S

SpR

Da

b

c

a

ARRAA

KSIEEcANAPRM

1

aevgvtcRsS

R

tR

0d

Molecular Immunology 48 (2011) 776–781

Contents lists available at ScienceDirect

Molecular Immunology

journa l homepage: www.e lsev ier .com/ locate /mol imm

hort communication

OCS3 suppresses the expression of IL-4 cytokine by inhibiting thehosphorylation of c-Jun through the ERK signaling pathway in rat mast cell lineBL-2H3

onghyun Kima, Seon-Hee Kimb, Sang-Heon Choc, Kichul Shinc, Sunyoung Kima,∗

School of Biological Sciences, Seoul National University, Seoul, Republic of KoreaViroMed Co. Ltd., Gwanak-gu, Seoul, Republic of KoreaDepartment of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea

r t i c l e i n f o

rticle history:eceived 20 January 2010eceived in revised form 19 October 2010ccepted 6 November 2010vailable online 17 December 2010

eywords:OCS3L-4RK

a b s t r a c t

SOCS3 is well known to negatively regulate various cytokine-mediated signaling responses, but its directrole in the expression of specific cytokines has not been clearly elucidated. To understand the role ofSOCS3 in the expression of IL-4, one of the key Th2 cytokines, RBL-2H3 cells (a rat mast cell line) wereengineered to express SOCS3 constitutively at a high level or at a lower level using shRNA. In RBL-2H3cells stably expressing SOCS3, the RNA and protein levels of IL-4 were significantly decreased, whileit was opposite in RBL-2H3 cells containing shRNA for SOCS3. Overexpression of SOCS3 was found toreduce the level of calcium ionophore-induced phosphorylation of ERK1/2 and c-Jun transcription factor.Consistent with this data, knockdown of SOCS3 increased the level of phosphorylated ERK1 and ERK2.Taken together, SOCS3 appears to play an important role as a negative feedback inhibitor in the expression

RK1/2-JunP-1egative feedbackllergyG102

of IL-4 by inhibiting serine phosphorylation of c-Jun via the ERK signaling pathway.© 2010 Elsevier Ltd. All rights reserved.

BL-2H3ast cell

. Introduction

Suppressor of cytokine signaling (SOCS) molecules act as a neg-tive feedback regulator in the JAK/STAT signaling cascade (Crokert al., 2003; Krebs and Hilton, 2001; Starr et al., 1997). STATs acti-ated by various stimulations induce the transcription of SOCSenes, while such induced SOCS proteins feedback-inhibit the acti-ation of STATs by blocking the activity of JAK or preventinghe interaction of STATs with the intracellular domain of various

ytokine receptors (Croker et al., 2003; Krebs and Hilton, 2001;atthe et al., 2007; Starr et al., 1997). The SOCS protein family con-ists of eight members [SOCS1 to 7 and CIS (cytokine-inducibleH2 domain-containing protein)], which share two conserved

Abbreviations: SOCS3, suppressor of cytokine signaling; shRNA, small hairpinNA; EGFP, enhanced green fluorescent protein.∗ Corresponding author at: Sunyoung Kim, School of Biological Sciences, Labora-

ory of Virology, Building 504, Seoul National University, Gwanak-gu, Seoul 151-742,epublic of Korea. Tel.: +82 2 880 8015; fax: +82 2 875 0907.

E-mail address: sunyoung@snu.ac.kr (S. Kim).

161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.molimm.2010.11.005

sequences, a Src homology 2 (SH2) domain and a unique SOCS box(Krebs and Hilton, 2001). These SOCS proteins play important rolesin the pathogenesis of various diseases (Krebs and Hilton, 2001;Ozaki et al., 2005; Seki et al., 2003; Starr et al., 1997). For example,SOCS3 is highly expressed in peripheral T cells of allergic patients,and its level correlates with the severity of the disease (Seki et al.,2003). However, it still remains to be proved whether the expres-sion of SOCS3 is a cause or a result in the allergic pathogenesis.In transgenic mice constitutively expressing SOCS3, it was shownto mediate the development of Th2 responses by inhibiting Th1responses and deteriorate the allergic symptoms (Ozaki et al., 2005;Seki et al., 2003). In the conditional knockout mouse model, how-ever, SOCS3 was shown to have little effect on the differentiationto the Th1 or Th2 subset (Chen et al., 2006).

We recently found that PG102, a water-soluble extract preparedfrom Actinidia arguta, exerts anti-allergic effects, in part by inhibit-

ing the expression of IL-4 (Kim et al., 2009a,b; Park et al., 2005,2006). The following microarray analysis demonstrated that PG102could highly increase the expression of SOCS3 at both the RNA andprotein levels (unpublished data). SOCS3 is known to inhibit theactivation of various STATs (Canfield et al., 2005; Starr et al., 1997),

mun

wteS

teSr(

2

2

itso(gta

2

tg(awtTGkcvtTf2pf

2

twPt1(4BC((Cpf(

D. Kim et al. / Molecular Im

hile one of the STAT members, namely STAT6, has been showno bind to the IL-4 promoter to increase its gene expression (Curielt al., 1997; Ratthe et al., 2007). It remained unclear as to whetherOCS3 is directly involved in the regulation of IL-4 expression.

In this study, we set out a series of experiments to investigatehe role of SOCS3 in the expression of IL-4. Using cells constitutivelyxpressing SOCS3 or its small hairpin RNA (shRNA), we showed thatOCS3 suppresses the expression of IL-4 by the control of phospho-ylation of c-Jun through the extracellular signal-regulated kinaseERK) signaling cascade.

. Materials and methods

.1. Plasmid

MIN5-SOCS3 plasmid expressing SOCS3 was constructed bynserting SOCS3 cDNA sequence (NM 053563) to MIN5 vec-or (Hahn et al., 2004; Lee et al., 2007). Plasmids encodinghRNA for SOCS3, RshSOCS3, were constructed by cloning theligonucleotide targeting nucleotides 654–692 of SOCS3 sequence5′-CCAGTATGATGCTCCACTT-3′) to pSUPER.retro.puro vector (Oli-oengine, Seattle, WA, USA) (Yang et al., 2007). The oligonucleotideargeting EGFP sequence (5′-GAACGGCATCAAGGTGAAC-3′) waslso cloned into the same vector as a control (Yang et al., 2007).

.2. Cell line, transfection and transduction

RBL-2H3 cell line was purchased from the American Type Cul-ure Collection (CRL-2256; Rockville, MD, USA). RBL-2H3 cells wererown in MEM (Sigma, St. Louis, MO, USA) containing 15% (v/v) FBSGibco, GrandIsland, NY, USA) at 37 ◦C under 5% CO2. To establishcell line expressing SOCS3, MIN5-SOCS3 plasmid was linearizedith ScaI (Takara Bio, Shiga, Japan) and 10 �g of the plasmid was

ransfected into 5 × 106 cells by electroporation (950 �F, 250 V).ransfected cells were cultured in the presence of G418 (1 mg/ml;ibco, GrandIsland, NY, USA) for 2 weeks. To prepare a SOCS3nockdown cell line, RshSOCS3 plasmid was transfected into 293Tells with pVM-gp and pVM-VSVG expressing the murine leukemiairus gag-pol and the vesicular stomatitis virus G protein, respec-ively, as described previously (Hahn et al., 2004; Kim et al., 1999).he resulting viral supernatants were collected, 48 h after trans-ection, filtered through a 0.45-�m filter, and used to transduce at× 105 cells on a 60 mm dish. Transduced cells were selected in theresence of puromycin (10 �g/ml; Calbiochem, San Diego, CA, USA)or 2 weeks.

.3. Western blot hybridization

Cells were plated at 2 × 106 cells in the 60 mm plate and cul-ured in the 2 ml complete culture media for 6 h. After treatmentith 1 �M of A23187 (Sigma), total cell lysates were prepared using

hosphosafe lysis buffer (Novagen, San Diego, CA, USA) at variousime points. Protein samples were subjected to SDS-PAGE on an1% polyacrylamide gel and transferred onto a PVDF membraneMillipore, Bedford, MA, USA). The membrane was incubated at◦C for overnight with antibodies specific for SOCS3 (Santa Cruziotechnology, Santa cruz, CA, USA), ERK1/2 (BD Sciences, San Jose,A, USA), phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA), c-JunSanta Cruz Biotechnology), phospho-c-Jun (Cell Signaling), �-actin

Sigma), or GAPDH (Advanced ImmunoChemical Inc., Long Beach,A, USA), then bound at room temperature for 1 h with horseradisheroxidase-conjugated anti-rabbit or anti-mouse IgG (Pierce, Rock-ord, IL, USA), and developed using chemiluminescence techniquesECL kit: Millipore).

ology 48 (2011) 776–781 777

2.4. Northern blot hybridization

Cells were seeded at 2 × 106 cells in the 60 mm plate, and incu-bated for 6 h, and then stimulated with 1 �M A23187. Three hourslater, total RNAs were prepared using Trizol (Invitrogen, Carlsbad,CA, USA), according to the manufacturer’s instruction. The DNAprobe specific for SOCS3 or IL-4 was used for determining its RNAlevel. As a RNA loading control, the same membrane was hybridizedwith a probe for GAPDH.

2.5. Real-time quantitative PCR

cDNAs were synthesized by using 500 ng of each total RNA,oligo(dt)12–18 primer, and Avian Myeloblastosis Virus reversetranscriptase (Roche Applied Science, Mannheim, Germany). Theresulting single-stranded cDNA was then used for a real-timeQPCR with specific primer sets and Power SYBR® Green PCRmaster Mix (Applied Biosciences, Warrington, UK) accordingto the manufacturer’s instruction by using the Thermal CyclerDice Real Time System (Takara Bio). PCR primer sets weresynthesized (COSMO Co. Ltd., Seoul, Korea) as follows: SOCS3(5′-TGCAGGAGAGCGGATTCT-3′; 5′-GTCACTCTGCAGCGAAAAGC-3′), IL-4 (5′-GTATCCACGGATGTAACGACAGC-3′;5′-AGGACTGCAAGTATTTCCCTCGTA-3′), and GAPDH (5′-AGCCTCGTCCCGTAGACAA-3′; 5′-AATCTCCACTTTGCCACTGC-3′).The PCR conditions were 95 ◦C for 10 min, followed by 45 cycleswith denaturation at 95 ◦C for 15 s and annealing and extensionat 60 ◦C for 1 min. Cycle threshold (Ct) of respective samples wasnormalized using the average Ct value of GAPDH. The differencein mRNA levels between SOCS3-expressing and control cell lineswas calculated from �Ct value as described previously (Kim et al.,2009a; Martell et al., 1999).

2.6. Cytokine assays

RBL-2H3 cells were stimulated with A23187 or IgE. For A23187stimulation, cells were plated at 2 × 105 cells/well in the 24-wellplate, and grown in 500 �l culture media for 6 h. The cells were incu-bated in the absence or the presence of 1 �M A23187 for 12 h, andthen the culture supernatant were taken. For IgE stimulation, cellswere dispensed into the 24-well plate at 2 × 105 cells/well in cul-ture media containing 50 ng/ml of anti-DNP mouse monoclonal IgE(Sigma), and incubated overnight at 37 ◦C under 5% CO2. They werethen washed with media, and treated with 100 ng/ml of DNP-BSA(Calbiochem). After 12 h, the culture supernatants were collected.The level of IL-4 or TNF-� was measured using the supernatants byELISA (R&D systems, Minneapolis, MN, USA; eBioscience Inc., SanDiego, CA, USA) according to the manufacturer’s instruction.

3. Results and discussion

3.1. Effect of constitutive expression of SOCS3 on the level of IL-4

To examine whether SOCS3 could affect the production of IL-4cytokine, a SOCS3 expression vector was constructed by insertingits cDNA sequence into MIN5 plasmid (Lee et al., 2007), resulting inMIN5-SOCS3 which produced the bacterial neo gene as a bicistronicmessage. MIN5-SOCS3 plasmid was linearized and transfected toRBL-2H3 cells, a rat mast cell line, by electroporation. Transfectedcells were cultured in the presence of G418 for 14 days, and the

drug-resistant population was obtained. A control cell line was alsoestablished, using MIN5 plasmid lacking the SOCS3 sequence. Thesecells were treated with calcium ionophore, A23187, and then totalcell lysates and culture supernatants were analysed by Western blotand ELISA to determine the level of SOCS3 and IL-4, respectively.

778 D. Kim et al. / Molecular Immunology 48 (2011) 776–781

Fig. 1. Effect of SOCS3 overexpression on IL-4 in RBL-2H3 cells. RBL-2H3 cells constitutively expressing SOCS3 were constructed as described in Section 2. (A) Cells werecultured in the presence or absence of 1 �M A23187, and total proteins were prepared after 9 h followed by Western blot hybridization. The �-actin protein was used as aloading control. The level of IL-4 (B and C) and TNF-� (D) was measured by ELISA using the culture supernatants after 12 h of treatment with A23187 or anti-DNP IgE andD tripli3 sed as d werc

2ta2tildicwcw

NP-BSA. Each value represents means ± SD measured in the duplicate (A23187) orh of stimulation with A23187 by Northern blot hybridization. GAPDH RNA was u

amples. *The RNA levels of SOCS3 and IL-4 were normalized with that of GAPDH anells. ND, not detectable.

The results from Western blot hybridization showed that RBL-H3 cells did not normally produce the SOCS3 protein, but thereatment with A23187 increased its level (Fig. 1A; compare lanes 1nd 3). As expected, a large amount of SOCS3 was expressed in RBL-H3 cells transfected with MIN5-SOCS3 plasmid, even before thereatment with A23187 (Fig. 1A; lane 2). The presence of calciumonophore did not significantly affect its level (Fig. 1A; compareanes 2 and 4). The result from ELISA showed that IL-4 was notetectable in normal RBL-2H3 cells, but treatment with A23187

ncreased the IL-4 level to 245 pg/ml (Fig. 1B). In RBL-2H3 cellsonstitutively expressing SOCS3, A23187-mediated increase of IL-4as decreased by approximately 2-fold, as compared with control

ells containing MIN5 (Fig. 1B). By contrast, when the level of TNF-�as measured by ELISA using the same supernatant, there was lit-

cate (IgE) microplate wells. (E) The RNA level of SOCS3 and IL-4 was analysed afters a loading control. (F) Real-time quantitative PCR was performed with same RNAe then expressed as a relative value to the RNA level in A23187-stimulated control

tle difference between SOCS3-overexpression and control cell lines(Fig. 1D). These data showed that the SOCS3 selectively decreasedthe production of IL-4 in RBL-2H3 cells.

To mimic a more physiological stimulation, RBL-2H3 cells weresensitized with anti-DNP IgE (antibody) overnight, and then chal-lenged with DNP-BSA (antigen) for 12 h. IgE-mediated stimulationinduced the production of IL-4 to 49.0 pg/ml in control cells contain-ing MIN5, but the constitutive expression of SOCS3 decreased thelevel of this cytokine by 3-fold (Fig. 1C). This result indicated that

the IL-4 inhibitory activity of SOCS3 was not limited to a specificstimulator.

To test at what level SOCS3 inhibits the IL-4 production, totalRNAs were prepared from various RBL-2H3 cells followed by North-ern hybridization and real-time quantitative PCR assay. RBL-2H3

D. Kim et al. / Molecular Immunology 48 (2011) 776–781 779

Fig. 2. Effect of SOCS3 knockdown on IL-4 in RBL-2H3 cells. RBL-2H3 cells expressing shRNA for SOCS3 were constructed as described in Section 2. The control cell lineh e presW he RNN el of ILN

cSceiccl

3

2rsid(tCttdtca2clsi

3

pMdK2

aving shRNA for EGFP was used as a negative control. (A) Cells were cultured in thestern blot hybridization. The �-actin protein was used as a loading control. (B) T

orthern blot hybridization. GAPDH RNA was used as a loading control. (C) The levD, Not detectable.

ells transfected with MIN5-SOCS3 plasmid produced a high level ofOCS3 RNA, regardless of the treatment with A23187 (Fig. 1E and F;ompare lane 1 with 2, and lane 3 with 4 in Fig. 1E and F). The stablexpression of SOCS3 resulted in the reduction of A23187-mediatedncreases of IL-4 RNA by approximately 3-fold, as compared withontrol cells (Fig. 1E and F; compare lanes 3 and 4). These data indi-ated that the SOCS3 suppressed the expression of IL-4 at the RNAevel.

.2. Effect of SOCS3 shRNA on the level of IL-4

To be certain that SOCS3 is a negative regulator of IL-4, a RBL-H3 cell line lacking in SOCS3 was established using shRNA. Aetroviral vector expressing shRNA specific for SOCS3 was con-tructed using pSUPER.retro.puro (Yang et al., 2007), resultingn RshSOCS3. As a control, another retroviral vector, RshEGFPesigned to produce shRNA for enhanced green fluorescent proteinEGFP), was used (Yang et al., 2007). In these retroviral constructs,he puromycin resistance gene was used as a selectable marker.ell-free retroviral vectors were prepared by the three-plasmidransfection method (Hahn et al., 2004; Kim et al., 1999), and usedo transduce RBL-2H3 cells followed by puromycin selection for 14ays. In RBL-2H3 cells transduced with retroviral vector RshSOCS3,he level of SOCS3 protein and RNA was lowered significantly, asompared with control cells having RshEGFP (Fig. 2A for proteinnd Fig. 2B for RNA; lanes 1 and 2 or lanes 3 and 4). In these RBL-H3 cells, the RNA level of IL-4 was significantly increased (Fig. 2B;ompare lanes 3 and 4). Consistent with this RNA data, the proteinevel of IL-4 in the culture supernatant was also increased, as mea-ured by ELISA (Fig. 2C). These data confirmed that SOCS3 couldndeed negatively regulate the expression of IL-4.

.3. Effect of SOCS3 on MAP kinase signaling pathway

It was previously reported that three MAP (mitogen-activated

rotein) kinase signaling pathways, namely, those of ERK1/2, p38APK, and JNK (c-Jun NH2-terminal kinase), play roles in the pro-

uction of IL-4 (Gibbs and Grabbe, 1999; Hirasawa et al., 2000;oranteng et al., 2004; So et al., 2007; Walczak-Drzewiecka et al.,005). To identify the pathway responsible for SOCS3-mediated

ence or absence of A23187, and total proteins were prepared after 9 h followed byA level of SOCS3 and IL-4 was measured, 3 h after treatment with 1 �M A23187, by-4 was measured using the culture supernatant after 12 h of stimulation by ELISA.

control of IL-4, the status of various MAPKs were studied in RBL-2H3 cells expressing SOCS3 at a high level or at a lower level. Cellswere stimulated by A23187 for the periods indicated in Fig. 3A andB, and then the level of total or phosphorylated MAPKs was mea-sured by Western blot using specific antibodies. The amount of totalERK1 and ERK2 was not affected by the level of SOCS3 as well as bythe treatment with A23187 (Fig. 3A and B). However, the level ofphophorylated ERK1 and ERK2 was sharply increased from 60 minafter the treatment by A23187 (Fig. 3A and B; compare lanes 1, 3, 5,7, 9, and 11) and it was affected by the level of SOCS3. In RBL-2H3cells constitutively expressing SOCS3, the level of phosphorylatedERK1 and ERK2 was significantly lowered, as compared with that incontrol cells at respective time points (Fig. 3A; compare lanes 7 and8, 9 and 10, and 11 and 12). However, when the level of SOCS3 wasdecreased by shRNA for SOCS3, the phosphorylation of ERK1 andERK2 was increased by contraries (for example, compare lanes 11and 12 in Fig. 3A and B). We also investigated the effects of SOCS3 onthe phosphorylation of p38 and JNK, but the phosphorylation statusof these proteins was not changed in RBL-2H3 cells constitutivelyexpressing SOCS3 (data not shown). Therefore, the phosphoryla-tion status of ERK1 and ERK2 appeared to be negatively regulatedby SOCS3 mainly using the ERK signaling pathway.

3.4. Effect of SOCS3 on phosphorylation of c-Jun

AP-1 is one of the most downstream molecules in the MAPKsignaling cascade, and acts as one of the key positive transcrip-tion factors for the expression of IL-4 (Karin, 1995; Li-Weber et al.,1997, 1998; Lorentz et al., 2003). The phosphorylation of c-Jun,a subunit of AP-1, at the position of Ser-73 and Ser-63, has beenshown to be important in its function as either a homodimer or aheterodimer with c-Fos (Karin, 1995). To determine the effect ofSOCS3 on the phosphorylation status of c-Jun, RBL-2H3 cells con-stitutively expressing SOCS3 were analysed by Western blot usingspecific antibodies that can detect phosphorylated c-Jun at the posi-

tion Ser-63 or Ser-73. The amount of total c-Jun was somewhatincreased in RBL-2H3 cells stably expressing SOCS3 (Fig. 3C; lanes3 and 4). However, the level of phosphorylated c-Jun was loweredin SOCS3-expressing cells, as compared with that in control cells(Fig. 3C; compare lanes 3 and 4). These results indicated that SOCS3

780 D. Kim et al. / Molecular Immun

Fig. 3. Effect of SOCS3 on ERKs and c-Jun in RBL-2H3 cells. Cells were treated with1 �M A23187, and total proteins were prepared at various time points, followed byWestern blot. The level of total or phosphorylated ERK1 and ERK2 was detected inRBL-2H3 cells expressing SOCS3 constitutively at a high level (A) or at a lower levelrrbc

cs

crctdmswv2itv

omFavatit

esulting from the use of shRNA for SOCS3 (B). (C) The level of total and phospho-ylated c-Jun was analysed in SOCS3-overexpressing cells after 2 h of stimulationy Western blot hybridization. �-actin and GAPDH proteins were used as a loadingontrol.

ould downregulate the expression of IL-4 cytokine by inhibitingerine phosphorylation of c-Jun via the ERK signaling pathway.

At present, the relationship between IL-4 and SOCS3 is highlyontroversial. Some investigators reported that SOCS3 is a positiveegulator of IL-4 (Ozaki et al., 2005; Seki et al., 2003), while otherslaimed the opposite (Nakaya et al., 2009). There was even a claimhat SOCS3 has no effect on IL-4 expression (Chen et al., 2006). Theseiscrepancies might have resulted from the use of different experi-ental systems, for example, different cell types, animal models or

timulators used to activate the relevant pathways. In this study,e manipulated the expression level of IL-4 by using a retroviral

ector, and tested its effects in a well known rat mast cell line, RBL-H3, because mast cells are one of key cell types in allergic diseases

nvolving IL-4. Our results indicated that SOCS3 negatively controlshe production of IL-4 by inhibiting serine phosphorylation of c-Junia the ERK pathway.

The role of SOCS3 is reported to be two extreme sides, dependingn experimental systems. Consequently, the proposed underlyingolecular mechanism also varies among different investigators.

or example, in Cacalano et al’s work, SOCS3 was reported to acts a positive regulator in the ERK signaling cascade turned on by

arious cytokines and growth factors, including EPO, IL-2, IL-3, EGFnd PDGF. In this case, tyrosine phosphorylated SOCS3 appearedo have been needed to maintain the activation of ERK by bind-ng to Ras inhibitor, p120 RasGAP (Cacalano et al., 2001). However,ogether with data from other reports (Ma et al., 2007; Riehle et al.,

ology 48 (2011) 776–781

2008; Woolson et al., 2009), our results indicated that the decreasedlevel of SOCS3, regardless of its phosphorylation status, resulted inthe increase in ERK1/2 phosphorylation, implying that tyrosine-phosphorylated SOCS3 might not interact with RasGAP. Consistentwith these data, a bulk of data showed that the inhibition of ERKactivation by SOCS3 led to the downregulation of IL-4 expression(Gibbs and Grabbe, 1999; Koranteng et al., 2004; So et al., 2007).

IL-4 is one of the key players involved in the differentiation ofTh1 and Th2 pathways and allergy pathogenesis. Any factors affect-ing the expression of IL-4 are destined to influence the immunebalance and subsequently the initiation and progression of relateddiseases. Given such importance of IL-4, further extensive inves-tigations are warranted to clarify the relationship between thiscytokine and SOCS3.

Acknowledgements

This study was funded by grants from the Plant DiversityResearch Center of 21C Frontier R&D Programs (Ministry of Edu-cation, Science and Technology; PF03211-00), the Korean Health21 R&D Project (Ministry of Health, Welfare and Family Affairs;A050440 and A060655) and the SRC program of KOSEF (R11-2005-009-06003-0).

References

Cacalano, N.A., Sanden, D., Johnston, J.A., 2001. Tyrosine-phosphorylated SOCS-3inhibits STAT activation but binds to p120 RasGAP and activates Ras. Nat. CellBiol. 3, 460–465.

Canfield, S., Lee, Y., Schroder, A., Rothman, P., 2005. Cutting edge: IL-4 induces sup-pressor of cytokine signaling-3 expression in B cells by a mechanism dependenton activation of p38 MAPK. J. Immunol. 174, 2494–2498.

Chen, Z., Laurence, A., Kanno, Y., Pacher-Zavisin, M., Zhu, B.M., Tato, C., Yoshimura, A.,Hennighausen, L., O’Shea, J.J., 2006. Selective regulatory function of Socs3 in theformation of IL-17-secreting T cells. Proc. Natl. Acad. Sci. U.S.A. 103, 8137–8142.

Croker, B.A., Krebs, D.L., Zhang, J.G., Wormald, S., Willson, T.A., Stanley, E.G., Robb,L., Greenhalgh, C.J., Forster, I., Clausen, B.E., Nicola, N.A., Metcalf, D., Hilton, D.J.,Roberts, A.W., Alexander, W.S., 2003. SOCS3 negatively regulates IL-6 signalingin vivo. Nat. Immunol. 4, 540–545.

Curiel, R.E., Lahesmaa, R., Subleski, J., Cippitelli, M., Kirken, R.A., Young, H.A., Ghosh,P., 1997. Identification of a Stat-6-responsive element in the promoter of thehuman interleukin-4 gene. Eur. J. Immunol. 27, 1982–1987.

Gibbs, B., Grabbe, J., 1999. Inhibitors of PI 3-kinase and MEK kinase differentiallyaffect mediator secretion from immunologically activated human basophils. J.Leukoc. Biol. 65, 883–890.

Hahn, W., Ho, S.H., Jeong, J.G., Hahn, E.Y., Kim, S., Yu, S.S., Kim, J.M., 2004. Viralvector-mediated transduction of a modified thrombospondin-2 cDNA inhibitstumor growth and angiogenesis. Gene Ther. 11, 739–745.

Hirasawa, N., Sato, Y., Fujita, Y., Ohuchi, K., 2000. Involvement of a phosphatidylinos-itol 3-kinase-p38 mitogen activated protein kinase pathway in antigen-inducedIL-4 production in mast cells. Biochim. Biophys. Acta 1456, 45–55.

Karin, M., 1995. The regulation of AP-1 activity by mitogen-activated protein kinases.J. Biol. Chem. 270, 16483–16486.

Kim, D., Kim, S.H., Park, E.J., Kang, C.Y., Cho, S.H., Kim, S., 2009a. Anti-allergic effectsof PG102, a water-soluble extract prepared from Actinidia arguta, in a murineovalbumin-induced asthma model. Clin. Exp. Allergy 39, 280–289.

Kim, D., Kim, S.H., Park, E.J., Kim, J., Cho, S.H., Kagawa, J., Arai, N., Jun, K., Kiyono, H.,Kim, S., 2009b. Suppression of allergic diarrhea in murine ovalbumin-inducedallergic diarrhea model by PG102, a water-soluble extract prepared from Actini-dia arguta. Int. Arch. Allergy Immunol. 150, 164–171.

Kim, S., Yu, S.S., Lee, I.S., Ohno, S., Yim, J., Kang, H.S., 1999. Human cytomegalovirusIE1 protein activates AP-1 through a cellular protein kinase(s). J. Gen. Virol. 80(Pt 4), 961–969.

Koranteng, R.D., Swindle, E.J., Davis, B.J., Dearman, R.J., Kimber, I., Flanagan, B.F.,Coleman, J.W., 2004. Differential regulation of mast cell cytokines by both dex-amethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitorSB203580. Clin. Exp. Immunol. 137, 81–87.

Krebs, D.L., Hilton, D.J., 2001. SOCS Proteins: negative regulators of cytokine signal-ing. Stem Cells 19, 378–387.

Lee, J.T., Yu, S.S., Kim, V.N., Kim, S., 2007. Control of splicing efficiency by the mousehistone H2a element in a murine leukemia virus-based retroviral vector. Mol.Ther. 15, 167–172.

Li-Weber, M., Giasi, M., Krammer, P.H., 1998. Involvement of Jun and Rel proteinsin up-regulation of interleukin-4 gene activity by the T cell accessory moleculeCD28. J. Biol. Chem. 273, 32460–32466.

Li-Weber, M., Salgame, P., Hu, C., Davydov, I., Krammer, P., 1997. Differential inter-action of nuclear factors with the PRE-I enhancer element of the human IL-4promoter in different T cell subsets. J. Immunol. 158, 1194–1200.

mun

L

M

M

N

O

P

P

D. Kim et al. / Molecular Im

orentz, A., Klopp, I., Gebhardt, T., Manns, M.P., Bischoff, S.C., 2003. Role of activatorprotein 1, nuclear factor-kappaB, and nuclear factor of activated T cells in IgEreceptor-mediated cytokine expression in mature human mast cells. J. AllergyClin. Immunol. 111, 1062–1068.

a, Z., Gong, Y., Patel, V., Karner, C.M., Fischer, E., Hiesberger, T., Carroll, T.J.,Pontoglio, M., Igarashi, P., 2007. Mutations of HNF-1� inhibit epithelial mor-phogenesis through dysregulation of SOCS-3. Proc. Natl. Acad. Sci. U.S.A. 104,20386–20391.

artell, M., Gomez, J., Esteban, J.I., Sauleda, S., Quer, J., Cabot, B., Esteban, R., Guardia,J., 1999. High-throughput real-time reverse transcription-PCR quantitation ofhepatitis C virus RNA. J. Clin. Microbiol. 37, 327–332.

akaya, M., Hashimoto, M., Nakagawa, R., Wakabayashi, Y., Ishizaki, T., Takada, I.,Komai, K., Yoshida, H., Yoshimura, A., 2009. SOCS3 in T and NKT cells nega-tively regulates Cytokine production and ameliorates ConA-induced hepatitis.J. Immunol. 183, 7047–7053.

zaki, A., Seki, Y., Fukushima, A., Kubo, M., 2005. The control of allergic conjunctivitisby suppressor of cytokine signaling (SOCS)3 and SOCS5 in a murine model. J.Immunol. 175, 5489–5497.

ark, E.-J., Kim, B., Eo, H., Park, K., Kim, Y., Lee, H.J., Son, M., Chang, Y.-S., Cho, S.-H.,

Kim, S., Jin, M., 2005. Control of IgE and selective TH1 and TH2 cytokines byPG102 isolated from Actinidia arguta. J. Allergy Clin. Immunol. 116, 1151–1157.

ark, E.-J., Park, K.C., Eo, H., Seo, J., Son, M., Kim, K.H., Chang, Y.-S., Cho, S.-H., Min,K.-U., Jin, M., Kim, S., 2006. Suppression of spontaneous dermatitis in NC/Ngamurine model by PG102 isolated from Actinidia arguta. J. Invest. Dermatol. 127,1154–1160.

ology 48 (2011) 776–781 781

Ratthe, C., Pelletier, M., Chiasson, S., Girard, D., 2007. Molecular mechanismsinvolved in interleukin-4-induced human neutrophils: expression and regula-tion of suppressor of cytokine signaling. J. Leukoc. Biol. 81, 1287–1296.

Riehle, K.J., Campbell, J.S., McMahan, R.S., Johnson, M.M., Beyer, R.P., Bammler, T.K.,Fausto, N., 2008. Regulation of liver regeneration and hepatocarcinogenesis bysuppressor of cytokine signaling 3. J. Exp. Med. 205, 91–103.

Seki, Y., Inoue, H., Nagata, N., Hayashi, K., Fukuyama, S., Matsumoto, K., Komine, O.,Hamano, S., Himeno, K., Inagaki-Ohara, K., Cacalano, N., O’Garra, A., Oshida, T.,Saito, H., Johnston, J.A., Yoshimura, A., Kubo, M., 2003. SOCS-3 regulates onsetand maintenance of T(H)2-mediated allergic responses. Nat. Med. 9, 1047–1054.

So, E.-Y., Oh, J., Jang, J.-Y., Kim, J.-H., Lee, C.-E., 2007. Ras/Erk pathway positivelyregulates Jak1/STAT6 activity and IL-4 gene expression in Jurkat T cells. Mol.Immunol. 44, 3416–3426.

Starr, R., Willson, T.A., Viney, E.M., Murray, L.J.L., Rayner, J.R., Jenkins, B.J., Gonda, T.J.,Alexander, W.S., Metcalf, D., Nicola, N.A., Hilton, D.J., 1997. A family of cytokine-inducible inhibitors of signalling. Nature 387, 917–921.

Walczak-Drzewiecka, A., Wyczolkowska, J., Dastych, J., 2005. c-Jun N-terminalkinase is involved in mercuric ions-mediated interleukin-4 secretion in mastcells. Int. Arch. Allergy Immunol. 136, 181–190.

Woolson, H.D., Thomson, V.S., Rutherford, C., Yarwood, S.J., Palmer, T.M., 2009. Selec-tive inhibition of cytokine-activated extracellular signal-regulated kinase bycyclic AMP via Epac1-dependent induction of suppressor of cytokine signalling-3. Cell Signal. 21, 1706–1715.

Yang, G., Xu, Y., Chen, X., Hu, G., 2007. IFITM1 plays an essential role in the antipro-liferative action of interferon-gamma. Oncogene 26, 594–603.