IL29 and IFN-α regulate the expression of MxA, 2′,5′OAS and PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells

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  Interferons (IFNs) can activate the PI3K-AKT and Raf-MEK-ERK signal pathways and induce antiviral proteins (MxA, 2′,5′-OAS and PKR) expression in specific cell lines. However, the relationship between those antiviral proteins expression and signal
  IL-29 and IFN- a  regulate the expression of MxA, 2 0 ,5 0 -OASand PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells Yu Chai  • Hai-Liang Huang  • Dao-Jun Hu  • Xin Luo  • Qian-Shan Tao  • Xiao-Ling Zhang  • Sheng-Quan Zhang Received: 9 October 2009/Accepted: 11 March 2010/Published online: 23 March 2010   Springer Science+Business Media B.V. 2010 Abstract  Interferons (IFNs) can activate the PI3K-AKTand Raf-MEK-ERK signal pathways and induce antiviralproteins (MxA, 2 0 ,5 0 -OAS and PKR) expression in specificcell lines. However, the relationship between those antiviralproteins expression and signal pathways remains unknownatpresent.Thusourexperimentsweredesignedtodeterminethe exact relationship in HepG2.2.15 cell line. The resultsdemonstrated that IFN- a  and IL-29 were both able toactivate PI3K-AKT and Raf-MEK-ERK signal pathways,and IFN- a  up-regulated the expression of MxA, 2 0 ,5 0 -OASand PKR whereas IL-29 increased mRNA expression of MxA and 2 0 ,5 0 -OAS and had no influence on PKR. Fur-thermore, MxA, 2 0 ,5 0 -OAS and PKR expression were down-regulated while PI3K-AKT signal pathway was blocked byLY294002. And MxA was up-regulated after Raf-MEK-ERK signal pathway being blocked by PD98059. Thesefindings indicate that the expression of MxA, 2 0 ,5 0 -OAS andPKR are up-regulate by PI3K-AKT signal pathway, andRaf-MEK-ERK signal pathway has a negative regulatoryeffect on the expression of MxA and no significant effect on2 0 ,5 0 -OAS and PKR. Keywords  Interleukin 29    MxA    2 0 ,5 0 -OAS   PKR    P-ERK     P-AKT Introduction Interferons (IFNs) belong to the large class of glycopro-teins known as cytokines that exhibit potent antiviral,antiproliferative, apoptotic and immunoregulatory activi-ties [1], which are classified into three major groups, type I,II and III. IFN- a  is a member of type I IFN family, whileIL-29 is one of type III [2, 3]. The IFNs of type I and type III can activate the Janus tyrosine Kinase (JAK)-SignalTransducer and Activator of Transcription (STAT) path-way [2–4]. It is known that JAKs and STATs, the Jak-1, Jak-2, Tyk-2 kinases and the Stat-1 and Stat-2 transcriptionfactors play central roles in mediating IFN-dependentbiological responses, including induction of the expressionof MxA, 2 0 ,5 0 -OAS and PKR [5–8]. However, the IFN- induced JAK-STAT signal alone is not sufficient to explainall the biological effects of IFNs [9]. It has been reported that IFNs can activate the PI3K-AKT and Raf-MEK-ERK signal pathways in specific typecells [10–13]. PI3K-AKT pathway transduces signals from extracellular stimuli to cellular pathways mediating a widevariety of biological responses, such as inhibition of apoptosis and stimulation of cell proliferation. LY294002,which is a highly selective inhibitor of PI3K, can block downstream pathways of PI3K, including AKT activation[14, 15]. Ras, Raf, mitogen-activated protein/extracellular signal-regulated kinases (MEK) and exogenously regulatedkinases (ERK) constitute a signaling pathway to mediatevarious cellular processes such as proliferation, differenti-ation, transcription regulation and development. PD98059(MEK1 Inhibitor) has been shown to act as a potent and Electronic supplementary material  The online version of thisarticle (doi:10.1007/s11033-010-0087-1) contains supplementarymaterial, which is available to authorized users.Y. Chai    H.-L. Huang    D.-J. Hu    X. Luo    Q.-S. Tao   X.-L. Zhang    S.-Q. Zhang ( & )Department of Biochemistry and Molecular Biology, AnhuiMedical University, 69 Meishan Road, Hefei, Anhui 230032,Chinae-mail:  1 3 Mol Biol Rep (2011) 38:139–143DOI 10.1007/s11033-010-0087-1  specific inhibitor of MEK1 activation and the MAP kinasecascade in vivo, which binds to the inactive forms of MEK1 and prevents activation by upstream activators suchas c-Raf  [16–18]. The HepG2.2.15 cell is a stably transfected HepG2(Human Hepatoblastoma) cell line which persistentlysecretes Hepatitis B Virus (HBV). The cell line is used as astable HBV-producing model in vitro [19]. However, whether the two signal pathways regulate these antiviralgenes expression in HepG2.2.15 cell line remainsunknown. Here, we report that PI3K-AKT and Raf-MEK-ERK signal pathways are activated by IFN- a  and IL-29 inHepG2.2.15 cells. Mechanistic studies show IL-29 andIFN- a  regulate the expression of MxA, 2 0 ,5 0 -OAS and PKRgenes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells. Materials and methods Cytokines and antibodiesRecombinant human IL-29 and IFN- a  were purchasedfrom PeproTech Inc (NJ, USA). P-ERK1/2, P-AKT, and b -ACTIN antibodies were received from Santa Cruz Bio-technology (Santa Cruz, CA, USA).Cell cultureHepG2.2.15 cells were incubated in complete Dulbecco’smodified Eagle’s medium (DMEM, containing 10% fetalbovine serum (FBS), 100 U/ml of penicillin and 100 U/mlof streptomycin) at 37  C in atmosphere of 5% CO 2 .Real-time PCR (relative quantification) analyzedexpression of 2 0 ,5 0 -OAS, MxA and PKR mRNAHepG2.2.15 cells in 12-well plates (density at 5  9  10 5 cells per well) were treated with IL-29 (100 ng/ml), IFN- a (10 ng/ml), PD98059 (100  l M) and LY294002 (50  l M)for 12 and 24 h. Cells were collected and the total RNA of the cells was isolated with Trizol from Invitrogen (Carls-bad, CA, USA). One microgram of cellular RNA was usedto conduct reverse transcription with a Promega RT kit andRandom primers (A3800, Madison, WI, USA). Then thereal-time PCR was carried out in a 20- l l reaction systemcontaining 200 nM of primers, 50 9  ROX and 2 9  PCRmaster mix (NJ, USA). All primers (Electronic Supple-mentary Material—Table 1) were purchased from TakaraCompany (Dalian, China). Samples were amplified in theApplied Biosystem 7500 real-time PCR System (FosterCity, CA, USA) for 40 cycles with the followingconditions: denaturation at 95  C for 15 s, annealing, andextension at 60  C for 40 s. Glyceraldehyde phosphatedehydrogenase (GAPDH) was used as control.Western blot analysis of phosphorylated ERK1/2and AKTAfter treatment, cells were washed twice with cold phos-phate buffered saline (PBS) and then incubated in a coldlysis buffer (1% Nonidet P40, 0.1% SDS, 150 mM Tris, pH7.4, 50 U/ml aprotinin and 1 mmol/l PMSF) for 20 min at4  C. Cellular lysate was centrifuged for 10 min at 12000 g at 4  C. The supernatants were collected as total cellularproteins. Quantitative analysis of the content by the LowryMethod, an equal amount of total proteins from eachsample was loaded on SDS-PAGE. After electrophoresis,the separated proteins were transferred to polyvinylidenedifluoride membrane (Millipore, USA) by electroblotting.And then, the membrane was blocked with Tris bufferedsaline (TBS) containing 5% fat-free milk for 2 h at roomtemperature (RT) followed by incubation in primary anti-body/TBS solution at 4  C overnight. After washing, themembrane was incubated in HRP linked secondary anti-body/TBS solution for 1 h at RT. Western Blot reactionproducts with chemiluminescence reagents (SuperSignal-West Femto Trial Kit; Pierce, Woburn, MA, USA) werevisualized by radiograph.Data analysisAll experiments were repeated at least three times andrepresentative results are presented. Data were expressed asmean  ±  standard deviation. Significance was considered at P \ 0.05. Results IL-29 and IFN- a  induced the activation of ERK1/2and AKT in HepG2.2.15 cellsIL-29 and IFN- a  binding to receptor complex activatesERK-1/2 and AKT in some type cells, so we analyzedwhether IL-29 and IFN- a  could activate the two signalpathways in HepG2.2.15 cells. The studies indicated thatIL-29 (100 ng/ml) and IFN- a  (10 ng/ml) could signifi-cantly up-regulate both P-ERK and P-AKT,  b -ACTIN wasused as control. And it was easy to find that ERK and AKTphosphorylation were clearly, respectively blocked byPD98059 (100  l M) and LY294002 (50  l M), even whenthe cells were stimulated by IL-29 and IFN- a  (Fig. 1). 140 Mol Biol Rep (2011) 38:139–143  1 3  IL-29, like IFN- a , up-regulated mRNA expressionof the antiviral proteins 2 0 ,5 0 -OAS and MxA, but notPKR in HepG2.2.15 cellsTo determine the influence of IL-29 and IFN- a  on thetranscriptional regulation of three antiviral proteins (2 0 ,5 0 -OAS, MxA and PKR) in HepG2.2.15 cells, the cells weretreated with IL-29 (100 ng/ml) and IFN- a  (10 ng/ml) for12 and 24 h, then the mRNA levels of the three genes wereanalyzed by real-time PCR (relative quantification). TheIFN- a  up-regulated mRNA expression of 2 0 ,5 0 -OAS, MxAand PKR by 2.0-, 1.7-, 1.5-folds at 12 h and 2.5-, 2.0-, 1.9-folds at 24 h, respectively ( P \ 0.01). However, IL-29only up-regulated MxA and 2 0 ,5 0 -OAS by 2.3-, 2.5-fold at12 h and 2.6-, 3.3-fold at 24 h, respectively ( P \ 0.01)(Fig. 2), but PKR remained unchanged.The antiviral proteins expression induced by IL-29or IFN- a  depended on the activation of PI3K-AKTsignal pathways in HepG2.2.15 cellsIn order to investigate the relationship between MxA, 2 0 ,5 0 -OAS, PKR and PI3K-AKT signal pathway in HepG2.2.15cells, the cells were pretreated with LY294002 (50  l M) for1 h, then added IFN- a  (10 ng/ml) and IL-29 (100 ng/ml).The mRNA levels of these genes were analyzed by real-time PCR (relative quantification). In Fig. 3, after the AKTpathway was blocked by LY294002, IFN-induced expres-sion of the MxA, 2 0 ,5 0 -OAS and PKR decreased. Thesedata suggest that the up-regulating gene expression of MxA, 2 0 ,5 0 -OAS and PKR by IL-29 and IFN- a  depends ontheir activated AKT pathway. However, LY294002 hasless effect on PKR than MxA and 2 0 ,5 0 -OAS. All theseresults indicate that PI3K-AKT pathway plays an importantrole in the regulation of the IFN-induced genes.The activation of Raf-MEK-ERK signal pathwaydecreased the expression of MxA, but no effecton the other two genesBecause the IL-29 and IFN- a  activate the Raf-MEK-ERK pathway, we explored whether the activation of ERK1/2takes part in the regulation of the IFNs-induced genes bythe following experiments. The cells were pretreated withPD98059 (100  l M) for 1 h, then added IFN- a  (10 ng/ml)and IL-29 (100 ng/ml). Results in Fig. 3 showed that theblock of ERK1/2 phosphorylation by PD98059 aloneresulted in about 3 times increase of MxA. MxA increasedapproximately 9–12-fold when treated by PD98059 com-bined with IL-29 or IFN- a , but there was only 2–3-foldincrease of MxA when treated by IL-29 or IFN- a Fig. 1  IL-29 and IFN- a  induced phosphorylation of ERK and AKT.HepG2.2.15 cells were pretreated with PD98059 (100  l M) andLY294002 (50  l M) for 1 h, then added IFN- a  (10 ng/ml) and IL-29(100 ng/ml) for 24 h. Total cellular proteins were extracted and usedto determine phosphorylated ERK and AKT by western blot analysisas described in ‘‘Materials and methods’’ section,  b -ACTIN was usedas control. Results are representative of three separate experiments Fig. 2  Regulation of the antiviral protein mRNA expression inHepG2.2.15 cells by IFN- a  and IL-29 HepG2.2.15 cells were treatedwith IFN- a  (10 ng/ml) and IL-29 (100 ng/ml) for 12 and 24 h. Totalcellular RNA was extracted and used to determine expression of MxA, 2 0 ,5 0 -OAS and PKR mRNA by real-time PCR, GAPDH wasused as control. All data are presented as the mean of threeexperiments. The error bars represent the standard deviation of themean Fig. 3  The effect of PD98059 or LY294002 on the expression of MxA, 2 0 ,5 0 -OAS and PKR HepG2.2.15 cells were pretreated withPD98059 (100  l M) and LY294002 (50  l M) for 1 h, then added IFN- a  (10 ng/ml) and IL-29 (100 ng/ml) for 24 h. Total cellular RNA wasextracted and used to detect expression of MxA, 2 0 ,5 0 -OAS and PKRmRNA by real-time PCR, GAPDH was used as control. Results arerepresentative of five separate experiments. The error bars representthe standard deviation of the meanMol Biol Rep (2011) 38:139–143 141  1 3  separately. These results suggest the activation of ERK1/2may function as a negative regulation factor of the MxAgene expression, but has no significant effect on 2 0 ,5 0 -OASand PKR. Discussion IL-29 is able to inhibit the replication of a broad range of viruses, including human HBV. We demonstrates that IFN- a  can induce the three antivirus proteins (MxA, 2 0 ,5 0 -OASand PKR) expression, and IL-29 increases only mRNAexpression of MxA and 2 0 ,5 0 -OAS, but has no effect onPKR. IL-29 and IFN- a  can up-regulate P-ERK and P-AKTat the same time.As reported, there is close relationship between MxA,2 0 ,5 0 -OAS, PKR genes expression and the JAK-STATsignal pathway [5, 20–23]. JAK activation occurs upon ligand-mediated receptor multimerization. And the acti-vated JAKs subsequently phosphorylate additional targets,including receptors and the major substrates, STATs.Phosphorylated STATs, together with the accessory factorIFN regulatory factor 9 (IRF-9; p48), form the transcriptionfactor complex known as IFN-stimulated gene factor 3(ISGF3) [24]. ISGF3 can recognize the IFN-stimulated response element (ISRE) and the Gamma-IFN activatedsite (GAS) sequences and up-regulates those genes whoseexpression is classically induced by type I IFNs and IL-29[24]. Analysis of the promotor regions of these IFN- induced antivirus proteins has revealed the presence of some regulatory DNA elements that are binding sites fortranscriptional regulators. Furthermore, several regulatoryelements, such as ISRE and GAS sites, have been found intheir promotor regions [25–28]. For these regulatory ele- ments, it is not surprising that the JAK-STAT signalpathway plays an important role in the process of inducingthe expression of these three genes.However, the IFN-induced JAK-STAT signal is notsufficient to explain all the biological effects of IFNs [9]. In our study, we have found that: Firstly, MxA, 2 0 ,5 0 -OAS andPKR are all down-regulated when PI3K-AKT signalpathway is blocked by LY294002, and up-regulated whenPI3K-AKT signal pathway is up-regulated by IL-29 andIFN- a  in HepG2.2.15 cells. These results indicate that theexpression of MxA, 2 0 ,5 0 -OAS and PKR is up-regulated byPI3K-AKT signal pathway. Secondly, MxA is up-regulatedwhen Raf-MEK-ERK signal pathway is blocked byPD98059. Therefore Raf-MEK-ERK signal pathway hasnegative regulatory effect on the expression of MxA inHepG2.2.15 cells. Thirdly, Raf-MEK-ERK signal pathwayhas no significant effort on 2 0 ,5 0 -OAS and PKR. Totallyspeaking, IL-29 and IFN- a  regulate the three genesexpression through a complex system, which may includeseveral intracellular pathways. But the molecular mecha-nism, by which two signal pathways affect the antiviralprotein expression, is not clear. Thus, in order to identifythe regulatory elements of the genes and the transcriptionalfactors involved in the two signal pathways, further studiesare needed.In conclusion, the present studies demonstrate the rela-tionship between these three antiviral proteins and the twosignal pathways in HepG2.2.15 cells for the first time. 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