- Open Access
Interferon alpha regulates MAPK and STAT1 pathways in human hepatoma cells
© Zhao et al; licensee BioMed Central Ltd. 2011
- Received: 2 September 2010
- Accepted: 6 April 2011
- Published: 6 April 2011
Signaling events triggered by interferon (IFN) account for the molecular mechanisms of antiviral effect. JAK-STAT pathway plays a critical role in IFN signaling, and other pathways are also implicated in IFN-mediated antiviral effect. Changes in mitogen-activated protein kinase (MAPK) and STAT1 pathways were evaluated in human hepatoma cells Huh7 and HepG2 upon IFN alpha treatment.
Phosphorylation of ERK was significantly and specifically up-regulated, whereas enhanced phosphorylation of upstream kinase MEK was unobservable upon IFN alpha treatment. A mild increase in p38 MAPK, SAPK/JNK and downstream target ATF-2 phosphorylation was detectable after exposure to IFN alpha, indicating differential up-regulation of the MAPK signaling cascades. Moreover, STAT1 phosphorylation was strongly enhanced by IFN alpha.
IFN alpha up-regulates MAPK and STAT1 pathways in human hepatoma cells, and may provide useful information for understanding the IFN signaling.
- Huh7 Cell
- Human Hepatoma Cell
- Antiviral Effect
- Venezuelan Equine Encephalitis Virus
- STAT1 Signaling Pathway
Interferon (IFN), the first cytokine discovered in 1957 , has been studied extensively and advances have been made in biochemical and molecular mechanisms underlying production and action of IFN system. IFN family is classified as types I, II, and III, and plays a pivotal role in innate defense toward virus infection. Being the first line of defense, type I IFN including IFN alpha (IFN α) and IFN beta, exert their potent antiviral activities immediately after virus infection by inhibiting viral replication and enhancing immune responses [2–4].
Signaling events triggered by IFN are involved in the molecular mechanisms of the antiviral effects. In particular, JAK-STAT pathway plays a critical role in the signaling events induced by IFN, and this pathway is initiated by IFN through interaction with cell surface specific receptors [5–7]. Upon IFN binding to its receptor, the receptor-associated tyrosine kinases JAK are phosphorylated and activated, the kinases subsequently activate transcription factors STAT, and the activated STAT translocate to nuclear followed by binding to IFN-stimulated response elements and modulating transcription of IFN-stimulated genes . The products of IFN-stimulated genes are known to be responsible for the antiviral effects of IFN . Apart from the JAK-STAT pathway, a number of pathways may also be important for the IFN-dependent biological responses. Mitogen-activated protein kinase (MAPK) signaling pathways are composed of three subfamilies including ERK, SAPK/JNK, and p38 MAPK, and activation of MAPK pathways contributes to some human diseases . MAPK pathways are activated by a wide variety of extracellular signals such as stress, growth factors, and cytokines. The p38 MAPK or ERK is rapidly phosphorylated and activated in response to IFN α treatment [11, 12]. However, involvement of MAPK pathways in the IFN signaling needs to be fully elucidated.
Hepatitis C virus (HCV) infection is the major cause of human liver diseases. IFN α is the current approved treatment for HCV infection . We reported that the MAPK signaling pathways triggered by HCV envelope protein E2 were involved in the viral pathogenesis [14–16]. Since certain signaling events triggered by IFN α account for its antiviral effect, we wondered whether the MAPK pathways were also affected and an association between MAPK and STAT1 pathways under IFN α treatment. Difference in cellular response was evaluated after exposure to IFN α. Human hepatoma cells are usually used as models for evaluation of the IFN signaling. Thus, the aim of this study is to reveal regulation of MAPK and STAT1 signaling pathways by IFN α in human hepatoma cells Huh7 and HepG2.
1. Influence of IFN α on MEK
2. Prevention of IFN α-induced ERK activation by U0126
Next, changes in downstream kinase ERK phosphorylation were estimated in Huh7 cells treated with the IFN α. As shown in Figure 1B, phosphorylation of ERK was increased after exposure to the IFN. Upon the treatment with IFN α at concentrations ranging from 100 to 800 U/ml, level of ERK phosphorylation was high after 15 min treatment compared to the treatment for 30 min, and a significant increase in ERK phosphorylation was observed at 60 min. In addition, MEK inhibitor U0126 was applied to estimate specific regulation of the kinase cascade by the IFN α. Huh7 was pretreated with 10 μM U0126 prior to the addition of 100 U/ml IFN α. Figure 1C showed that level of ERK phosphorylation was markedly decreased in the cells pretreated with the U0126, indicating that pretreatment with U0126 prevents the IFN α-induced ERK phosphorylation. Constant level of total ERK was also determined. GAPDH was determined as a control for protein loading. Thus, IFN α significantly and specifically up-regulates ERK phosphorylation.
3. Regulation of p38 MAPK, SAPK/JNK, and ATF-2 by IFN α
To evaluate effects of IFN on p38 MAPK and SAPK/JNK, cell lysates from Huh7 and HepG2 treated with the different concentrations of IFN α at the indicated time points were examined by Western blot analysis. In comparison with phosphorylation of p38 MAPK without the IFN α treatment, level of p38 MAPK phosphorylation was slightly increased following the IFN α treatment (Figure 2). As for SAPK/JNK, differential profiles were observable in response to the IFN α treatment between HepG2 and Huh7. In HepG2 cells, the IFN α had no obvious effect on SAPK/JNK phosphorylation. Whereas SAPK/JNK phosphorylation was enhanced by the IFN α in a time dependent-manner in Huh7 cells, and the IFN α treatment for 60 min led to a significant increase in SAPK/JNK phosphorylation. GAPDH was determined as a control for protein loading.
4. Regulation of STAT1 by IFN α
Being the effective therapy for HCV infection, IFN α has been focused to reveal the molecular mechanisms underlying antiviral effect. Among the multiple mechanisms, modulation of the signaling events by IFN α is particularly important for the antiviral effect. In this report, we have investigated regulation of the MAPK and STAT1 signaling pathways by IFN α in human hepatoma cells. Our data show that the phosphorylation of ERK, p38 MAPK, SAPK/JNK, and ATF-2 is up-regulated by IFN α, whereas IFN α seems to be no enhanced effect on the MEK phosphorylation. In response to IFN α treatment, the STAT1 phosphorylation is also enhanced. The profiles of kinetic kinase phosphorylation are shown.
IFN is believed to exert its antiviral effect predominantly through JAK-STAT signaling pathway. IFN sequentially phosphorylates and activates JAK kinases and STAT transcription factors to stimulate the transcription of IFN-stimulated genes. STAT family has seven known members, and STAT1 and STAT2 play central roles in induction of the IFN-dependent antiviral state . Studies show that some viruses have evolved strategies to evade the host immune responses and set up successful infections by targeting STAT pathway. Ebola virus, rotavirus, Venezuelan equine encephalitis virus, Sindbis virus, and Marburg virus are shown to prevent the nuclear translocation and phosphorylation of STAT1, resulting in the impairment of the IFN α-dependent antiviral effect [20–23]. As for HCV, the interference of HCV proteins with IFN α-induced JAK-STAT pathway has been proposed to be an escape strategy of HCV . For instance, HCV core protein inhibits IFN α-induced activation of STAT1 in hepatic cells . IFN α-mediated STAT activation is blocked in Huh7 cells containing the HCV genomic replicon . HCV nonstructural protein 5A inhibits IFN α signaling through suppression of STAT1 phosphorylation in hepatocyte-derived cells . Consistent with these findings, here we document that the STAT1 phosphorylation is strongly enhanced by IFN α in human hepatoma cells. Our data show that the phosphorylation of STAT1 is enhanced upon the IFN α treatment not only for a short time period but also for a long time period. These results are confirmed by using the IFN α-2a and 2b.
Changes in the MAPK signaling pathways induced by the HCV proteins contribute to HCV pathogenesis. In this regards, it is interesting to examine whether MAPK pathways are also interfered by IFN α and the association between MAPK pathways and JAK-STAT pathway triggered by IFN α. In the present study, we addressed the regulation of MEK, ERK, p38 MAPK, SAPK/JNK, and ATF-2 by the IFN α. The cellular response was evaluated upon the different concentrations and time periods of the IFN α treatment. The ERK pathway is activated by growth factors, cytokines, and virus infection. Interestingly, we found that the phosphorylation of ERK was significantly and specifically up-regulated by the IFN α, whereas enhanced phosphorylation of upstream kinase MEK was unobservable. The p38 MAPK and SAPK/JNK are the stress-related signal transduction pathways. The IFN α treatment led to the mild increase in p38 MAPK and SAPK/JNK phosphorylation, and also resulted in the enhancement of downstream target ATF-2 phosphorylation. Our data indicate that IFN α is capable of differentially up-regulating MAPK pathways in human hepatoma cells. Under the same condition of IFN α treatment, Huh7 appears to be susceptible to the IFN α compared with HepG2, implying that the regulation of MAPK pathways by IFN α may depend on cell types. In support of our data, reports show that the p38 MAPK, JNK, and ERK are activated by IFN gamma or the type I IFN [28–30]. Thus, in addition to classical JAK-STAT pathway, MAPK pathways are also activated by IFN α and involved in the IFN signaling. Recent studies illustrate an important role for the IFN signaling pathways in triggering the host antiviral responses to HCV infection [31–34]. Our results suggest that the up-regulation of MAPK and STAT1 pathways by IFN α might be implicated in its antiviral effect, although we have not ruled out the possible involvement of other pathways in the signaling events induced by IFN α. Further studies are still necessary to determine the downstream effects of these phosphorylation events, including the double-stranded RNA-activated protein kinase, 2'-5' oligoadenylate synthetase, and Mx.
In conclusion, our results demonstrate that IFN α up-regulates MAPK and STAT1 signaling pathways in human hepatoma cells, and provide useful information for understanding the IFN signaling events.
Recombinant human IFN α-2a and 2b were obtained from PBL Interferon Source (Piscataway, NJ) and Huaxin High Biotechnology (Shanghai, China), respectively. Alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG were purchased from Vector Lab (Burlingame, CA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Invitrogen (Camarillo, CA). Chemiluminescent detection reagents were from Millipore (Billerica, MA). 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium were obtained from Sigma (St. Louis, MO). MEK inhibitor U0126 and antibodies specific for MEK, ERK, STAT1, phospho-MEK (Ser217/221), phospho-ERK (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-ATF-2 (Thr71), or phospho-STAT1 (Tyr701) were purchased from Cell Signaling Technology (Beverly, MA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were from HyClone.
2. IFN treatment
Human hepatoma cells Huh7 and HepG2 were grown in DMEM plus 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2. Cells at 80% confluence were washed twice with phosphate-buffered saline and then maintained for 24 hrs in serum-free DMEM before treatment with IFN α-2b. For concentration- and time-dependent stimulation of IFN, cells serum-starved were treated with increasing concentrations of IFN α-2b dissolved in 0.1% bovine serum albumin (0, 100, 200, 400, 800 U/ml) for 15, 30, or 60 min. For long time treatment of IFN, Huh7 cells were serum starved for 12 hrs and then cultured in serum-free DMEM containing 800 U/ml IFN α-2a for 12, 24, 48, and 72 hrs, respectively. Following the above treatments, cells were washed with phosphate-buffered saline, collected by centrifugation at 2,000 rpm for 5 min, lysed in sodium dodecyl sulfate sample buffer on ice, and heated for 5 min at 100°C. Cell lysates were centrifuged at 12,000 rpm for 10 min to remove cellular debris, mixed with sample loading buffer, boiled, and that equal amounts of protein extracts were subjected to Western blot analysis.
3. U0126 pretreatment
Huh7 cells serum-starved were pretreated for 1 h with 10 μM U0126 dissolved in dimethylsulfoxide prior to treatment with 100 U/ml IFN α-2b for another 1 h. Cells with or without the U0126 pretreatment were harvested and lysed for measurement of ERK by Western blotting.
4. Western blotting
Phosphorylated and total levels of signal molecules were assayed by Western blotting following the manufacturer's protocol with some modification. In brief, cell lysates were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were separated and transferred onto nitrocellulose membranes. After blocking in 5% nonfat milk for 1 h, membranes were incubated overnight at 4°C with antibodies against MEK, ERK, STAT1, phospho-MEK, phospho-ERK, phospho-SAPK/JNK, phospho-p38 MAPK, phospho-ATF-2, or phospho-STAT1 at the recommended dilutions, followed by incubation with alkaline phosphatase or horseradish peroxidase-conjugated secondary antibodies. The blots were developed with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium substrates or chemiluminescent detection reagents. Glyceraldehyde-3-phosphate dehydrogease (GAPDH) was determined as a control for protein loading.
This work was supported by the National Natural Science Foundation of China Grants (30771928, 30600529, 30921006), National Key Basic Research and Development (973) Program of China (2009CB522503), National S&T Major Project for Infectious Diseases (2008ZX10002-13), and the Shanghai Leading Academic Discipline Project (B901).
- Isaacs A, Lindenmann J: Virus interference. I. The interferon. Proc R Soc Lond B Biol Sci 1957, 147: 258-267. 10.1098/rspb.1957.0048View ArticlePubMedGoogle Scholar
- Baron S, Dianzani F: The interferons: a biological system with therapeutic potential in viral infections. Antiviral Res 1994, 24: 97-110. 10.1016/0166-3542(94)90058-2View ArticlePubMedGoogle Scholar
- Capobianchi MR, Abbate I, Cappiello G, Solmone M: HCV and interferon: viral strategies for evading innate defence mechanisms in the virus-host battle. Cell Death Differ 2003,10(Suppl 1):S22-24. 10.1038/sj.cdd.4401142View ArticlePubMedGoogle Scholar
- Akira S, Uematsu S, Takeuchi O: Pathogen recognition and innate immunity. Cell 2006, 124: 783-801. 10.1016/j.cell.2006.02.015View ArticlePubMedGoogle Scholar
- Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994, 264: 1415-1421. 10.1126/science.8197455View ArticlePubMedGoogle Scholar
- Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD: How cells respond to interferons. Annu Rev Biochem 1998, 67: 227-264. 10.1146/annurev.biochem.67.1.227View ArticlePubMedGoogle Scholar
- de Weerd NA, Samarajiwa SA, Hertzog PJ: Type I interferon receptors: biochemistry and biological functions. J Biol Chem 2007, 282: 20053-20057. 10.1074/jbc.R700006200View ArticlePubMedGoogle Scholar
- Sarasin-Filipowicz M, Oakeley EJ, Duong FH, Christen V, Terracciano L, Filipowicz W, Heim MH: Interferon signaling and treatment outcome in chronic hepatitis C. Proc Natl Acad Sci USA 2008, 105: 7034-7039. 10.1073/pnas.0707882105PubMed CentralView ArticlePubMedGoogle Scholar
- Samuel CE: Antiviral actions of interferons. Clin Microbiol Rev 2001, 14: 778-809. 10.1128/CMR.14.4.778-809.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Johnson GL, Lapadat R: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002, 298: 1911-1912. 10.1126/science.1072682View ArticlePubMedGoogle Scholar
- Uddin S, Majchrzak B, Woodson J, Arunkumar P, Alsayed Y, Pine R, Young PR, Fish EN, Platanias LC: Activation of the p38 mitogen-activated protein kinase by type I interferons. J Biol Chem 1999, 274: 30127-30131. 10.1074/jbc.274.42.30127View ArticlePubMedGoogle Scholar
- Arora T, Floyd-Smith G, Espy MJ, Jelinek DF: Dissociation between IFN-alpha-induced anti-viral and growth signaling pathways. J Immunol 1999, 162: 3289-3297.PubMedGoogle Scholar
- Hoofnagle JH, di Bisceglie AM: The treatment of chronic viral hepatitis. N Engl J Med 1997, 336: 347-356. 10.1056/NEJM199701303360507View ArticlePubMedGoogle Scholar
- Zhao LJ, Wang L, Ren H, Cao J, Li L, Ke JS, Qi ZT: Hepatitis C virus E2 protein promotes human hepatoma cell proliferation through the MAPK/ERK signaling pathway via cellular receptors. Exp Cell Res 2005, 305: 23-32. 10.1016/j.yexcr.2004.12.024View ArticlePubMedGoogle Scholar
- Zhao LJ, Zhang XL, Zhao P, Cao J, Cao MM, Zhu SY, Liu HQ, Qi ZT: Up-regulation of ERK and p38 MAPK signaling pathways by hepatitis C virus E2 envelope protein in human T lymphoma cell line. J Leukoc Biol 2006, 80: 424-432. 10.1189/jlb.0106014View ArticlePubMedGoogle Scholar
- Zhao LJ, Zhao P, Chen QL, Ren H, Pan W, Qi ZT: Mitogen-activated protein kinase signaling pathways triggered by the hepatitis C virus envelope protein E2: implications for the prevention of infection. Cell Proliferat 2007, 40: 508-521. 10.1111/j.1365-2184.2007.00453.xView ArticleGoogle Scholar
- Rosa D, Saletti G, De Gregorio E, Zorat F, Comar C, D'Oro U, Nuti S, Houghton M, Barnaba V, Pozzato G, Abrignani S: Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc Natl Acad Sci USA 2005, 102: 18544-18549. 10.1073/pnas.0509402102PubMed CentralView ArticlePubMedGoogle Scholar
- Choi SH, Park KJ, Ahn BY, Jung G, Lai MM, Hwang SB: Hepatitis C virus nonstructural 5B protein regulates tumor necrosis factor alpha signaling through effects on cellular IkappaB kinase. Mol Cell Biol 2006, 26: 3048-3059. 10.1128/MCB.26.8.3048-3059.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Huang Y, Chen XC, Konduri M, Fomina N, Lu J, Jin L, Kolykhalov A, Tan SL: Mechanistic link between the anti-HCV effect of interferon gamma and control of viral replication by a Ras-MAPK signaling cascade. Hepatology 2006, 43: 81-90. 10.1002/hep.21011View ArticlePubMedGoogle Scholar
- Basler CF, Amarasinghe GK: Evasion of interferon responses by Ebola and Marburg viruses. J Interferon Cytokine Res 2009, 29: 511-520. 10.1089/jir.2009.0076PubMed CentralView ArticlePubMedGoogle Scholar
- Sherry B: Rotavirus and reovirus modulation of the interferon response. J Interferon Cytokine Res 2009, 29: 559-567. 10.1089/jir.2009.0072PubMed CentralView ArticlePubMedGoogle Scholar
- Simmons JD, White LJ, Morrison TE, Montgomery SA, Whitmore AC, Johnston RE, Heise MT: Venezuelan equine encephalitis virus disrupts STAT1 signaling by distinct mechanisms independent of host shutoff. J Virol 2009, 83: 10571-10581. 10.1128/JVI.01041-09PubMed CentralView ArticlePubMedGoogle Scholar
- Yin J, Gardner CL, Burke CW, Ryman KD, Klimstra WB: Similarities and differences in antagonism of neuron alpha/beta interferon responses by Venezuelan equine encephalitis and Sindbis alphaviruses. J Virol 2009, 83: 10036-10047. 10.1128/JVI.01209-09PubMed CentralView ArticlePubMedGoogle Scholar
- Heim MH, Moradpour D, Blum HE: Expression of hepatitis C virus proteins inhibits signal transduction through the Jak-STAT pathway. J Virol 1999, 73: 8469-8475.PubMed CentralPubMedGoogle Scholar
- Bode JG, Ludwig S, Ehrhardt C, Albrecht U, Erhardt A, Schaper F, Heinrich PC, Häussinger D: IFN-alpha antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-3. FASEB J 2003, 17: 488-490.PubMedGoogle Scholar
- Luquin E, Larrea E, Civeira MP, Prieto J, Aldabe R: HCV structural proteins interfere with interferon-alpha Jak/STAT signalling pathway. Antiviral Res 2007, 76: 194-197. 10.1016/j.antiviral.2007.06.004View ArticlePubMedGoogle Scholar
- Lan KH, Lan KL, Lee WP, Sheu ML, Chen MY, Lee YL, Yen SH, Chang FY, Lee SD: HCV NS5A inhibits interferon-alpha signaling through suppression of STAT1 phosphorylation in hepatocyte-derived cell lines. J Hepatol 2007, 46: 759-767. 10.1016/j.jhep.2006.11.013View ArticlePubMedGoogle Scholar
- Valledor AF, Sánchez-Tilló E, Arpa L, Park JM, Caelles C, Lloberas J, Celada A: Selective roles of MAPKs during the macrophage response to IFN-gamma. J Immunol 2008, 180: 4523-4529.View ArticlePubMedGoogle Scholar
- Jeon YJ, Choi JS, Lee JY, Yu KR, Kim SM, Ka SH, Oh KH, Kim KI, Zhang DE, Bang OS, Chung CH: ISG15 modification of filamin B negatively regulates the type I interferon-induced JNK signalling pathway. EMBO Rep 2009, 10: 374-380. 10.1038/embor.2009.23PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou Z, Hamming OJ, Ank N, Paludan SR, Nielsen AL, Hartmann R: Type III interferon (IFN) induces a type I IFN-like response in a restricted subset of cells through signaling pathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases. J Virol 2007, 81: 7749-7758. 10.1128/JVI.02438-06PubMed CentralView ArticlePubMedGoogle Scholar
- He Y, Katze MG: To interfere and to anti-interfere: the interplay between hepatitis C virus and interferon. Viral Immunol 2002, 15: 95-119. 10.1089/088282402317340260View ArticlePubMedGoogle Scholar
- Ishida H, Ohkawa K, Hosui A, Hiramatsu N, Kanto T, Ueda K, Takehara T, Hayashi N: Involvement of p38 signaling pathway in interferon-alpha-mediated antiviral activity toward hepatitis C virus. Biochem Biophys Res Commun 2004, 321: 722-727. 10.1016/j.bbrc.2004.07.015View ArticlePubMedGoogle Scholar
- Helbig KJ, Yip E, McCartney EM, Eyre NS, Beard MR: A screening method for identifying disruptions in interferon signaling reveals HCV NS3/4a disrupts Stat-1 phosphorylation. Antiviral Res 2008, 77: 169-176. 10.1016/j.antiviral.2007.11.008View ArticlePubMedGoogle Scholar
- Murao K, Imachi H, Yu X, Cao WM, Nishiuchi T, Chen K, Li J, Ahmed RA, Wong NC, Ishida T: Interferon alpha decreases expression of human scavenger receptor class BI, a possible HCV receptor in hepatocytes. Gut 2008, 57: 664-671. 10.1136/gut.2006.111443View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.