Recently, we reported that β-carotene, vitamin D2, and linoleic acid inhibited hepatitis C virus (HCV) RNA replication in hepatoma cells. Interestingly, in the course of the study, we found that the antioxidant vitamin E negated the anti-HCV activities of these nutrients. These results suggest that the oxidative stress caused by the three nutrients is involved in their anti-HCV activities. However, the molecular mechanism by which oxidative stress induces anti-HCV status remains unknown. Oxidative stress is also known to activate extracellular signal-regulated kinase (ERK). Therefore, we hypothesized that oxidative stress induces anti-HCV status via the mitogen activated protein kinase (MAPK)/ERK kinase (MEK)–ERK1/2 signaling pathway. In this study, we found that the MEK1/2-specific inhibitor U0126 abolished the anti-HCV activities of the three nutrients in a dose-dependent manner. Moreover, U0126 significantly attenuated the anti-HCV activities of polyunsaturated fatty acids, interferon-γ, and cyclosporine A, but not statins. We further demonstrated that, with the exception of the statins, all of these anti-HCV nutrients and reagents actually induced activation of the MEK–ERK1/2 signaling pathway, which was inhibited or reduced by treatment not only with U0126 but also with vitamin E. We also demonstrated that phosphorylation of ERK1/2 by cyclosporine A was attenuated with N-acetylcysteine treatment and led to the negation of inhibition of HCV RNA replication. We propose that a cellular process that follows ERK1/2 phosphorylation and is specific to oxidative stimulation might lead to down-regulation of HCV RNA replication. Conclusion: Our results demonstrate the involvement of the MEK–ERK1/2 signaling pathway in the anti-HCV status induced by oxidative stress in a broad range of anti-HCV reagents. This intracellular modulation is expected to be a therapeutic target for the suppression of HCV RNA replication. (HEPATOLOGY 2009.)
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Hepatitis C virus (HCV), which belongs to the family Flaviviridae, is a single-stranded positive-sense RNA virus of approximately 9.6 kb.1, 2 Persistent infection with HCV causes chronic hepatitis, which often leads to liver cirrhosis and hepatocellular carcinoma.3 Therefore, HCV infection is a major health problem worldwide. Interferon (IFN)-based therapies, including the combination of pegylated IFN with ribavirin, are the current standard strategies for chronic hepatitis, but their sustained virological response rates are unsatisfactory.4, 5 There is thus an urgent need for novel partners with IFN or more effective reagents that may improve the sustained virological response rate.
Following the development in 1999 of a cell culture system to support efficient HCV RNA replication,6 numerous studies have identified reagents that inhibit HCV RNA replication and enhance the effect of IFN treatment.7–9 Some of these reagents are already available for clinical use. Previously, we also developed a genome-length HCV RNA (strain O of genotype 1b) replication system (OR6) with Renilla luciferase (RL) as a reporter in hepatoma cell lines.10 Using this OR6 assay system, we found that mizoribine,11 as an immunosuppressant, and fluvastatin (FLV) and pitavastatin (PTV),9, 12 as the reagents for hypercholesterolemia, suppressed genome-length HCV RNA replication. Furthermore, in a recent study13 in which we comprehensively analyzed the activities of ordinary nutrients on HCV RNA replication, three nutrients, β-carotene (BC), vitamin D2 (VD2), and linoleic acid (LA), were found to suppress HCV RNA replication and enhance the antiviral activity of IFN-α or cyclosporine A (CsA) in an additive or a synergistic manner. Because the anti-HCV activities of these three nutrients, as well as CsA, were canceled by treatment with antioxidants such as vitamin E (VE) or selenium, we suggested that oxidative stress might be involved in the anti-HCV activities of these three nutrients and CsA. However, the detailed molecular mechanism via which the oxidative effects of these three nutrients and CsA suppress HCV RNA replication has not been explored.
The production of reactive oxygen species (ROS) plays a pivotal role in various cellular processes, including cell proliferation, differentiation, and apoptosis.14 Whereas high-level production of ROS resulting from external stimuli is recognized as an important component of the pathogenesis of inflammatory and cancerous diseases, endogenously produced ROS at low concentrations are shown to function as signaling mediators of cellular responses.15, 16 Emerging evidence indicates that these ROS-triggered responses are mediated primarily via cellular signaling cascades, including a signaling pathway of extracellular signal-regulated kinase (ERK)1/2, namely p44/42 mitogen-activated protein kinase (MAPK), which belongs to the MAPK family.17, 18
Several studies have revealed that certain viral proteins initiate activation of the MAPK/ERK kinase (MEK)–ERK1/2 signaling pathway, which may facilitate the viral replication and infectivity in the infected cells.19, 20 The HCV core protein21 and the envelope protein22 have also been reported to up-regulate this signaling pathway. However, another study reported that the HCV nonstructural 5A (NS5A) protein suppressed activating protein-1 activation by inhibiting the phosphorylation of ERK1/2 in replicon cells.23 Moreover, recent studies using an inhibitor specific to the MEK–ERK1/2 signaling pathway reported that the direct anti-HCV activities of IFN-γ24 and acetylsalicylic acid25 are mediated in part through the induction of this cascade.
We demonstrate that the activation of MEK–ERK1/2 signaling plays a significant role in the anti-HCV activity caused by oxidative stress in a broad range of anti-HCV reagents.
Dimethyl sulfoxide (DMSO), BC, VD2, VE, LA, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and IFN-γ were purchased from Sigma Aldrich (St. Louis, MO), and CsA, FLV, U0126, PD98059, SB203580, and c-Jun N-terminal kinase inhibitor II were obtained from Calbiochem (San Diego, CA). Epidermal growth factor (EGF) was purchased from Toyobo (Osaka, Japan). PTV was purchased from Kowa Company, Ltd. (Tokyo, Japan). Anti-HCV core antibody (CP11) was purchased from the Institute of Immunology (Tokyo, Japan), and anti-HCV NS5A antibody was the generous gift of Dr. A. Takamizawa (Research Foundation for Microbial Diseases, Osaka University). Antibodies specific to ERK1/2 (p44/42 MAPK), MEK1/2, and phosphorylated (S217/221) MEK1/2 were purchased from Cell Signaling Technology (Beverly, MA), and anti-phosphorylated (T202/Y204) ERK1/2 antibody was obtained from BD Biosciences (San Jose, CA). Anti–β-actin antibody was purchased from Sigma Aldrich.
The cell lines OR6 and sO were cloned from ORN/C-5B/KE RNA and subgenomic replicon RNA (ON/3-5B)–replicating cells, respectively (Fig. 1). These cells were derived from the hepatoma cell line HuH-7, cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and 300 μg/mL of G418 (Geneticin; Invitrogen, Carlsbad, CA), and passaged twice a week at a 5:1 split ratio. ORN/C-5B/KE and ON/3-5B were derived from HCV-O (strain O of genotype 1b).10
OR6 Reporter Assay.
For the RL assay, 1.0-1.5 × 104 OR6 cells were plated onto 24-well plates in triplicate and precultured for 24 hours. The cells were pretreated with DMSO or a specific inhibitor for 1 hour and then were treated with each anti-HCV nutrient or compound in either the absence (DMSO) or presence of a specific inhibitor for 72 hours. After the treatment, the cells were harvested with Renilla lysis reagent (Promega, Madison, WI) and subjected to RL assay according to the manufacturer's protocol.
Western Blot Analysis.
For analysis of the effect of a specific inhibitor on the anti-HCV activity, 6.0-6.5 × 104 OR6 cells were plated onto 6-well plates and precultured for 24 hours. The pretreatment with DMSO or a specific inhibitor for 1 hour and subsequent treatment for 72 hours was performed in the same manner as for the OR6 reporter assay. For analysis of the activities of each anti-HCV nutrient or reagent on the MEK–ERK1/2 signaling pathway, 1.0 × 105 OR6 or sO cells were plated onto 6-well plates and precultured in 10% FBS-containing medium for 24 hours. After the preculture, the culture medium was changed to FBS-free medium and the cells were cultured for 48 hours prior to treatment with each nutrient or reagent. When the effect of a specific inhibitor or VE on ERK1/2 phosphorylation was analyzed, the cells were pretreated with the specific inhibitor or VE for 1 hour prior to each treatment. Preparation of the cell lysates, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblotting were then performed as described.26
Measurement of ROS.
OR6 cells in 24-well plates were left untreated or were treated with hydrogen peroxide (1 mM), LA (200 μM), and CsA (15 μg/mL) for 30 minutes and then incubated with dihydrodichlorocarboxyfluorescein diacetate (Invitrogen) (5 μμ) for 15 minutes. Fluorescence was measured with a FLUOROSKAN ASCENT fluorescence plate reader (Thermo Fisher Scientific, Waltham, MA) at an excitation wavelength of 485 nm and emission wavelength of 535 nm.
Cell Growth Assay.
To examine the activity of EGF on OR6 cell growth, 6.0-6.5 × 104 OR6 cells were plated onto 6-well plates in triplicate and were pre-cultured for 24 hours. The cells were treated with or without EGF for 72 hours, and the number of viable cells was counted after trypan blue dye treatment as described.11
Statistical comparison of the luciferase activities between the various treatment groups was performed using the Student t test. P values of less than 0.05 were considered statistically significant.
Effects of MEK1/2-Specific Inhibitors on the Anti-HCV Activities of BC, VD2, and LA in OR6 Cells.
Our recent study suggested the involvement of oxidative stress in the suppressive mechanism of three anti-HCV nutrients: BC, VD2, and LA.13 Because there have been reports of negative regulation of HCV RNA replication via the MEK–ERK1/2 signaling pathway,24, 25 which is one of the oxidative stress-induced cellular signaling pathways, we hypothesized that the suppression of HCV RNA replication by these three nutrients might be mediated via this cascade (Supporting Fig. 1). To test this hypothesis, we first used an OR6 assay system to examine the effects of U0126 and PD98059, inhibitors specific to MEK1/2, on the three anti-HCV nutrients at 60% inhibitory concentration. As shown in Fig. 2A, treatment with either 5 μM of U0126 or 10 μM of PD98059 slightly enhanced HCV RNA replication in comparison with the control. However, U0126 attenuated the anti-HCV activities of the three nutrients more clearly than PD98059 (Fig. 2A,B). U0126 prevented the anti-HCV activities of the three nutrients in a significant and dose-dependent manner and exerted complete inhibition against the anti-HCV activities of BC and LA (Fig. 2C,D), while the inhibitory effect of PD98059 was more mild (Fig. 2E,F). As shown in Fig. 2G, we also found that U0126 treatment restored the expressions of HCV proteins, core, and NS5A in a dose-dependent manner. We further demonstrated that knockdown of MEK1 or MEK2 by small interfering RNA negated the anti-HCV activity of LA (Supporting Fig. 2A-C). These inhibitions by U0126 against the anti-HCV activities of the three nutrients were not due to the enhancement of encephalomyocarditis virus/internal ribosomal entry site–driven RL activity, because this activity was not increased by U0126 (data not shown). Moreover, treatment with neither SB203580 (an inhibitor specific to p38 MAPK) nor c-Jun N-terminal kinase inhibitor, both of which belong to the same cascade family as MEK–ERK1/2, significantly affected the anti-HCV activities of the three nutrients (data not shown). These results imply that the activation of the MEK–ERK1/2 signaling pathway might be required for the suppression of genome-length HCV RNA replication by the three nutrients in cell culture.
Effect of U0126 on the Suppressive Effects of Polyunsaturated Fatty Acids and Anti-HCV Reagents in OR6 Cells.
Previous studies using a cell culture system have shown that polyunsaturated fatty acids (PUFAs), including LA, act as anti-HCV nutrients.27, 28 A recent study reported that lipid peroxidation of PUFAs was correlated with their anti-HCV activities, which were prevented by treatment with VE.29 This result coincides with our previous observations on the effects of LA.13 We proposed that the MEK–ERK1/2 signaling pathway might be involved in the anti-HCV activity of PUFAs, including LA, because lipid peroxidation is known to be a ROS-triggered cellular modification. 16 As expected, treatment with U0126 attenuated the anti-HCV activities of four representative PUFAs in a significant and dose-dependent manner (Fig. 3A,B).
Moreover, because the anti-HCV activities of BC, VD2, LA, and CsA, but not FLV, were found to be negated by VE,13 we were also interested in the potent role of the MEK–ERK1/2 signaling pathway in the anti-HCV mechanism of CsA. Furthermore, the previous study using a subgenomic replicon system had already shown the partial involvement of this cascade in the antiviral activity of IFN-γ.24 Therefore, we examined the effects of U0126 on various anti-HCV reagents: IFN-γ, CsA, and statins (FLV and PTV). We confirmed that also in genome-length HCV RNA replication cells, U0126 significantly inhibited the anti-HCV activity of IFN-γ (Fig. 3C,D). Interestingly, consistent with the effects of treatment with VE,13 the anti-HCV activity of CsA was completely abrogated by U0126 in a significant and dose-dependent manner, whereas statins were unaffected (Fig. 3C,D).
U0126 restored the reduced expression of HCV proteins by PUFAs, IFN-γ, and CsA in a dose-dependent manner, whereas statins were unaffected (Fig. 3E,F). These results were supported by additional real-time reverse-transcription polymerase chain reaction and immunofluorescence analyses (Supporting Fig. 3A-C). We also observed that knockdown of MEK1 or MEK2 by small interfering RNA did not affect the anti-HCV activity of PTV (Supporting Fig. 2A-C). Collectively, these findings suggest that the MEK–ERK1/2 signaling pathway may play a critical role in the negative regulation of HCV RNA replication by the anti-HCV nutrients BC and VD2, PUFAs, and the anti-HCV reagents IFN-γ and CsA, but not statins.
Activation of the MEK–ERK1/2 Signaling Pathway by Anti-HCV Nutrients and Reagents.
To further ensure the involvement of the MEK–ERK1/2 signaling pathway in the suppressive mechanisms of anti-HCV nutrients and reagents, we next examined whether these nutrients and reagents could actually initiate the activation of this signaling pathway. After treating the HCV RNA replicating cells with each of the nutrients and reagents, we performed immunoblotting specific to the phosphorylation of ERK1/2 and MEK1/2. In the same way as EGF, a potent activator of these kinases, the three anti-HCV nutrients (BC, VD2, and LA) enhanced the phosphorylation of ERK1/2 and MEK1/2 in both genome-length and subgenomic HCV RNA replication cells (Fig. 4A,B). IFN-γ, CsA, and all of the PUFAs also up-regulated this cascade in OR6 cells (Fig. 4C,D). The increase in phosphorylation of ERK1/2 was not observed after either statin treatment (Fig. 4D). The activation of MEK–ERK1/2 by the three anti-HCV nutrients was apparent until 1 hour after their application and subsequently attenuated, although EGF exhibited persistent enhancement of MEK–ERK1/2 phosphorylation (Fig. 4E). Because the experiments regarding ERK1/2 phosphorylation were performed in FBS-free conditions, we checked the anti-HCV activity of PTV, CsA, and LA in FBS-free medium. The results revealed that these anti-HCV reagents and nutrients also inhibited HCV RNA replication in FBS-free conditions (Supporting Fig. 4). Taken together, these findings indicate that the anti-HCV nutrients and reagents activated the MEK–ERK1/2 signaling pathway in HCV RNA replicating cells, providing further confirmation that this signaling cascade might be involved in their anti-HCV activities.
MEK1/2-Specific Inhibitors Attenuated the Increased Phosphorylation of ERK1/2 by Anti-HCV Nutrients/Reagents and EGF.
We next tested whether MEK1/2-specific inhibitors could prevent not only the suppression of HCV RNA replication but also the activation of ERK1/2 by the anti-HCV nutrients BC, VD2, and PUFAs and the anti-HCV reagents IFN-γ and CsA. Consistent with the inhibitory effects on their anti-HCV activities, U0126 more markedly abrogated the increase in ERK1/2 phosphorylation by anti-HCV nutrients, reagents, and EGF than did PD98059 (Fig. 5A,B). As shown in Fig. 5C, the enhanced ERK1/2 phosphorylation by the three nutrients and EGF was reduced by U0126 in a dose-dependent manner.
VE Attenuated the Increased Phosphorylation of ERK1/2 by Anti-HCV Nutrients/Reagents and EGF.
Because the suppression of HCV RNA replication by BC, VD2, LA, and CsA were completely negated by the treatment with VE in our recent study,13 we investigated whether VE could also inhibit ERK1/2 activation by anti-HCV nutrients and reagents. As expected, VE also attenuated the enhanced phosphorylation of ERK1/2 by not only anti-HCV nutrients and CsA but also IFN-γ and EGF (Fig. 6A,B). We also demonstrated that phosphorylation of ERK1/2 by CsA was attenuated with N-acetylcysteine treatment and led to the negation of inhibition of HCV RNA replication (Supporting Fig. 5A-C). The anti-HCV nutrients and reagents, whose activities were negated by U0126, were also inhibited by VE. In contrast, the anti-HCV activities of statins were not negated by U0126 or VE. We also demonstrated that LA and CsA induce ROS (Fig. 7). Collectively, these results suggest that these nutrients and reagents induce ROS as an oxidant in HCV RNA replicating cells, leading to activation of the MEK–ERK1/2 signaling pathway and suppression of HCV RNA replication.
The Effects of EGF on HCV RNA Replication were Different than Those of the Anti-HCV Nutrients/Reagents.
Because the study by Huang et al.24 showed that EGF time-dependently suppressed the expressions of HCV nonstructural proteins in subgenomic replicon-harboring cells, we wondered whether EGF could suppress genome-length HCV RNA replication. EGF inhibited HCV RNA replication by approximately 25% at a concentration of 100 ng/mL. This anti-HCV activity was weaker than that of the anti-HCV nutrients and reagents tested in this study. However, as shown in the cell growth assay, EGF promoted OR6 cell proliferation in a dose-dependent manner (Supporting Fig. 6). These cell growth effects of EGF may have caused us to underestimate the actual anti-HCV activity of EGF. The other reagents and nutrients did not affect cell proliferation compared with EGF (Supporting Fig. 7).
The previous studies using the MEK1/2-specific inhibitor and subgenomic replicon system showed that induction of the MEK–ERK1/2 signaling pathway might be required for the suppression of HCV RNA replication by some reagents.24, 25 In agreement with the study by Huang et al.,24 we also confirmed that U0126 inhibited the anti-HCV activity of IFN-γ in OR6 cells stably replicating genome-length HCV RNA. Although they did not identify the direct activation of the MEK–ERK1/2 signaling pathway by IFN-γ, we demonstrated that IFN-γ could stimulate this cascade in HCV RNA replication cells. Moreover, this stimulation was not only inhibited by U0126 but also by antioxidant VE. This result indicates the involvement of oxidative stress in the anti-HCV activity of IFN-γ as well as the MEK–ERK1/2 signaling pathway. IFNs induce the transcription of IFN-stimulated genes through the JAK–STAT pathway, but the induction of IFN-stimulated genes by IFN-γ has been far more complex than that by IFN type I.30 A study using a macrophage cell line revealed that IFN-γ activated ERK1/2, followed by the expression of IFN-γ–stimulated genes downstream of the JAK–STAT signaling pathway.31 Another study reported that the defensive activity of IFN-γ against hepatitis B virus in hepatoblastoma cells was mediated through the induction of oxidative stress.32 Furthermore, ROS itself has been reported to suppress HCV RNA replication in human hepatoma cells.33 These reports support our proposal regarding anti-HCV activity of oxidative stress that the generation of intracellular ROS inhibits HCV RNA replication through activation of the MEK–ERK1/2 signaling pathway. Waris and Siddiqui34 reported that calcium-dependent ROS generation induced cyclooxygenase-2 and prostaglandin E(2) via the activation of nuclear factor kappa B, leading to the suppression of HCV RNA replication. Choi et al.35 also demonstrated that elevated calcium suppressed HCV RNA replication. The activation of nuclear factor kappa B by ROS was mediated through the MEK–ERK1/2 signaling pathway. Therefore, we suggest that the oxidative reagents and nutrients in this study also may induce anti-HCV status by calcium-dependent ROS generation.
In the course of our study of the anti-HCV activities of these three nutrients, we found that treatment with U0126 more strongly inhibited their anti-HCV activities than treatment with PD98059. U0126 has been shown to possess approximately 100-fold-higher MEK1/2-specific inhibitory activity than PD98059.36 This different potential between the two inhibitors was considered to cause a gap in their effects on anti-HCV activities. We further found that, much like EGF, all three nutrients enhanced the phosphorylation of ERK1/2 and MEK1/2, which was reduced by treatment with U0126 or VE. In addition, the present study was the first to observe that BC, which has been shown to produce ROS,37 activates the MEK–ERK1/2 signaling pathway, an action that VD238 and LA39 have already been shown to exhibit in leukemia cell and dendritic cell lines, respectively. Furthermore, we found the involvement of the MEK–ERK1/2 signaling pathway in the anti-HCV mechanism of the three nutrients as well as various PUFAs, which were reported to be mediated through lipid peroxidation.29 These results suggest that the anti-HCV nutrients BC, VD2, and PUFAs, including LA, as well as IFN-γ may suppress HCV RNA replication via activation of the MEK–ERK1/2 signaling pathway in response to ROS production.
We also investigated the involvement of the MEK–ERK1/2 signaling pathway in the suppressive mechanism of anti-HCV reagents other than IFN-γ. In our previous study, the anti-HCV activity of CsA, but not FLV, was prevented by VE.13 Consequently, these results implied that CsA, but not statins, could be potent activators of the MEK–ERK1/2 signaling pathway as oxidants, leading to down-regulation of HCV RNA replication. CsA has been demonstrated to bind to cyclophilins and suppress HCV RNA replication by abolishing their interaction with NS5B polymerase.40 This CsA binding to cyclophilins, especially cyclophilin A (CyPA), has been shown to result in the generation of ROS through inhibition of the peptidylprolyl-cis-trans-isomerase–like activity of CyPA.41 Moreover, CyPA was reported to be secreted in response to oxidative stress,42 and to bind to a cell surface receptor, CD147, followed by ERK1/2 activation.43 These reports and our results suggest that CsA, acting as an oxidant, may trigger activation of the MEK–ERK1/2 signaling pathway, both directly by producing ROS by way of interaction with CyPA in the early phase, and indirectly by secreting CyPA in the late phase. Both activations could lead to an inhibition of HCV RNA replication. Thus, CyPA may play a critical role as an intermediator in the oxidative anti-HCV activity of CsA. In the latest study, CyPA was identified as the most essential cellular cofactor of HCV RNA replication among cyclophilins.44 Further studies will be needed to clarify whether CyPA is required for the oxidative suppressive mechanism of anti-HCV nutrients/reagents other than CsA.
Although we expected that strong activation of the MEK–ERK1/2 signaling pathway would suppress HCV RNA replication, EGF exhibited only slight anti-HCV activity in OR6 cells. The promotion of cell growth by EGF might prevent its primary inhibitory effect on HCV RNA replication. A portion of the ERK1/2 phosphorylation by EGF was also reduced by treatment with VE (Fig. 6A), suggesting that EGF might stimulate the MEK–ERK1/2 signaling pathway, in part, as an oxidant, and that this oxidative activity of EGF could exhibit its slight anti-HCV activity.
In this study, using MEK1/2 specific inhibitors, we revealed that the MEK–ERK1/2 signaling pathway is involved in the oxidative antiviral mechanism of the anti-HCV nutrients BC, VD2, and PUFAs and the anti-HCV reagents IFN-γ and CsA. Our results suggest that this oxidative induction of the MEK–ERK1/2 signaling pathway could be a novel therapeutic strategy for the eradication of HCV infection. Although oxidants themselves cause liver damage, they may work as anti-HCV factors during therapy in patients with chronic hepatitis C.
In conclusion, this study suggests that the anti-HCV activity of oxidative stress is closely linked to the activation of the MEK–ERK1/2 signaling pathway.
The authors thank Atsumi Morishita for technical assistance.