL and TRAIL. FasL is the most harmful to hepatocytes. Moreover, sensitivity of cells to FasL and toxicity to adjacent liver cells increases with release of cell contents [97]. TRAIL which is a homologue of TNF-α, induces apoptosis in hepatocytes, a process which is independent of direct activation of caspases but is dependent on the activation of JNK and Bim. Therefore, inhibition of JNK and Bim protects hepatocytes against death induced by FasL or TNF-α [97-99]. Furthermore, p-JNK phosphorylates Bim [100], Bax and Bak whereas Bcl-2 and Bcl-xL are inhibited during APAP-induced hepatotoxicity. Therefore, these findings imply that TRAIL induces apoptosis in hepatocytes through the JNK-Bim axis. Previous studies report that extramitochondrial depletion of GSH alters the thiol-disulfide redox state, leading to inhibition of transactivation of NF-kB and sustained activation of JNK, which ultimately induce sensitivity to TNF-α-induced apoptosis [13]. The JNK pro-apoptosis pathway, P38 and NF-kB pro-survival signaling pathways are simultaneously activated [101]. Sustained activation of JNK plays an essential role in primary hepatocytes sensitive to TNFα-induced apoptosis by inhibiting NF-kB [93]. Therefore, JNK plays an important role in promoting apoptosis in liver cells, induced by death receptor signaling during APAP hepatotoxicity. Additionally, sensitivity to TNFα-induced apoptosis in liver cells treated with APAP may promote APAP-induced liver damage. This explains why the dose of APAP required to induce acute liver injury in patients with basic liver diseases such as chronic hepatitis B, alcoholic liver and non-alcoholic fatty liver, is lower compared with that of patients without underlying diseases.
JNK plays a role in stress-induced apoptosis through the mitochondrial pathway [4]. Defect of JNK-deficient fibroblasts in stress-induced apoptosis is mainly located in the mitochondria [59]. Previous studies report that translocation of p-JNK to the mitochondria induces release of cytochrome C and SMAC from the mitochondrial intermembrane space, leading to apoptosis [65, 102]. Notably, release of cytochrome C from the mitochondria is inhibited by the absence of JNK [103]. Additionally, sustained activation of JNK promotes cell damage or death [93, 104]. The potential targets for p-JNK for regulation of release of cytochrome C are the apoptotic regulatory members of the Bcl-2 family[4]. In addition, p-JNK translocation to the mitochondria promotes translocation of Bax to the mitochondria thus activating caspase 9, leading to apoptosis through the intrinsic pathway. Moreover, the anti-apoptotic proteins Bcl-2 and Bcl-xL are phosphorylated by JNK [105, 106]. Therefore, apoptosis through the JNK-dependent but transcription-independent pathway, (the mitochondrial/caspase9 pathway), is implicated in APAP-induced cell death.
Notably, only sustained rather than transient activation of JNK is associated with apoptosis [107], which is consistent with the mechanism of APAP-induced liver injury. For instance, TNF-α causes transient JNK activation with no apoptotic response [13, 108]. Although several studies report that JNK-mediated apoptosis is involved in APAP-induced liver injury, the relationship between JNK-mediated apoptosis and APAP-induced liver injury has not been fully explored. Several studies report that apoptosis is not involved in APAP-induced liver injury because caspases, especially caspase 3, is not activated during APAP hepatotoxicity [109]. Furthermore, a previous study reported that the levels of activated caspase 3 did not increase in human hepatocytes after an APAP overdose [110]. Moreover, previous studies report that pan-caspase inhibitors do not alleviate APAP-induced liver damage [86, 111]. However, TRAIL induces apoptosis in hepatocytes and in a process that is not dependent on the direct activation of caspases but dependent on activation of JNK and Bim [97]. This observation partially explains why caspase 3 is not activated during APAP-induced liver injury as reported by some studies. However, recent studies report that caspase 3 is activated during APAP-induced liver damage [42, 112, 113]. In addition, hydrogen sulfide inhibits apoptosis through the JNK pathway, thus alleviating APAP-induced liver damage [114]. Several compounds protects liver cells against APAP-induced toxicity through anti-apoptotic mechanisms [115, 116]. Previous in vitro studies report a decrease in cell viability and a significant increase in levels of apoptotic cells after APAP administration [74]. Notably, treatment with caspase 3 inhibitors completely blocks APAP-induced apoptosis [47].
Necrosis:
Necrosis is the main form of liver-cell death during APAP-induced liver injury. Previous studies report that APAP-induced liver injury does not involve apoptosis [117], as mentioned above. Studies report that JNK inhibitors block APAP-induced necrotic cell death [13]. Notably, APAP-induced death of liver cells is not characterized by cell shrinkage, nuclear pyknosis and other common cytological features of apoptosis. On the contrary, cytological manifestations APAP-induced liver damage mainly include extensive mitochondrial dysfunction and nuclear lysis accompanied by swelling of cells and organelles and release of cellular contents. These features show that APAP-induced cell death is a oncotic necrosis process [86].Mitochondrial respiration is inhibited after translocation of p-JNK to the mitochondria and ATP production is significantly reduced, leading to insufficient energy for activation of caspase and formation of apoptotic bodies. Previous studies report that when fructose and glycine are administered along with APAP, a significant increase in ATP production is observed and, cells are protected from necrosis although MPT was still normal and an increase in apoptosis was observed [47]. In addition, endonuclease G and the Apoptosis-inducing Factor (AIF) are released from the mitochondria, translocated to the nucleus, where they induce nuclear DNA fragmentation after JNK activation and ultimately initiate necrosis in cells [118]. In addition, apoptosis requires a reduced environment for caspase activation and the redox state of liver cells may affect apoptosis during APAP-induced liver injury [11, 119, 120].Furthermore, Damage-associated Molecular Pattern (DAMP) which induces inflammation, is released and interacts with pattern recognition receptors including toll-like receptors(TLR) on inflammatory cells. These events lead to release of pro-inflammatory cytokines (such as TNFαand INF-γ) and induction of an inflammatory response [91, 92]. These are generally the characteristics of necrosis since apoptosis is not characterized by release of cellular contents. TRAIL-JNK-Bim axis is important in APAP-induced cell necrosis, and deletion of TRAIL or Bim protects hepatocytes and sinusoidal endothelial cells from necrosis [100, 121]. Therefore, ability of TRAIL-JNK-Bim axis in amplification of necrosis in liver cells is greater compared with the ability of the Bcl-2 family members to induce apoptosis through the mitochondrial pathway [97].
Previous studies also report that hepatocytes undergo both necrotic and apoptotic cell death in APAP-induced hepatotoxicity [47, 122]. In addition, upstream events such as JNK activation and translocation to the mitochondria, induction of pro-apoptotic Bcl-2 homologs and increase in MPT with subsequent release of cytochrome C and AIF, are indicators of apoptotic cell death. However, inhibition of mitochondrial respiration and ATP synthesis cannot solely induce apoptosis. Moreover, JNK and MPT mediate apoptosis and necrosis. Therefore, apoptosis is the main form of cell death in the early stages of APAP hepatotoxicity whereas necrosis mainly occurs in the late stages [113, 123]. A previous study reports presence of caspase-cleaved cytokeratin-18 in patients with an APAP overdose, indicating that apoptosis of hepatocytes occurred in the early stages of APAP-induced acute liver injury [124].
However, several studies challenged the widely accepted conclusion that JNK mediates necrotic or apoptotic cell death in APAP-induced liver injury. For instance, knockout of Gst-pi, a negative regulator of JNK, protects mice from APAP-induced liver damage [125]. In addition, simultaneous activation of JNK1 and JNK2 in mice protected them against APAP-induced necrotic cell death by regulating oxidative stress response. Moreover, lack of JunD activation in hepatocytes with specifical knockout of JNK1 and JNK2 (Jnk∆hepa), shows that JNK-JunD-dependent mechanism may be involved in protection of liver cells against APAP-induced liver damage [109]. Nevertheless, JNK signaling pathway plays a central role as it modulates necrotic or apoptotic cell death in APAP-induced liver injury probably through a mechanism that accelerates and amplifies oxidative damage.
In summary, JNK-mediated necrotic and apoptotic cell death are involved in APAP-induced liver injury and the specific form of death may depend on different conditions.
Necroptosis and other Forms of Cell Death
Several signaling pathways involved in stress initiation, amplification, expansion and ultimately cell death, have been identified in APAP-induced liver injury thus promoting the use of the term, programmed necrosis [126]. Previous studies report that RIPK1 and RIPK3 combine and translocate to the mitochondria to mediate necroptosis [67, 127]. In addition, RIPK3 and MLKL are up-regulated after APAP injection, in a time and dose-dependent manner similar to JNK [128]. Notably, JNK is a biomarker of UPR and ER stress. However, other studies report that necroptosis does not affect cell death in APAP-induced toxicity [68]. Moreover, additional studies report several forms of cell death during APAP hepatotoxicity including initial necrosis followed by pyroptosis, apoptosis and necroptosis [129].
In summary, JNK-mediated death of liver cells is necessary for APAP-induced liver toxicity and several protective effects are exhibited through inhibition of JNK. For instance, the tumor suppressor, P53, protects liver cells against APAP-induced liver damage by inhibiting JNK activation [130]. Moreover, 4MP inhibits JNK activation and p-JNK translocation to the mitochondria, thus protecting liver cells against APAP-induced liver damage [131, 132]. Furthermore, the Gadd45β agonist inhibits phosphorylation of MKK4 and JNK [133] whereas Metformin inhibits JNK thus exhibiting protective effects against APAP through Gadd45β [134, 135]. Additionally, quercetin offers protection against APAP-induced liver injury by reducing JNK activation [136, 137]. Inhibition or inactivation of JNK can protect liver cells against APAP-induced liver injury [89, 138, 139].