Activated JNK signaling pathway exacerbates oxidative stress.
Depletion of GSH and formation of protein adducts in mitochondria damages the electron transport chain(ETC) participating in ATP synthesis. The damaged ETC results in production of more ROS[30]. Furthermore, depletion of GSH and formation of protein adducts lead to damage of the mitochondrial antioxidant defense system and inhibits elimination of ROS [30].In addition, NAPQI binds to mitochondrial respiratory compounds thus inhibiting electron transfer in the mitochondrial respiratory chain [31]. Moreover, JNK signaling cascade is activated to amplify oxidative stress [32].
JNK signaling pathway is an important part of the MAPK cascade where MAP Kinase Kinase Kinase (MAPKKK) is first activated to form activated ASK-1, MLK3 and GSK3β[33-36]. This is followed by modification through phosphorylation thus activating MAP Kinase Kinase (MAPKK) which ultimately activates JNK [37]. Phosphorylated JNK and activated GSK-3β then translocate to the mitochondria [34, 36]. ASK-1, is inhibited through binding to thioredoxin in the cytoplasm and mitochondria [38], is activated by dissociating from thioredoxin due to oxidation of sulfhydryl groups in thioredoxin by ROS [39]. MLK3 induces JNK phosphorylation within 1 hour of APAP injection [28]. A previous study reports that ULK1/2 activates JNK through MKK4/7 and phosphorylation of Ser403 site of MKK7 by ULK1/2 is necessary for phosphorylation of Ser271/Thr275 site of MKK7 by MAP3K in APAP-induced liver injury. Therefore, ULK1/2-dependent phosphorylation of MKK7 is a crucial step in APAP-induced JNK activation [40]. Furthermore, MKK4 and MKK7 exhibit a synergistic effect in JNK activation [40]. Notably, activity of mTORC1 is inhibited when after administration of an overdose APAP [41], consequently inhibits phosphorylation of ULK1/2. This enhances activity of ULK1/2 and activation of JNK [40]. A previous study divided JNK phosphorylation into the early and late stages [42]. The study reported that phosphorylated-GSK-3β/JNK axis is a major source of APAP-induced liver injury during the early phase whereas the ASK-1/JNK axis is implicated in liver injury during the late stages [42]. Therefore, inhibition of these upstream molecules of JNK pathway attenuates JNK activation and protects the liver against APAP-induced toxicity.
Mitochondria act as the upstream organelle for JNK activation and a downstream target for activated JNK. Therefore, inhibition of JNK prevents mitochondrial injury in APAP-induced liver injury [43]. Moreover, downstream targets for p-JNK are mitochondria under oxidative stress and not healthy mitochondria [11].
In addition, phosphorylated JNK translocates to the mitochondria and binds to the anchor protein, Sab, located in the outer mitochondrial membrane [44]. The effect of p-JNK is gradually amplified after translocation and binding to Sab. Proteins in the intermembrane space of mitochondria are released into the cytoplasm following translocation of p-JNK and binding to Sab. Moreover, protein tyrosine phosphatase nonreceptor type 6 (SHP1) is released into the cytoplasm from the mitochondrial Sab channel [45]. Notably, Sab is required for sustained activation of JNK and silencing Sab protects against APAP [46]. Moreover, JNK-mitochondrial signaling loop is a vicious circle that continuously amplifies APAP-induced liver damage [23].
The main molecular targets for p-JNK are members of the Bcl-2 family. Translocation of Bax to the mitochondria is a downstream response for JNK activation after APAP overdose [47] which has been reported several models [48]. P-JNK promotes translocation of Bax to the mitochondria by directly phosphorylating Bax or by phosphorylating 14-3-3 anchoring Bax in the cytoplasm [48, 49] thus inducing mitochondrial Membrane Permeability Transition (MPT). MPT is a common and important mechanism for different forms of hepatotoxicity. The inner mitochondrial membrane is permeable to small molecular solutes and the proton gradient of oxidative phosphorylation is disrupted when the mitochondria membrane is depolarized [50]. Loss of mitochondrial membrane potential results to uncoupling of oxidative phosphorylation [20] and swelling of the mitochondria [18, 51, 52]. Bax is necessary for MPT [2] implying that JNK and Bax have a synergistic effect. A previous study reports that mitochondria are still polarized 3.5 hours post APAP injection but were depolarized after 4.5 hours [47]. This observation implies that MPT is a downstream step in JNK activation. Previous studies divided MPT into two open modes, namely, ① regulatory MPT, which was inhibited by the MPT inhibitor, CsA and occurred following exposure to a low dose of APAP or a short time and ② non-regulatory MPT, which occurred when a high dose of APAP was administered [53]. These modes explain different outcomes from injection of the above-mentioned high or low doses of APAP, partially or completely. Release of ROS including superoxide from the mitochondria, results in oxidation of sulfhydryl groups in MPT pores which is a key factor for MPT. This is concept was proven through reduction of disulfides by dithiothreitol reduced thus preventing occurrence of MPT [18, 54]. However, studies report contradicting findings on the roles of Bid and Bcl-xL,the downstream members of Bcl-2 family of p-JNK. Studies report that the levels of Bax and Bid in the mitochondria increase after treatment with APAP. However, truncated Bid (tBid) may not have significant effect on APAP-induced liver injury. In addition, effect of Bax is blocked by JNK inhibitors although Bid and Bcl-xL are not affected by these inhibitors [11]. A different study reports that Bax and Bid translocate to the mitochondria after an APAP overdose and inhibition of caspases prevents cleavage of Bid [55]. Furthermore, a previous study reports that translocation of Bax to the mitochondria inactivates Bcl-xL, which is an anti-apoptotic member of the Bcl-2 family [56]. Moreover, loss of mitochondrial membrane potential reduces levels of Bcl-2 [31]. This implies that anti-apoptotic members of the Bcl-2 family in the mitochondria are inhibited despite their role in blocking MPT and levels of members of Bcl-2 family are affected by APA administration.
Bax, a downstream molecule of JNK activation forms channels by oligomerization or binding to MPT pores to promote release of cytochrome C [23]. Therefore, after the continuously activated JNK translocates to the mitochondria, mitochondrial respiration is severely inhibited and production of ROS increases [7, 57]. Translocation of phosphorylated JNK and subsequent loss of mitochondrial membrane potential have been reported in human liver cells [58] and in a model of UV-induced oxidative stress [59]. In addition to cytochrome C, other intermembrane proteins such as endonuclease G and the Apoptosis-inducing Factor (AIF) are released which then translocate to the nucleus, leading to fragmentation of nuclear DNA [54, 60]. The blockage of release or translocation to the nucleus of these mitochondrial proteins, downstream molecules of the p-JNK has protective effect [61]. Moreover, ROS are further released thus reacting with the NO produced in this process to form peroxynitrite and nitrotyrosine. Peroxynitrite is a strong oxidant that is released into the cytoplasm through passive diffusion or VDAC anion channel [62].
Protein Kinase Cα(PKCα) is an important target for p-JNK[63]. Its activity increases after APAP overdose and is inhibited by the conventional JNK inhibitor, SP600125 [64]. A previous study reports that after APAP overdose, the levels of PKC-α increase then PKC-α is translocated to the mitochondria. In the mitochondria it also phosphorylates mitochondrial proteins and promotes phosphorylation and translocation of JNK to the mitochondria, subsequently promoting liver damage [63]. Moreover, JNK and PKC-α act synergistically to regulate mitochondrial respiration and mitochondrial-mediated necrotic and apoptotic cell death [21, 65]. Therefore, PKC-α and JNK interact with each other synergistically to participate in feed forward regulation of APAP-induced liver injury [63].
Grb2-associated binder 1 (Gab1) adaptor protein plays an important role in controlling the balance between death and compensatory proliferation of hepatocytes during APAP-induced liver injury[66]. Notably, silencing Gab1 induces activation of p-JNK and increases translocation of p-JNK to the mitochondria. Moreover, silencing Gab1 promotes release of mitochondrial enzymes into the cytoplasm and induces DNA fragmentation [66].
Previous studies report that the Dynamin related protein 1(Drp1), a downstream molecule of p-JNK, is translocated to the mitochondria, thus mediating mitochondrial division [67, 68]. RIPK3, an upstream molecule of JNK mediates production of ROS in mitochondria [69]. Silencing RIPK3 inhibits mitochondrial translocation of Drp1, and prevents release of mitochondrial AIF and DNA fragmentation [67]. In addition, RIPK1 plays a crucial role in sustained activation of JNK[70]. Notably, inhibition of RIPK1 prevents translocation of Drp1 to the mitochondria [68]. Furthermore, inhibition, knockout and/or silencing of ASK1, MLK3, GSK3b, PKCα, JNK, Sab and cyclophilin D (CypD) a mitochondrial permeability transition pore regulator[71], have protective effects against APAP [68]. P-AMPK signaling pathway which induces autophagy inhibits downstream events of p-JNK although it is inhibited after APAP administration. Up-regulation of p-AMPK induced by a PKC inhibitor, exhibits protective effects against APAP toxicity despite sustained JNK activation [63].This is similar to blocking the release and translocation of endonuclease G and the Apoptosis-inducing Factor (AIF) to the nucleus (downstream events of p-JNK), which exhibit a protective effect.
Nrf2 and its Antidote Response Element (ARE) are important antioxidants in the body. Downstream target genes for Nrf2, including HO-1, NQO1 and GSH protects the body from oxidative stress [72, 73], implying that Nrf2 alleviates APAP hepatotoxicity. A previous study reports that upregulation of Nrf2 and its ARE inhibits JNK activation and protects against APAP-induced oxidative stress [74]. Furthermore, some compounds alleviates oxidative stress through AMPK/Akt/Nrf2 in APAP-induced liver injury [31, 75, 76]. However, p-JNK can target Nrf2 to promote its degradation [31, 75]. Biochemical analysis showed that p-JNK directly interacts with the Nrf2-ECH homology (Neh) 1 domain of Nrf2 and phosphorylates the serine-aspartate-serine motif 1 (SDS1) region in the Neh6 domain of Nrf2 [77]. Conversely, a recent report indicated that the protection of flagellin-induced Nrf2 against APAP was dependent upon the activation of TLR5-JNK/p38 pathways [78].
In addition, ER stress is an important mechanism for APAP-induced hepatotoxicity. JNK is an ER stress factor and two key proteins implicated in ER stress including CHOP and Bim are downstream molecules of JNK. 4-PBA, an inhibitor of ER stress effectively prevents JNK activation when administered before APAP injection [6]. c-Jun, a p-JNK downstream transcription factor, binds to the 5’regulatory region of CHOP gene [79, 80] and mediates transcription of the gene. Parkin is a post-translational regulation factor of CHOP that induces degeneration of CHOP through one of the branches of UPR, the PERK/eIF2α/ATF4 pathway. Notably, c-Jun can competitively bind to the binding site of ATF4 in the Parkin gene promoter[74]. Therefore, JNK/c-Jun can inhibit expression of Parkin [81].
Inhibition of JNK-dependent p53 up-regulated Modulator of Apoptosis (PUMA), a downstream molecule of JNK, significantly improves hepatocyte necrosis [82]. NF-κB response gene products inhibit JNK [83] but the process is blocked by depletion of GSH [17].
Some studies report that only JNK2 is involved in amplifying the effect of oxidative stress by translocating to the mitochondria whereas JNK1 has not significant effects on oxidative stress [84]. However, other studies report that both JNK1 and JNK2 translocate to the mitochondria, play a role in amplifying oxidative stress and participate in activation of downstream events. In addition, JNK1 has a greater effect on mitochondrial bioenergetics compared with JNK2 [11]. Therefore, the specific role of the two regioisomers of JNK should be explored further.
In summary, after depletion of mitochondrial GSH by NAPQI and formation of covalent adducts with mitochondrial proteins, oxidative stress, (amplified by the JNK signaling pathway and JNK-related signal transduction pathways) exerts a “second hit”[26] on damage of liver cells. Therefore, this process is a synergistic event that leads to decreased mitochondrial respiration and bioenergetics, mitochondrial dysfunction and eventually severe liver damage.