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.