JNK mediates hepatic ischemia reperfusion injury

Journal of Hepatology 42 (2005) 850–859 www.elsevier.com/locate/jhep

JNK mediates hepatic ischemia reperfusion injury Tetsuya Uehara1, Brydon Bennett2, Steve T. Sakata2, Yoshitaka Satoh2, Graham K. Bilter2, John K. Westwick2, David A. Brenner3,* 1

Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 2 Signal Research Division, Celgene Corporation, San Diego, CA, USA 3 Department of Medicine, Columbia University, 630 W. 168th Street, PH 8 E Room 105, New York, NY, USA

Background/Aims: Hepatic ischemia followed by reperfusion (I/R) is a major clinical problem during transplantation, liver resection for tumor, and circulatory shock, producing apoptosis and necrosis. Although several intracellular signal molecules are induced following I/R including NF-kB and c-Jun N terminal kinase (JNK), their roles in I/R injury are largely unknown. The aim of this study is to assess the role of JNK during warm I/R injury using novel selective JNK inhibitors. Methods: Male Wistar rats (200G25 g) are pretreated with vehicle or with one of three compounds (CC0209766, CC0223105, and CC-401), which are reversible, highly selective, ATP-competitive inhibitors of JNK. In the first study, rats are assessed for survival using a model of ischemia to 70% of the liver for 90 min followed by 30% hepatectomy of the non-ischemic lobes and then reperfusion. In the second study, rats are assessed for liver injury resulting from 60 or 90 min of ischemia followed by reperfusion with analysis over time of hepatic histology, serum ALT, hepatic caspase-3 activation, cytochrome c release, and lipid peroxidation. Results: In the I/R survival model, vehicle-treated rats have a 7-day survival of 20–40%, while rats treated with the three different JNK inhibitors have survival rates of 60–100% (P!0.05). The decrease in mortality correlates with improved hepatic histology and serum ALT levels. Vehicle treated rats have pericentral necrosis, neutrophil infiltration, and some apoptosis in both hepatocytes and sinusoidal endothelial cells, while JNK inhibitors significantly decrease both types of cell death. JNK inhibitors decrease caspase-3 activation, cytochrome c release from mitochondria, and lipid peroxidation. JNK inhibition transiently blocks phosphorylation of c-Jun at an early time point after reperfusion, and AP-1 activation is also substantially blocked. JNK inhibition blocks the upregulation of the pro-apoptotic Bak protein and the degradation of Bid. Conclusions: Thus, JNK inhibitors decrease both necrosis and apoptosis, suggesting that JNK activity induces cell death by both pathways. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; Necrosis; Tumor necrosis factor a; c-Jun; Liver 1. Introduction Ischemia reperfusion (I/R) injury is an important clinical problem for several organs including brain, heart, kidney, and liver. Hepatic I/R injuries occur during transplantation, liver resection for tumor, and circulatory shock [1]. Possible Received 8 November 2004; received in revised form 4 January 2005; accepted 24 January 2005; available online 7 April 2005 * Corresponding author. Tel.: C1 212 305 5838; fax: C1 212 305 9822. E-mail address: [email protected] (D.A. Brenner). Abbreviations: I/R, ischemia reperfusion; JNK, c-Jun N terminal kinase; ALT, serum alanine aminotransferase; AFC, carbobenzoxy-Asp-Glu-Val-Asp-7amino-4-trifluoromethyl coumarin; HNE, 4-hydroxynonenal-modified proteins; ROS, reactive oxygen species; MPT, mitochondrial permeability transition; NFkB, nuclear factor kB; TNFa, tumor necrosis factor a; PVDF, polyvinylidene fluoride; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunoassay; IL-8, interleukin-8; ICAM-1, intercellular adhesion molecule-1; ATP, adenosine triphosphate; ASK1, apoptosis stimulating kinase 1. 0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.01.030

T. Uehara et al. / Journal of Hepatology 42 (2005) 850–859

consequences of hepatic I/R injury include liver failure and/ or multi-organ system failures, resulting in morbidity and mortality [2]. Hepatic I/R injury produces two types of cell death, apoptosis and necrosis in hepatocytes and nonparenchymal cells [3–5]. Several intracellular signaling molecules are activated by I/R including NFkB and c-Jun N terminal kinase (JNK) [6]. However, their role in the molecular pathogenesis of hepatic I/R injury is largely unknown. JNK is phosphorylated and activated by several types of stresses, including stimulation by cytokines, such as TNFa [7] and IL-1 [8], and environmental stresses such as radiation and oxidant stress [9,10]. Substrates for JNK include the transcription factors c-Jun and ATF-2. Recent studies have proposed that activated JNK may also directly affect mitochondria through undefined substrates leading to apoptosis [11–14]. JNK is strongly induced during warm hepatic I/R injury [8] and during the cold ischemia/warm repetition injury of liver transplantation [6,15]. Using selective JNK inhibitors, we now report that JNK blockade suppresses liver injury in a rat model of hepatic warm I/R. These are the first data from animal studies directly demonstrating that JNK plays a harmful role during liver I/R injury.

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by hepatectomy. Survival of the animals is dependent solely on the remaining 70% of the liver, which had been subjected to the ischemic injury. This model allows for maintenance of portal decompression while the liver is rendered ischemia and thus avoids both the use of temporary portacaval bypass and production of intestinal congestion. Animals surviving for 7 days after surgery are considered survivors. With this model, survival rates for vehicle treatment are 77.8 (nZ9), 33.3 (nZ30), and 0% (nZ7) with the ischemic period for 60, 90, and 120 min, respectively. The time of 90 min of ischemia was chosen for the survival study.

2.4. Model of partial hepatic ischemia (liver functional study) A non-lethal model of partial (70%) hepatic ischemia was developed. Under anesthesia using Ketamine and Acepromazine, rats are subjected to 70% ischemia for 60 or 90 min as described above, without resecting the remaining lobes. Rats are sacrificed at different periods after reperfusion for tissue and blood samples. Each JNK inhibitor or vehicle control is intravenously injected at 15 min before the start of ischemia.

2.5. Serum ALT levels after I/R injury Serum alanine aminotransferase (ALT) levels are measured 6 h after reperfusion by automatic analysis by the Pathology Department, University of North Carolina. A period of 6 h after reperfusion is chosen because it corresponds to peak serum enzyme concentrations after ischemic liver injury in the rat [20].

2.6. Histology and cell counting for apoptosis and necrosis 2. Materials and methods 2.1. Reagents Specific JNK inhibitors [16–19], CC0209766, CC0223105, and CC-401 were synthesized by Signal Pharmaceuticals, Inc. Each JNK inhibitor is dissolved in vehicle before injection (5% 1-methyl-2-pyrrolidone, 30% PEG-400, 25% PEG-200, 20% propylene glycol, USP, 20% 0.9% sodium chloride for injection, USP). JNK inhibitors (3–20 mg/kg rat, dissolved in 0.6–8.0 mg/ml vehicle) or equivalent volume of vehicle only are administrated intravenously at 15 min before starting ischemia. Some rats are given JNK inhibitors twice at 15 min before ischemia and at 4 h after reperfusion.

2.2. Animal experiments Male Wistar rats (Harlan, Indianapolis, IN) weighing 175–225 g are used for all experiments. Rats are allowed free access to rat chow and water before and after surgical procedures. All experiments are conducted in compliance with the University of North Carolina Institutional Animal Care and Use Committee.

2.3. Model of total hepatic ischemia (survival study) Liver ischemia followed by partial hepatectomy of the non-involved liver is performed by a modification of the technique described by Kohli et al. [3]. Briefly, rats are anesthetized by intramuscular injection of Ketamine (83.3 mg/kg rat) and Acepromazine (0.03 mg/kg rat). After a midline laparotomy, the portal triad is exposed and all structures (hepatic artery, portal vein, and bile duct) to the left and median liver lobes are occluded with a soft vascular clamp (Diethrich Bulldog Clamp, Fine Science Tools). Some rats receive an injection of dye solution into inflow vessels to ensure complete occlusion. Preservation of perfusion of the remaining liver prevents mesenteric venous congestion during ischemia. During ischemia the abdominal wall is closed with sutures. Immediately after reperfusion, the non-ischemic lobes (right and caudate) are removed

Livers are fixed in 10% Formalin for 24 h at room temperature, washed with water, stored in 70% ethanol at 4 8C, and embedded in paraffin. Sections of 5 mm are stained with Hematoxylin and Eosin. The number of apoptotic and necrotic cells are counted in 10 high power fields (400!) on each slide and their percentages with respect to all hepatocytes are determined (Olympus IX70). Apoptotic hepatocytes and sinusoidal endothelial cells are identified by standard morphological criteria (cell shrinkage, chromatin condensation, and apoptotic body). Necrotic cells are identified by standard morphological criteria (loss of architecture, vacuolization, karyolysis, and increased eosinophilia). The number of neutrophils is counted as described above.

2.7. Caspase 3 measurement Caspase-3 activity is measured with an in vitro fluorogenic peptide substrate carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (AFC) using the FluorAce kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacture’s instructions. Frozen samples of ischemic lobes obtained at 6 h after reperfusion are homogenized in 200 ml of lysis buffer (10 mM Hepes, pH 7.4, 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A, 10 mg/ml aprotinin, and 20 mg/ml leupeptin). Samples are centrifuged at 16,000g in a microfuge for 10 min at 4 8C. The protein concentrations of supernatants are measured using the Bradford assay (Bio-Rad). Supernatants (250 mg) are incubated with 25 mmol/l z-DEVD-AFC (Enzyme and Systems Products, Livermore, CA) at room temperature. The change in fluorescence (excitation at 370 nm and emission at 490 nm) is monitored after 60 and 120 min of incubation time and expressed as picomoles of AFC release per microgram of protein.

2.8. Western blotting for phospho-cJun Liver lobes subjected to 60 min of ischemia and then obtained at 0, 15, 30, or 60 min of reperfusion are prepared for whole cell extraction. Liver samples are homogenized in lysis buffer (10 mM Hepes, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5% NP-40, and 25% glycerol) with protease and phosphatase inhibitors at 4 8C, followed by rotating the tubes for 30 min at 4 8C. After centrifugation, cleared tissue lysates are collected

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and stored at K80 8C for later analysis. Lysates containing 50 mg of protein are separated by electrophoresis on 10% acrylamide SDS gels and transferred to nitrocellulose membranes (Schleicher & Schuell). Equal loading is confirmed by Ponceau S staining. Phospho-c-Jun is detected using rabbit anti-p-c-Jun antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Blots are blocked in blocking buffer (5% non-fat dry milk in PBST) for 1 h, incubated 1 h at room temperature in primary antibody, diluted 1:1000 in blocking buffer, and then 1 h with horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology), diluted 1:1000 in blocking buffer. Proteins are detected with ECL detection reagents (Amersham Corp., Arlington Heights, IL).

2.9. Nuclear extract preparation and electrophoretic mobility shift assay for AP-1 Nuclear proteins are prepared from fresh liver tissues using previously described methods [21]. Protein-DNA binding reactions are carried out for 20 min on ice, using 5 mg nuclear extract, 10 pg of 32 P-labeled DNA probes for the AP-1 consensus binding site [22] with or without 1 mg (100!) unlabeled competitor probe. Complexes are separated by electrophoresis on non-denaturing 4% acrylamide gels and assayed by autoradiography and PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). Pretreatment with c-Jun antibody eliminated the AP-1 complex.

2.13. Immunohistochemical detection of 4-hydroxynonenalmodified proteins Paraffin-embedded sections of liver tissue are deparaffinized, rehydrated, and stained immunohistochemically for the presence of an in vivo marker of lipid peroxidation, 4-hydroxynonenal (HNE) protein adducts, by sequential incubation with a polyclonal antibody (Alpha Diagnostic International, San Antonio, TX), diluted in 1:500 in PBS (pH 7.4) containing 1% Tween 20 and 1% bovine serum albumin. Peroxidase-linked secondary antibody and diaminobenzidine (Peroxidase Envision kit, DAKO, Carpinteria, CA) are used to detect specific binding. The slides are rinsed twice with PBS–0.1% Tween 20 between all incubations, and sections are counterstained with hematoxylin. To control for non-specific binding of the secondary antibody, sections from the same animals are processed without the primary antibody, followed by the procedure detailed above. HNE positive cells are quantified by Image Analyzer.

2.14. Statistics The proportion of surviving rats between the treatments groups is compared using Fisher’s exact test. Mean values for serum ALT, TNFa and percent positive cells for HNE staining are compared using the Student’s t test. Mean values for caspase-3 activities are compared using the Student’s t test after normalizing AFC releases as the percentage of normal rats values. Error bars in figures represent standard errors.

2.10. Western blot analysis for cytochrome c The preparation of cytosolic S100-fractions and Western blot analysis is performed as described previously [6]. Livers subjected to 90 min of ischemia followed by 180 min of reperfusion are prepared freshly for this assay. Untreated rat livers are used as controls. Briefly, S-100 fractions are prepared from fresh liver samples by differential centrifugation in buffer containing 250 mM sucrose. Lysates containing 25 mg of protein are separated by electrophoresis on 15% acrylamide SDS gels and transferred into nitrocellulose membranes. Equal loading is confirmed by Ponceau S staining. Cytochrome c is detected using primary monoclonal anticytochrome c antibody, diluted 1:1000 in blocking buffer (Pharmingen, San Diego, CA) and secondary anti-mouse horseradish peroxidaseconjugated antibody, diluted 1:1000 in blocking buffer (Santa Cruz Biotechnology). Proteins are detected with ECL detection reagents (Amersham Pharmacia Biotech).

2.11. Rat liver K-PASA and Western blots Whole rat liver extracts were run on 4–12% Bis-Tris NuPAGE gradient gels (Invitrogen; Carlsbad, CA) with 15–40 mg per lane (Western blots) or 400 mg per gel (2D gels), and transferred onto PVDF membranes. Immunoblotting was performed with TBSC0.5% Tween 20 and 5% nonfat dry milk. Primary antibodies were: Akt total (BD Biosciences; San Diego, CA), Akt Ser 473 (Cell Signaling Technology; Beverly, MA), PDK1 Ser 241 (Cell Signaling Technology), FKHRL1 Thr 32 (Cell Signaling Technology), GSK-3b Ser 9 (Cell Signaling Technology), Bak total (Biosource; Camarillo, CA), Bid total (BD Biosciences). Blots were evaluated with enhanced chemiluminescence (ECL-Plus, Amersham). Membranes were stripped with Restoree Western Blot Stripping Buffer before reprobing.

2.12. TNFa levels in tissue Livers subjected to 90 min of ischemia followed by 60 and 180 min of reperfusion are prepared for this assay (NZ4, each). Untreated rat livers are used as controls (NZ4). Whole liver tissue is lysed in buffer (25 mM Hepes, pH 7.4, 0.1% CHAPS, 5 mM MgCl2, 1.3 mM EDTA, 1.0 mM EGTA) containing protease and phosphatase inhibitors. Cleared lysates are measured by ELISA (R&D Systems) according to the manufacture’s instructions and adjusted to total protein concentration.

3. Results 3.1. JNK inhibitors improve survival in model of hepatic I/R injury The effect of JNK inhibitors on animal survival was evaluated using a model of total hepatic I/R in which survival depends on recovery of the ischemic liver after reperfusion. Rats (nZ10 per group) were injected intravenously with vehicle, CC0209766 (20 mg/kg rat), or CC0223105 (20 mg/kg rat) 15 min prior to 90 min ischemia and 4 h after reperfusion (Fig. 1A). Vehicle treated rats have a 20% survival rate, whereas CC0209766 or CC0223105 treated rats have a 60 or 70% survival rate, respectively. In another series, rats (nZ5) were injected with vehicle only or CC-401 at different dosage (20 or 10 mg/kg rat) (Fig. 1B). Vehicle treated rats have a 40% survival whereas CC-401 treated rats have an 80% (10 mg/kg rat) and 100% (20 mg/kg rat) survival. In a final series, rats (nZ15 for vehicle, nZ5 each for CC-401 at 10 or 20 mg/kg) were injected with a single administration of vehicle only or CC-401 at 15 min before starting ischemia (Fig. 1C). Vehicle treated rats have a 40% survival whereas CC-401 treated rats have 100% survival (10 or 20 mg/kg rat) (P!0.01). All deaths occurred within the first 72 h after reperfusion. All autopsies show neither signs of intraperitoneal hemorrhage nor macro-thrombosis in the portal vein and IVC. Histological evaluations on autopsies showed both massive necrosis in the liver and inflammatory cells infiltrating the lungs (data not shown), consistent with previous reports [23].

T. Uehara et al. / Journal of Hepatology 42 (2005) 850–859 CC020976620mg/kg CC022310520mg/kg Vehicle

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Fig. 1. JNK inhibitors increase survival in hepatic I/R. Animal survival is evaluated using a model of total hepatic ischemia. Rats (nZ10) are injected intravenously with vehicle, CC0209766 (20 mg/kg), or CC0223105 (20 mg/kg) 15 min before 90 min ischemia and 4 h after reperfusion (A). In a second series, rats (nZ5) are injected with vehicle only or CC-401 at different dosage (20, 10 mg/kg) (B). In a third series, rats are injected with CC-401 only 15 min before ischemia at several dosages (20, 10, 5, 3 mg/kg) (C). Animals surviving for 7 days after surgery are considered survivors.

3.2. JNK inhibitors decrease both hepatic necrosis and apoptosis Liver function was evaluated using a model of partial hepatic I/R. Animals injected with the inhibitors have significant lower levels of ALT at 6 h after reperfusion (CC0223105, 362.5G108, CC0209766, 366.0G54) than those receiving vehicle only (634.3G66) (Fig. 2) (P!0.05). Hematoxylin and eosin staining is performed on ischemic liver lobes from rats treated with vehicle (Fig. 3A,B), CC0223105 (Fig. 3C,D), or CC0209766 (Fig. 3E,F) at 6 h after reperfusion (!100, !400). Rat livers treated with vehicle show hepatocyte pericentral necrosis, increased eosinophilia, and vacuolization

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(Fig. 3A,B). Some hepatocytes and sinusoidal endothelial cells show apoptotic change (Fig. 3B). These morphologic changes are significantly inhibited by JNK inhibitors. The percent of necrotic and apoptotic cells were evaluated on

Fig. 2. JNK inhibitors decrease serum ALT levels at 6 h after reperfusion. Liver injury is assessed in a rat model of partial hepatic ischemia followed by reperfusion. Rats injected with the inhibitors have significant lower levels of ALT than those receiving vehicle only (nZ4, meanGSE; P!0.05, Student’s t test).

Fig. 3. JNK inhibitors decrease histological liver injury assessed at 6 h after reperfusion. (A–F) Hematoxylin and eosin staining is performed on ischemic liver lobes from vehicle (A, B), CC0223105 (C, D), and CC0209766 (E, F) at 6 h after reperfusion (!100, !400). Rat liver treated with vehicle shows pericentral necrosis, increased eosinophilia, and vacuolization (A, B). Some hepatocytes and sinusoidal endothelial cell show apoptotic change (B, arrow). These morphologic changes seen are significantly inhibited by JNK inhibitors (D).

T. Uehara et al. / Journal of Hepatology 42 (2005) 850–859

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Fig. 4. JNK inhibitors decrease necrosis. Rat livers are subjected to partial ischemia followed by 6 h of reperfusion. The percentage of necrotic cells are evaluated on HE stained sections by using general morphological criteria from vehicle, CC0223105 and CC0209766 treated I/R livers, and non-ischemic control livers. The percentage of necrotic cells is significantly reduced by JNK inhibitors. JNK inhibitors also decrease the number of neutrophils as compared to treatment with vehicle alone (*P!0.05, **P!0.01).

HE stained sections using morphological criteria. Rats treated with vehicle have necrotic cells (14.2G4.8%) at 6 h after reperfusion. In contrast, CC0223105 or CC0209766 treatment resulted in significantly fewer necrotic cells (1.82G1.3, 1.45G1.1%) (Fig. 4A) (each, P!0.05), respectively. Neutrophil counts are also increased at 6 h after reperfusion (vehicle, 1.45G0.3%), which is also decreased by JNK inhibitors (CC0223105, 0.27G0.1%; CC0209766, 0.24G0.1%) (Fig. 4B) (P!0.01, P!0.05). Rats treated with vehicle also had apoptotic cells (2.53G0.2%) at 6 h after reperfusion. In contrast, treatment with JNK inhibitors (CC0223105 and CC0209766) significantly decreases apoptotic cells (0.59G0.1, 0.54G0.1%) (each, P!0.0001) (Fig. 5B). Caspase-3 activity was measured in ischemic liver lobes from vehicle, CC0223105, and CC0209766 treated rats at 6 h after reperfusion. Untreated rat livers served as control (88.5G23). Caspase-3 activity shows about a 5-fold increase at 6 h after reperfusion (479.8G87.2) in vehicle treated rats. In contrast, both CC0223105 and CC0209766 treatment significantly decreased caspase-3 activity (146.4G9.8, 121.0G23) (Fig. 5A) (each, P!0.05).

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Fig. 5. JNK inhibitors decrease apoptosis. Rat livers are subjected to partial ischemia followed by 6 h of reperfusion. The percentage of apoptotic cells is evaluated on HE stained sections by use of general morphological criteria from vehicle, CC0223105 and CC0209766 treated I/R livers, and no treatment liver. The percentage of apoptotic cells is significantly reduced by JNK inhibitors (#P!0.0001). Caspase-3 activity is measured in ischemic liver lobes from vehicle, CC0223105, and CC0209766 at 6 h after reperfusion. Non-ischemic rat livers are used as control. Caspase-3 activation is inhibited by these JNK inhibitors (*P!0.05).

3.3. JNK induces mitochondrial damage with release of cytochrome c into the cytoplasm The apopotic pathway may produce mitochondrial damage, resulting in the release of intermembrane proteins cytochrome c and SMAC/Diablo into the cytoplasm [24,25]. Western blotting was performed for cytochrome c using the hepatic cytosolic fraction (Fig. 6) and mitochondrial fraction (Fig. 6) from non-frozen livers obtained after 90 min of ischemia followed by 180 min of reperfusion. Cytosolic fractions from control liver show minimal cytochrome c release, perhaps due to mechanical damage during homogenization. Vehicle treated I/R rats show No I/R

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60min Ischemia Fig. 7. Transient inhibition of phosphorylation of c-Jun and AP-1 binding activity by JNK inhibitor. Western blotting is performed with phospho-cJun antibody (1:1000) for ischemic liver lobes obtained at 0, 15, 30, and 60 min after reperfusion (A). Phosphorylation of c-Jun is seen at 15 min after reperfusion, and increased up to 60 min after reperfusion. Treatment with CC0209766 blocks this increase at 15 min. AP-1 binding activity is measured with 5 mg of nuclear extracts from vehicle or CC0209766 treated liver obtained at 0, 15, 30, and 60 min after reperfusion (B). Cultured rat hepatocyte treated with TNFa is used as a positive control. Increased AP-1 binding activity is seen in the vehicle treatment, whereas SP9766 decrease this activation.

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Since the inhibitors reversibly bind JNK [16], the standard in vitro kinase assay [6] does not detect their effect. Instead, JNK activity may be detected indirectly by measuring the phosphorylation of its endogenous substrate c-Jun. Western blotting was performed for phospho-c-Jun for ischemic liver lobes obtained at 0, 15, 30, and 60 min after reperfusion. C-Jun is phosphorylated at the 15-min time point after reperfusion and increases up to 60 min after reperfusion. In contrast, treatment with CC0209766 blocks phosphorylation of c-Jun at 15 min after reperfusion, but phospho-c Jun is still present from 30 min after reperfusion (Fig. 7A). EMSA for AP-1 was performed on the liver nuclear extracts obtained at 0, 15, 30, or 60 min after reperfusion. AP-1 activity was increased in the reperfused liver nucleus treated with vehicle alone, while this is substantially blocked by CC0209766 (Fig. 7B). In addition to blocking c-Jun phosphorylation and AP-1 activity, JNK may have additional effects mediated by

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unknown substrates. JNK inhibition had no effect on the phosphorylation and activation of the signaling kinases Akt, PDK1, Bax or GSK3b (Fig. 8 and data not shown). However, JNK inhibitors blocked the induction of Bak and the degradation of Bid (Fig. 8), two components of the apoptotic pathway.

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marked cytochrome c release at 3 h of reperfusion following 90 min of ischemia, which was blocked by JNK inhibitor (CC-401). There are no differences between each group on cytochrome c levels in the mitochondrial fraction, reflecting that most mitochondria remained intact. Thus, JNK activation is required for the release of cytochrome c into the mitochondria, an integral component of the mitochondrial pathway of apoptosis.

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Fig. 8. JNK inhibition blocks Bak induction and Bid degradation. Western blotting is performed with primary antibodies against Akt total (BD Biosciences; San Diego, CA), Akt Ser 473 (Cell Signaling Technology; Beverly, MA), PDK1 Ser 241 (Cell Signaling Technology), FKHRL1 Thr 32 (Cell Signaling Technology), GSK-3b Ser 9 (Cell Signaling Technology), Bak total (Biosource; Camarillo, CA), Bid total (BD Biosciences).

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TNFa levels were measured by ELISA assays on liver tissue after 90 min of ischemia and 60 and 180 min of reperfusion with prior treatment with vehicle or CC-401 (Fig. 9). Untreated rat livers served as control (59.5G18.2). Vehicle treatment shows 3.8 and 4.1-fold increases of TNFa at 60 and 180 min post-reperfusion time (217.6G23.2, 242.9G39.7), while CC-401 significantly decreased tissue TNFa (143.3G11.9, 123.5G6.8) (P!0.05) (Fig. 9). 3.6. JNK inhibitors decrease lipid peroxidation Immunohistochemical staining was performed with antibody to 4-hydroxynonenal-modified proteins (HNE) (brown color) on normal untreated rat livers (Fig. 10A) or after 60 min of ischemia and 6 h of reperfusion with prior treatment with vehicle (Fig. 10B), CC0223105 (Fig. 10C), or CC0209766 (Fig. 10D) (!200). HNE positive cells were quantified by Image Analyzer (Fig. 10E). Both the cytoplasm and nuclei were positively stained in vehicle treated I/R livers (Fig. 10B), while treatment with a JNK inhibitor substantially blocks lipid peroxidation (Fig. 10C,D).

4. Discussion JNK is rapidly activated during the reperfusion phase of liver transplantation and following warm ischemia [6,8,26], although the functional consequences of JNK activation were unknown. Specific JNK inhibitors have been developed that inhibit the JNK1, JNK2, and JNK3 isoforms

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Fig. 9. JNK inhibitor decreases hepatic TNFa level. Hepatic TNFa is measured by ELISA in livers from normal rats, or ischemic livers after 60 and 180 min of reperfusion treated with vehicle or CC-401. Hepatic TNFa is increased with vehicle treatment at both time points, while JNK inhibitor decreases tissue TNFa (*P!0.05 vs NT, **P!0.01 vs NT, #P!0.05 vs vehicle, nZ4).

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Fig. 10. JNK inhibitors decrease lipid peroxidation. Immunohistochemical staining is performed for 4-hydroxynonenal-modified proteins (brown) in livers from normal rats (A), or ischemic livers after 6 h of reperfusion treated with vehicle (B), CC0223105 (C), CC0209766 (D). HNE positive cells are quantified by Image Analyzer (E). Both the cytoplasm and nuclei are positively stained in vehicle treated rats (B), while treatment with JNK inhibitor blocks lipid peroxidation (*P!0.05, **P!0.01).

through specific competition for their ATP binding sites [16]. These inhibitors block c-Jun phosphorylation, expression of inflammatory genes in cultured cells, proliferation of cultured cells, and TNFa production by LPS in vivo [16]. The present study demonstrates that JNK inhibitors protect from hepatic injury induced by warm I/R as measured by increased animal survival, reduction in the release of hepatic transaminases, and decreased histological damage. The JNK inhibitors decreased both necrosis and apoptosis, suggesting that JNK activity mediates cell death by both pathways. 4.1. I/R injury induces apoptosis and necrosis I/R injury occurs in the brain [27], heart [28], kidney [29,30], and liver [1,31]. Hepatic I/R induces cell death through both apoptosis and necrosis [3,4,32]. Perhaps the two types of cell death in hepatocytes represent a spectrum that share common intracellular signaling pathways but differing in ATP levels [33]. The mechanism of hepatic I/R injury consists of two phases [34]. Activation of Kupffer cells in the initial phase produces proinflammatory cytokines including

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TNFa and IL-12 [35]. In the second phase, neutrophils are recruited by CXC chemokines including IL-8 and infiltrate by cellular adhesion molecules including ICAM-1. These activated neutrophils release reactive oxygen species (ROS) leading to hepatocellular necrosis [4]. Other studies emphasize the role of apoptotic cell death in hepatic I/R injury. For example, inhibitors of caspases attenuate hepatocyte apoptosis and necrosis in warm I/R [36]. Moreover, TNFa, not Fas, is a key initiator for inducing hepatocyte apoptosis in hepatic I/R [37]. Our study demonstrates JNK inhibitors decrease both hepatocyte apoptosis and necrosis and sinusoidal endothelial cell apoptosis induced by I/R. In addition to morphological criteria, release of cytochrome c into the cytoplasm and caspase-3 activity provide corroborative evidence for hepatocyte apoptosis. JNK inhibitors also block these markers of apoptosis. A unifying model is that apoptosis and necrosis share common pathways, including the mitochondrial permeability transition (MPT) and that the specific type of cell death is dictated by the ATP levels in cells [38]. 4.2. JNK signaling in hepatic I/R In general, several upstream stimuli including cytokines, endotoxin, UV radiation, radicals, and hypoxia activate JNK [9,26,39–41]. In hepatic I/R, JNK is activated rapidly after reperfusion [6,8]. TNFa, a critical cytokine in hepatic I/R injury [42], perhaps mediated by the generation of ROS and the activation of the signaling kinase ASK1 [9,10,40]. JNK inhibitors prevent TNFa-induced apoptosis of cultured hepatocytes [39,43]. However, it is unknown how activated JNK induces cell death in hepatic I/R (this study) or in liver transplantation [44]. Liver expresses both JNK1 and JNK2, which appear to have distinct roles in the regulation of c-Jun phorphorylation [45]. Since the JNK inhibitors block both isoforms, I/R studies adopted to the JNK knock-out mice might reveal the roles of JNK isotypes in I/R injury. The best-characterized JNK substrates are the transcription factors c-Jun and ATF2 [46]. Consistent with JNK activation of nuclear transcription factors, JNK is activated and translocates to the nucleus in cardiac reperfusion injury [47]. Since JNK activation parallels c-Jun phosphorylation after hepatic reperfusion, in our study, one possible target for JNK is c-Jun. JNK inhibitors block this c-Jun phosphorylation at early, but not later time points after reperfusion. A possible target for phospho c-Jun in the AP-1 transcription factor is the TNFa gene itself, which contains an AP-1 binding site on its promoter [48] and mediates hepatic I/R injury [38,49]. Hepatic TNFa levels are increased during I/R injury and this induction is partially blocked by the JNK inhibitors. Therefore, a component of the beneficial effect of JNK inhibition on hepatic I/R injury may be to block the pathway of activated JNK, phosphorylating c-Jun, which in turn induces TNFa gene expression, leading to the hepatotoxic cytokine TNFa.

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Thus, the JNK inhibitors may be protective via inhibiting cytokine or reactive oxygen species production in Kupffer cells [50], preventing dysfunction in sinusoidal endothelial cells [51], or directly protecting hepatocytes [39,43]. 4.3. New target for JNK mediated cell death In addition to inducing transcription of cytotoxic cytokines, JNK may have additional substrates that more directly mediate cell death. For example, Aoki et al. has recently shown that JNK translocates to mitochondria leading to the activation of apoptotic signaling in oxidative stress [12]. During some apoptotic pathways, a critical amplification step is the induction of the MPT with the subsequent cytocylic release of cytochrome c. Cytochrome c then acts as a co-factor to activate caspase-9 and the downstream executionary caspases. The MPT may be induced directly, such as by reactive oxygen species or intracellular calcium, or alternatively may result from activation of death receptors leading to caspase-8 activation and the cleavage of Bid. Our study demonstrates that hepatic I/R induces Bid degradation, the induction of the proapoptotic Bak, the cytosolic release of caspase-3, and caspase-3 activation. JNK decreased all of these mediators of the apoptotic pathway. Thus, JNK may have a substrate that directly acts on the mitochondrial apoptotic pathway during hepatic I/R. Excess ROS accumulation can determine the degree of liver I/R injury [50,52]. HNE is a major product of endogenous lipid peroxidation. The W-6-family of polyunsaturated fatty acids (linolieic and arachidonic acids) may produce HNE as a result of free radical attack. Free radicals produced cell death both through the induction of the MPT and through direct intercellular damage of proteins, lipids and DNA. HNE positive staining occurs predominantly in nuclei of hepatocytes and even in cytoplasm in prolonged ischemia [53]. Our study demonstrates HNE positive cells in nuclei and some cytoplasm of hepatocytes at 6 h after reperfusion, whereas these changes are substantially blocked by JNK inhibitors (Fig. 9). Thus, JNK may regulate ROS generation during hepatic warm I/R. 4.4. Role of JNK during hepatic I/R JNK inhibitors increase survival in a model of hepatic warm I/R. I/R results in production of both apoptosis and necrosis, and JNK inhibitors decrease hepatic injury, lipid peroxidation, and cytochrome c release. JNK may phosphorylate transcription factors such as c-Jun, or may directly phosphorylate new substrates involved in the mitochondrial apoptotic pathway, such as Bak and Bid. This interaction may induce mitochondrial damage. Thus, JNK inhibitors may block this cell death pathway at the mitochondrial level, and decrease ROS generation, cytochrome c release, apoptosis, and necrosis, and protect from warm hepatic I/R injury.

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Acknowledgements Grant support: National Institutes of Health.

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