Activation of ASK-1 and downstream MAP kinases in cytochrome P4502E1 potentiated tumor necrosis factor α liver injury

Tumor necrosis factor alpha (TNFα), plays an important role in alcoholic liver injury [1–3]. Chronic alcohol consumption promotes the permeability of bacteria through the intestinal epithelia into the bloodstream [4]. The elevation in lipopolysarcharide (LPS) induces activation of Kupffer cells and release of cytokines such as TNFα [5–7]. Alcohol-induced liver injury is prevented by destruction of Kupffer cells, by antibodies which neutralize TNFα, and in TNFα receptor 1 knockout mice [3, 8, 9]. Chronic alcohol consumption also induces cytochrome P4502E1 (CYP2E1) [10], an important pathway by which ethanol elevates oxidative stress [11–12]. CYP2E1 is a loosely coupled cytochrome P450, producing superoxide and hydrogen peroxide during its catalytic cycle [13]. In general, TNFα is not toxic to hepatocytes because it also activates NF-κB, with the subsequent production of survival factors [14]. Besides elevating TNFα, alcohol is believed to sensitize the liver to become susceptible to TNFα [6]. CYP2E1 may be one of these sensitizing factors. For example, in-vitro combined treatment with ethanol plus TNFα was toxic to hepatocytes and HepG2 E47 cells which express high levels of CYP2E1 [15]. RALA hepatocytes with increased CYP2E1 were sensitized to TNFα mediated cell death [16]. These results suggest that increased oxidant stress from CYP2E1 may sensitize isolated hepatocytes to TNFα-induced toxicity. Administration of TNFα to mice pre-treated with pyrazole to induce CYP2E1 caused significant liver injury, under conditions in which pyrazole alone, or TNFα administered to saline-treated mice did not cause liver injury [17]. This injury was mediated through a pathway involving elevated oxidative stress and activation of JNK and p38 MAPK [17, 18] and was blocked by inhibitors/scavengers of ROS, iNOS, JNK or p38 MAPK [18]. The potentiation by PY was blunted in CYP2E1−/− mice [17], suggesting an important role for CYP2E1 in the potentiated injury by TNFα plus PY. The upstream mediators of JNK and p38 MAPK activation were not identified in these previous studies. For mechanistic and therapeutic implications, it would be important to evaluate the MAP kinase kinase kinase and MAP kinase kinase which activate JNK and p38 MAPK in this PY plus TNFα model. Apoptosis signal-regulating kinase 1 (ASK-1) is a member of the MAP3K family which is responsive to stress-induced cell damage. Activation of ASK-1 can determine cell fate by regulation of both the MKK4/MKK7-JNK and the MKK3/MKK6-p38 MAPK signaling cascades [19]. ASK-1 is activated by oxidative stress, ER stress, and inflammatory cytokines such as TNFα [20]. In resting cells, ASK-1 forms an inactive complex with reduced thioredoxin (Trx). Under conditions of stress by TNFα or ROS, ASK-1 dissociates from Trx and becomes activated [21]. Oxidation of Trx by ROS causes dissociation of ASK-1 from the oxidized Trx which switches the inactive form of ASK-1 to the active kinase. The Trx-ASK complex is thought to be a redox sensor, which functions as a molecular switch turning the cellular redox state into a MAP kinase signaling pathway [22]. Activated ASK-1 then promotes activation (phosphorylation) of the downstream MAPKK, MKK4/MKK7 which can activate JNK, and MKK3/MKK6 which can activate p38 MAPK [19–22]. Since previous studies showed that in mice treated with TNFα and PY, JNK and p38 MAPK were activated [17, 18], we evaluated whether CYP2E1 plus TNFα induced ROS promote release of ASK-1 from the Trx-ASK1 complex and activate ASK-1 followed by the phosphorylation of MKK4/MKK7 and/or MKK3/MKK6 which subsequently regulate the phosphorylation of JNK and p38 MAPK and contribute to the liver injury. Previous studies were all carried out in samples collected 24h after administration of TNFα. As a secondary goal, we determined the time course for development of oxidative stress, activation of MAPK, induction of CYP2E1, in relation to the development of liver injury in order to understand the sequence of events which occur when TNFα is added to mice pretreated with pyrazole to induce CYP2E1.