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].