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Thymic stromal lymphopoietin promotes the proliferation of human trophoblasts via phosphorylated STAT3-mediated c-Myc upregulation
Placenta 33 (2012) 387e391
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Thymic stromal lymphopoietin promotes the proliferation of human trophoblasts via phosphorylated STAT3-mediated c-Myc upregulation H.-H. Pu a, b, J. Duan a, Y. Wang a, D.-X. Fan a, D.-J. Li a, *, L.-P. Jin a, * a
Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, 413 Zhaozhou Road, Shanghai 200011, China b Department of Obstetrics & Gynecology, Xuhui Central Hospital of Shanghai, Shanghai 200031, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 17 January 2012
Our previous study has demonstrated that thymic stromal lymphopoietin (TSLP) stimulates trophoblast proliferation and invasion, suggesting TSLP plays an important role in the placentation in early human pregnancy, but the intracellular molecular mechanism is currently unknown. The present study is undertaken to investigate whether the STAT3-c-Myc signaling pathway is involved in TSLP-mediated trophoblast proliferation. Primary human first-trimester trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The levels of STAT3 tyrosine phosphorylation and c-Myc expression were determined by using Western blot analysis, and the proliferation of trophoblasts was analyzed by BrdU cell proliferation assay. JEG-3 cells were transfected with siRNA targeting to c-Myc, and the proliferation was determined in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc silencing. It was revealed that treatment with TSLP significantly enhanced STAT3 phosphorylation and c-Myc expression in human trophoblasts. The effect of TSLP upregulation on trophoblast proliferation was abrogated completely by either STAT3 inhibitor or c-Myc siRNA silence. We further found that the upregulation of TSLP on c-Myc expression was abrogated completely by the STAT3 inhibitor, which suggests that the intracellular STAT3 phosphorylation is an upstream signal of c-Myc expression in the TSLP-stimulated trophoblast proliferation. These results suggest that TSLP may upregulate c-Myc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: TSLP c-Myc STAT3 pathway Trophoblast Proliferation
1. Introduction As a critical cell player in the human placenta, fetal cytotrophoblasts play an important role in successful pregnancy. Their proliferation and differentiation as well as invasion during implantation occur through a series of tightly controlled processes of intercellular signaling mediated by growth factors, cytokines and hormones [1]. It involves extensive cross-talk between the trophoblast cells and the receptive endometrium. Lack of any crucial embryonic or maternal signal at the site of implantation may result in a shallow or failure of embryo implantation. The c-Myc oncogene is the prototypical member of the Myc/ Mad/ Max transcription factor network, which regulates divergent cellular functions such as proliferation, differentiation, and
Abbreviations: TSLP, thymic stromal lymphopoietin; STAT, signal transducer and activator of transcription; DCs, dendritic cells; BrdU, Bromo deoxy Uridine. * Corresponding authors. Tel.: þ86 21 63455050 8288; fax: þ86 21 63457331. E-mail addresses: [email protected] (D.-J. Li), [email protected] (L.-P. Jin). 0143-4004/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2012.01.015
apoptosis. In mammalian cells, Myc expression is rapidly induced following mitogenic or cytokine stimulation. One of its more obvious biological effects is to drive cellular proliferation, which is sufficient to allow quiescent cells to reenter the cell cycle [2]. In an effort to further dissect the molecular pathways regulated by Myc, several genome-wide screens have been used to identify transcriptional targets of Myc. Several studies have revealed that expression of up to 10e15% of all genes may be affected by c-Myc, and one of the more consistent categories of genes up-regulated by Myc is cell growth and protein synthesis [3,4]. Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers the dendritic cell-mediated Th2 response and regulatory T cell expansion. TSLP signals via TSLP receptor expressed on a wide range of cell types in the adaptive and innate immune system. TSLP exerts profound influence on the polarization of dendritic cells (DCs) to drive T helper (Th) 2 cytokine production, and mediates allergic inflammation such as asthma and atopic dermatitis [5e7]. It has been reported that TSLP-activated myeloid DCs might be critical in the positive selection of medium-to-high-affinity selfreactive thymocytes into the regulatory T cell (Treg cell) lineage [8].
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Both human and mouse TSLPeTSLPR interactions activate similar signaling pathways. TSLP activates the transcription factor signal transducer and activator of transcription 3 (STAT3) in human and STAT5 in mouse and human [9,10]. Our previous studies have shown that human first-trimester trophoblasts secrete soluble TSLP and express the functional TSLP receptor complex TSLP receptor and IL-7 receptor-a, which may render trophoblasts a target for autocrine and paracrine regulation by the locally produced TSLP. We have also demonstrated that TSLP is in fact an effective inducer to proliferation and invasion of human first-trimester trophoblasts, which indicates that the TSLPeTSLPR interaction plays an important role in the development of the human placenta [11]. However, the mechanism by which TSLP promotes the proliferation of human trophoblasts is currently unknown. 2. Methods 2.1. Isolation and primary culture of human first-trimester trophoblast First-trimester human villous tissues were obtained from placentas of 5 clinically normal pregnancies [age: 28.20 þ 3.38 years, gestational age at sampling 8.69 þ 1.56 weeks (mean S.D.)], which were terminated for nonmedical reasons. Each subject completed a signed, written consent form approved by Human Investigation Committee in Hospital of Obstetrics & Gynecology, Fudan University. Villous tissues obtained from patients enrolled in the study were digested by four 10 min incubations with 0.25% trypsin and 1500 IU/ml of DNase type I (AppliChem GmbH, Germany) at 37 C with gentle agitation. The digested cell suspensions were pooled and carefully layered over a discontinuous Percoll gradient (65e20%, graduated in 5% steps) and centrifuged at 1000 g for 15 min. The middle layer (density 1.042e1.068 g/ml) was recovered and washed with DMEM-highglucose medium. The cells were seeded on matrigel-precoated cell culture plates at 3 105 cells/well in DMEM-high-glucose complete medium (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 2 mM glutamine, 25 mM HEPES, 100 U/ml of penicillin, 100 mg/ml of streptomycin and 20% fetal bovine serum (FBS) and incubated in 5% CO2 at 37 C for 12 h, and then the medium was renewed by DMEMhigh-glucose complete medium supplemented with 1% FBS. After further cultivation for 12 h, the cells were stimulated with recombinant human TSLP. We analyzed the purity of the primary trophoblasts by immunocytochemistry. Trophoblasts are cytokeratin 7 positive, and vimentin negative. The cell purity of the primary trophoblasts was above 90% (data not shown). 2.2. Cell lines and reagents The human choriocarcinoma cell line JEG-3 obtained from the American Type Culture Collection (Manassas, VA, USA), were routinely grown in DMEM culture solution (GIBCO BRL, Gaithersburg, MD, USA) containing 10% FBS in 5% CO2 at 37 C. 2.3. TSLP treatment Recombinant human TSLP was purchased from PeproTech (Rocky Hill, UK). Primary human trophoblasts were treated with 400 ng/ml TSLP for 48 h. Then the level of c-Myc protein in primary human trophoblasts was detected by Western blot analysis. Primary human trophoblasts were treated with 400 ng/ml TSLP for 0, 5, 15, 30 and 60 min and subsequently analyzed for tyrosine phosphorylation of STAT3 by Western blot analysis. 2.4. Treatment with STAT3, STAT5, AKT or ERK pathway inhibitor Primary human trophoblasts were cultured as described above, and 20 mmol/l AKT pathway inhibitor LY294002, 20 mmol/l ERK pathway inhibitor U0126 (both from Calbiochem, CA), 40 mmol/l STAT3 pathway inhibitor D4071 or 20 mmol/l STAT5 pathway inhibitor M1323 (both from Sigma, St. Louis, USA) were added to the culture supernatants. After 30 min, 400 ng/ml TSLP was added to the cells, and PBS was used as a control. The proteins were extracted from the cells 30 min later for STAT3 phosphorylation assay, or 48 h later for c-Myc protein assay and BrdU cell proliferation assay. 2.5. c-Myc siRNA transfection c-Myc siRNA was procured from Invitrogen (Carlsbad, CA). The siRNA sequence targeting c-Myc is 50 -CAGTTGCCACTTCCACATA-30 . A random siRNA (50 ACTACCGTTGTTATAGGTG-30 ) which does not have any target region in human genes served as negative control. c-Myc siRNA transfection was performed according to the manufacturer’s instructions. One day before transfection, plate JEG-3 cells in 500 ml of growth medium without antibiotics such that they will be 30e50% confluent at the time of transfection. The siRNA oligonucleotides targeting c-Myc and
Lipofectamine 2000 were mixed in OPTIMEM and then added to the cells at room temperature, with nontargeting siRNA oligonucleotides as control. After 6 h incubation, the cells were incubated in DMEM for a further 72 h in 5% CO2 at 37 C, and the protein expression of c-Myc in JEG-3 cells was determinated by Western blot analysis. 2.6. Western blot analysis Cells were lysed in RIPA lysis buffer containing proteinase inhibitor for 30 min. The samples were boiled for 10 min, centrifuged, and the supernatants collected. Each sample (25 mg) was loaded on a 10% SDS-PAGE gel. After electrophoresis, the proteins were transferred onto a polyvinyl difluoride membrane (Bio-Rad), and the membrane incubated in blocking buffer containing 5% non-fat dry milk for 1 h at room temperature. Then the membrane was probed with a primary antibody in blocking buffer [1 mg/ml anti-c-Myc mouse mAb (Santa Cruz Biotechnology Inc.; St. Louis, MO) and 0.1 mg/ml anti-GAPDH mouse mAb (Sigma) as the internal marker] overnight at 4 C, followed by probing with anti-mouse Ig-HRP conjugates (Amersham Biosciences; Buckinghamshire, UK) in blocking buffer for 2 h at 37 C. The signals were recorded on HyperFilm MP (Amersham Pharmacia Biotech) and developed in a Kodak X-Omat film developer. Results were scanned and densitometrically analyzed using Scion Image software (Scion Inc). 2.7. BrdU cell proliferation assay After different treatment described above, 20 ml of Bromo deoxy Uridine (BrdU) reagent (Sigma) was added to each well and incubated for 12 h 37 C. The media was aspirated and the adherent trophoblasts were incubated with the Fixing Solution (200 mL/well). After 30 min, anti-BrdU monoclonal antibody (100 mL/well) was added and the incubation was continued for an additional hour at room temperature. After addition of goat anti-mouse IgG-peroxidase conjugated secondary antibody, substrate and stop solution, the amount of BrdU was determined by reading the sample at dual wavelength of 450/550 nm using a spectrophotometer microplate reader. The result was expressed as the ratio of the OD value of cells with treatment to that without treatment (control). 2.8. Matrigel invasion assay The upper surface of the filter in transwell plates was coated with 15 ml of pure matrigel and the filter was air-dried under sterile conditions. Before use, the matrigel was rehydrated with 100 ml of warm DMEM for 2 h. The trophoblasts or JEG-3 cells treated with NME1 siRNA or NME1 overexpression (2 105 resuspended in 200 ml of RPMI1640 medium supplemented with 10% FBS) were seeded in the upper chamber. TSLP (400 ng/ml) or TSLP combined with different signaling inhibitors (STAT3i, STAT5i, AKTi, ERKi) were added. The cells were allowed to invade and held for 48 h in 5% CO2 at 37 C. Thereafter, the cells attached to the upper surface of the filter were removed by scrubbing with a cotton swab, and the cells remaining on the lower surface were fixed in methanol for 20 min at room temperature and stained with hematoxylin. For quantification, the cells that had migrated to the lower surface were counted under a light microscope in five random fields at a magnification of 200. The results were expressed as a percentage of the controls. 2.9. Statistical analysis Data were analyzed with aid of SPSS database, and statistical significance was determined using the Student’s t test with P values of <0.05 being considered statistically significant.
3. Results 3.1. STAT3 inhibitor abrogated TSLP-mediated trophoblast proliferation Our previous study has revealed that TSLP stimulates the proliferation of human trophoblast [11]. To define the signal pathway transmitted by TSLP receptor in TSLP-mediated trophoblast proliferation, primary human trophoblasts were treated with TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, ERK), and then human trophoblast proliferation was analyzed by BrdU cell proliferation assay. As shown in Fig. 1, TSLP-induced human trophoblast proliferation (P < 0.05), and the stimulatory effect of TSLP was abrogated by STAT3 inhibitor (P < 0.05), but not affected by other inhibitors (P > 0.05). Our results demonstrated that STAT3 pathway was involved in TSLP-mediated trophoblast proliferation.
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Fig. 1. The STAT3 pathway was involved in TSLP regulation on trophoblast proliferation. Primary human trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK) for 48 h. The proliferation of trophoblasts was determined by BrdU cell proliferation assay. Results were highly reproducible in five independent experiments.
3.2. TSLP-induced tyrosine phosphorylation of STAT3 in human first-trimester trophoblasts To further test the effect of TSLP on STAT3 phosphorylation in human trophoblasts, primary human trophoblasts were treated with TSLP for 0, 5, 15, 30 and 60 min, and subsequently analyzed for tyrosine phosphorylation of STAT3 by Western blot analysis employing an antibody to STAT3 phosphorylated at Tyr 705. The results in Fig. 2A showed that TSLP stimulation evoked a timedependent phosphorylation of STAT3, reaching a maximum at 30 min. We investigated the impact of STAT3 inhibitor on the TSLPmediated STAT3 tyrosine phosphorylation. Primary human trophoblasts were treated with TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, ERK), and then tyrosine phosphorylation of STAT3 in human trophoblasts was detected by Western blot analysis. As shown in Fig. 2B, pSTAT3 expression in human trophoblasts increased after treatment with TSLP, and this upregulation of pSTAT3 was abrogated by STAT3 inhibitor (P < 0.05) and was not affected by other inhibitors (P > 0.05).
RNA interference (siRNA) on the cell proliferation and invasion in JEG-3 cells. Western blot analysis showed that c-Myc protein expression in JEG-3 cells was silenced successfully (Fig. 4A). We investigated the effect of c-Myc silence on the cell proliferation. Treatment with TSLP induced the proliferation of JEG-3 cells, and the stimulatory effect of TSLP was abrogated by c-Myc silence (P < 0.05) (Fig. 4B). We also analyzed the effect of c-Myc silence on the cell invasion. Treatment with TSLP significantly increased the invasion of JEG-3 cells, and c-Myc silence slightly decreased the
3.3. STAT3 inhibitor abrogated TSLP-mediated c-Myc expression We further investigated the impact of STAT3 pathway on the TSLP-mediated c-Myc expression. Primary human trophoblasts were treated with TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, ERK), and then c-Myc protein expression in human trophoblasts was detected by Western blot analysis. Data were presented in Fig. 3 that c-Myc expression in human trophoblasts significantly increased after treatment with TSLP, and this upregulation of c-Myc protein was abrogated by STAT3 signaling inhibitor (P < 0.05) and was not affected by other inhibitors (P > 0.05). Our results suggest that TSLP can upregulate c-Myc expression via STAT3 pathway. 3.4. The c-Myc siRNA interference abrogated TSLP-mediated JEG-3 cell proliferation Based on the experimental results described above, we further determined the influence of knockdown of c-Myc expression by
Fig. 2. TSLP-induced tyrosine phosphorylation of STAT3 in human first-trimester trophoblasts. (A) Primary human trophoblasts were either left untreated (“0”) or incubated with TSLP for 5, 15, 30, or 60 min. (B) Primary human trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). Cells were then lysed and subjected to Western blot analysis. The blots were first probed with an antibody recognizing STAT3 phosphorylated at Tyr 705 (top). After washing off this antibody, the blot was re-probed with an antibody toward STAT3 to confirm protein integrity and comparable loading (bottom). The experiments were performed at least three times and representative result was presented.
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Fig. 3. The STAT3 pathway was involved in TSLP regulation on c-Myc expression. Primary human trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The level of c-Myc protein expression was determined by using Western blot analysis with an anti-c-Myc antibody. GAPDH was used as loading control. The experiments were performed at least three times and representative result was presented.
invasion of JEG-3 cells, however, no significant difference appeared (P > 0.05) (Fig. 4C). Our observations suggest that TSLPstimulated increase in trophoblast proliferation might be dependent on c-Myc expression.
4. Discussion Several studies have demonstrated that TSLP plays an important role in the cross-talk between various epithelial cells and dendritic cells to induce or modulate physiological and pathological immune responses [12]. In our previous studies, we have reported that the pregnant-associated hormones result in a significant increase in TSLP mRNA and protein expression in human trophoblasts [11]. The trophoblast-derived TSLP activates decidual DCs with increased costimulatory molecules, major histocompatibility complex class II, and OX-40L expression, and the TSLP-activated decidual DCs prime decidual CD4 þ T cells for TH2 cell differentiation, which might play an important role in establishing and maintaining maternalefetal immunotolerance [13]. Therefore, human trophoblasts may contribute to maternal-fetal tolerance by instructing dDCs to induce regulatory TH2 bias in early human pregnancy via TSLP secretion [13]. Interestingly, the trophoblast-secreted TSLP induces the proliferation and invasion of trophoblasts by autocrine and paracrine regulation, which may contribute to the development of human placenta and the establishment of normal pregnancy [11]. However, the mechanism by which TSLP regulates the proliferation of trophoblasts is poorly understood. Myc regulates divergent cellular functions such as proliferation, differentiation, and apoptosis. In mammalian cells, Myc regulates all aspects of protein synthesis, increasing components of ribosome biogenesis and tRNA levels and key factors involved in translation initiation and elongation. The findings that loss of Myc severely impairs cell growth in both Drosophila and mammalian cells [14], whereas its overexpression clearly increases overall protein production, most notably in resting B lymphocytes [15], suggest that the regulation of protein synthesis is controlled by Myc. Thus, we hypothesized that TSLP may affect trophoblast proliferation by regulating c-Myc expression. In this study, the effect of TSLP stimulation on c-Myc expression was examined using Western blot analysis. We showed that treatment with TSLP significantly enhanced c-Myc expression in human trophoblasts. To confirm the role of c-Myc expression in TSLP-induced trophoblast proliferation, we knocked down c-Myc expression by RNA interference (RNAi) in JEG-3 cells, and re-evaluated the effect of TSLP on cell proliferation.
Fig. 4. The c-Myc siRNA interference abrogated TSLP-mediated JEG-3 cell proliferation. JEG-3 cells were transfected with siRNA targeting to c-Myc. (A): Characterization of c-Myc siRNA. c-Myc expression was detected by Western blot analysis with GAPDH as the loading control. (B): c-Myc was involved in TSLP regulation on the cell proliferation. The proliferation was determined by BrdU cell proliferation assay in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc siRNA. (C): c-Myc was not involved in TSLP regulation on the cell invasion. The invasion was determined by Matrigel invasion assay in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc siRNA. Results were highly reproducible in five independent experiments.
We found that the effect of TSLP upregulation of proliferation was abrogated by c-Myc siRNA. These data suggest that the stimulatory effect of TSLP on trophoblast proliferation depends on the c-Myc pathway. Thus, c-Myc may serve as a major contributing factor in this pathway. Unlike all other known members of the cytokine receptor family, engagement of the TSLP receptor complex leads to activation of a Stat protein (Stat) in the absence of Jak involvement. Both human and mouse TSLPeTSLPR interactions activate similar signaling pathways. Like IL-7, TSLP activates the transcription factor STAT3 in human and STAT5 in mouse and human. However, the mechanisms that TSLP and IL-7 use to activate STAT are distinctly different. TSLPmediated signal transduction does not require Jak1 and Jak2, as
H.-H. Pu et al. / Placenta 33 (2012) 387e391
evidenced by the observation that dominant negative forms of Jak1 and Jak2 are unable to block TSLP-mediated STAT activation, in contrast, IL-7 signaling is inhibited by dominant negative Jak1 [10,16]. In this study, to define which signaling transduction pathway mediates TSLP-stimulated trophoblast proliferation, human trophoblasts were treated with TSLP combined with different signaling inhibitor (STAT3, STAT5, AKT, and ERK). Our results showed that the stimulatory effect of TSLP on trophoblast proliferation was abrogated by STAT3 inhibitor and was not affected by other inhibitors. We also found TSLP stimulation evoked a timedependent phosphorylation of STAT3 in human trophoblasts. These data suggest that STAT3 pathway is involved in TSLP-mediated trophoblast proliferation. To further assess the role of STAT3 signal transduction pathway in regulation of TSLP on c-Myc expression, we treated human trophoblasts with STAT3, STAT5, AKT, or ERK signal transduction pathway inhibitor and re-evaluated the effect of TSLP on the expression of c-Myc. We found that the upregulation of c-Myc expression induced by TSLP was abrogated by the STAT3 signaling inhibitor and was not affected by other inhibitors. We concluded that TSLP upregulated the expression of cMyc via activation of the STAT3 phosphorylation signaling. Thus it is suggested that the intracellular signal transduction pathway STAT3 signaling was an upstream signal in TSLP regulation on cMyc expression in human trophoblasts. Taken together, our results indicate that TSLP may stimulate cMyc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation. The results of the current study help to better understand the regulation and the possible mechanisms of the effect of TSLP on trophoblast proliferation. Further studies to understand the role of TSLP in maintenance of normal pregnancy are necessary at cellular and molecular level. Acknowledgments This work is supported by National Natural Science Foundation of China No. 31170870 (to L-P Jin), Research Fund for Doctoral Program from Education Ministry of China 200802461019 (to L-P Jin), Natural Science Foundation of Shanghai 10ZR1405100 (to L-P Jin), National Basic Research Program of China 2006CB944007 (to D-J Li), Key Project (30730087) and Major International Joint Research Project (30910103909) of NSFC (to D-J. Li), NSFC 30670787
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(to D-J. Li), and Program for Outstanding Medical Academic Leader of Shanghai (to D-J Li). References [1] Fitzgerald JS, Germeyer A, Huppertz B, Jeschke U, Knofler M, Moser G, et al. Governing the invasive trophoblast: current aspects on intra- and extracellular regulation. Am J Reprod Immunol 2010;63:492e505. [2] Grandori C, Cowley SM, James LP, Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu Rev Cell Dev Biol 2000; 16:653e99. [3] Fernandez PC, Frank SR, Wang L, Schroeder M, Liu S, Greene J, et al. Genomic targets of the human c-Myc protein. Genes Dev 2003;17:1115e29. [4] Orian A, van Steensel B, Delrow J, Bussemaker HJ, Li L, Sawado T, et al. Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev 2003;17:1101e14. [5] Soumelis V, Reche PA, Kanzler H, Yuan W, Edward G, Homey B, et al. Human epithelial cells trigger dendritic cell-mediated allergic inflammation by producing TSLP. Nat Immunol 2002;3:673e80. [6] Ying S, O’Connor B, Ratoff J, Meng Q, Mallett K, Cousins D, et al. Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting ahemokines and disease severity. J Immunol 2005;174:8183e90. [7] Yoo J, Omori M, Gyarmati D, Zhou B, Aye T, Brewer A, et al. Spontaneous atopic dermatitis in mice expressing an inducible thymic stromal lymphopoietin transgene specifically in the skin. J Exp Med 2005;202:541e9. [8] Watanabe N, Wang YH, Lee HK, Ito T, Cao W, Liu YJ. Hassall’s corpuscles instruct dendritic cells to induce CD4 þ CD25þ regulatory T cells in human thymus. Nature 2005;436:1181e5. [9] Isaksen DE, Baumann H, Zhou B, Nivollet S, Farr AG, Levin SD, et al. Uncoupling of proliferation and Stat5 activation in thymic stromal lymphopoietinmediated signal transduction. J Immunol 2002;168:3288e94. [10] He R, Geha RS. Thymic stromal lymphopoietin. Ann N Y Acad Sci 2010;1183: 13e24. [11] Wu HX, Guo PF, Jin LP, Liang SS, Li DJ. Functional regulation of thymic stromal lymphopoietin on proliferation and invasion of trophoblasts in human first-trimester pregnancy. Hum Reprod 2010;25:1146e52. [12] Liu YJ, Soumelis V, Watanabe N, Ito T, Wang YH, Malefyt RW, et al. TSLP: an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic cell maturation. Annu Rev Immunol 2007;25:193e219. [13] Guo PF, Du MR, Wu HX, Lin Y, Jin LP, Li DJ. Thymic stromal lymphopoietin from trophoblasts induces dendritic cell-mediated regulatory TH2 bias in the decidua during early gestation in humans. Blood 2010;116:2061e9. [14] Mateyak MK, Obaya AJ, Adachi S, Sedivy JM. Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell Growth Differ 1997;8:1039e48. [15] Iritani BM, Eisenman RN. c-Myc enhances protein synthesis and cell size during B lymphocyte development. Proc Natl Acad Sci U S A 1999;96: 13180e5. [16] Isaksen DE, Baumann H, Trobridge PA, Farr AG, Levin SD, Ziegler SF. Requirement for Stat5 in thymic stromal lymphopoietin-mediated signal transduction. J Immunol 1999;163(11):5971e7.
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Thymic stromal lymphopoietin promotes the proliferation of human trophoblasts via phosphorylated STAT3-mediated c-Myc upregulation H.-H. Pu a, b, J. Duan a, Y. Wang a, D.-X. Fan a, D.-J. Li a, *, L.-P. Jin a, * a
Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, 413 Zhaozhou Road, Shanghai 200011, China b Department of Obstetrics & Gynecology, Xuhui Central Hospital of Shanghai, Shanghai 200031, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 17 January 2012
Our previous study has demonstrated that thymic stromal lymphopoietin (TSLP) stimulates trophoblast proliferation and invasion, suggesting TSLP plays an important role in the placentation in early human pregnancy, but the intracellular molecular mechanism is currently unknown. The present study is undertaken to investigate whether the STAT3-c-Myc signaling pathway is involved in TSLP-mediated trophoblast proliferation. Primary human first-trimester trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The levels of STAT3 tyrosine phosphorylation and c-Myc expression were determined by using Western blot analysis, and the proliferation of trophoblasts was analyzed by BrdU cell proliferation assay. JEG-3 cells were transfected with siRNA targeting to c-Myc, and the proliferation was determined in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc silencing. It was revealed that treatment with TSLP significantly enhanced STAT3 phosphorylation and c-Myc expression in human trophoblasts. The effect of TSLP upregulation on trophoblast proliferation was abrogated completely by either STAT3 inhibitor or c-Myc siRNA silence. We further found that the upregulation of TSLP on c-Myc expression was abrogated completely by the STAT3 inhibitor, which suggests that the intracellular STAT3 phosphorylation is an upstream signal of c-Myc expression in the TSLP-stimulated trophoblast proliferation. These results suggest that TSLP may upregulate c-Myc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: TSLP c-Myc STAT3 pathway Trophoblast Proliferation
1. Introduction As a critical cell player in the human placenta, fetal cytotrophoblasts play an important role in successful pregnancy. Their proliferation and differentiation as well as invasion during implantation occur through a series of tightly controlled processes of intercellular signaling mediated by growth factors, cytokines and hormones [1]. It involves extensive cross-talk between the trophoblast cells and the receptive endometrium. Lack of any crucial embryonic or maternal signal at the site of implantation may result in a shallow or failure of embryo implantation. The c-Myc oncogene is the prototypical member of the Myc/ Mad/ Max transcription factor network, which regulates divergent cellular functions such as proliferation, differentiation, and
Abbreviations: TSLP, thymic stromal lymphopoietin; STAT, signal transducer and activator of transcription; DCs, dendritic cells; BrdU, Bromo deoxy Uridine. * Corresponding authors. Tel.: þ86 21 63455050 8288; fax: þ86 21 63457331. E-mail addresses: [email protected] (D.-J. Li), [email protected] (L.-P. Jin). 0143-4004/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2012.01.015
apoptosis. In mammalian cells, Myc expression is rapidly induced following mitogenic or cytokine stimulation. One of its more obvious biological effects is to drive cellular proliferation, which is sufficient to allow quiescent cells to reenter the cell cycle [2]. In an effort to further dissect the molecular pathways regulated by Myc, several genome-wide screens have been used to identify transcriptional targets of Myc. Several studies have revealed that expression of up to 10e15% of all genes may be affected by c-Myc, and one of the more consistent categories of genes up-regulated by Myc is cell growth and protein synthesis [3,4]. Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers the dendritic cell-mediated Th2 response and regulatory T cell expansion. TSLP signals via TSLP receptor expressed on a wide range of cell types in the adaptive and innate immune system. TSLP exerts profound influence on the polarization of dendritic cells (DCs) to drive T helper (Th) 2 cytokine production, and mediates allergic inflammation such as asthma and atopic dermatitis [5e7]. It has been reported that TSLP-activated myeloid DCs might be critical in the positive selection of medium-to-high-affinity selfreactive thymocytes into the regulatory T cell (Treg cell) lineage [8].
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Both human and mouse TSLPeTSLPR interactions activate similar signaling pathways. TSLP activates the transcription factor signal transducer and activator of transcription 3 (STAT3) in human and STAT5 in mouse and human [9,10]. Our previous studies have shown that human first-trimester trophoblasts secrete soluble TSLP and express the functional TSLP receptor complex TSLP receptor and IL-7 receptor-a, which may render trophoblasts a target for autocrine and paracrine regulation by the locally produced TSLP. We have also demonstrated that TSLP is in fact an effective inducer to proliferation and invasion of human first-trimester trophoblasts, which indicates that the TSLPeTSLPR interaction plays an important role in the development of the human placenta [11]. However, the mechanism by which TSLP promotes the proliferation of human trophoblasts is currently unknown. 2. Methods 2.1. Isolation and primary culture of human first-trimester trophoblast First-trimester human villous tissues were obtained from placentas of 5 clinically normal pregnancies [age: 28.20 þ 3.38 years, gestational age at sampling 8.69 þ 1.56 weeks (mean S.D.)], which were terminated for nonmedical reasons. Each subject completed a signed, written consent form approved by Human Investigation Committee in Hospital of Obstetrics & Gynecology, Fudan University. Villous tissues obtained from patients enrolled in the study were digested by four 10 min incubations with 0.25% trypsin and 1500 IU/ml of DNase type I (AppliChem GmbH, Germany) at 37 C with gentle agitation. The digested cell suspensions were pooled and carefully layered over a discontinuous Percoll gradient (65e20%, graduated in 5% steps) and centrifuged at 1000 g for 15 min. The middle layer (density 1.042e1.068 g/ml) was recovered and washed with DMEM-highglucose medium. The cells were seeded on matrigel-precoated cell culture plates at 3 105 cells/well in DMEM-high-glucose complete medium (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 2 mM glutamine, 25 mM HEPES, 100 U/ml of penicillin, 100 mg/ml of streptomycin and 20% fetal bovine serum (FBS) and incubated in 5% CO2 at 37 C for 12 h, and then the medium was renewed by DMEMhigh-glucose complete medium supplemented with 1% FBS. After further cultivation for 12 h, the cells were stimulated with recombinant human TSLP. We analyzed the purity of the primary trophoblasts by immunocytochemistry. Trophoblasts are cytokeratin 7 positive, and vimentin negative. The cell purity of the primary trophoblasts was above 90% (data not shown). 2.2. Cell lines and reagents The human choriocarcinoma cell line JEG-3 obtained from the American Type Culture Collection (Manassas, VA, USA), were routinely grown in DMEM culture solution (GIBCO BRL, Gaithersburg, MD, USA) containing 10% FBS in 5% CO2 at 37 C. 2.3. TSLP treatment Recombinant human TSLP was purchased from PeproTech (Rocky Hill, UK). Primary human trophoblasts were treated with 400 ng/ml TSLP for 48 h. Then the level of c-Myc protein in primary human trophoblasts was detected by Western blot analysis. Primary human trophoblasts were treated with 400 ng/ml TSLP for 0, 5, 15, 30 and 60 min and subsequently analyzed for tyrosine phosphorylation of STAT3 by Western blot analysis. 2.4. Treatment with STAT3, STAT5, AKT or ERK pathway inhibitor Primary human trophoblasts were cultured as described above, and 20 mmol/l AKT pathway inhibitor LY294002, 20 mmol/l ERK pathway inhibitor U0126 (both from Calbiochem, CA), 40 mmol/l STAT3 pathway inhibitor D4071 or 20 mmol/l STAT5 pathway inhibitor M1323 (both from Sigma, St. Louis, USA) were added to the culture supernatants. After 30 min, 400 ng/ml TSLP was added to the cells, and PBS was used as a control. The proteins were extracted from the cells 30 min later for STAT3 phosphorylation assay, or 48 h later for c-Myc protein assay and BrdU cell proliferation assay. 2.5. c-Myc siRNA transfection c-Myc siRNA was procured from Invitrogen (Carlsbad, CA). The siRNA sequence targeting c-Myc is 50 -CAGTTGCCACTTCCACATA-30 . A random siRNA (50 ACTACCGTTGTTATAGGTG-30 ) which does not have any target region in human genes served as negative control. c-Myc siRNA transfection was performed according to the manufacturer’s instructions. One day before transfection, plate JEG-3 cells in 500 ml of growth medium without antibiotics such that they will be 30e50% confluent at the time of transfection. The siRNA oligonucleotides targeting c-Myc and
Lipofectamine 2000 were mixed in OPTIMEM and then added to the cells at room temperature, with nontargeting siRNA oligonucleotides as control. After 6 h incubation, the cells were incubated in DMEM for a further 72 h in 5% CO2 at 37 C, and the protein expression of c-Myc in JEG-3 cells was determinated by Western blot analysis. 2.6. Western blot analysis Cells were lysed in RIPA lysis buffer containing proteinase inhibitor for 30 min. The samples were boiled for 10 min, centrifuged, and the supernatants collected. Each sample (25 mg) was loaded on a 10% SDS-PAGE gel. After electrophoresis, the proteins were transferred onto a polyvinyl difluoride membrane (Bio-Rad), and the membrane incubated in blocking buffer containing 5% non-fat dry milk for 1 h at room temperature. Then the membrane was probed with a primary antibody in blocking buffer [1 mg/ml anti-c-Myc mouse mAb (Santa Cruz Biotechnology Inc.; St. Louis, MO) and 0.1 mg/ml anti-GAPDH mouse mAb (Sigma) as the internal marker] overnight at 4 C, followed by probing with anti-mouse Ig-HRP conjugates (Amersham Biosciences; Buckinghamshire, UK) in blocking buffer for 2 h at 37 C. The signals were recorded on HyperFilm MP (Amersham Pharmacia Biotech) and developed in a Kodak X-Omat film developer. Results were scanned and densitometrically analyzed using Scion Image software (Scion Inc). 2.7. BrdU cell proliferation assay After different treatment described above, 20 ml of Bromo deoxy Uridine (BrdU) reagent (Sigma) was added to each well and incubated for 12 h 37 C. The media was aspirated and the adherent trophoblasts were incubated with the Fixing Solution (200 mL/well). After 30 min, anti-BrdU monoclonal antibody (100 mL/well) was added and the incubation was continued for an additional hour at room temperature. After addition of goat anti-mouse IgG-peroxidase conjugated secondary antibody, substrate and stop solution, the amount of BrdU was determined by reading the sample at dual wavelength of 450/550 nm using a spectrophotometer microplate reader. The result was expressed as the ratio of the OD value of cells with treatment to that without treatment (control). 2.8. Matrigel invasion assay The upper surface of the filter in transwell plates was coated with 15 ml of pure matrigel and the filter was air-dried under sterile conditions. Before use, the matrigel was rehydrated with 100 ml of warm DMEM for 2 h. The trophoblasts or JEG-3 cells treated with NME1 siRNA or NME1 overexpression (2 105 resuspended in 200 ml of RPMI1640 medium supplemented with 10% FBS) were seeded in the upper chamber. TSLP (400 ng/ml) or TSLP combined with different signaling inhibitors (STAT3i, STAT5i, AKTi, ERKi) were added. The cells were allowed to invade and held for 48 h in 5% CO2 at 37 C. Thereafter, the cells attached to the upper surface of the filter were removed by scrubbing with a cotton swab, and the cells remaining on the lower surface were fixed in methanol for 20 min at room temperature and stained with hematoxylin. For quantification, the cells that had migrated to the lower surface were counted under a light microscope in five random fields at a magnification of 200. The results were expressed as a percentage of the controls. 2.9. Statistical analysis Data were analyzed with aid of SPSS database, and statistical significance was determined using the Student’s t test with P values of <0.05 being considered statistically significant.
3. Results 3.1. STAT3 inhibitor abrogated TSLP-mediated trophoblast proliferation Our previous study has revealed that TSLP stimulates the proliferation of human trophoblast [11]. To define the signal pathway transmitted by TSLP receptor in TSLP-mediated trophoblast proliferation, primary human trophoblasts were treated with TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, ERK), and then human trophoblast proliferation was analyzed by BrdU cell proliferation assay. As shown in Fig. 1, TSLP-induced human trophoblast proliferation (P < 0.05), and the stimulatory effect of TSLP was abrogated by STAT3 inhibitor (P < 0.05), but not affected by other inhibitors (P > 0.05). Our results demonstrated that STAT3 pathway was involved in TSLP-mediated trophoblast proliferation.
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Fig. 1. The STAT3 pathway was involved in TSLP regulation on trophoblast proliferation. Primary human trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK) for 48 h. The proliferation of trophoblasts was determined by BrdU cell proliferation assay. Results were highly reproducible in five independent experiments.
3.2. TSLP-induced tyrosine phosphorylation of STAT3 in human first-trimester trophoblasts To further test the effect of TSLP on STAT3 phosphorylation in human trophoblasts, primary human trophoblasts were treated with TSLP for 0, 5, 15, 30 and 60 min, and subsequently analyzed for tyrosine phosphorylation of STAT3 by Western blot analysis employing an antibody to STAT3 phosphorylated at Tyr 705. The results in Fig. 2A showed that TSLP stimulation evoked a timedependent phosphorylation of STAT3, reaching a maximum at 30 min. We investigated the impact of STAT3 inhibitor on the TSLPmediated STAT3 tyrosine phosphorylation. Primary human trophoblasts were treated with TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, ERK), and then tyrosine phosphorylation of STAT3 in human trophoblasts was detected by Western blot analysis. As shown in Fig. 2B, pSTAT3 expression in human trophoblasts increased after treatment with TSLP, and this upregulation of pSTAT3 was abrogated by STAT3 inhibitor (P < 0.05) and was not affected by other inhibitors (P > 0.05).
RNA interference (siRNA) on the cell proliferation and invasion in JEG-3 cells. Western blot analysis showed that c-Myc protein expression in JEG-3 cells was silenced successfully (Fig. 4A). We investigated the effect of c-Myc silence on the cell proliferation. Treatment with TSLP induced the proliferation of JEG-3 cells, and the stimulatory effect of TSLP was abrogated by c-Myc silence (P < 0.05) (Fig. 4B). We also analyzed the effect of c-Myc silence on the cell invasion. Treatment with TSLP significantly increased the invasion of JEG-3 cells, and c-Myc silence slightly decreased the
3.3. STAT3 inhibitor abrogated TSLP-mediated c-Myc expression We further investigated the impact of STAT3 pathway on the TSLP-mediated c-Myc expression. Primary human trophoblasts were treated with TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, ERK), and then c-Myc protein expression in human trophoblasts was detected by Western blot analysis. Data were presented in Fig. 3 that c-Myc expression in human trophoblasts significantly increased after treatment with TSLP, and this upregulation of c-Myc protein was abrogated by STAT3 signaling inhibitor (P < 0.05) and was not affected by other inhibitors (P > 0.05). Our results suggest that TSLP can upregulate c-Myc expression via STAT3 pathway. 3.4. The c-Myc siRNA interference abrogated TSLP-mediated JEG-3 cell proliferation Based on the experimental results described above, we further determined the influence of knockdown of c-Myc expression by
Fig. 2. TSLP-induced tyrosine phosphorylation of STAT3 in human first-trimester trophoblasts. (A) Primary human trophoblasts were either left untreated (“0”) or incubated with TSLP for 5, 15, 30, or 60 min. (B) Primary human trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). Cells were then lysed and subjected to Western blot analysis. The blots were first probed with an antibody recognizing STAT3 phosphorylated at Tyr 705 (top). After washing off this antibody, the blot was re-probed with an antibody toward STAT3 to confirm protein integrity and comparable loading (bottom). The experiments were performed at least three times and representative result was presented.
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Fig. 3. The STAT3 pathway was involved in TSLP regulation on c-Myc expression. Primary human trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The level of c-Myc protein expression was determined by using Western blot analysis with an anti-c-Myc antibody. GAPDH was used as loading control. The experiments were performed at least three times and representative result was presented.
invasion of JEG-3 cells, however, no significant difference appeared (P > 0.05) (Fig. 4C). Our observations suggest that TSLPstimulated increase in trophoblast proliferation might be dependent on c-Myc expression.
4. Discussion Several studies have demonstrated that TSLP plays an important role in the cross-talk between various epithelial cells and dendritic cells to induce or modulate physiological and pathological immune responses [12]. In our previous studies, we have reported that the pregnant-associated hormones result in a significant increase in TSLP mRNA and protein expression in human trophoblasts [11]. The trophoblast-derived TSLP activates decidual DCs with increased costimulatory molecules, major histocompatibility complex class II, and OX-40L expression, and the TSLP-activated decidual DCs prime decidual CD4 þ T cells for TH2 cell differentiation, which might play an important role in establishing and maintaining maternalefetal immunotolerance [13]. Therefore, human trophoblasts may contribute to maternal-fetal tolerance by instructing dDCs to induce regulatory TH2 bias in early human pregnancy via TSLP secretion [13]. Interestingly, the trophoblast-secreted TSLP induces the proliferation and invasion of trophoblasts by autocrine and paracrine regulation, which may contribute to the development of human placenta and the establishment of normal pregnancy [11]. However, the mechanism by which TSLP regulates the proliferation of trophoblasts is poorly understood. Myc regulates divergent cellular functions such as proliferation, differentiation, and apoptosis. In mammalian cells, Myc regulates all aspects of protein synthesis, increasing components of ribosome biogenesis and tRNA levels and key factors involved in translation initiation and elongation. The findings that loss of Myc severely impairs cell growth in both Drosophila and mammalian cells [14], whereas its overexpression clearly increases overall protein production, most notably in resting B lymphocytes [15], suggest that the regulation of protein synthesis is controlled by Myc. Thus, we hypothesized that TSLP may affect trophoblast proliferation by regulating c-Myc expression. In this study, the effect of TSLP stimulation on c-Myc expression was examined using Western blot analysis. We showed that treatment with TSLP significantly enhanced c-Myc expression in human trophoblasts. To confirm the role of c-Myc expression in TSLP-induced trophoblast proliferation, we knocked down c-Myc expression by RNA interference (RNAi) in JEG-3 cells, and re-evaluated the effect of TSLP on cell proliferation.
Fig. 4. The c-Myc siRNA interference abrogated TSLP-mediated JEG-3 cell proliferation. JEG-3 cells were transfected with siRNA targeting to c-Myc. (A): Characterization of c-Myc siRNA. c-Myc expression was detected by Western blot analysis with GAPDH as the loading control. (B): c-Myc was involved in TSLP regulation on the cell proliferation. The proliferation was determined by BrdU cell proliferation assay in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc siRNA. (C): c-Myc was not involved in TSLP regulation on the cell invasion. The invasion was determined by Matrigel invasion assay in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc siRNA. Results were highly reproducible in five independent experiments.
We found that the effect of TSLP upregulation of proliferation was abrogated by c-Myc siRNA. These data suggest that the stimulatory effect of TSLP on trophoblast proliferation depends on the c-Myc pathway. Thus, c-Myc may serve as a major contributing factor in this pathway. Unlike all other known members of the cytokine receptor family, engagement of the TSLP receptor complex leads to activation of a Stat protein (Stat) in the absence of Jak involvement. Both human and mouse TSLPeTSLPR interactions activate similar signaling pathways. Like IL-7, TSLP activates the transcription factor STAT3 in human and STAT5 in mouse and human. However, the mechanisms that TSLP and IL-7 use to activate STAT are distinctly different. TSLPmediated signal transduction does not require Jak1 and Jak2, as
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evidenced by the observation that dominant negative forms of Jak1 and Jak2 are unable to block TSLP-mediated STAT activation, in contrast, IL-7 signaling is inhibited by dominant negative Jak1 [10,16]. In this study, to define which signaling transduction pathway mediates TSLP-stimulated trophoblast proliferation, human trophoblasts were treated with TSLP combined with different signaling inhibitor (STAT3, STAT5, AKT, and ERK). Our results showed that the stimulatory effect of TSLP on trophoblast proliferation was abrogated by STAT3 inhibitor and was not affected by other inhibitors. We also found TSLP stimulation evoked a timedependent phosphorylation of STAT3 in human trophoblasts. These data suggest that STAT3 pathway is involved in TSLP-mediated trophoblast proliferation. To further assess the role of STAT3 signal transduction pathway in regulation of TSLP on c-Myc expression, we treated human trophoblasts with STAT3, STAT5, AKT, or ERK signal transduction pathway inhibitor and re-evaluated the effect of TSLP on the expression of c-Myc. We found that the upregulation of c-Myc expression induced by TSLP was abrogated by the STAT3 signaling inhibitor and was not affected by other inhibitors. We concluded that TSLP upregulated the expression of cMyc via activation of the STAT3 phosphorylation signaling. Thus it is suggested that the intracellular signal transduction pathway STAT3 signaling was an upstream signal in TSLP regulation on cMyc expression in human trophoblasts. Taken together, our results indicate that TSLP may stimulate cMyc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation. The results of the current study help to better understand the regulation and the possible mechanisms of the effect of TSLP on trophoblast proliferation. Further studies to understand the role of TSLP in maintenance of normal pregnancy are necessary at cellular and molecular level. Acknowledgments This work is supported by National Natural Science Foundation of China No. 31170870 (to L-P Jin), Research Fund for Doctoral Program from Education Ministry of China 200802461019 (to L-P Jin), Natural Science Foundation of Shanghai 10ZR1405100 (to L-P Jin), National Basic Research Program of China 2006CB944007 (to D-J Li), Key Project (30730087) and Major International Joint Research Project (30910103909) of NSFC (to D-J. Li), NSFC 30670787
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