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Apoptosis signal-regulating kinase 1 is crucial for oxidative stress-induced but not for osmotic stress-induced hepatocyte cell death
Life Sciences 83 (2008) 859–864
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / l i f e s c i e
Apoptosis signal-regulating kinase 1 is crucial for oxidative stress-induced but not for osmotic stress-induced hepatocyte cell death Kenji Taki 1,2, Rieko Shimozono 1,2, Hajime Kusano 1, Nobutaka Suzuki 1, Katsuhiro Shinjo 1, Hiroyuki Eda ⁎,1 Discovery Biology-3, Nagoya Laboratories, Pfizer Global Research and Development, Pfizer Japan Inc., 5-2 Taketoyo, Aichi, 470-2393, Japan
a r t i c l e
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Article history: Received 7 April 2008 Accepted 3 October 2008 Keywords: Apoptosis signal-regulating kinase 1 (ASK1) Oxidative stress Osmotic stress JNK p38 Reactive oxygen species (ROS) Hepatocyte Liver
a b s t r a c t Aims: In this study, we investigated the involvement of apoptosis signal-regulating kinase 1 (ASK1) in oxidative stress and osmotic stress-induced hepatocyte death. Main methods: Activation of ASK1–JNK/p38 cascade and resulting cell death induced by oxidative and osmotic stress was investigated by Western immunoblot analysis and cell toxicity assay using human hepatoma cell lines, Huh7 expressing high level of ASK1 and HepG2 cells expressing low level of ASK1. Gene knock-down of ASK1 using shRNA against ASK1 was conducted using mouse hepatocyte cell line, AML12. Key findings: Activation of ASK1–JNK/p38 cascade and cell death in Huh7 expressing high level of ASK1 was markedly induced by the oxidative stress. HepG2 expressing low level of ASK1 was resistant to oxidative stress while cell death induced by osmotic stress was comparable between Huh7 and HepG2 cells. Although the phosphorylation of ASK1 was not observed by osmotic stress, the phosphorylation of p38 and JNK and resulting cell death was induced in both cell lines. The phosphorylation of ASK1 and p38/JNK in the mouse primary hepatocyte were also increased by oxidative stress. Knock-down of ASK1 mRNA in AML12 in vitro significantly reduced oxidative stress-induced cell death, however, knock-down of ASK1 in cells did not affect the osmotic stress-induced cell death. Significance: This study revealed that ASK1 regulates oxidative stress- but not osmotic stress-induced hepatocyte death, suggesting ASK1 plays a critical role in oxidative-stress induced hepatocyte death. These results raise the possibility that an ASK1 may be a promising therapeutic target for liver diseases caused by oxidative stress. © 2008 Elsevier Inc. All rights reserved.
Introduction Mitogen-activated protein kinase (MAPK) signaling pathways respond to endogenous and exogenous stresses such as oxidative stress, osmotic stress, proinflammatory cytokines, heat shock, ultraviolet irradiation, and infection (Kyriakis and Avruch, 2001). Following exposure to these stresses stimuli, MAPKs are activated and regulate cellular responses such as cell growth, differentiation, survival and death. MAPK signaling pathways consist of three kinases, MAP3K, MAP2K, and MAPK that form a sequential activation pathway from MAP3K though MAP2K to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is one of the MAP3K that activates p38 and JNK via activating the MAP2Ks, MKK4/MKK7 and MKK3/MKK6 (Ichijo et al., 1997; Wang et al., 1996). ASK1 is activated by a variety of stresses including calcium
⁎ Corresponding author. St. Louis Laboratories, Pfizer Global Research and Development, Pfizer Inc., 700 Chesterfield Parkway West, mail zone AA3C, Chesterfield, MO 63017, USA. Tel.: +1 636 247 2166; fax: +1 636 247 5300. E-mail address: hiroyuki.eda@pfizer.com (H. Eda). 1 Tel.: +81 569 74 4866; fax: +81 569 74 4606. 2 First two authors contributed equally to the study. 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.10.004
influx, endoplasmic reticulum (ER) stress, lipopolysaccharide (LPS), reactive oxygen species (ROS), and tumor necrosis factor (Matsuzawa et al., 2005; Nishitoh et al., 1998; Saitoh et al., 1998; Takeda et al., 2004). These stresses, especially ROS, are critically involved in the pathogenesis of many degenerative and life-style related diseases (Ames et al., 1993; Stadtman, 1992). ASK1 has been shown to play a key role in such ROS-mediated diseases as Alzheimer's disease, diabetes mellitus, ischemia-reperfusion injury (Fujiwara et al., 2007; Kadowaki et al., 2005; Nishikawa and Araki 2007; Watanabe et al., 2005). These studies indicate that the relationship between ASK1induced cell death and diseases mediated by oxidative stress. ROS are involved in the pathogenesis and progression of both viral infectious and non-infectious liver diseases (Arteel, 2003; Miñana et al., 2002; Okuda et al., 2002; Videla et al., 2004; Wang and Weinman, 2006). In infectious liver diseases, chronic hepatitis C patients manifest hepatic oxidative stress that is associated with the progression of liver fibrosis (Wang and Weinman, 2006). HCV core protein alters mitochondria function and results in the increase of ROS and, as a consequence, leads to hepatocyte injury (Okuda et al., 2002). In non-infectious liver diseases, hepatocyte apoptosis induced by ROS also plays an important role in the progression of alcohol-induced liver disease (Arteel, 2003; Miñana et al., 2002) and non-alcoholic
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steatohepatitis (Videla et al., 2004). Several reports have shown a critical role for sustained activation of JNK/p38 in hepatocyte apoptosis that leads to liver diseases and inhibition of p38 kinase attenuates ischemia-reperfusion injury of liver (Crenesse et al., 2000; Yoshinari et al., 2001). Given that fibroblasts derived from ASK1−/− embryo were resistant to oxidative stress-induced JNK and p38 activation and the subsequent apoptosis (Tobiume et al., 2001), these data indicate that ASK1 may be involved in the hepatocyte death mediated by oxidative stress in liver diseases. Indeed, Gilot et al. (2002) reported that inhibition of ASK1 by a free radical scavenging molecule prevents apoptosis in primary rat hepatocyte as well as thioacetamide-induced liver injury in vivo. However, it remains to be determined if there is the direct relationship between ASK1 and oxidative stress in hepatocyte death by gene knock-down of ASK1. Thus, we investigated the involvement of ASK1 in hepatocyte death to determine the direct relationship between ASK1 and oxidative stressinduced hepatocyte death.
Materials and methods Animals Male BALB/c mice were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). All experimental procedures used in this study were approved by the local ethics committee based on international guidelines (Institutional Animal Care and Use Committee) and adherence to the Pfizer policy.
Cell culture HepG2 and AML12 cell lines were purchased from the American Type Culture Collection (Manassas, VA). Huh7 cell line was purchased from Health Science Research Resources Bank (Osaka, Japan). HepG2 cell line was cultured in MEM culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 μg/ml penicillin and streptomycin. Huh7 cell line was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 100 μg/ml penicillin and streptomycin. AML12 cell line was cultured in DMEM/F-12 1:1 mixture medium supplemented with 10% heat-inactivated FBS, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethazone, 100 μg/ml penicillin and streptomycin.
Isolation of hepatocyte from mouse Livers were perfused for 5 min from a portal vein using the Liver Perfusion Medium (Invitrogen, Carlsbad, CA) in anesthetized mouse. After 5 min perfusion, the Liver Perfusion Medium was changed to the Liver Digest Medium (Invitrogen) and perfusion was continued further 15 min and then the livers were removed. Livers were minced in Hepatocyte Wash Medium (Invitrogen), filtered using Cell strainer (BD Bioscience, Franklin Lakes, NJ), then centrifuged at 54 ×g for 1 min. Cell pellet was resuspended into the Hepatocyte Wash Medium and the centrifugation step repeated twice. The pelleted cells were cultured in the plating medium (William's E medium, 5% heat-inactivated FBS, 4 µg/ml insulin, 1.0 µM dexamethazone, 100 μg/ml penicillin and streptomycin) for 5 h followed by overnight culture in incubation medium (William's E medium, 5% heat-inactivated FBS, 4 µg/ml insulin, 0.1 µM dexamethazone, 100 μg/ml penicillin and streptomycin). The following day, culture medium was changed to the culture medium (William's E medium, ITS+ premix (BD Bioscience), 0.1 µM dexamethazone, 100 μg/ml penicillin and streptomycin).
Quantitative RT-PCR The total cellular RNA was isolated using TRIzol® Reagent (Invitrogen) and purified using RNeasy kit (Qiagen, Valencia, CA). The TaqMan® probes were purchased from Applied Biosystems (Foster City, CA). The primers for each gene are as follows, human ASK1: Hs00178726_m1, mouse ASK1: Mm00434883_m1, and mouse GAPDH: Mm99999915_g1. Human GAPDH was purchased from Applied Biosystems (Pre-Developed TaqMan® Assay Reagents). Reverse transcription reaction was carried out using a SuperScript™ III First Strand Synthesis System (Invitrogen). Real-time PCR was carried out using Platinum® qPCR SuperMix-UDG with ROX (Invitrogen) on a real-time PCR (ABI PRISM7900, Applied Biosystems) with the default reaction conditions. Measurement of cell death A single suspension of Huh7 and HepG2 cells (2.5 × 104 cells) was plated into a 96-well culture plate and cultured for 24 h. Then cells were exposed to hydrogen peroxide (H2O2), menadion, and sorbitol for 24 h. Cell viability was measured with the LDH cytotoxic test kit (Wako Pure Chemical, Osaka, Japan) or the Cell Counting Kit-8 (DOJINDO, Osaka, Japan). Western blot analysis Cells were washed twice with cold PBS, and then incubated for 5 min on ice in the M-PER lysis buffer (PIERCE, Rockford, IL) including the Complete Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IL). Cell lysate was sonicated and centrifuged at 14,000 ×g for 10 min to remove insoluble material. Samples (30 μg each) were separated by 7% SDS-PAGE and electro-transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Nonspecific binding was blocked with the Blocking One-P blocking buffer (Nacalai Tesque, Kyoto, Japan) then incubated overnight on 4 °C with primary antibody in the CanGetSignal® solution 1 (TOYOBO, Osaka, Japan). The filters were washed three times with TBS with 0.1% Tween-20 (T-TBS) at the room temperature (RT) and incubated with appropriate secondary antibody conjugated to horseradish peroxidase in the CanGetSignal® solution 2 (TOYOBO) for 1 h at RT. Following three times washing with T-TBS, the proteins of interest were visualized with the ECL™ Western Blotting Reagent (GE Healthcare, Waukesha, WI) and filters were scanned with the LumiVisionPRO 400EX (AISIN, Aichi, Japan). Protein concentration was measured by using the Protein Quantification Kit-Rapid (DOJINDO). Anti-ASK1, anti-p38, anti-JNK antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-ASK1-Thr845, antiphospho-p38, and anti-phospho-JNK antibody were from Cell Signaling (Danvers, MA). Secondary antibodies were sheep anti-mouse IgGHRP (GE healthcare), goat anti-rabbit IgG-HRP, and donkey anti-goat IgG-HRP (Santa Cruz). shRNA-expressed adenoviral vector construction, adenoviral infection and gene-knockdown Recombinant adenoviral vectors harboring shRNA against mouse ASK1 (shASK1-Ad) were constructed from replication-deficient adenovirus type 5 with deletions in the E1 and E3 genes obtained using the Gateway ViraPower™ adenovirus vector system (Invitrogen). The ASK1 shRNA sequence was decided to using the BLOCK-iT™ RNAi Designer and forward and reverse oligonucleotides sequences were synthesized by Invitrogen. The sequence of 64-nucleotide encoding mouse ASK1-specific shRNA was as follows: 5′-TGCTGATCCGAAGCAACTTGCTCTTCGTTTTGGCCACTGACTGACGAAGAGCATTGCTTCGGAT-3′ and 5 ′- C CT GAT C C GAAGC AAT G CT C T TC GTC AGT CAGT GG CC A AAA CGAAGAGCAAGTTGCTTCGGATC-3′). These oligonucleotides were
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of 100 and 300. After 24 h of transfection, cells were washed with fresh medium and were further incubated for 48 h. Results Expression of ASK1 in Huh7 and HepG2 cells Expression level of ASK1 in human hepatoma cell lines, Huh7 and HepG2 cells, was compared using RT-PCR and Western immunoblot. The expression level of ASK1 mRNA in Huh7 was more than 5-times higher than that of HepG2 cell line (Fig. 1A). Western blot analysis revealed that the ASK1 protein was expressed in Huh7 cells but not but in HepG2 cells (Fig. 1B). In contrast, protein expression level of JNK and p38, the downstream kinases of ASK1, were quite similar in both cell lines (Fig. 1B).
Fig. 1. MAPK expression in Huh7 and HepG2 cell lines. A: Expression of ASK1 mRNA in Huh7 and HepG2 cell lines measured by RT-PCR. ASK1 mRNA levels were normalized using GAPDH. The quantity of ASK1 mRNA was expressed as relative quantity to that of Huh7. Data were expressed as mean ± SD (n = 2). B: Western blot analysis of ASK1, p38, and JNK expression in Huh7 and HepG2 cell lines.
annealed and subcloned to the pcDNA™ 6.2-GW/miR, then further subcloned to pDONR™ 221 and pAd/CMV/V5-DEST with the Gateway system to develop pAd/CMV/V5-DEST-shASK1. Clones were selected and amplified by transforming into DH5α E. coli cells. PacI linearized adenoviral DNA was transfected into packaging cells (293A) using jetPEI™ (Polyplus-transfection SA, Illkirch, France). Recombinant crude viruses were infected and propagated in 293A cells. After 36–48 h of infection, the cells were lysed by freezing–thawing and viruses were collected from the cells. All viral preparations were purified by the Vivapure® AdenoPACK (Sartorius AG, Goettingen, Germany). Titers of virus were determined by the plaque assay using 293A cells. The AML12 cells were transfected with shASK1-Ad at multiplicity of infection (MOI)
Table 1 Oxidative stress- and osmotic stress-induced cell death in Huh7 and HepG2 cell lines Stimulation (mM) H2O2 (0.3) H2O2 (1.0) H2O2 (3.0) Menadion (0.02) Menadion (0.05) Sorbitol (200) Sorbitol (500) Sorbitol (800)
Cell viability (%) HepG2
Huh7
100 ± 3.2a 92 ± 5.9 79 ± 5.0 96 ± 1.0 85 ± 6.5 76 ± 1.4 60 ± 3.8 39 ± 2.9
35 ± 2.9 0.44 ± 0.48 0.89 ± 0.14 104 ± 2.4 0.0 ± 0.20 70 ± 8.3 53 ± 5.2 45 ± 1.7
Cells were exposed to H2O2, menadion, and sorbitol for 24 h. Cell viability was measured with the Cell Counting Kit-8. a Mean ± SD (n = 3).
Fig. 2. Time course of phosphorylation of ASK1, p38, and JNK on Huh7 and HepG2 cells treated with H2O2 and sorbitol. Cells are exposed to 0.3 mM H2O2 (A) or 500 mM sorbitol (B). MAPK signaling induced by each stimulus was measured using Western blot analysis. P-ASK1: phosphorylated ASK1, P-p38: phosphorylated p38, P-JNK: phosphorylated JNK.
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concentration-dependent and significant cell death in Huh7. On the other hand, HepG2 was resistant to those stimuli even at the highest concentrations. In contrast, cell death induced by osmotic stress was comparable between these two cell lines. To further understand the effect of ASK1 expression on stressinduced cell death, the MAPK signaling induced by each stimulus was investigated using Western blot analysis. Phosphorylation of ASK1 and its downstream signals, JNK and p38 was detected in Huh7 cells exposed by H2O2 at the concentration of 0.3 mM (Fig. 2A). On the other hand, phosphorylation of these kinases was not induced in HepG2 cells by H2O2 at the same concentration. In contrast, the phosphorylation of ASK1 was not observed after exposure to sorbitol at the concentration of 500 mM, although the phosphorylation of p38 and JNK was induced in both cell lines (Fig. 2B). Knockdown of ASK1 attenuates cell death induced by oxidative stress To confirm these results, gene knock-down of ASK1 using shRNA against ASK1 was conducted using mouse hepatocyte cell line, AML12. Infection of mouse ASK1 shRNA-expressing adenovirus (shASK1-Ad) at a MOI of 100 significantly reduced the expression level of ASK1 mRNA in AML12 cells by up to 45% compared to that of control adenovirus infection (Fig. 3A). We then investigated whether ASK1 knock-down affected oxidative and osmotic stress-induced cell death using the ASK1 shRNA-expressing adenovirus infected AML12 cells, (Fig. 3B). Cell death induced by H2O2 was dramatically attenuated in the AML12 cells infected with the ASK1 shRNA-expressing adenovirus compared with that of control virus. However, ASK1 knock-down did not affect cell death induced by sorbitol, confirming that ASK1 regulates the cell death induced by oxidative but not osmotic stress in hepatocyte. Phosphorylation of ASK1, p38 and JNK in mouse primary hepatocytes Finally, we investigated using mouse primary hepatocytes whether ASK1 is involved in the hepatocyte death induced by oxidative stress (Fig. 4). The phosphorylation of ASK1 and its downstream signaling
Fig. 3. ASK1 knockdown attenuated H2O2-induced cell death in AML12 cells. A: ASK1 gene-knockdown in AML12 cells. ASK1 mRNA was normalized using GAPDH. The quantity of ASK1 mRNA expression was expressed relative to that of AML12 with no infection. Data were expressed as mean ± SD (n = 3). ⁎: p b 0.0001 vs. Control Ad (Student's t-test) B: Reduction of cell death induced by oxidative stress in AML12 cells transfected with ASK1 shRNA. AML12 was infected with the mouse ASK1 shRNAexpressing adenoviruses at MOI of 100. After 24 h of transfection, cells were washed with fresh medium and were further incubated for 48 h. Then, cells were exposed with H2O2 or sorbitol for 24 h. Cell death was measured by LDH-Cytotoxic Test. Data were expressed as mean ± SD. ⁎: p b 0.0001 vs. Control Ad (Student's t-test).
Phosphorylation of ASK1, p38 and JNK and cell death induced by oxidative stress Next, cell death induced by oxidative and osmotic stress was investigated by exploiting the differences in ASK1 expression between these cell lines (Table 1). Hydrogen peroxide (H2O2) and menadion (2methyl-1,4-naphthoquinone), a quinone that undergoes redox cycles leading to the formation of superoxide radicals, were used as an oxidative stress and sorbitol was used for an osmotic stress. As shown in Table 1, cell death in Huh7 was markedly induced by the oxidative stress stimuli, H2O2 and menadion. Exposure to H2O2 resulted in
Fig. 4. H2O2-induced ASK1, p38 and JNK phosphorylation in primary cultured BALB/c hepatocyte. Cells are exposed to 3 mM H2O2. MAPK signaling induced by each stimulus was measured using Western blot analysis. P-ASK1: phosphorylated ASK1, P-p38: phosphorylated p38, P-JNK: phosphorylated JNK.
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p38 and JNK phosphorylation were increased in the mouse primary hepatocyte after exposure to H2O2. Cell death was confirmed after the exposure of H2O2 (data not shown). Discussion In this report we have investigated the role of ASK1 in hepatocyte cell death induced by oxidative and osmotic stress. We found that Huh7 cells expressing high level of ASK1 were sensitive to oxidative stress that resulted in cell death. In contrast, HepG2 cells expressing low level of ASK1 were resistant to these stress stimuli. Oxidative stress leads to cell death by activation of ASK1/JNK-p38 (Matsuzawa et al., 2005; Saitoh et al., 1998). Expression levels of ASK1 mRNA and protein in Huh7 are significantly higher than that of HepG2 cells. On the other hand, expression level of JNK and p38, downstream kinases of ASK1 were quite similar in both cell lines. These results indicate that high expression level of ASK1 attribute to the sensitivity to the oxidative stress in Huh7 cells. We observed similar difference in the activation of ASK1–JNK/p-38 and resulting cell death between these two cell lines using tunicamyin (data not shown). Tunicamyicn is an inhibitor of N-glycosylation in ER and induces cell death via ER stressmediated activation of ASK1 (Nishitoh et al., 2002). This also suggests that high expression levels of ASK1 induce cell death in Huh7 by these stresses. Contrary to our results, Yang et al. (2002) reported that ASK1 activation is required for cell survival using HepG2 cell line. In their study, HepG2 cells, transfected with ASK1, became more resistant to Fas-mediated cell death compared with cells transfected with kinasedefective mutant ASK1. These opposite results may be explained by the differences in the expression level of ASK1 which could determine the cell fate. Indeed, Takeda et al. (2000) reported that moderate expression of constitutive active ASK1 in PC12 cells induced differentiation and survival, whereas excessive expression of ASK1 induced apoptosis. This indicates that intrinsic expression level of ASK1 in Huh7 cells is sufficient enough to induce cell death induced by oxidative stress. In contrast, differential expression of ASK1 did not affect sensitivity to sorbitol-induced osmotic stress in these two cell lines. Following exposure to the osmotic stress, JNK and p38 were activated and resulted in cell death in both cell lines, clearly indicating that ASK1 activation is not required for the cell death induced by osmotic stress. Some reports have described activation of JNK and p38 in response to osmotic stress in hepatocytes (Häussinger et al., 2006). Osmotic stress induced CD95 trafficking to the plasma membrane, which involves JNK-dependent mechanisms and sensitizes hepatocytes toward CD95L-mediated apoptosis (Reinehr et al., 2002). However, CD95 trafficking to the plasma membrane is not sufficient enough to induce full-blown apoptosis in hepatocytes and exogenous CD95L is required to execute apoptosis in this study. It is currently unknown which MAP3K is involved in osmotic stress-induced hepatocyte death. MEKKs, TAK1, or MLK are possible kinases that could regulate the activation of JNK and p38 and resulting cell death (Gotoh et al., 2001; HuangFu et al., 2006; Uhlik et al., 2003). These studies investigated that the activation of MAP3K–JNK/p38 pathway in response to osmotic stress. These MAPK (MEKKs, TAK1, and MLK) activate JNK and/or p38 in cells exposed to osmotic stress but not induce apoptosis. Nevertheless, further studies are required to elucidate which MAP3K is involved in osmotic stress-induced hepatocyte death. We have shown that knock-down of ASK1 in hepatocyte in vitro significantly reduced oxidative stress-induced hepatocyte death. This result clearly demonstrates that ASK1 regulates cell death induced by oxidative stress in hepatocytes. Oxidative stress-induced JNK and p38 activation and the subsequent apoptosis were observed in fibroblasts derived from ASK1−/− embryo (Tobiume et al., 2001). Our results suggest that oxidative stress induced cell death is regulated by ASK1 even in hepatocytes. We confirmed the increase in the phosphorylation of ASK1, its downstream signaling p38 and JNK phosphorylation
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and resulting cell death in the mouse primary hepatocyte after exposure to H2O2. Further analysis using ASK1 gene knock-down is to be needed to confirm the correlation between the activation of ASK1 and cell death in the primary hepatocyte. Given that ROS-mediated apoptosis is critical for pathogenesis of liver diseases, ASK1 may have a crucial role in liver diseases. Indeed, the role of AKS1 in ROS-mediated hepatocyte death has been reported. Gilot et al. (2002) reported that dimethyl sulfoxide, a free radical scavenging molecule, inhibited ASK1/JNK-p38 kinase activities and resulting hepatocyte apoptosis, while overexpression of ASK1 restored hepatocyte apoptosis. Using immortalized human hepatocytes, Lim et al. (2008) also reported that troglitazone produces intramitochondrial oxidant stress that activates ASK1, leading to mitochondrial-mediated apoptosis. Furthermore, Hsieh and Papaconstantinou used hepatocyte cultures to show that the longevity of Snell dwarf mice could be attributed to their resistance to oxidative stress by the combined decrease in ASK1 pool levels and corresponding increase in the level of thioredoxin– ASK1 complex (Hsieh et al., 2006). These preclinical results suggest that ASK1 has a critical role in the oxidative stress-induced liver injury. ROS are involved in the pathogenesis and progression of viral infectious and non-infectious liver diseases such as chronic hepatitis C, ALD, and NASH (Arteel, 2003; Miñana et al., 2002; Okuda et al., 2002; Videla et al., 2004; Wang and Weinman, 2006). Taken together, ASK1 could have a pathophysiological role in liver diseases in which ROS-mediated hepatocyte apoptosis correlates to disease progression. In conclusion, this study confirmed that ASK1 regulates oxidative stress- but not osmotic stress-induced hepatocyte death. We confirmed that ASK1 regulates oxidative but not osmotic stressinduced hepatocyte death. Furthermore, knock-down of ASK1 in hepatocyte in vitro significantly reduced oxidative but not osmotic stress-induced hepatocyte death. These results provide evidence that ASK1 plays a critical role in oxidative-stress induced hepatocyte death. Furthermore, these results suggest that ASK1 may be a promising therapeutic target for liver diseases caused by oxidative stress. Acknowledgments We are grateful to Professor Hidenori Ichijo of University of Tokyo for the scientific discussion. We thank Dr. Medra M. Hardy for critical reading of the article. References Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy Sciences of the United States of America 90 (17), 7915–7922. Arteel, G.E., 2003. Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterology 124 (3), 778–790. Crenesse, D., Gugenheim, J., Hornoy, J., Tornieri, K., Laurens, M., Cambien, B., Lenegrate, G., Cursio, R., De Souza, G., Auberger, P., Heurteaux, C., Rossi, B., Schmid-Alliana, A., 2000. Protein kinase activation by warm and cold hypoxia-reoxidation in primarycultured rat hepatocytes-JNK1/SAPK1 involvement in apoptosis. Hepatology 32 (5), 1029–1036. Fujiwara, T., Takeda, K., Ichijo, H., 2007. ASK family proteins in stress response and disease. Molecular Biotechnology 37 (1), 13–18. Gilot, D., Loyer, P., Corlu, A., Glaise, D., Lagadic-Gossmann, D., Atfi, A., Morel, F., Ichijo, H., Guguen-Guillouzo, C., 2002. Liver protection from apoptosis requires both blockage of initiator caspase activities and inhibition of ASK1/JNK pathway via glutathione Stransferase regulation. Journal of Biological Chemistry 277 (51), 49220–49229. Gotoh, I., Adachi, M., Nishida, E., 2001. Identification and characterization of a novel MAP kinase kinase kinase, MLTK. Journal of Biological Chemistry 276 (6), 4276–4286. Häussinger, D., Reineh, R., Schliess, F., 2006. The hepatocyte integrin system and cell volume sensing. Acta Physiologica (Oxf) 187 (1–2), 249–255. Hsieh, C.C., Papaconstantinou, J., 2006. Thioredoxin-ASK1 complex levels regulate ROSmediated p38 MAPK pathway activity in livers of aged and long-lived Snell dwarf mice. FASEB Journal 20 (2), 259–268. HuangFu, W.C., Omori, E., Akira, S., Matsumoto, K., Ninomiya-Tsuji, J., 2006. Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-κB activation. Journal of Biological Chemistry 281 (39), 28802–28810. Ichijo, H., Nishida, E., Irie, K., Ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., Gotoh, Y., 1997. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275 (5296), 90–94.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / l i f e s c i e
Apoptosis signal-regulating kinase 1 is crucial for oxidative stress-induced but not for osmotic stress-induced hepatocyte cell death Kenji Taki 1,2, Rieko Shimozono 1,2, Hajime Kusano 1, Nobutaka Suzuki 1, Katsuhiro Shinjo 1, Hiroyuki Eda ⁎,1 Discovery Biology-3, Nagoya Laboratories, Pfizer Global Research and Development, Pfizer Japan Inc., 5-2 Taketoyo, Aichi, 470-2393, Japan
a r t i c l e
i n f o
Article history: Received 7 April 2008 Accepted 3 October 2008 Keywords: Apoptosis signal-regulating kinase 1 (ASK1) Oxidative stress Osmotic stress JNK p38 Reactive oxygen species (ROS) Hepatocyte Liver
a b s t r a c t Aims: In this study, we investigated the involvement of apoptosis signal-regulating kinase 1 (ASK1) in oxidative stress and osmotic stress-induced hepatocyte death. Main methods: Activation of ASK1–JNK/p38 cascade and resulting cell death induced by oxidative and osmotic stress was investigated by Western immunoblot analysis and cell toxicity assay using human hepatoma cell lines, Huh7 expressing high level of ASK1 and HepG2 cells expressing low level of ASK1. Gene knock-down of ASK1 using shRNA against ASK1 was conducted using mouse hepatocyte cell line, AML12. Key findings: Activation of ASK1–JNK/p38 cascade and cell death in Huh7 expressing high level of ASK1 was markedly induced by the oxidative stress. HepG2 expressing low level of ASK1 was resistant to oxidative stress while cell death induced by osmotic stress was comparable between Huh7 and HepG2 cells. Although the phosphorylation of ASK1 was not observed by osmotic stress, the phosphorylation of p38 and JNK and resulting cell death was induced in both cell lines. The phosphorylation of ASK1 and p38/JNK in the mouse primary hepatocyte were also increased by oxidative stress. Knock-down of ASK1 mRNA in AML12 in vitro significantly reduced oxidative stress-induced cell death, however, knock-down of ASK1 in cells did not affect the osmotic stress-induced cell death. Significance: This study revealed that ASK1 regulates oxidative stress- but not osmotic stress-induced hepatocyte death, suggesting ASK1 plays a critical role in oxidative-stress induced hepatocyte death. These results raise the possibility that an ASK1 may be a promising therapeutic target for liver diseases caused by oxidative stress. © 2008 Elsevier Inc. All rights reserved.
Introduction Mitogen-activated protein kinase (MAPK) signaling pathways respond to endogenous and exogenous stresses such as oxidative stress, osmotic stress, proinflammatory cytokines, heat shock, ultraviolet irradiation, and infection (Kyriakis and Avruch, 2001). Following exposure to these stresses stimuli, MAPKs are activated and regulate cellular responses such as cell growth, differentiation, survival and death. MAPK signaling pathways consist of three kinases, MAP3K, MAP2K, and MAPK that form a sequential activation pathway from MAP3K though MAP2K to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is one of the MAP3K that activates p38 and JNK via activating the MAP2Ks, MKK4/MKK7 and MKK3/MKK6 (Ichijo et al., 1997; Wang et al., 1996). ASK1 is activated by a variety of stresses including calcium
⁎ Corresponding author. St. Louis Laboratories, Pfizer Global Research and Development, Pfizer Inc., 700 Chesterfield Parkway West, mail zone AA3C, Chesterfield, MO 63017, USA. Tel.: +1 636 247 2166; fax: +1 636 247 5300. E-mail address: hiroyuki.eda@pfizer.com (H. Eda). 1 Tel.: +81 569 74 4866; fax: +81 569 74 4606. 2 First two authors contributed equally to the study. 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.10.004
influx, endoplasmic reticulum (ER) stress, lipopolysaccharide (LPS), reactive oxygen species (ROS), and tumor necrosis factor (Matsuzawa et al., 2005; Nishitoh et al., 1998; Saitoh et al., 1998; Takeda et al., 2004). These stresses, especially ROS, are critically involved in the pathogenesis of many degenerative and life-style related diseases (Ames et al., 1993; Stadtman, 1992). ASK1 has been shown to play a key role in such ROS-mediated diseases as Alzheimer's disease, diabetes mellitus, ischemia-reperfusion injury (Fujiwara et al., 2007; Kadowaki et al., 2005; Nishikawa and Araki 2007; Watanabe et al., 2005). These studies indicate that the relationship between ASK1induced cell death and diseases mediated by oxidative stress. ROS are involved in the pathogenesis and progression of both viral infectious and non-infectious liver diseases (Arteel, 2003; Miñana et al., 2002; Okuda et al., 2002; Videla et al., 2004; Wang and Weinman, 2006). In infectious liver diseases, chronic hepatitis C patients manifest hepatic oxidative stress that is associated with the progression of liver fibrosis (Wang and Weinman, 2006). HCV core protein alters mitochondria function and results in the increase of ROS and, as a consequence, leads to hepatocyte injury (Okuda et al., 2002). In non-infectious liver diseases, hepatocyte apoptosis induced by ROS also plays an important role in the progression of alcohol-induced liver disease (Arteel, 2003; Miñana et al., 2002) and non-alcoholic
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steatohepatitis (Videla et al., 2004). Several reports have shown a critical role for sustained activation of JNK/p38 in hepatocyte apoptosis that leads to liver diseases and inhibition of p38 kinase attenuates ischemia-reperfusion injury of liver (Crenesse et al., 2000; Yoshinari et al., 2001). Given that fibroblasts derived from ASK1−/− embryo were resistant to oxidative stress-induced JNK and p38 activation and the subsequent apoptosis (Tobiume et al., 2001), these data indicate that ASK1 may be involved in the hepatocyte death mediated by oxidative stress in liver diseases. Indeed, Gilot et al. (2002) reported that inhibition of ASK1 by a free radical scavenging molecule prevents apoptosis in primary rat hepatocyte as well as thioacetamide-induced liver injury in vivo. However, it remains to be determined if there is the direct relationship between ASK1 and oxidative stress in hepatocyte death by gene knock-down of ASK1. Thus, we investigated the involvement of ASK1 in hepatocyte death to determine the direct relationship between ASK1 and oxidative stressinduced hepatocyte death.
Materials and methods Animals Male BALB/c mice were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). All experimental procedures used in this study were approved by the local ethics committee based on international guidelines (Institutional Animal Care and Use Committee) and adherence to the Pfizer policy.
Cell culture HepG2 and AML12 cell lines were purchased from the American Type Culture Collection (Manassas, VA). Huh7 cell line was purchased from Health Science Research Resources Bank (Osaka, Japan). HepG2 cell line was cultured in MEM culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 μg/ml penicillin and streptomycin. Huh7 cell line was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 100 μg/ml penicillin and streptomycin. AML12 cell line was cultured in DMEM/F-12 1:1 mixture medium supplemented with 10% heat-inactivated FBS, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethazone, 100 μg/ml penicillin and streptomycin.
Isolation of hepatocyte from mouse Livers were perfused for 5 min from a portal vein using the Liver Perfusion Medium (Invitrogen, Carlsbad, CA) in anesthetized mouse. After 5 min perfusion, the Liver Perfusion Medium was changed to the Liver Digest Medium (Invitrogen) and perfusion was continued further 15 min and then the livers were removed. Livers were minced in Hepatocyte Wash Medium (Invitrogen), filtered using Cell strainer (BD Bioscience, Franklin Lakes, NJ), then centrifuged at 54 ×g for 1 min. Cell pellet was resuspended into the Hepatocyte Wash Medium and the centrifugation step repeated twice. The pelleted cells were cultured in the plating medium (William's E medium, 5% heat-inactivated FBS, 4 µg/ml insulin, 1.0 µM dexamethazone, 100 μg/ml penicillin and streptomycin) for 5 h followed by overnight culture in incubation medium (William's E medium, 5% heat-inactivated FBS, 4 µg/ml insulin, 0.1 µM dexamethazone, 100 μg/ml penicillin and streptomycin). The following day, culture medium was changed to the culture medium (William's E medium, ITS+ premix (BD Bioscience), 0.1 µM dexamethazone, 100 μg/ml penicillin and streptomycin).
Quantitative RT-PCR The total cellular RNA was isolated using TRIzol® Reagent (Invitrogen) and purified using RNeasy kit (Qiagen, Valencia, CA). The TaqMan® probes were purchased from Applied Biosystems (Foster City, CA). The primers for each gene are as follows, human ASK1: Hs00178726_m1, mouse ASK1: Mm00434883_m1, and mouse GAPDH: Mm99999915_g1. Human GAPDH was purchased from Applied Biosystems (Pre-Developed TaqMan® Assay Reagents). Reverse transcription reaction was carried out using a SuperScript™ III First Strand Synthesis System (Invitrogen). Real-time PCR was carried out using Platinum® qPCR SuperMix-UDG with ROX (Invitrogen) on a real-time PCR (ABI PRISM7900, Applied Biosystems) with the default reaction conditions. Measurement of cell death A single suspension of Huh7 and HepG2 cells (2.5 × 104 cells) was plated into a 96-well culture plate and cultured for 24 h. Then cells were exposed to hydrogen peroxide (H2O2), menadion, and sorbitol for 24 h. Cell viability was measured with the LDH cytotoxic test kit (Wako Pure Chemical, Osaka, Japan) or the Cell Counting Kit-8 (DOJINDO, Osaka, Japan). Western blot analysis Cells were washed twice with cold PBS, and then incubated for 5 min on ice in the M-PER lysis buffer (PIERCE, Rockford, IL) including the Complete Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IL). Cell lysate was sonicated and centrifuged at 14,000 ×g for 10 min to remove insoluble material. Samples (30 μg each) were separated by 7% SDS-PAGE and electro-transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Nonspecific binding was blocked with the Blocking One-P blocking buffer (Nacalai Tesque, Kyoto, Japan) then incubated overnight on 4 °C with primary antibody in the CanGetSignal® solution 1 (TOYOBO, Osaka, Japan). The filters were washed three times with TBS with 0.1% Tween-20 (T-TBS) at the room temperature (RT) and incubated with appropriate secondary antibody conjugated to horseradish peroxidase in the CanGetSignal® solution 2 (TOYOBO) for 1 h at RT. Following three times washing with T-TBS, the proteins of interest were visualized with the ECL™ Western Blotting Reagent (GE Healthcare, Waukesha, WI) and filters were scanned with the LumiVisionPRO 400EX (AISIN, Aichi, Japan). Protein concentration was measured by using the Protein Quantification Kit-Rapid (DOJINDO). Anti-ASK1, anti-p38, anti-JNK antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-ASK1-Thr845, antiphospho-p38, and anti-phospho-JNK antibody were from Cell Signaling (Danvers, MA). Secondary antibodies were sheep anti-mouse IgGHRP (GE healthcare), goat anti-rabbit IgG-HRP, and donkey anti-goat IgG-HRP (Santa Cruz). shRNA-expressed adenoviral vector construction, adenoviral infection and gene-knockdown Recombinant adenoviral vectors harboring shRNA against mouse ASK1 (shASK1-Ad) were constructed from replication-deficient adenovirus type 5 with deletions in the E1 and E3 genes obtained using the Gateway ViraPower™ adenovirus vector system (Invitrogen). The ASK1 shRNA sequence was decided to using the BLOCK-iT™ RNAi Designer and forward and reverse oligonucleotides sequences were synthesized by Invitrogen. The sequence of 64-nucleotide encoding mouse ASK1-specific shRNA was as follows: 5′-TGCTGATCCGAAGCAACTTGCTCTTCGTTTTGGCCACTGACTGACGAAGAGCATTGCTTCGGAT-3′ and 5 ′- C CT GAT C C GAAGC AAT G CT C T TC GTC AGT CAGT GG CC A AAA CGAAGAGCAAGTTGCTTCGGATC-3′). These oligonucleotides were
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of 100 and 300. After 24 h of transfection, cells were washed with fresh medium and were further incubated for 48 h. Results Expression of ASK1 in Huh7 and HepG2 cells Expression level of ASK1 in human hepatoma cell lines, Huh7 and HepG2 cells, was compared using RT-PCR and Western immunoblot. The expression level of ASK1 mRNA in Huh7 was more than 5-times higher than that of HepG2 cell line (Fig. 1A). Western blot analysis revealed that the ASK1 protein was expressed in Huh7 cells but not but in HepG2 cells (Fig. 1B). In contrast, protein expression level of JNK and p38, the downstream kinases of ASK1, were quite similar in both cell lines (Fig. 1B).
Fig. 1. MAPK expression in Huh7 and HepG2 cell lines. A: Expression of ASK1 mRNA in Huh7 and HepG2 cell lines measured by RT-PCR. ASK1 mRNA levels were normalized using GAPDH. The quantity of ASK1 mRNA was expressed as relative quantity to that of Huh7. Data were expressed as mean ± SD (n = 2). B: Western blot analysis of ASK1, p38, and JNK expression in Huh7 and HepG2 cell lines.
annealed and subcloned to the pcDNA™ 6.2-GW/miR, then further subcloned to pDONR™ 221 and pAd/CMV/V5-DEST with the Gateway system to develop pAd/CMV/V5-DEST-shASK1. Clones were selected and amplified by transforming into DH5α E. coli cells. PacI linearized adenoviral DNA was transfected into packaging cells (293A) using jetPEI™ (Polyplus-transfection SA, Illkirch, France). Recombinant crude viruses were infected and propagated in 293A cells. After 36–48 h of infection, the cells were lysed by freezing–thawing and viruses were collected from the cells. All viral preparations were purified by the Vivapure® AdenoPACK (Sartorius AG, Goettingen, Germany). Titers of virus were determined by the plaque assay using 293A cells. The AML12 cells were transfected with shASK1-Ad at multiplicity of infection (MOI)
Table 1 Oxidative stress- and osmotic stress-induced cell death in Huh7 and HepG2 cell lines Stimulation (mM) H2O2 (0.3) H2O2 (1.0) H2O2 (3.0) Menadion (0.02) Menadion (0.05) Sorbitol (200) Sorbitol (500) Sorbitol (800)
Cell viability (%) HepG2
Huh7
100 ± 3.2a 92 ± 5.9 79 ± 5.0 96 ± 1.0 85 ± 6.5 76 ± 1.4 60 ± 3.8 39 ± 2.9
35 ± 2.9 0.44 ± 0.48 0.89 ± 0.14 104 ± 2.4 0.0 ± 0.20 70 ± 8.3 53 ± 5.2 45 ± 1.7
Cells were exposed to H2O2, menadion, and sorbitol for 24 h. Cell viability was measured with the Cell Counting Kit-8. a Mean ± SD (n = 3).
Fig. 2. Time course of phosphorylation of ASK1, p38, and JNK on Huh7 and HepG2 cells treated with H2O2 and sorbitol. Cells are exposed to 0.3 mM H2O2 (A) or 500 mM sorbitol (B). MAPK signaling induced by each stimulus was measured using Western blot analysis. P-ASK1: phosphorylated ASK1, P-p38: phosphorylated p38, P-JNK: phosphorylated JNK.
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concentration-dependent and significant cell death in Huh7. On the other hand, HepG2 was resistant to those stimuli even at the highest concentrations. In contrast, cell death induced by osmotic stress was comparable between these two cell lines. To further understand the effect of ASK1 expression on stressinduced cell death, the MAPK signaling induced by each stimulus was investigated using Western blot analysis. Phosphorylation of ASK1 and its downstream signals, JNK and p38 was detected in Huh7 cells exposed by H2O2 at the concentration of 0.3 mM (Fig. 2A). On the other hand, phosphorylation of these kinases was not induced in HepG2 cells by H2O2 at the same concentration. In contrast, the phosphorylation of ASK1 was not observed after exposure to sorbitol at the concentration of 500 mM, although the phosphorylation of p38 and JNK was induced in both cell lines (Fig. 2B). Knockdown of ASK1 attenuates cell death induced by oxidative stress To confirm these results, gene knock-down of ASK1 using shRNA against ASK1 was conducted using mouse hepatocyte cell line, AML12. Infection of mouse ASK1 shRNA-expressing adenovirus (shASK1-Ad) at a MOI of 100 significantly reduced the expression level of ASK1 mRNA in AML12 cells by up to 45% compared to that of control adenovirus infection (Fig. 3A). We then investigated whether ASK1 knock-down affected oxidative and osmotic stress-induced cell death using the ASK1 shRNA-expressing adenovirus infected AML12 cells, (Fig. 3B). Cell death induced by H2O2 was dramatically attenuated in the AML12 cells infected with the ASK1 shRNA-expressing adenovirus compared with that of control virus. However, ASK1 knock-down did not affect cell death induced by sorbitol, confirming that ASK1 regulates the cell death induced by oxidative but not osmotic stress in hepatocyte. Phosphorylation of ASK1, p38 and JNK in mouse primary hepatocytes Finally, we investigated using mouse primary hepatocytes whether ASK1 is involved in the hepatocyte death induced by oxidative stress (Fig. 4). The phosphorylation of ASK1 and its downstream signaling
Fig. 3. ASK1 knockdown attenuated H2O2-induced cell death in AML12 cells. A: ASK1 gene-knockdown in AML12 cells. ASK1 mRNA was normalized using GAPDH. The quantity of ASK1 mRNA expression was expressed relative to that of AML12 with no infection. Data were expressed as mean ± SD (n = 3). ⁎: p b 0.0001 vs. Control Ad (Student's t-test) B: Reduction of cell death induced by oxidative stress in AML12 cells transfected with ASK1 shRNA. AML12 was infected with the mouse ASK1 shRNAexpressing adenoviruses at MOI of 100. After 24 h of transfection, cells were washed with fresh medium and were further incubated for 48 h. Then, cells were exposed with H2O2 or sorbitol for 24 h. Cell death was measured by LDH-Cytotoxic Test. Data were expressed as mean ± SD. ⁎: p b 0.0001 vs. Control Ad (Student's t-test).
Phosphorylation of ASK1, p38 and JNK and cell death induced by oxidative stress Next, cell death induced by oxidative and osmotic stress was investigated by exploiting the differences in ASK1 expression between these cell lines (Table 1). Hydrogen peroxide (H2O2) and menadion (2methyl-1,4-naphthoquinone), a quinone that undergoes redox cycles leading to the formation of superoxide radicals, were used as an oxidative stress and sorbitol was used for an osmotic stress. As shown in Table 1, cell death in Huh7 was markedly induced by the oxidative stress stimuli, H2O2 and menadion. Exposure to H2O2 resulted in
Fig. 4. H2O2-induced ASK1, p38 and JNK phosphorylation in primary cultured BALB/c hepatocyte. Cells are exposed to 3 mM H2O2. MAPK signaling induced by each stimulus was measured using Western blot analysis. P-ASK1: phosphorylated ASK1, P-p38: phosphorylated p38, P-JNK: phosphorylated JNK.
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p38 and JNK phosphorylation were increased in the mouse primary hepatocyte after exposure to H2O2. Cell death was confirmed after the exposure of H2O2 (data not shown). Discussion In this report we have investigated the role of ASK1 in hepatocyte cell death induced by oxidative and osmotic stress. We found that Huh7 cells expressing high level of ASK1 were sensitive to oxidative stress that resulted in cell death. In contrast, HepG2 cells expressing low level of ASK1 were resistant to these stress stimuli. Oxidative stress leads to cell death by activation of ASK1/JNK-p38 (Matsuzawa et al., 2005; Saitoh et al., 1998). Expression levels of ASK1 mRNA and protein in Huh7 are significantly higher than that of HepG2 cells. On the other hand, expression level of JNK and p38, downstream kinases of ASK1 were quite similar in both cell lines. These results indicate that high expression level of ASK1 attribute to the sensitivity to the oxidative stress in Huh7 cells. We observed similar difference in the activation of ASK1–JNK/p-38 and resulting cell death between these two cell lines using tunicamyin (data not shown). Tunicamyicn is an inhibitor of N-glycosylation in ER and induces cell death via ER stressmediated activation of ASK1 (Nishitoh et al., 2002). This also suggests that high expression levels of ASK1 induce cell death in Huh7 by these stresses. Contrary to our results, Yang et al. (2002) reported that ASK1 activation is required for cell survival using HepG2 cell line. In their study, HepG2 cells, transfected with ASK1, became more resistant to Fas-mediated cell death compared with cells transfected with kinasedefective mutant ASK1. These opposite results may be explained by the differences in the expression level of ASK1 which could determine the cell fate. Indeed, Takeda et al. (2000) reported that moderate expression of constitutive active ASK1 in PC12 cells induced differentiation and survival, whereas excessive expression of ASK1 induced apoptosis. This indicates that intrinsic expression level of ASK1 in Huh7 cells is sufficient enough to induce cell death induced by oxidative stress. In contrast, differential expression of ASK1 did not affect sensitivity to sorbitol-induced osmotic stress in these two cell lines. Following exposure to the osmotic stress, JNK and p38 were activated and resulted in cell death in both cell lines, clearly indicating that ASK1 activation is not required for the cell death induced by osmotic stress. Some reports have described activation of JNK and p38 in response to osmotic stress in hepatocytes (Häussinger et al., 2006). Osmotic stress induced CD95 trafficking to the plasma membrane, which involves JNK-dependent mechanisms and sensitizes hepatocytes toward CD95L-mediated apoptosis (Reinehr et al., 2002). However, CD95 trafficking to the plasma membrane is not sufficient enough to induce full-blown apoptosis in hepatocytes and exogenous CD95L is required to execute apoptosis in this study. It is currently unknown which MAP3K is involved in osmotic stress-induced hepatocyte death. MEKKs, TAK1, or MLK are possible kinases that could regulate the activation of JNK and p38 and resulting cell death (Gotoh et al., 2001; HuangFu et al., 2006; Uhlik et al., 2003). These studies investigated that the activation of MAP3K–JNK/p38 pathway in response to osmotic stress. These MAPK (MEKKs, TAK1, and MLK) activate JNK and/or p38 in cells exposed to osmotic stress but not induce apoptosis. Nevertheless, further studies are required to elucidate which MAP3K is involved in osmotic stress-induced hepatocyte death. We have shown that knock-down of ASK1 in hepatocyte in vitro significantly reduced oxidative stress-induced hepatocyte death. This result clearly demonstrates that ASK1 regulates cell death induced by oxidative stress in hepatocytes. Oxidative stress-induced JNK and p38 activation and the subsequent apoptosis were observed in fibroblasts derived from ASK1−/− embryo (Tobiume et al., 2001). Our results suggest that oxidative stress induced cell death is regulated by ASK1 even in hepatocytes. We confirmed the increase in the phosphorylation of ASK1, its downstream signaling p38 and JNK phosphorylation
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and resulting cell death in the mouse primary hepatocyte after exposure to H2O2. Further analysis using ASK1 gene knock-down is to be needed to confirm the correlation between the activation of ASK1 and cell death in the primary hepatocyte. Given that ROS-mediated apoptosis is critical for pathogenesis of liver diseases, ASK1 may have a crucial role in liver diseases. Indeed, the role of AKS1 in ROS-mediated hepatocyte death has been reported. Gilot et al. (2002) reported that dimethyl sulfoxide, a free radical scavenging molecule, inhibited ASK1/JNK-p38 kinase activities and resulting hepatocyte apoptosis, while overexpression of ASK1 restored hepatocyte apoptosis. Using immortalized human hepatocytes, Lim et al. (2008) also reported that troglitazone produces intramitochondrial oxidant stress that activates ASK1, leading to mitochondrial-mediated apoptosis. Furthermore, Hsieh and Papaconstantinou used hepatocyte cultures to show that the longevity of Snell dwarf mice could be attributed to their resistance to oxidative stress by the combined decrease in ASK1 pool levels and corresponding increase in the level of thioredoxin– ASK1 complex (Hsieh et al., 2006). These preclinical results suggest that ASK1 has a critical role in the oxidative stress-induced liver injury. ROS are involved in the pathogenesis and progression of viral infectious and non-infectious liver diseases such as chronic hepatitis C, ALD, and NASH (Arteel, 2003; Miñana et al., 2002; Okuda et al., 2002; Videla et al., 2004; Wang and Weinman, 2006). Taken together, ASK1 could have a pathophysiological role in liver diseases in which ROS-mediated hepatocyte apoptosis correlates to disease progression. In conclusion, this study confirmed that ASK1 regulates oxidative stress- but not osmotic stress-induced hepatocyte death. We confirmed that ASK1 regulates oxidative but not osmotic stressinduced hepatocyte death. Furthermore, knock-down of ASK1 in hepatocyte in vitro significantly reduced oxidative but not osmotic stress-induced hepatocyte death. These results provide evidence that ASK1 plays a critical role in oxidative-stress induced hepatocyte death. Furthermore, these results suggest that ASK1 may be a promising therapeutic target for liver diseases caused by oxidative stress. Acknowledgments We are grateful to Professor Hidenori Ichijo of University of Tokyo for the scientific discussion. We thank Dr. Medra M. Hardy for critical reading of the article. References Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy Sciences of the United States of America 90 (17), 7915–7922. Arteel, G.E., 2003. Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterology 124 (3), 778–790. Crenesse, D., Gugenheim, J., Hornoy, J., Tornieri, K., Laurens, M., Cambien, B., Lenegrate, G., Cursio, R., De Souza, G., Auberger, P., Heurteaux, C., Rossi, B., Schmid-Alliana, A., 2000. Protein kinase activation by warm and cold hypoxia-reoxidation in primarycultured rat hepatocytes-JNK1/SAPK1 involvement in apoptosis. Hepatology 32 (5), 1029–1036. Fujiwara, T., Takeda, K., Ichijo, H., 2007. ASK family proteins in stress response and disease. Molecular Biotechnology 37 (1), 13–18. Gilot, D., Loyer, P., Corlu, A., Glaise, D., Lagadic-Gossmann, D., Atfi, A., Morel, F., Ichijo, H., Guguen-Guillouzo, C., 2002. Liver protection from apoptosis requires both blockage of initiator caspase activities and inhibition of ASK1/JNK pathway via glutathione Stransferase regulation. Journal of Biological Chemistry 277 (51), 49220–49229. Gotoh, I., Adachi, M., Nishida, E., 2001. Identification and characterization of a novel MAP kinase kinase kinase, MLTK. Journal of Biological Chemistry 276 (6), 4276–4286. Häussinger, D., Reineh, R., Schliess, F., 2006. The hepatocyte integrin system and cell volume sensing. Acta Physiologica (Oxf) 187 (1–2), 249–255. Hsieh, C.C., Papaconstantinou, J., 2006. Thioredoxin-ASK1 complex levels regulate ROSmediated p38 MAPK pathway activity in livers of aged and long-lived Snell dwarf mice. FASEB Journal 20 (2), 259–268. HuangFu, W.C., Omori, E., Akira, S., Matsumoto, K., Ninomiya-Tsuji, J., 2006. Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-κB activation. Journal of Biological Chemistry 281 (39), 28802–28810. Ichijo, H., Nishida, E., Irie, K., Ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., Gotoh, Y., 1997. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275 (5296), 90–94.
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