Background
Interferon (IFN), the first cytokine discovered in 1957 [
1], 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 [
8]. The products of IFN-stimulated genes are known to be responsible for the antiviral effects of IFN [
9]. 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 [
10]. 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 [
13]. 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.
Discussion
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 [
9]. 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 [
24]. For instance, HCV core protein inhibits IFN α-induced activation of STAT1 in hepatic cells [
25]. IFN α-mediated STAT activation is blocked in Huh7 cells containing the HCV genomic replicon [
26]. HCV nonstructural protein 5A inhibits IFN α signaling through suppression of STAT1 phosphorylation in hepatocyte-derived cells [
27]. 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.
Methods
1. Materials
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.
Acknowledgements
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).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LJZ designed and performed research, analyzed and interpreted data, and wrote the manuscript. XH, SFH, and HR performed research. ZTQ has given final approval for the version to be published. All authors read and approved the final manuscript.