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Cytoplasmic Retention of Peroxide-Activated ERK Provides Survival in Primary Cultures of Rat Hepatocytes Carola M. Rosseland,1,2 Lene Wierød,1,2 Morten P. Oksvold,1,2 Heidi Werner,1,2 Anne Carine Østvold,2 G. Hege Thoresen,3 Ragnhild E. Paulsen,2,3 Henrik S. Huitfeldt,1,2 and Ellen Skarpen1,2 Reactive oxygen species (ROS) are implicated in tissue damage causing primary hepatic dysfunction following ischemia/reperfusion injury and during inflammatory liver diseases. A potential role of extracellular signal-regulated kinase (ERK) as a mediator of survival signals during oxidative stress was investigated in primary cultures of hepatocytes exposed to ROS. Hydrogen peroxide (H2O2) induced a dose-dependent activation of ERK, which was dependent on MEK activation. The ERK activation pattern was transient compared with the ERK activation seen after stimulation with epidermal growth factor (EGF). Nuclear accumulation of ERK was found after EGF stimulation, but not after H2O2 exposure. A slow import/rapid export mechanism was excluded through the use of leptomycin B, an inhibitor of nuclear export sequence– dependent nuclear export. Reduced survival of hepatocytes during ROS exposure was observed when ERK activation was inhibited. Ribosomal S6 kinase (RSK), a cytoplasmic ERK substrate involved in cell survival, was activated and located in the nucleus of H2O2-exposed hepatocytes. The activation was abolished when ERK was inhibited with U0126. In conclusion, our results indicate that activity of ERK in the cytoplasm is important for survival during oxidative stress in hepatocytes and that RSK is activated downstream of ERK. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index. html). (HEPATOLOGY 2005;42:200-207.)

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eactive oxygen species (ROS) have been implicated in the pathogenesis of degenerative and inflammatory diseases, aging, and cancer. In the liver, high levels of ROS are produced during liver growth, inflammatory injury, and upon exposure to hepatotoxins.1 ROS are defined as partially reduced metabolites of molecular oxygen and include superoxide anion (O2•⫺), hydrogen peroxide (H2O2), and hydroxyl radical (OH•). They are highly reactive, and may cause oxidative Abbreviations: ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; EGF, epidermal growth factor; RSK, ribosomal S6 kinase; LAP2, lamina-associated polypeptide 2. From the 1Laboratory for Toxicopathology, Institute of Pathology, Rikshospitalet University Hospital, the 2Center for Cellular Stress Responses, and the 3Institute of Pharmacy, University of Oslo, Norway. Received December 23, 2004; accepted April 22, 2005. Address reprint requests to: Ellen Skarpen, Institute of Pathology, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail: ellen.skarpen@labmed. uio.no; fax: (47) 23071511. Copyright © 2005 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.20762 Potential conflict of interest: Nothing to report. 200

damage to cellular macromolecules.2 In normal hepatocytes, the major source of ROS production is toxic byproducts of oxidative phosphorylation in mitochondria. In addition, detoxification of hepatotoxins in the endoplasmic reticulum or peroxisome compartments induces an endogenous production of ROS. To protect against the potentially damaging effects of ROS, hepatocytes possess several antioxidant enzymatic and nonenzymatic defense systems. When the cellular production of ROS exceeds the antioxidant capacity of the cell, oxidative damage of lipids, proteins, and DNA will occur, resulting in oxidative stress, hepatic injury, and possibly cell death.1 Oxidative stress is thought to contribute to liver damage during ischemia and reperfusion injury by the release of cytokines and ROS from activated Kupffer cells and recruited leukocytes. This stress will affect neighboring hepatocytes, altering their functions and damaging their integrity.3 Until recently, intracellular ROS production was regarded solely as damaging by-products of enzymatic reactions. However, increasing evidence suggests a physiological role of endogenously produced ROS as

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mediators in signal transduction pathways.2,4 Thus, intracellular ROS-production may also be a highly regulated process. As evidence for this, a significant increase in ROS has been shown after growth factor stimulation.5 Furthermore, the growth factor signals depended on intracellular ROS to mediate their physiological effects.6 Although ROS appear to be important for cellular signaling, the signaling molecules targeted by intracellular ROS are less clear. However, exogenous hydrogen peroxide exposure has been shown to activate or inactivate several proteins involved in cellular signaling, including receptor tyrosine kinases, phosphatases, and members of the mitogen-activated protein kinase (MAPK) family.4 Mitogen-activated protein kinases are activated in response to several stimuli, such as growth factors, cytokines, and cellular stress. They include MAPK extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), which are regulators of various cellular processes including cell growth, differentiation, and cell survival. In hepatocytes, epidermal growth factor (EGF) binding to the EGF receptor initiates activation of ERK through the Raf/MEK/ERK pathway followed by nuclear accumulation of ERK.7 In the nucleus, ERK phosphorylates substrates important for growth progression.8 However, ERK also phosphorylates substrates in other cellular compartments, including membrane proteins and proteins in the cytoplasm.9 Oxidative stress activates ERK in several cell types,2 including hepatocytes.10-13 In addition, ROS activate the EGF receptor,10,13,14 leading to activation of the Raf/MEK/ERK phosphorylation cascade.10,13,15 However, the signal transduction pathway leading to activation of ERKs may be different among cell types. Some authors have suggested the Src family tyrosine kinases are the primary targets of H2O2 leading to a subsequent phosphorylation of EGF receptor at its Src-regulatory site Y845.16,17 Oxidative stress by ROS may cause severe damage and cell death. However, ROS may also induce adaptive responses through activation of proteins that mediate the survival of cells. ERK is an essential mediator of survival signals through phosphorylation of ribosomal S6 kinase (RSK), which positively influence the transcription of pro-survival genes.18 A role for ERK in survival after ROS exposure has been shown in several cell lines15,19,20 and in recent studies in hepatocytes.11,13 Whether ERK is activated in a highly regulated manner or through interference with growth regulatory pathways is still an unresolved matter. We have studied the potential role of ERK during adaptation of hepatocytes to oxidative stress in a hydrogen peroxide environment.

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Materials and Methods Materials. William’s medium E, Dulbecco’s modified Eagle medium, penicillin, and streptomycin were obtained from Gibco (Grand Island, NY). Hydrogen peroxide was purchased from Fluka (Buchs, Switzerland). Collagenase (type IV, C-5138), collagen (type 1 from rat tail), insulin, and receptor-grade EGF from mouse submaxillary glands were purchased from Sigma Aldrich Co. (St. Louis, MO). Rabbit anti-ERK1 and rabbit antiMEK1 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Mouse anti–lamina-associated polypeptide (LAP2) was obtained from Transduction Laboratories (Lexington, KY). Rabbit anti-phosphorylated ERK, anti-phosphorylated MEK, and anti-phosphorylated RSK (Ser380) were obtained from Cell Signaling Technology Inc. (Beverly, MA). Mouse anti–␤tubulin and peroxidase-conjugated goat anti-rabbit IgG was purchased from Sigma Aldrich (St. Louis, MO). Peroxidase-conjugated donkey anti-mouse IgG, CY2, and rhodamine redX-conjugated donkey antibody to mouse and rabbit IgG were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). The MEK inhibitor U0126 was purchased from Promega (Madison, WI). Leptomycin B was kindly provided by Dr. Minoru Yoshida (Saitama, Japan). Cell Isolation and Culture. Young adult male Wistar rats (Møllergaard and Blomhoff, Odense, Denmark) with a living weight of 200 to 220 g were kept on a 12-hour light/dark cycle and fed water ad libitum. Hepatocytes were isolated and seeded as previously described.7 The cultures were kept in 95% air and 5% CO2 at 37°C. Cell death was measured via trypan blue exclusion. Hepatocytes were exposed to 10 nM EGF or 1 mmol/L H2O2, both for 10 minutes, unless otherwise stated. U0126 was added at concentrations of 10 or 20 ␮mol/L and incubated for 30 minutes. Leptomycin B was incubated for 30 minutes at a concentration of 2 ng/mL. Western Blot Analysis and Nuclear Fractionation. Western blot analysis was performed essentially as previously described.7 Equal protein loading was controlled with an antibody to ␤-tubulin. For nuclear fractionation, hepatocytes were lysed in 10 mmol/L sodium phosphate buffer (pH 7.4) containing 10 mmol/L NaCl, 5 mmol/L MgCl2, 0.1% NP40, 0.2 mmol/L AEBSF, 20 ␮mol/L leupeptin, 200 U/mL aprotinin, 65 ␮mol/L sodium orthovanadate, and 10 mmol/L ␤-glycerophosphate. The cells were lysed for 10 minutes on ice and homogenized by 20 strokes with a tight-fitting homogenizer. Nuclei were collected via centrifugation at 900g for 10 minutes at 4°C and prepared for Western blot analysis. The supernatant

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containing the cytoplasmic proteins was also prepared for Western blotting. Confocal Immunofluorescence Microscopy. Immunofluorescence staining was performed essentially as previously described.7 The immunostained cells were examined with a Leica TCS SP confocal microscope (Leica, Heidelberg, Germany) equipped with TCS NT software and Ar (488 nm) and two He/Ne (543 and 633 nm) lasers. A Plan apochromat 100X 1.4 NA oil objective was used. All multilabeled images were acquired sequentially. Image series recorded with the confocal microscope were exported as single-image files in TIF format using analySIS 3.1 (Soft Imaging System, Mu¨nster, Germany) or Photoshop 5.0.2 (Adobe, Mountain View, CA). MAP Kinase Assay and Measurement of DNA Synthesis. Both the measurement of MAP kinase activities and the DNA synthesis were performed as previously described.7

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Hydrogen Peroxide Exposure Induced Phosphorylation and Kinase Activation of ERK in Primary Hepatocytes. ERK phosphorylation at increasing concentrations of H2O2 was compared with ERK activation

Fig. 2. Phosphorylation of MEK and effect of MEK inhibition during H2O2 exposure. (A) Primary cultures of hepatocytes were stimulated with increasing amounts of H2O2 and EGF for 10 minutes and were subjected to Western immunoblot analysis with antibodies against active MEK. Hepatocytes were incubated with or without 10 ␮mol/L U0126 for 30 minutes, and stimulated with 1 mmol/L H2O2 or 10 nmol/L EGF for 10 minutes. (B) The cells were harvested and subjected to Western immunoblotting with antibodies recognizing active MEK and phosphorylated ERK. In both panel A and panel B, the protein expression of ␤-tubulin was used as a loading control. Representative examples of at least three experiments are shown. EGF, epidermal growth factor; pMEK, phosphorylated MEK; pERK, phosphorylated ERK.

Fig. 1. Effect of various concentrations of H2O2 and EGF on phosphorylation and kinase activity of ERK. Primary cultures of hepatocytes were stimulated with increasing amounts of H2O2 and EGF for 10 minutes. (A) The cells were harvested, and Western immunoblot analyses were performed on lysates with an antibody that recognizes phosphorylated ERK and an anti-ERK1 antibody recognizing ERK1 and ERK2. The protein expression of ␤-tubulin was used as a loading control. A representative example of at least three experiments is shown. (B) ERK activity was measured 10 minutes after stimulation with H2O2 or EGF (SEM; n ⫽ 4-6). EGF, epidermal growth factor; pMEK, phosphorylated MEK; pERK, phosphorylated ERK.

in control cells and in hepatocytes stimulated with EGF, a potent ERK activator. As shown in Fig. 1A, increased ERK phosphorylation was observed from 0.1 mmol/L H2O2 and peaked at 5 mmol/L, where the level of phosphorylation was equal to hepatocytes stimulated with the highest dose of EGF. To compare the level of ERK phosphorylation with the total amount of ERK protein, we performed Western immunoblotting on the same hepatocyte lysates using an anti-ERK1 antibody recognizing both ERK1 and ERK2. Only small variations in protein levels were observed. The biological effects of ERK are mediated through phosphorylation of target proteins. Thus, the ERK activity during H2O2 exposure was measured (Fig. 1B). The kinase activities correlated well with the ERK phosphorylation pattern seen after exposure of hepatocytes with various doses of H2O2. A slight increase in ERK activity was observed at 0.1 mmol/L H2O2. It was further increased up to 5 mmol/L H2O2. ERK Was Activated By MEK After Hydrogen Peroxide Exposure. The physiological activator of ERK is MEK. Activation of MEK was observed from a concentration of 0.1 mmol/L H2O2 and was further increased with increasing doses of peroxide exposure (Fig. 2A). To confirm that MEK was an activator of ERK after H2O2 exposure, hepatocytes were treated with the MEK inhibitor U0126 before stimulation with EGF or H2O2. As

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shown in Fig. 2B, U0126 nearly abolished the activation induced by both EGF and H2O2. ERK Activation After Hydrogen Peroxide Stimulation Was Independent of EGF Receptor Phosphorylation. EGF receptor activation and its putative role as a signalling mediator of ERK after H2O2 exposure were studied. Compared to control cells, enhanced phosphorylation of the EGF receptor was observed at 0.5 mmol/L H2O2 (Supplemental Fig. 1A; available at the HEPATOLOGY website: http://www.interscience.wiley.com/jpages/ 0270-9139/suppmat/index.html). A further increase in the phosphorylation level was found with increasing concentrations of H2O2. However, when EGF receptor signalling was blocked using the specific EGF receptor kinase inhibitor PD153035, no inhibition of ERK activity was observed in H2O2– exposed hepatocytes (Supplemental Fig. 1B). Comparably, a dose-dependent decrease in ERK activation was found with increasing doses of PD153035 after EGF-treatment. Thus, ERK activation by H2O2 appeared to be EGF receptor independent. Phosphorylation of the SRC regulatory site Y845 on the EGF receptor plays a vital role in the induction of growth regulatory genes.16 In hepatocytes exposed to H2O2 phosphorylation of the Y845-site was not observed. However, EGF-stimulation induced strong phosphorylation of Y845 (Supplemental Fig. 1C), which was reduced in the presence of the Src inhibitor PP1 (data not shown). Furthermore, PP1 showed no effect on H2O2 mediated ERKactivation (data not shown). Ligand activation of the EGF receptor induces sorting of phosphorylated EGF receptor molecules to endosomes for lysosomal degradation.22 Downregulation of the EGF receptor was not observed at any time points after H2O2 treatment, contrary to what was seen with EGF (Supplemental Fig. 1D). Furthermore, immunostaining experiments showed EGF receptor in plasma membranes in control (Supplemental Fig. 2A) and H2O2-exposed (Supplemental Fig. 2C) cells. In contrast, EGF receptor colocalized with EEA1-positive vesicles in cytoplasmic compartments after EGF-stimulation (Supplemental Fig. 2B). Hydrogen Peroxide–Activated ERK Was Located in the Cytoplasm. Intracellular localization of ERK after hydrogen peroxide exposure was examined by two-colour immunofluorescence staining combining antibodies against ERK and nuclear LAP2 (Fig. 3). In control cells, ERK was evenly distributed throughout the cytoplasm (Fig. 3Aa-b). Upon stimulation with EGF, ERK was accumulated in the nuclei of hepatocytes (Fig. 3Ac-d), whereas in H2O2-exposed cells ERK was not redistributed to the nucleus (Fig 3Ae-f). Similar results were obtained from repeating experiments (Fig. 3B). Nuclear accumulation was not induced even at very high concentrations of

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Fig. 3. Immunofluorescence staining of ERK after stimulation with EGF and H2O2. (A) Hepatocytes were stained with an anti-ERK (green) combined with an antibody to LAP2 (red), a marker of nuclear membranes. Single confocal laser scan images of ERK (a,c,e) and merged images of ERK and LAP2 (b,d,f) in control cells (a,b), hepatocytes treated with 10 nmol/L EGF for 10 minutes (c,d), or cells stimulated with 1 mmol/L H2O2 for 10 minutes (e,f) are shown. Representative images of at least three experiments are shown. (B) ERK nuclear localization from 100 cells in three independent experiments (SEM; n ⫽ 3).EGF, epidermal growth factor; LAP2, lamina-associated polypeptide.

H2O2 (data not shown), nor by increasing the exposure time (up to 2 hours; data not shown). The intracellular localization of ERK after hydrogen peroxide exposure was further studied via nuclear enrichment of hepatocyte lysates (Fig. 4). In hepatocytes exposed to H2O2, active ERK was not found in the nuclear fraction. In contrast, EGF-stimulated hepatocytes displayed high levels of active ERK in the nuclei. ERK and MEK are actively exported from the nucleus by the receptor CMR1/

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Fig. 4. Subcellular distribution of pERK. Hepatocyte lysates from control cells or from hepatocytes stimulated with 10 nmol/L EGF or 1 mmol/L H2O2 for 10 minutes were separated in nuclear and supernatant fractions. Western immunoblotting was performed on the fractions with antibodies against active ERK, LAP2, which is enriched in nuclear membranes, and ␤-tubulin, which is located in the cytoplasm. A representative example of at least three experiments is shown. EGF, epidermal growth factor; LAP, lamina-associated polypeptide; pERK, phosphorylated ERK.

exportin1. Leptomycin B, a specific inhibitor of CRM1/ exportin, was used to reveal a potential rapid export of H2O2-activated ERK. Treatment of hepatocytes with leptomycin B did not change the intracellular localization of ERK in either unstimulated cells (Fig. 5A), nor in hepatocytes stimulated with EGF (Fig. 5B) or H2O2 (Fig. 5C). Damage of cellular membranes and proteins by peroxides may cause interference with signaling pathways and intracellular transport. Nuclear import impairment of ERK was not a result of oxidative damage by H2O2, because nuclear accumulation was found after EGF-stimulation of hepatocytes exposed to 1 mmol/L H2O2. Hydrogen Peroxide–Induced ERK Activation Promoted Survival But Not Proliferation in Primary Hepatocytes. Potent mitogens induce nuclear accumulation and a persistent activation of ERK. Following exposure to H2O2, the ERK phosphorylation was transient and declined to control levels after 30 minutes, whereas EGF-induced posphorylation remained above control levels at all time points examined (Supplemental Fig. 4). In the same lysates, the level of ERK proteins was examined with an anti-ERK1 antibody recognizing ERK1 and ERK2. Only small variations in protein levels were ob-

Fig. 5. Immunofluorescence staining of ERK after treatment of cells with leptomycin B. Hepatocytes were treated with 2 ng/mL leptomycin B and epidermal growth factor or H2O2 before immunostaining with antiERK. Confocal laser scan images of ERK are shown. (A) Control hepatocytes. (B) 10 nmol/L epidermal growth factor for 10 minutes. (C) 1 mmol/L H2O2 for 10 minutes. Representative images of at least three experiments are shown.

Fig. 6. Survival of hepatocytes exposed to H2O2. Primary cultures of hepatocytes were exposed to increasing doses of H2O2 with or without preincubation with 10 ␮mol/L U0126 for 30 minutes. Trypan blue exclusion assay was performed on cells 24 hours after exposure. The number of surviving cells was compared with the total cell number. The results represent a combination of four separate experiments (SEM; n ⫽ 4).

served (lower panel). Next, hepatocyte proliferation was studied in primary cultures of hepatocytes after stimulation with H2O2 and EGF. As shown in Supplemental Fig. 5, increased DNA synthesis was not observed after stim-

Fig. 7. Phosphorylation of RSK and effect of ERK inhibition during H2O2 exposure. (A) Primary cultures of hepatocytes were stimulated with increasing amounts of H2O2 and EGF for 10 minutes. The cells were harvested, and Western immunoblot analyses were performed on lysates with an antibody that recognizes phosphorylated RSK. (B) Hepatocytes were incubated with or without 10 or 20 ␮mol/L U0126 for 30 minutes, and stimulated with 1 mmol/L H2O2 or 10 nmol/L EGF for 10 minutes. The cells were harvested and subjected to Western immunoblotting with an antibody recognizing active RSK (B). In both panel A and panel B, the protein expression of ␤-tubulin was used as a loading control. Representative examples of at least three experiments are shown.

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ulation with H2O2. Rather, a dose-dependent reduction in DNA synthesis was found. In contrast, the potent mitogen EGF induced strong proliferation of hepatocytes at 1 and 10 nmol/L concentrations. The cytotoxic effect of H2O2 on primary cultures of hepatocytes was determined via trypan blue exclusion assay. As presented in Fig. 6 (white columns), high percentage of cell survival was found at up to 1 mmol/L concentration of H2O2. At 5 mmol/L H2O2, only 50% of the cells survived 24 hours of exposure to H2O2. A lactate dehydrogenase assay showed similar results (data not shown). The H2O2-induced cell death was caspase-3–independent, because cleavage of the fluorometric caspase-3 substrate Ac-DEVD-AMC was not found (data not shown). In addition, the caspase-3 inhibitor Ac-DEVD-CMK did not reduce H2O2-induced cell death (data not shown). Next, a potential role of ERK in hepatocyte survival during oxidative stress was examined. The effect of ERK inhibition on cell survival after peroxide exposure was studied in hepatocyte cultures. Cells were pretreated with the MEK inhibitor U0126 before exposure to increasing doses of H2O2. A strong reduction in the survival of hepatocytes was observed when ERK was inhibited (Fig. 6, dark columns). At 5 mmol/L H2O2, hepatocyte survival was reduced from 50% to below 10% in the presence of U0126. This result indicates that ERK promotes cell survival during cytotoxic stress. Hydrogen Peroxide Exposure Induced RSK Activation via ERK. RSK is a cytoplasmic substrate of ERK and a mediator of cell survival.18,21 We therefore examined the activation of RSK during oxidative stress. RSK phosphorylation at increasing concentrations of H2O2 was compared with RSK activation in control cells and in hepatocytes stimulated with EGF. As shown in Fig. 7A, RSK phosphorylation was observed after exposure to 0.5 mmol/L H2O2. Maximal RSK activation was observed at 5 mmol/L, where the level of phosphorylation was equal to hepatocytes stimulated with the highest dose of EGF. To confirm that activation of RSK was mediated via ERK, hepatocytes were treated with the MEK inhibitor U0126 before stimulation with H2O2 or EGF. As shown in Fig. 7B, U0126 abolished the RSK activation induced by both H2O2 and EGF. Hydrogen Peroxide–Activated RSK Was Located in the Nucleus. Relocalization of active RSK mediates transcriptional regulation of pro-survival genes. Thus intracellular localization of active RSK after hydrogen peroxide exposure was examined via nuclear enrichment of hepatocyte lysates (Fig. 8). In hepatocytes exposed to H2O2, active RSK was found in the nuclear fraction. Furthermore, EGF-stimulated hepatocytes displayed high levels of active RSK in the nuclei.

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Fig. 8. Subcellular distribution of pRSK. The nuclear fraction was enriched from hepatocyte lysates from control cells, or from hepatocytes stimulated with 10 nmol/L EGF or 1 mmol/L H2O2 for 10 minutes. Western immunoblotting was performed on the nuclear fractions compared with whole cell lysates with antibodies against active RSK, and LAP2, which is enriched in nuclear membranes, and with ␤-tubulin, which is located in the cytoplasm. A representative example of at least three experiments is shown. EGF, epidermal growth factor; LAP, laminaassociated polypeptide; pRSK, phosphorylated RSK.

Discussion Inflammatory responses caused by ischemia/reperfusion and liver diseases promote generation of superoxide and hydrogen peroxide from resident and recruited phagocytes, which may cause oxidative stress to surrounding hepatocytes. In addition, ROS may be produced intracellularly in hepatocytes in response to cytokines, also released from phagocytes.3,22 In cholestatic liver disease, accumulation of bile acids within hepatocytes induces ROS-mediated cell death.1 In a recent study, cultured hepatocytes exposed to hydrophobic bile acids showed increased cell death, which was counteracted by treatment with ursodeoxycholic acid, a medicament used by patients with cholestatic liver disease. The increased survival of hepatocytes via this treatment depended upon activation of ERK.23 In our study, oxidative stress was induced via the addition of H2O2 to primary cultures of hepatocytes. H2O2 is soluble in both lipid and aqueous environments; therefore, it is capable of crossing cell membranes and reacting with intracellular targets.2 During H2O2 exposure, we found a strong activation of ERK, which provided protection against cell death. Our results are in line with previous studies.11,13 Active ERK did not accumulate in the nucleus but was strictly located in the cytoplasm; thus the survival signals from ERK were transduced from the cytoplasm during oxidative stress in hepatocytes. Accumulating evidence suggests that ROS regulate susceptibility to cell death through a direct interaction with cell signaling pathways, which controls programmed cell death, growth, and survival. Thus the balance be-

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tween death and survival may be determined by ROS in a highly regulated manner through the activation or inactivation of signal molecules. In hepatocytes, growth and survival signals are initiated by EGF binding and mediated through the EGF receptor/Ras/Raf/MEK/ERK signaling pathway. MEK is the upstream activator of ERK in the signaling pathway from the EGF receptor. Several reports show activation of MEK after ROS exposure. We found MEK to be the activator of ERK in hepatocytes as well. However, ERK was not redistributed to the nucleus after ROS-stimulated activation as seen after EGF. Thus, although ROS activate individual signal proteins in the growth factor–regulated signaling pathway, there are major differences in signal regulation when hepatocytes are exposed to H2O2 compared with EGF. The mechanisms responsible for promoting nuclear translocation of ERK and controlling its durability are largely unknown. In quiescent cells, ERK is located in the cytoplasm, in complex with its activator MEK.24 Because of its nuclear export sequence, MEK localizes in the cytoplasm irrespectively of its activation state.25 ERK itself does not contain a nuclear export sequence or a nuclear import sequence.26 In spite of this, ERK accumulates in the nucleus upon activation by a potent mitogen.8 Nuclear export of dephosphorylated ERK is also mediated by MEK, which shuttles in and out of the nucleus as a result of its nuclear export sequence.27 Nuclear export sequence– dependent nuclear export is mediated through the nuclear export receptor CRM1, a process that is inhibited by leptomycin B.27 Activation of ERK in hepatocytes by H2O2 did not promote nuclear translocation of active ERK. Furthermore, to exclude the possibility of a rapid export of the ERK/MEK complex, H2O2-exposed cells were pretreated with leptomycin B. Also in this case, ERK was exclusively located in the cytoplasm. Thus, ERK activated by H2O2 did not enter the nucleus, but was arrested in the cytosol. Specific intracellular localization of ERK has been shown to determine cell faith. Generally, ERK trapped in the cytoplasm blocks DNA synthesis and cell proliferation, whereas forcing an active ERK into the nucleus promotes proliferation and oncogenic transformation.28-30 Because nuclear accumulation was not found after stimulation with H2O2, the survival effects of ERK during H2O2 exposure of hepatocytes must be mediated through regulation of cytoplasmic targets. This finding is supported by a recent study in which cytoplasmic ERK protected against apoptosis in leukemia cells induced by serum starvation.31 RSK is a central mediator of ERK during cell survival.18 It enhances transcription of the survival proteins Bcl and Bcl-xl and neutralizes the proapoptotic protein Bad. RSK is primarily located in the

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cytoplasm but is relocated to the nucleus upon stimulation with growth factors.21,32 Both oxidative stress and growth factor stimulation induced strong activation of RSK via ERK and the presence of RSK in the nucleus. Thus survival may be mediated by ERK in the cytoplasmic compartment through activation and nuclear transport of RSK. In conclusion, we found that ROS mediated adaptive responses to environmental stress through activation of ERK, providing increased cell survival. ERK activated by ROS was restricted to the cytoplasm, suggesting that the physiological role of ERK in survival during oxidative stress is mediated from the cytoplasmic compartment. Acknowledgment: We thank the Department of Comparative Medicine at Rikshospitalet University Hospital for expert animal guidance. Leptomycin B was a kind gift from Dr. Minoru Yoshida, Chemical Genetics Laboratory, Riken, Wako, Saitama, Japan.

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