TNFα–induced ROS plays an important role in alcoholic liver injury [1
], but TNFα alone usually fails to induce liver injury as TNFα activation of NF-κB blunts TNFα induced liver damage [14
]. The induction of CYP2E1 by ethanol is one important pathway by which ethanol induces oxidative stress [10
]. Increasing CYP2E1 by pyrazole treatment sensitizes the liver to TNFα-induced liver injury. We used a concentration of TNFα which by itself was too low such that TNFα signaling and injury was minimal which allows the potentiation of TNFα actions by induction of CYP2E1 to be studied. In this model, liver injury is associated with activation of JNK and p38 MAPK and elevated production of ROS and RNS [17
]. JNK and p38 MAPK inhibitors such as SP600123 and SB203580 and ROS or RNS inhibitors such as NAC or 1400W blocked the CYP2E1 plus TNFα liver injury. How JNK or p38 MAPK were activated and the upstream signaling MAPKKKs and MAPKKs, are not known and were a major focus of the current study. A time course of in vivo liver injury induced by PY plus TNFα was carried out to determine the sequence of events and relationships between induction of CYP2E1, oxidative stress, the activation of ASK-1, MKK3/MKK6, MKK4/MKK7, p38 MAPK and JNK with the development of liver injury. Another goal was to evaluate whether apoptosis occurs in this injury model. summaries a comparison of the effects of TNFα plus PY relative to TNFα alone on what we believe to be important contributors to mechanisms by which TNFα plus PY produce liver injury in WT mice, but not in CYP2E1 knockout mice. The table shows time courses for changes in liver parameters produced by TNFα plus PY at 4, 8 or 12h after administration of TNFα to PY-treated mice relative to any changes produced when TNFα was given to saline-treated mice. The liver injury occurs at 8 to 12h after the addition of TNFα. It would therefore be expected that increases (or decreases) in critical components for this injury would likely change prior to the liver injury and this is the basis for the discussion below.
Comparison of the effects of TNFα plus pyrazole as compared to TNFα alone on liver parameters
Since ROS is postulated to be a critical factor in the mechanism by which TNFα plus PY induce liver injury, development of ROS should precede the liver injury. Indeed, hepatic GSH levels were decreased and TBARS levels were elevated 4h after administration of TNFα to PY-treated mice. Thus oxidative stress precedes the liver injury. This is consistent with the ability of the antioxidant NAC to blunt the liver injury [18
]. A likely contributor to the increase in oxidative stress is the induction of CYP2E1 by the pyrazole treatment as no injury or oxidative stress was observed in CYP2E1 knockout mice. CYP2E1 levels were already elevated at the time of TNFα administration (0h) since the mice were treated for two days prior to this injection of TNFα on day 3.
ASK-1, a member of the MAPKKK family, activates both MKK4/MKK7-JNK and MKK3/MKK6-p38 MAPK signaling cascades. ASK-1 is activated in cells subjected to stresses such as ROS or TNFα [25
]. In non-stressed cells, ASK-1 forms an inactive high-molecular-mass complex with Trx [30
]. Trx is an oxidation/reduction (redox) regulatory protein, which inhibits the kinase activity of ASK-1. Upon ROS stress, the oxidized Trx is dissociated from ASK-1, resulting in activation of ASK-1 [21
]. ASK-1 was activated in PY-treated mice at 4h after the administration of TNFα. Immunoprecipitation analysis showed that ASK-1 was dissociated from the inactive Trx-ASK complex at 4h, consistent with the activation of ASK-1 at 4h. In CYP2E1−/− mice, pyrazole plus TNFα treatment failed to activate ASK-1 and ASK-1 was not dissociated from the Trx-ASK1 complex. TNFα alone failed to activate ASK-1 or release ASK-1 from the inhibitory Trx complex. If CYP2E1 generated ROS is important for the release and activation of ASK-1, elevation of CYP2E1 and in oxidative stress should occur as early events. Increases in CYP2E1 and ROS occur at 4h, at least consistent with the activation of ASK-1 at 4h, although future experiments with shorter time intervals will be necessary to evaluate these relationships in more detail. Recently, ASK-1 was shown to be important for acetaminophen-induced liver injury [31
]. Our results implicate a role for ASK-1 in CYP2E1 potentiation of TNFα-induced liver injury. Future experiments with ASK-1 knockout mice [31
] are planned to further validate the role of ASK-1 in the PY/TNFα model.
JNK or p38 MAPK activities are increased upon phosphorylation by MAPK kinase (MKK4/MKK7 or MKK3/MKK6) [24
]. The activity of ASK-1 modulates and regulates the phosphorylation of MKK4/MKK7 and MKK3/MKK6. PY plus TNFα treatment increased MKK4 phosphorylation at 4, 8 and 12h while activation of MKK7 was delayed until 12h. MKK3 and MKK6 phosphorylation were also increased at 4 to 8h. In CYP2E1−/− mice, no MAPKK was activated at any observation time point. TNFα alone did not significantly activate the MAPKK in wild type or CYP2E1−/− mice. The activation of MKK4 and MKK3/6 (4–8h) occur prior to the onset of liver injury (8–12h).
Activated MAPK can be inactivated through dephosphorylation of threonine and/or tyrosine residues within the activation loop. The dephosphorylation can be catalyzed by serine/threonine phosphatase, tyrosine phosphatase and dual-specificity phosphatases or by MAP kinase phosphatase (MKP) [27
]. In the MKP family, MKP-1 plays a critical role in the negative regulation of JNK and p38 MAPK in response to stress including oxidative stress [32
]. MKP-1 levels and activity are very sensitive to ROS [28
]. This raised the question as to whether CYP2E1-generated ROS activated MAPKKK, MAPKK, and MAPK not only via removal of Trx from the Trx-ASK1 complex, but also by decreasing MPK-1. PY plus TNFα treatment slightly increased levels of MKP-1 at 4 and 8h. MKP-1 has been shown to be induced by moderate oxidative stress [27
]. Perhaps the modest increase in MKP-1 is an adaptive response to CYP2E1 plus TNFα oxidative stress. However, MKP-1 is decreased by more pronounced oxidative stress. This may explain the large decrease at 12h after administration of TNFα to PY-treated mice. It is interesting to speculate that the ROS induced decrease of MKP-1 at 12h and 24h may play a role in the sustained activation of JNK and p38 MAPK found 24h after addition of TNFα to PY-treated mice [17
TNFα induced cell apoptosis involves activation of the caspase 8 pathway [34
]. Pyrazole plus TNFα treatment activated caspase 8 and caspase 3 at 12h, occurring after the increase in oxidative stress (4h). The large increases in TUNEL staining at 12h occur in association with the activation of caspase 8 and 3 at this time point. The smaller increases in TUNEL at 4 and 8h are not associated with increases in caspase 8 and 3 activity and possibly may be caspase-independent or may reflect non-apoptotic DNA fragmentation. In CYP2E1−/− mice, pyrazole plus TNFα treatment do not increase TUNEL-positive cells or activate caspases 8 and 3. Thus, combined treatment with TNFα plus PY leads to cell death with features of either apoptosis or necrosis or both, a form of cell death termed necroptosis [35
]. An extensive signaling network regulating necroptosis, including Bcl-2 family members, is required for TNFα mediated death-receptor-induced necroptosis [37
]. Whether liver injury in the PY plus TNFα model reflects a model of necroptosis will require further study, e.g. does inhibition of apoptosis inhibit necrosis?
Bcl-2, a major anti apoptotic protein was decreased as early as 4h and remained at low levels at 8 and 12h after TNFα administration to the PY mice. Bax, a major pro-apoptotic protein was increased at 4, 8 and 12h. Levels of cFLIPL
which prevent TNFα induced complex II formation and block the activation of caspase 8 [29
] were decreased at 4h. Thus, the TNFα plus PY treatment results in an early decline in antiapoptotic factors such as Bcl-2, and c-FLIP and an increase in pro-apoptotic factors such as Bax at 4 to 12h. These changes, which would promote activation of caspases, occur prior to the increases in caspase 8 and 3 activity (12h). Treatment of mice with PY plus TNFα was previously shown to induce mitochondrial damage, causing a decline in membrane potential and an increase in membrane permeability transition [17
]. Whether the decline in Bcl-2 and/or the increase in Bax contribute to the mitochondrial dysfunction remains to be determined.
The role of CYP2E1 in the activation of ASK-1, MKK4/MKK7 or MKK3/MKK6 is apparent, since TNFα treatment only induced such activations in wild type mice treated with PY to induce CYP2E1 but not in CYP2E1−/− mice. We hypothesize that TNFα alone-or CYP2E1 alone- generated ROS stress is not sufficient to trigger the dissociation of ASK-1 from the Trx-ASK complex. The CYP2E1 sensitization of TNFα induced liver injury may occur through a synergistic effect with TNFα to produce an enhanced ROS stress consistent with the so call —Two Hit hypothesis. ASK-1 can activate NF-κB in a redox-dependent manner and this may serve as a survival signal in hepatocytes early during injury. We previously found that while TNFα alone caused some activation of NF-κB and increased production of NF-κB-dependent survival factors such manganese SOD2, this upregulation was blocked in the livers from the TNFα plus pyrazole treated mice [18
]. Thus, a possible TNFα-ASK-1-increase in the NF-κB survival pathway is blunted by the increase in CYP2E1 (and ROS) produced by the pyrazole treatment.
The scheme shown in summarizes mechanisms that we believe play a role in the potentiation of TNFα liver injury by CYP2E1. We hypothesize that CYP2E1-derived ROS are critical to the TNFα–induced dissociation of ASK-1 from the Trx-ASK complex and lead to the activation of ASK-1. The fact that TNFα alone added to wild type mice, or TNFα plus PY added to CYP2E1−/− mice failed to activate ASK-1, and ability of the ROS/RNS inhibitors NAC and 1400W to block the activation of JNK or p38 MAPK [18
] suggests that TNFα plus CYP2E1 derived ROS/RNS are —Two Hits to activate MAPKKKs like ASK-1, followed by activation of the downstream MAPKK and subsequently JNK and p38 MAPK. CYP2E1 plus TNFα also induced the degradation of MKP-1, a MAPKK or MAPK negative regulator. Levels of MKP-1 remain low after 12 or 24h treatment, which may contribute to sustained activation of JNK or p38 MAPK by the PY plus TNFα treatment. The decrease of Bcl-2 and the increase of Bax suggest that the ASK-1-MKK3/6, MKK4/7-p38 MAPK, JNK signaling pathway may target the mitochondria and produce mitochondrial dysfunction and subsequently liver injury. cFLIP blocks the ability of TNFα induced complex I to recruit caspase 8 to form complex II followed by activation of caspase 8. The decrease in cFLIP produced by the TNFα plus PY treatment may result in enhancement of caspase 8 activity. Activated caspase 8 may increase proapoptotic Bcl-2 family members such as Bax and Bid which causes mitochondria to release apoptosis factors to activate caspase 3 and induce apoptosis. Although further studies to explore aspects of this scheme are required, this study may offer possible mechanisms to explain how two independent major risk factors in alcohol-induced liver injury, TNFα and CYP2E1, interact to promote liver injury.
Scheme. CYP2E1 sensitizes TNFα to activate ASK-1 and its downsteam signaling targets