CLS-08090; No of Pages 9 Cellular Signalling xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
Xiao-Ming Dong a,b,1, Rong-Hua Yin b,1, Yang Yang c,1, Zhi-Wei Feng b, Hong-Mei Ning d, Lan Dong e, Wei-Wei Zheng b, Liu-jun Tang b, Jian Wang b, Yu-Xin Jia a, Yi-Nan Jiang f, En-Dong Liu f, Hui Chen b, Yi-Qun Zhan b, Miao Yu b, Chang-Hui Ge b, Chang-Yan Li b,f,⁎, Xiao-Ming Yang a,b,⁎
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GATA-2 inhibits transforming growth factor-β signaling pathway through interaction with Smad4
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School of Chemical Engineering and Technology, Department of Pharmaceutical Engineering, Tianjin University, Tianjin 300072, China State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China c Purdue University, Department of Biological Sciences, 915W. State Street, West Lafayette, IN 47907-2054, United States d Department of Hematopoietic Stem Cell Transplantation, Affiliated Hospital to Academy of Military Medical Sciences, Beijing 100071, China e Department of Anesthesiology, General Hospital of Chinese People's Armed Police Forces, Beijing, China f An Hui Medical University, Hefei 230032, China
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Article history: Received 13 January 2014 Accepted 28 January 2014 Available online xxxx
GATA-2, a member of zinc finger GATA transcription factor family, plays key role in the hematopoietic stem cells self-renewal and differentiation. The transforming growth factor-β (TGFβ) signaling pathway is a major signaling network that controls cell proliferation, differentiation and tumor suppression. Here we found that GATA-2 negatively regulated TGF-β signaling pathway in Smad4-dependent manner. GATA-2 specifically interacts with Smad4 with its N-terminal while the zinc finger domain of GATA-2 is essential for negative regulation of TGFβ. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the complex Smad2/3/4 formation in response to TGFβ, the DNA binding activity of Smad4 was decreased significantly by GATA-2 overexpression. Overexpression of GATA-2 in K562 cells led to reduced TGFβ-induced erythroid differentiation while knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation. All these results suggest that GATA-2 is a novel negative regulator of TGFβ signal pathway. © 2014 Published by Elsevier Inc. 22
Keywords: GATA-2 Smad4 TGF-β signaling pathway Erythroid differentiation
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GATA-2 is a member of the GATA family transcription factors and plays essential roles in hematopoietic stem and progenitor cell compartments [1–3]. GATA-2 knock-out mice die around embryonic days 9.5–10.5 due to a pan-hematopoietic defect, providing evidence that GATA-2 is a key regulator of development, maintenance, and/or function of hematopoietic stem cells [4,5]. The in vitro experiment using GATA-2−/− embryonic stem cells showed that GATA-2 was necessary for proliferation/survival of early hematopoietic cells [6]. Ectopic expression of GATA-2 in erythroid precursors promotes proliferation and blocks erythroid differentiation [7,8]. Expression of GATA-2 precedes that of another family member, GATA-1, and must decrease as GATA-1 expression increases to enable erythroid differentiation [9,10]. These
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1. Introduction
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Abbreviations: DMEM, Dulbecco's modified Eagle's medium; Epo, erythropoietin; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HDAC3, histone deacetylase 3; siRNA, small interfering RNA; TGF-β, transforming growth factor-β. ⁎ Corresponding authors at: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China. Tel./fax: +86 10 68176833. E-mail addresses: [email protected] (C.-Y. Li), [email protected] (X.-M. Yang). 1 These authors contributed equally to this work.
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data indicate that GATA-2 is essential for appropriate expansion and survival of early hematopoietic cells, and plays a negative role in erythroid differentiation. TGF-β signaling plays an important role in maintaining normal hematopoiesis. In human hematopoietic cells, blocking TGF-β signaling enhanced the survival, proliferation, and growth kinetics of human HSCs [11]. Homozygous deletion of TGF-β resulted in embryonic lethality with defective yolk sac vasculogenesis secondary to abnormal endothelial differentiation and defective hematopoiesis with a reduced yolk sac erythroid cell number [12]. Using hematopoietic cell lines as models, TGF-β has been shown to be an inducer of erythroid differentiation, even stronger than Epo at the cellular level [13]. In human cord blood CD36+ erythroid progenitor cells, TGF-β markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression, while inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle [14]. Moreover, in immature adult hematopoietic progenitor cells, phospho-Smad2/3 associates with TIF1γ to promote erythroid differentiation [15]. Given the key roles played by GATA-2 and TGF-β signal pathway in the erythropoiesis, it is postulated that there could be an important functional interaction between them. A previous study on Evi-1 suggests an association between GATA-2 and TGFβ signal pathway.
0898-6568/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.cellsig.2014.01.028
Please cite this article as: X.-M. Dong, et al., Cell. Signal. (2014), http://dx.doi.org/10.1016/j.cellsig.2014.01.028
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2.1. Cell lines and reagents
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HEK293T cells, HuH78 cells and HepG2 cells were maintained in DMEM (Gibco Invitrogen, CA) with 10% fetal calf serum (FCS), while K562 cells were cultured in RPMI 1640 medium (Gibco) with 10% FCS. All the cells were cultured in a 37 °C incubator with 5% CO2 in the presence of 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 g/l sodium bicarbonate and 10 mM HEPES. TGF-β was purchased from Peprotech (100-21C).
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2.2. Plasmid constructions
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Serial deletions of GATA-2 were amplified by PCR, using a full-length cDNA encoding GATA-2 as template and the PCR products were cloned into plasmid vector pFLAG-CMV-2. The human full-length Smad4 (pcDNA3.1-Smad4 (his/myc)) and the reporter vectors including 3 × CAGA-Luc, PAI-Luc, and 3TP were kindly gifted from Dr. Qinong Ye (Beijing Institute of Biotechnology, Beijing, China).
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2.3. Co-immunoprecipitation
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Cells were washed once in PBS, and lysed in 1 ml of lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), and centrifuged for 15 min at 12,000 rpm at 4 °C. The supernatant was transferred to a fresh tube, and immunoprecipitations were performed with anti-Flag affinity gel (A2220, Sigma) or anti-Myc antibody (Santa Cruz) followed by adsorption to protein A/G plus-agarose beads (sc-2003, Santa Cruz). After SDS-PAGE, the samples were transferred onto polyvinylidene difluoride membranes (Amersham life science) and probed with a variety of antibodies. For detecting interaction of endogenous GATA-2 with Smad4, K562 cells were lysed in 0.5 ml lysis buffer and immunoprecipitated with anti-Smad4 antibody or control serum (Santa Cruz). After extensive washing with the lysis buffer, the immunoprecipitates were resolved by SDS-PAGE, followed by Western blot analysis using the anti-GATA-2 antibody.
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2.5. Subcellular localization assays
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To analyze the subcellular location of exogenous GATA-2 and Smad4, HepG2 cells were seeded in 6-well plates, cultured in DMEM supplemented with 10% fetal bovine serum and transfected with the pEGFP-N1- or pDsRED-N1-based vectors as indicated. Twenty four hours later, the cells were fixed for 30 min at room temperature with 4% paraformaldehyde in PBS and stained with Hoechst 33258. Confocal imaging was performed using Zeiss 510 META system. The green fluorescence was excited at 488 nm with 505–530 nm barrier filter and red fluorescence was simultaneously excited at 543 nm with 560 nm barrier filter.
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2.6. Lentiviral vector construction and production
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For construction of lentivirus-mediated GATA-2 overexpression, GATA-2 full length was cloned into the pBPLV vector to generate pBPLV-EDAG recombinant vector expressing GATA-2-GFP fusion protein. For construction of lentivirus-mediated RNA interference, two siRNA oligos against EDAG were synthesized in GenePharma Biotechnology, the sequences are as follows: si GATA-2-1: 5′-GGC TCG TTC CTG TTC AGA ATT-3′ and si GATA-2-2: 5′-GGA GGA GGA TTG TGF TGA TTT-3′. The siRNA sequences were cloned into a psicoR-GFP vector to generate siGATA-2 lentivirus. The siGATA-2 lentivirus expresses CMV promoter-driven GFP protein and U6 promoter-driven siRNA targeting GATA-2. A negative control siRNA was cloned into psicoR-GFP as a control. For production of lentivirus, HEK293T cells were cotransfected with transfer vectors pBPLV-GATA-2 or psicoR-GFP-si GATA-2 with the packaging vectors including pLP1, pLP2 and pLP-VSVG. Lentiviruses were harvested 72 h after transfection, passed through a 0.45-μm filter, and concentrated 100-fold by ultracentrifugation through 20% sucrose cushion (100,000 g for 90 min; 4 °C). Titers of viral stock were determined on K562 cells transduced by spinoculation (1000 g for 90 min; 30 °C) and analyzed by FACS 48 h later. GFP-positive cells were sorted using fluorescence-activated cell sorter (FACS; BD Biosciences).
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2.7. Colony formation assay
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Cells were transfected with indicated plasmids with Lipofectamine 2000 (Invitrogen), and then plated on 60-mm tissue culture dish (2 × 103 cells per well) and cultured in DMEM supplemented with 10% FBS. The cells were allowed to grow for 10 days in the presence of neomycin with medium changes every 2 days, after which they were washed, fixed with methanol, and stained with Giemsa (Sigma). Colonies 2-mm or greater in size were scored. All experiments were conducted three times.
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2.8. MTT assay
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1 × 103 cells were seeded in each well of 96-well plates and left overnight to adhere. Absorbance was determined by using CellTiter 96 Aqueous One Solution Reagent (Promega) according to the manufacturer's protocol at indicated time points. All experiments were conducted three times.
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For Western blotting, cells were lysed with M-PER® Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA). Then, Western blot analysis was performed according to standard procedures. Antibodies were used at the following concentrations: GATA-2 antiserum, 1: 1000; Smad4 (sc-47725, Santa Cruz), 1:1000; Myc (sc-40, Santa Cruz), 1:1000; Flag (F 3165, Sigma), 1:5000; GAPDH (sc-47778, Santa Cruz), 1:1000. Chemiluminescent detection was conducted using supersignal substrate (Pierce) according to the manufacturer's specifications.
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Evi-1-deficient P-Sp-derived cells showed a severely decreased colony forming capacity and in these cells the expression level of GATA-2 is up-regulated. Blocking of TGF-β signaling is also able to recover the hematopoietic defect of Evi-1-deficient P-Sp cells [16]. These findings suggest that Evi-1 promotes hematopoietic stem/progenitor expansion during embryogenesis through up-regulation of GATA-2 and inhibition of TGF-β signaling [16]. However, whether GATA-2 regulates signaling and the molecular mechanism remains unknown. In a study about building a global atlas of combinatorial transcriptional regulation in mouse and man, Smad4 was found to interact with GATA-2 [17]. In our recent study on the protein interaction network of the human liver, we also identified the interaction between Smad4 and GATA-2 [18]. Since Smad4 is the co-Smads of TGF-β signal pathway [19], it is easy to ask whether GATA-2 regulates TGF-β signal pathway. In the present study, we identified that GATA-2 interacts directly with Smad4, not with other Smad proteins such as Smad1,2, 3, 5 and Smad8, suggesting a specific interaction between GATA-2 and Smad4. GATA-2 negatively regulated TGF-β signaling and attenuates the growth inhibition induced by TGF-β. Furthermore, we found that GATA-2 could recruit HDAC3 to Smad4 and decreased the DNAbinding activity of Smad4. In a erythroleukemia cell line K562 cells, overexpression of GATA-2 inhibited the erythroid differentiation induced by TGF-β while knockdown of endogenous GATA-1 accelerated the TGF-β-induced erythroid differentiation. These results suggested a novel role of GATA-2 in regulating TGF-β signaling pathway.
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Total RNA was reverse-transcribed and amplified using reverse transcription and PCR kits, respectively (Promega Corp., Madison, WI, U.S.). Real-time RT-PCR was performed by Bio-Rad IQ5 (Bio-Rad, U.S.). The abundance of mRNA of each gene was normalized to GAPDH. The sequences of the primers are provided in Supplementary Table S1.
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2.11. Benzidine staining analysis
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Erythroid differentiation of K562 cells was assessed by the benzidine cytochemical test and the benzidine-positive cells represented more mature erythroid cells. Briefly, cells were washed twice with ice-cold PBS and then resuspended in ice-cold PBS. The benzidine solution (3 ml), which contained hydrogen peroxide (final concentration 0.0012%), was added and incubated for 10 min at room temperature. Benzidine-positive cells were quantitated under the microscope. At least 100 cells were counted in triplicate for each condition.
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2.12. Statistical analysis
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All experiments were performed at least three times. Data were reported as means ± SD and the statistical significance was assessed by one-way analysis of variance followed by the Students–Newman– Keuls tests. A value of p ≤ 0.05 was considered to be significant.
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3.1. GATA-2 is a negative regulator of TGFβ signaling pathway
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We first investigated the effect of GATA-2 on the TGF-β signaling pathway. Cells were transiently transfected with the luciferase gene driven by the CAGA element (3 × CAGA-Luc) and GATA-2 expression vector in the presence or absence of TGF-β1. As shown in Fig. 1A, TGFβ1 induced the luciferase activity dramatically, reaching ~ 4.5fold at the concentration of 5 ng/ml. This increase was significantly reduced when GATA2 was cotransfected, in a manner dependent on the GATA2 dose. We then tested whether the similar repression was seen when R-Smads (Smad2, Smad3) or co-Smad (Smad4) proteins was overexpressed. The CAGA-Luc activity was highly enhanced when the R-Smads or co-Smads were cotransfected with CAGA-Luc. However, GATA2 dramatically repressed the transactivation by these Smads (Fig. 1B). We further investigated the effect of GATA-2 on Smad4mediated transcription in detail. GATA-2 inhibited Smad4-mediated transcription in a dose-dependent manner (Fig. 1C). Another GATA family member GATA-1 had no effect on the CAGA-Luc activity (Supplementary Figure S1), suggesting a specific effect of GATA-2 on TGF-β signaling pathway. It is well known that 3TP-Luc, a TGF-β1-responsive reporter plasmid carrying the plasminogen-activator inhibitor-1 promoter, is highly responsive to overexpression Smad2/3/4 and TGF-β in HepG2 cells. We used this system to evaluate the effect of GATA2 on transactivation by overexpressed Smad2/3/4 (Fig. 1D). The results clearly showed that co-transfection of GATA2 repressed the 3TP-Luc induced by Smad2/3/4 almost to the control level. Next, we examined whether GATA2 also repressed the transcriptional response induced by TGFβ1 stimulation. Just as Fig. 1E shows, GATA2 repressed the transcriptional activities induced by TGF-β1 stimulation. Real time PCR analysis
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3.2. GATA2 physically interacts with Smad4 and TGF-β1 treatment enhances the interaction between them
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Our previous study suggested that GATA-2 is a candidate binding protein of Smad4. To assess whether the interaction between GATA-2 and Smad4 is specific, we investigated the interaction between GATA2 and Smad proteins including Smad1, 2, 3, 4, 5 and Smad7 using yeast two hybrid. As Supplementary Figure S3 shows, GATA2 was detected to be a binding protein of Smad4 not other Smad proteins. To evaluate the interaction between GATA-2 and Smad4 in mammalian cells, cell lysates from HEK293T cells co-transfected with GATA-2FLAG and Myc epitope-tagged Smad4 expression vectors were subjected to immunoprecipitation with anti-Flag antibody. The efficiency of each transfection was monitored by GFP cotransfection and all of the transfections displayed similar efficiency (data not shown). As shown in Fig. 2A, Smad4 was specifically immunoprecipitated with GATA-2 by anti-Flag antibody. In contrast, no Smad4 protein was observed in FLAG antibody immunoprecipitates from cells transfected with Smad4-Myc and pCMV-FLAG. The interaction between GATA-2 and Smad4 was confirmed by reverse co-IP in HEK293T cells (Fig. 2B). The interaction between endogenous GATA-2 and Smad4 was further confirmed at physiological conditions in K562 cells. Immunoprecipitations were performed with anti-Smad4 antibody using normal mouse IgG as control. As shown in Fig. 2C, endogenous GATA2 can be immunoprecipitated by anti-Smad4 antibody. The possibility of ribosomal RNA or DNA mediating the binding was ruled out since the binding was not altered when RNase A and DNase were included throughout the cell lysis and IP. Given the observed physical interaction between overexpressed GATA-2 and Smad4 in HEK293T cells, we examined the intracellular location of the endogenous proteins in HepG2 cells in the presence or absence of TGF-β1. In the absence of TGF-β1, endogenous Smad4 mainly localizes in the cytoplasm, whereas GATA-2 is a nuclear diffuse protein, which was consistent with the previous studies (Supplementary Figure S4). After TGF-β1 treatment, Smad4 was induced to a remarkable nuclear translocation, which showed a complete co-localization with GATA-2 (Fig. 2D). Co-immunoprecipitation assay suggested that TGFβ1 treatment enhanced the interaction between GATA-2 and Smad4 (Fig. 2E). In K562 cells which express both GATA-2 and Smad4 endogenously, the same results were obtained (data not shown).
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3.3. Mapping the binding region of GATA-2 and Smad4
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Luciferase assays were carried out at 24 h posttransfection with the Dual-Luciferase Reporter Assay System (Promega). Transfection efficiencies were normalized using cotransfected plasmid pRL-TK measured by Renilla luciferase activity (Promega). Luciferase activity was measured 24 h later.
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also suggested that GATA-2 repressed the gene expression induced by TGF-β1 (Fig. 1F). Using lentivirus-mediated transfer, the full length coding sequence of GATA2 was stably expressed in human epithelial cell line HaCaT. The cells were treated with TGF-β1 and the proliferation was assessed by MTS analysis and colony formation assay. Just as shown in Fig. 1G–H, the growth of control HaCaT cells was significantly inhibited in the presence of TGF-β1, and GATA2 expressing HaCaT cells was resistant to the inhibition of TGF-β1. To investigate the effect of endogenous GATA-2 on TGF-β signaling, K562 cells were infected with lentivirus-based GATA-2 siRNA or universal scramble siRNA (siControl). Then the GFP positive cells were purified using FACS sorter and then transfected with CAGA-Luc reporter vector. As shown in Supplementary Figure S2, GATA-2 siRNA lentivirus decreased the endogenous GATA-2 protein level compared to the control cells and knockdown of endogenous GATA-2 led to increased activity of TGF-β signaling (Fig. 1I). Taken together, these results suggested that GATA2 inhibited the TGF-β1 signaling pathway.
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To determine which domain of GATA-2 is required for binding 290 Smad4, full-length GATA-2 and deletion mutants of GATA-2 were cloned 291 into vector pCMV-FLAG, expressing Flag-tagged proteins. HEK293T cells 292
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Fig. 1. GATA-2 negatively regulates TGF-β signaling pathway. (A) 293T cells were transfected with 0.2 μg of CAGA-Luc vector and different amounts of GATA-2 expression vector as indicated. Then 8 h later the cells were treated with or without 5 ng/ml TGF-β1 and 24 h later the luciferase activity was measured. (B) 293T cells were co-transfected with 0.2 μg of CAGA-Luc vector, 50 ng of the Smad protein expression vectors as indicated, and 200 ng of the expression vector for GATA2. The luciferase activity was measured 24 h later. (C) 293T cells were co-transfected with 0.2 μg CAGA-Luc, 50 ng Smad4 expression plasmid and different doses of GATA2 expression vector. Then cells were treated with or without 5 ng/ml TGF-β1 and the luciferase activity was measured 24 h later. (D) HepG2 cells were co-transfected with CAGA-Luc and the expression vectors as indicated. Then 24 h later cells were harvested for luciferase activity assay. (E) HepG2 cells were co-transfected with 0.2 μg p3TP-Lux vector and 200 ng GATA2 expression vector in the absence or presence of 5 ng/ml TGF-β1. Then the luciferase activity was measured 24 h later. (F) HaCaT cells were transfected with GATA-2 expression vector and then treated with or without TGF-β1 for 24 h. Total RNA was extracted for real-time PCR analysis and the values were normalized to GAPDH levels with the value obtained from untreated cells set as 1. (G) HaCaT infected with GATA-2 overexpression lentivirus or control lentivirus was treated with 5 ng/ml TGF-β1 and then cell proliferation was measured at the indicated time by MTS assay. The colony formation was measured 7 days later (H). (I) K562 cells were infected with lentivirus-based GATA-2 siRNA or universal scramble siRNA (siControl). Then the GFP positive cells were purified using FACS sorter and then transfected with CAGA-Luc reporter vector. Cells were treated with or without 5 ng/ml TGF-β1 and the luciferase activity was measured. Data are mean ± SD (n = 3). Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
were co-transfected with Myc tagged-Smad4 and GATA-2 deletion mutants or control vector. Immunoprecipitations were performed with anti-Flag antibody. As shown in Fig. 3A, GATA-2(N387) and GATA2(N270) can be co-immunoprecipated by Smad4, while no signal was detected in the immunoprecipitates from cells transfected with C-terminal deletion mutants of GATA-2 containing two zinc fingers. This result implicated that the GATA2 N-terminus domain (1-270aa) is required for Smad4 binding. To determine which region of Smad4 is required for GATA-2 binding, Myc-tagged full-length Smad4 and deletion mutants were co-transfected into HEK293T cells with GATA-2 expression vector. Immunoprecipitations were performed using anti-Myc antibody. As shown in Fig. 3B, all the Smad4 deletion mutants failed to interact
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3.4. GATA-2 represses TGF-β signaling through Smad4
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To investigate whether GATA-2 repressed TGF-β signaling through binding to Smad4, the Smad4-deficient breast cancer cell line MDAMB-468 cells were co-transfected with GATA-2 expression vector and CAGA-Luc reporter vector. Then the luciferase activity was measured. As shown in Fig. 4A, GATA-2 failed to repress the reporter activity in MDA-MB-468 cells, implicating that GATA-2 repressed TGF-β signaling depending on Smad4 protein.
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Fig. 2. GATA2 physically interacts with Smad4. (A) 293T cells were co-transfected with the expression vectors as indicated. After 24 h, cells lysates were prepared, treated with RNase and DNase and subjected to immunoprecipitation with anti-Flag antibody (A) or anti-Myc antibody (B). The immunoprecipitates were analyzed with the indicated antibodies. (C) K562 cell lysates were prepared and immunoprecipitations were performed with anti-Smad4 antibody using normal mouse IgG as control in the presence of RNase A and DNase I. The immunoprecipitates were analyzed with anti-GATA-2 antibody and anti-Smad4 antibody. (D) HepG2 cells were co-transfected with GFP-Smad4 and RFP-GATA-2 vectors in the presence or absence of 5 ng/ml TGF-β1. Nucleus was stained with DAPI. The locations of Smad4 (green) and GATA2 (red) were observed through confocal imaging. (E) 293T cells were transfected with the expression vectors as indicated in the presence or absence of 5 ng/ml TGF-β1. Cell lysates were immunoprecipitated by anti-Flag antibody, and the immunoprecipitates were analyzed with the indicated antibodies.
Fig. 3. Mapping the binding region of GATA-2 and Smad4. (A) HEK293 cells were transiently co-transfected with the expression vectors as indicated. After 24 h, cell lysates were prepared and immunoprecipitations were performed with anti-Flag antibody. Immunoprecipitates were analyzed with anti-Flag antibody and anti-Myc antibody. The schematic illustration of GATA-2 and its truncated fragments and the relative binding of Smad4 were shown in the upper panel. (B) HEK293 cells were co-transfected with GATA-2 and the deletion mutants of Smad4 with Myc tag. After 24 h of transfection, cell lysates were prepared and immunoprecipitations were performed with anti-Flag antibody. Immunoprecipitates were analyzed with anti-Flag antibody and anti-Myc antibody.
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Fig. 4. GATA-2 represses TGF-β signaling through Smad4. (A) The Smad4-deficient breast cancer cell line MDA-MB-468 cells were co-transfected with CAGA-Luc vector and different doses of GATA2 expression vector as indicated. Then the luciferase activity was measured. (B) 293T cells were co-transfected with CAGA-Luc vector and full length or deletion mutants of GATA2. Then the cells were harvested and analyzed for luciferase activity. (C) 293T cells were co-transfected with CAGA-Luc vector, full length GATA2 expression vector and different doses of GATA-2 ZNF deletion mutant. Then the luciferase activity was measured. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
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3.5. GATA-2 decreased the DNA-binding activity of Smad4 and recruited HDAC3
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To examine how GATA-2 regulates TGF-β signaling through Smad4, we determined the effect of GATA-2 on the phosphorylation of Smad2/3 and formation of the Smad2/3/4 complex induced by TGF-β. As shown in Fig. 5A–B, TGF-β induced a significant increase of Smad2/3 phosphorylation, and the Smad2/3/4 complex was enhanced. However, forced expression of GATA-2 did not affect the phosphorylation of Smad2/3 and the Smad2/3/4 complex formation. To test whether the effect of GATA-2 on Smad-transactivation is through direct competition with
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To define the full picture of the GATA2 domains that contribute to inhibition of TGF-β signaling, we employed a series of deletion mutants of GATA2 for specific regions and tested them for their repression activities. As shown in Fig. 4B, the deletion mutant GATA-2(1–387) severely represses the TGF-β signaling just as the wtGATA-2 did, and the C-terminal deletion mutants failed to repress TGF-β signaling. Interestingly, the deletion mutant GATA-2(N270) abolished the activity of repression although containing the binding domain with Smad4. This result indicated that the region between amino acids 270 and 387 of GATA2 containing two zinc fingers, although does not contribute to binding to Smad4, is required for repression of TGF-β signaling and some other co-repressor proteins might bind to this domain. To confirm this hypothesis, the deletion mutant containing two GATA-2 zinc finger (GATA-2(ZNF)) was co-transfected with GATA-2 expression vector and reporter vectors into HEK293T cells and the luciferase activity was measured. As Fig. 4C shows, GATA-2(ZNF) attenuated the inhibition effect of GATA-2 on TGF-β signaling activity in a dosedependent manner, suggesting that GATA-2(ZNF) might compete with full length GATA-2 to bind some repressors and released the effect of GATA-2.
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p300 for binding to Smads or a quenching of Smad transactivation by binding up of p300, the binding of p300 to Smad4 was determined. The result suggested that GATA-2 did not affect the binding between p300 and Smad4 (Fig. 5C). To determine whether GATA-2 altered the DNA binding activity of Smad proteins, EMSA was performed. As seen in Fig. 5D, incubation of the labeled Smad binding sites oligonucleotides with nuclear extract from Smad4-transfected HEK293T cells formed DNA–protein complex. However, when GATA-2 was co-transfected, the DNA-binding activity was decreased in a dose-dependent manner. This result suggested that GATA-2 decreased the DNA-binding activity of Smad proteins. To investigate how GATA-2 inhibited TGF-β signaling, histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) were used. As shown in Fig. 5E, TSA treatment attenuated the repression activity of GATA-2, while no obvious effect was observed with 5-Aza-dC treatment (Fig. 5F). Previous study reported that HDAC3 binds to the zinc finger domain of GATA-2 and plays key role in the transcriptional repression activity of GATA-2 [20]. Using IP analysis, we also found that HDAC3 can be recruited by GATA-2 on Smad4 and the GATA-2/Smad4/HDAC3 forms a complex in HEK293T cells (Supplementary Figure S5). These data suggested that GATA-2 negatively regulates TGF-β signaling through recruiting HDAC3.
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3.6. GATA-2 inhibited erythroid differentiation of K562 cells induced by 369 TGF-β signaling 370 Previously studies suggest that Activin A, a member of the TGF-β superfamily, accelerates the terminal erythroid differentiation assessed by up-regulation of hemoglobin expression in an erythroleukemia cell line K562 [21]. Because GATA-2 seems to repress TGF-β signaling, we tried to determine if GATA-2 affects TGF-β-dependant cell
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Fig. 5. GATA-2 decreased the DNA-binding activity of Smad4 and recruited histone deacetylase. (A) HaCaT cells were transfected with GATA-2 expression vector or control vector and then cells were treated with 5 ng/ml TGF-β1 for the indicated time. Lysates were prepared for Western blotting analysis with indicated antibodies. (B) 293T cells were co-transfected with the expression vectors as indicated in the presence or absence of 5 ng/ml TGF-β1. Two hours later cell lysates were immunoprecipitated with anti-Smad4 monoclonal antibody, and the immunoprecipitates were immunoblotted with indicated antibodies. (C) 293T cells were co-transfected with the expression vectors as indicated in the presence or absence of 5 ng/ml TGF-β1. Cell lysates were immunoprecipitated with anti-HA antibody, and the immunoprecipitates were then immunoblotted with antibodies indicated. (D) 293T cells were co-transfected with Myc-Smad4 and Flag-GATA2 in the presence or absence of 5 ng/ml TGF-β1 and the nuclear extracts were prepared for EMSA. Then 10 μg nuclear protein was combined with biotin-labeled SBE. The arrowhead pointed to the specifically shifted bands. For competition experiments, 100-fold excess of unlabeled oligonucleotides was added. (E) 293T cells were co-transfected with CAGA-Luc and 200 ng GATA2 expression vector or control vector. Then cells were treated with 100 ng/mL TSA or 40 μM 5-Aza-CdR (F) and 8 h later the luciferase activity was measured. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
differentiation. The lentiviruses expressing GATA-2 or control GFP were transduced into K562 cells and stable cell lines were constructed (Supplementary Figure S6). Then the cells were treated with Activin A and the hemoglobin production was measured by benzidine staining. In the control cells, Activin A treatment led to increased number of benzidine positive cells and hemoglobin production. However, GATA2 overexpression attenuates the erythroid differentiation induced by Activin A (Fig. 6A–B). Luciferase activity assay also confirmed that GATA-2 overexpression in K562 cells led to attenuated Activin A-induced TGF-β signaling pathway (Supplementary Figure S7). To confirm the effect of GATA2 on the TGF-β signaling pathway, we used lentivirus-mediated RNAi against GATA2 to knockdown endogenous GATA2 in K562 cells. As Fig. 6C–D shows, when GATA2 was
knockdown, the cells were more responsive to Activin A treatment, the percentage of benzidine positive cells and hemoglobin production were higher than the control cells, suggesting a negative role of GATA2 in the erythroid differentiation induced by TGF-β signaling pathway activation.
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Although a previous study on Evi-1 indicates an association between GATA-2 and TGFβ signal pathway [16], whether and how GATA-2 regulates TGFβ signal pathway remain unknown. Our present study provides direct evidence that GATA-2 negatively regulates TGFβ signal pathway through binding to Smad4 and then decreasing its DNA-
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binding activity. We found that GATA-2 reduced TGFβ-dependent transcription and cell growth inhibition. In Smad4-deficient cells, the repression activity on TGFβ signaling of GATA-2 was abolished. The N-terminal 270aa is required for GATA-2 to bind full length of Smad4, and the zinc finger domain of GATA-2 might recruit some co-repressors to decrease TGFβ signaling. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the formation of Smad2/3/4 complex, the DNA-binding activity of Smad4 was significantly reduced by GATA-2. Overexpression of GATA-2 in K562 cells inhibited the TGFβ-mediated erythroid differentiation and knockdown of GATA-2 accelerated the erythroid differentiation induced by TGFβ signaling activation. All these results indicated that GATA2 negatively regulate TGFβ signaling dependent on GATA-2/Smad4 interaction. Propagation of TGF-β signaling within the cell depends on a highly regulated combination of phosphorylation, complex formation, and nuclear translocation of the Smad proteins [22]. Many transcription factors and cofactors are known to be involved in the Smad signaling through directly associating with various Smad proteins. c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-β-dependent manner. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3
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Fig. 6. GATA-2 inhibited erythroid differentiation of K562 cells induced by TGF-β signaling. (A) K562 cells were infected with lentivirus-based expressing GATA-2 or control lentivirus and then GFP positive cells were purified by FACS sorter. Then the cells were treated with 50 ng/ml Activin A for the indicated time. Then the erythroid differentiation was investigated with benzidine staining. Total RNA was extracted from cells treated for 8 days and the expression level of β-hemoglobin (HBB) was analyzed by real-time PCR (B). (C) K562 cells were infected with lentivirus-based GATA-2 siRNA (siGATA-2-1, siGATA-2-2) or universal scramble siRNA (siControl). Cells were treated with 50 ng/ml Activin A for the indicated time. Then the erythroid differentiation was investigated with benzidine staining. Total RNA were extracted from cells treated for 8 days and the expression level of β-hemoglobin (HBB) was analyzed by real-time PCR (D). Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-β signaling [23]. Another oncogene v-ErbA was also found to associate with the Smad4, sequester Smad4 in the cytoplasm, and then antagonizing TGF-β-induced cell growth inhibition [24]. The TGF-β inducible protein Tsc-22 was reported to enhance TGF-β signaling by associating with Smad4 and then induce erythroid cell differentiation. Tsc-22 does not seem to affect subcellular translocation or phosphorylation of Smad proteins. Tsc-22 might enhance Smad activity by recruiting various transcriptional regulators including p300 and HDAC [25]. Furthermore, the homeoprotein DLX1 interacts with Smad4 and blocks a signaling pathway from Activin A in hematopoietic cells probably through sequestering transcriptional activator complex from the Smad3/Smad4 complex [26]. In our present study, we found that although GATA-2 significantly decreased the DNA-binding activity of Smad protein, GATA-2 did not affect the Smad2/3 phosphorylation, the Smad2/3/4 complex formation, the nuclear translocation of Smad4, and the binding between Smad4 and p300 in response to TGFβ, suggesting that GATA-2 inhibits TGFβ signaling through recruiting some co-repressors. Furthermore, we found that the regions containing 2 zinc finger (270aa–387aa) is essential for the repression activity of GATA-2, suggesting that this region might contribute to recruiting some co-repressors to suppress Smad4 activity. Very interestingly, a previous study suggests that HDAC3 interacts with GATA-2 and repressed
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All authors declare no competing financial interests.
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Authors' contributions
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Xiao-Ming Dong, Rong-Hua Yin, Yang Yang, Zhi-Wei Feng, HongMei Ning, Lan Dong, Wei-Wei Zheng, Yu-Xin Jia,Yi-Nan Jiang, En-Dong Liu, Hui Chen, Yi-Qun Zhan, Miao Yu, and Chang-Hui Ge performed the experiments. Dong Li, Liu-Jun Tang and Jian Wang analyzed the data. Chang-Yan Li designed and performed experiments, and contributing to writing the manuscript. Xiao-Ming Yang designed the experiments and contributed to writing the manuscript.
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Acknowledgment
We thank Dr. Qinong Ye (Beijing Institute of Biotechnology, Beijing, 512 China) for providing the human full-length Smad4 (pcDNA3.1-Smad4 513 (his/myc)), 3 × CAGA-Luc, and 3TP-Luc plasmids. 514 515
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[1] N.P. Rodrigues, A.J. Tipping, Z. Wang, T. Enver, Int. J. Biochem. Cell Biol. 44 (2012) 457–460. [2] C. Vicente, A. Conchillo, M.A. Garcia-Sanchez, M.D. Odero, Crit. Rev. Oncol. Hematol. 82 (2012) 1–17. [3] C. Heyworth, K. Gale, M. Dexter, G. May, T. Enver, Genes Dev. 13 (1999) 1847–1860. [4] F.Y. Tsai, G. Keller, F.C. Kuo, M. Weiss, J. Chen, M. Rosenblatt, F.W. Alt, S.H. Orkin, Nature 371 (1994) 221–226. [5] K. Ohneda, M. Yamamoto, Acta Haematol. 108 (2002) 237–245. [6] F.Y. Tsai, S.H. Orkin, Blood 89 (1997) 3636–3643. [7] K. Kitajima, M. Masuhara, T. Era, T. Enver, T. Nakano, EMBO J. 21 (2002) 3060–3069. [8] K. Briegel, K.C. Lim, C. Plank, H. Beug, J.D. Engel, M. Zenke, Genes Dev. 7 (1993) 1097–1109. [9] C. Perry, H. Soreq, Eur. J. Biochem. 269 (2002) 3607–3618. [10] R. Ferreira, K. Ohneda, M. Yamamoto, S. Philipsen, Mol. Cell. Biol. 25 (2005) 1215–1227. [11] X. Fan, G. Valdimarsdottir, J. Larsson, A. Brun, M. Magnusson, S.E. Jacobsen, P. ten Dijke, S. Karlsson, J. Immunol. 168 (2002) 755–762. [12] M.C. Dickson, J.S. Martin, F.M. Cousins, A.B. Kulkarni, S. Karlsson, R.J. Akhurst, Development 121 (1995) 1845–1854. [13] Y. Zermati, B. Varet, O. Hermine, Exp. Hematol. 28 (2000) 256–266. [14] Y. Zermati, S. Fichelson, F. Valensi, J.M. Freyssinier, P. Rouyer-Fessard, E. Cramer, J. Guichard, B. Varet, O. Hermine, Exp. Hematol. 28 (2000) 885–894. [15] X. Bai, J. Kim, Z. Yang, M.J. Jurynec, T.E. Akie, J. Lee, J. LeBlanc, A. Sessa, H. Jiang, A. DiBiase, Y. Zhou, D.J. Grunwald, S. Lin, A.B. Cantor, S.H. Orkin, L.I. Zon, Cell 142 (2010) 133–143. [16] T. Sato, S. Goyama, E. Nitta, M. Takeshita, M. Yoshimi, M. Nakagawa, M. Kawazu, M. Ichikawa, M. Kurokawa, Cancer Sci. 99 (2008) 1407–1413. [17] T. Ravasi, H. Suzuki, C.V. Cannistraci, S. Katayama, V.B. Bajic, K. Tan, A. Akalin, S. Schmeier, M. Kanamori-Katayama, N. Bertin, P. Carninci, C.O. Daub, A.R. Forrest, J. Gough, S. Grimmond, J.H. Han, T. Hashimoto, W. Hide, O. Hofmann, A. Kamburov, M. Kaur, H. Kawaji, A. Kubosaki, T. Lassmann, E. van Nimwegen, C.R. MacPherson, C. Ogawa, A. Radovanovic, A. Schwartz, R.D. Teasdale, J. Tegner, B. Lenhard, S.A. Teichmann, T. Arakawa, N. Ninomiya, K. Murakami, M. Tagami, S. Fukuda, K. Imamura, C. Kai, R. Ishihara, Y. Kitazume, J. Kawai, D.A. Hume, T. Ideker, Y. Hayashizaki, Cell 140 (2010) 744–752. [18] J. Wang, K. Huo, L. Ma, L. Tang, D. Li, X. Huang, Y. Yuan, C. Li, W. Wang, W. Guan, H. Chen, C. Jin, J. Wei, W. Zhang, Y. Yang, Q. Liu, Y. Zhou, C. Zhang, Z. Wu, W. Xu, Y. Zhang, T. Liu, D. Yu, L. Chen, D. Zhu, X. Zhong, L. Kang, X. Gan, X. Yu, Q. Ma, J. Yan, L. Zhou, Z. Liu, Y. Zhu, T. Zhou, F. He, X. Yang, Mol. Syst. Biol. 7 (2011) 536. [19] S. Itoh, F. Itoh, M.J. Goumans, P. Ten Dijke, Eur. J. Biochem. 267 (2000) 6954–6967. [20] Y. Ozawa, M. Towatari, S. Tsuzuki, F. Hayakawa, T. Maeda, Y. Miyata, M. Tanimoto, H. Saito, Blood 98 (2001) 2116–2123. [21] Y. Fukuchi, M. Kizaki, K. Yamato, C. Kawamura, A. Umezawa, J. Hata, T. Nishihara, Y. Ikeda, Oncogene 20 (2001) 704–713. [22] J. Massague, Y.G. Chen, Genes Dev. 14 (2000) 627–644. [23] S. Akiyoshi, H. Inoue, J. Hanai, K. Kusanagi, N. Nemoto, K. Miyazono, M. Kawabata, J. Biol. Chem. 274 (1999) 35269–35277. [24] R.A. Erickson, X. Liu, Mol. Biol. Cell 20 (2009) 1509–1519. [25] S.J. Choi, J.H. Moon, Y.W. Ahn, J.H. Ahn, D.U. Kim, T.H. Han, Mol. Cell. Biochem. 271 (2005) 23–28. [26] S. Chiba, K. Takeshita, Y. Imai, K. Kumano, M. Kurokawa, S. Masuda, K. Shimizu, S. Nakamura, F.H. Ruddle, H. Hirai, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 15577–15582. [27] A. Moustakas, S. Souchelnytskyi, C.H. Heldin, J. Cell Sci. 114 (2001) 4359–4369. [28] N.O. Fortunel, A. Hatzfeld, J.A. Hatzfeld, Blood 96 (2000) 2022–2036. [29] H. Kamesaki, G.Y. Michaud, S.G. Irving, N. Suwabe, S. Kamesaki, M. Okuma, J. Cossman, Blood 87 (1996) 999–1005. [30] L. Attisano, J.L. Wrana, Science 296 (2002) 1646–1647. [31] L.M. Wakefield, A.B. Roberts, Curr. Opin. Genet. Dev. 12 (2002) 22–29. [32] J. Massague, Nat. Rev. Mol. Cell Biol. 1 (2000) 169–178. [33] M.S. Kumar, D.C. Hancock, M. Molina-Arcas, M. Steckel, P. East, M. Diefenbacher, E. Armenteros-Monterroso, F. Lassailly, N. Matthews, E. Nye, G. Stamp, A. Behrens, J. Downward, Cell 149 (2012) 642–655.
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GATA-2-mediated transcription, suggesting a potential role of GATA-2/ HDAC3 complex in gene repression [20]. The region containing whole 2 zinc fingers was required for HDAC3 binding [20]. Our result suggests that HDAC3 can be recruited to Smad4 by GATA-2 and a broad spectrum inhibitor of histone deacetylation transferase TSA attenuated the repression activity of GATA-2, suggesting an involvement of HDAC proteins. Whether HDAC3 is essential for GATA-2 to suppress Smad4 activity remains to be determined. Since Smad4 is the co-Smad protein in TGFβ signal pathway, it is easy to suppose that GATA-2 repressed the TGFβ signal pathway induce by various members of TGFβ superfamily such as TGFβ1, Activin A, and BMPs. In our present study, we found that GATA-2 inhibited TGFβ1induced transcription and Activin A-mediated erythroid differentiation. Whether GATA-2 regulates BMP-dependent TGFβ signaling pathway needs further investigation. In addition, the family of Smad proteins have MH1 and MH2 domains in common [27]. Our study suggests that GATA-2 binds to the full length of Smad4 protein but not any of the deletion mutants. Also we showed that GATA-2 specifically interacts with Smad4, but does not interact with other Smad proteins including Smad1, 2, 3, 5, and 7. Interestingly, we also investigated the effect of GATA-1 on TGFβ signaling pathway and no effect was observed. These data indicated that the negative regulation of TGFβ signaling pathway is specific for GATA-2, but not other GATA family member, and the interaction between GATA-2 and Smad4 is specific for Smad4, but not other Smad proteins. The low homology at the N-terminal between GATA-1 and GATA-2 protein might contribute to the difference. But how GATA-2 specifically interacts with Smad4 remains further investigation. TGFβ is one of the key regulators of erythropoiesis because it is involved in the control of both early and later stage of erythroid progenitor cell development [28]. GATA-2 is a negative regulator of erythropoiesis and makes major contributions to the proliferation of hematopoietic stem cells [29]. Our present study suggests that overexpression of GATA-2 inhibited TGFβ-induced erythroid differentiation and knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation in K562 cells. However, whether GATA-2/Smad4 interaction plays a role in erythroid differentiation in vivo needs further investigation. More importantly, the TGF-β signaling pathway is a major signaling that controls cell proliferation, differentiation, and tumor suppression [30–32]. GATA-2 negatively regulates TGF-β signaling pathway in HaCaT cells and attenuates the growth inhibition of TGF-β, indicating a potential role of GATA-2 in tumor development. In fact, a recent study suggests that the GATA-2 transcriptional network is requisite for Ras oncogene-driven non-small cell lung cancer and gata2 loss dramatically reduced tumor development [33]. Whether GATA-2/Smad4 interaction plays roles in tumor development remains an interesting question. Taken together, we provide direct evidence that GATA-2 is a negative regulator of TGFβ signaling pathway through binding to Smad4.
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Contents lists available at ScienceDirect
Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
Xiao-Ming Dong a,b,1, Rong-Hua Yin b,1, Yang Yang c,1, Zhi-Wei Feng b, Hong-Mei Ning d, Lan Dong e, Wei-Wei Zheng b, Liu-jun Tang b, Jian Wang b, Yu-Xin Jia a, Yi-Nan Jiang f, En-Dong Liu f, Hui Chen b, Yi-Qun Zhan b, Miao Yu b, Chang-Hui Ge b, Chang-Yan Li b,f,⁎, Xiao-Ming Yang a,b,⁎
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GATA-2 inhibits transforming growth factor-β signaling pathway through interaction with Smad4
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School of Chemical Engineering and Technology, Department of Pharmaceutical Engineering, Tianjin University, Tianjin 300072, China State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China c Purdue University, Department of Biological Sciences, 915W. State Street, West Lafayette, IN 47907-2054, United States d Department of Hematopoietic Stem Cell Transplantation, Affiliated Hospital to Academy of Military Medical Sciences, Beijing 100071, China e Department of Anesthesiology, General Hospital of Chinese People's Armed Police Forces, Beijing, China f An Hui Medical University, Hefei 230032, China
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Article history: Received 13 January 2014 Accepted 28 January 2014 Available online xxxx
GATA-2, a member of zinc finger GATA transcription factor family, plays key role in the hematopoietic stem cells self-renewal and differentiation. The transforming growth factor-β (TGFβ) signaling pathway is a major signaling network that controls cell proliferation, differentiation and tumor suppression. Here we found that GATA-2 negatively regulated TGF-β signaling pathway in Smad4-dependent manner. GATA-2 specifically interacts with Smad4 with its N-terminal while the zinc finger domain of GATA-2 is essential for negative regulation of TGFβ. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the complex Smad2/3/4 formation in response to TGFβ, the DNA binding activity of Smad4 was decreased significantly by GATA-2 overexpression. Overexpression of GATA-2 in K562 cells led to reduced TGFβ-induced erythroid differentiation while knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation. All these results suggest that GATA-2 is a novel negative regulator of TGFβ signal pathway. © 2014 Published by Elsevier Inc. 22
Keywords: GATA-2 Smad4 TGF-β signaling pathway Erythroid differentiation
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GATA-2 is a member of the GATA family transcription factors and plays essential roles in hematopoietic stem and progenitor cell compartments [1–3]. GATA-2 knock-out mice die around embryonic days 9.5–10.5 due to a pan-hematopoietic defect, providing evidence that GATA-2 is a key regulator of development, maintenance, and/or function of hematopoietic stem cells [4,5]. The in vitro experiment using GATA-2−/− embryonic stem cells showed that GATA-2 was necessary for proliferation/survival of early hematopoietic cells [6]. Ectopic expression of GATA-2 in erythroid precursors promotes proliferation and blocks erythroid differentiation [7,8]. Expression of GATA-2 precedes that of another family member, GATA-1, and must decrease as GATA-1 expression increases to enable erythroid differentiation [9,10]. These
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Abbreviations: DMEM, Dulbecco's modified Eagle's medium; Epo, erythropoietin; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HDAC3, histone deacetylase 3; siRNA, small interfering RNA; TGF-β, transforming growth factor-β. ⁎ Corresponding authors at: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China. Tel./fax: +86 10 68176833. E-mail addresses: [email protected] (C.-Y. Li), [email protected] (X.-M. Yang). 1 These authors contributed equally to this work.
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data indicate that GATA-2 is essential for appropriate expansion and survival of early hematopoietic cells, and plays a negative role in erythroid differentiation. TGF-β signaling plays an important role in maintaining normal hematopoiesis. In human hematopoietic cells, blocking TGF-β signaling enhanced the survival, proliferation, and growth kinetics of human HSCs [11]. Homozygous deletion of TGF-β resulted in embryonic lethality with defective yolk sac vasculogenesis secondary to abnormal endothelial differentiation and defective hematopoiesis with a reduced yolk sac erythroid cell number [12]. Using hematopoietic cell lines as models, TGF-β has been shown to be an inducer of erythroid differentiation, even stronger than Epo at the cellular level [13]. In human cord blood CD36+ erythroid progenitor cells, TGF-β markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression, while inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle [14]. Moreover, in immature adult hematopoietic progenitor cells, phospho-Smad2/3 associates with TIF1γ to promote erythroid differentiation [15]. Given the key roles played by GATA-2 and TGF-β signal pathway in the erythropoiesis, it is postulated that there could be an important functional interaction between them. A previous study on Evi-1 suggests an association between GATA-2 and TGFβ signal pathway.
0898-6568/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.cellsig.2014.01.028
Please cite this article as: X.-M. Dong, et al., Cell. Signal. (2014), http://dx.doi.org/10.1016/j.cellsig.2014.01.028
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HEK293T cells, HuH78 cells and HepG2 cells were maintained in DMEM (Gibco Invitrogen, CA) with 10% fetal calf serum (FCS), while K562 cells were cultured in RPMI 1640 medium (Gibco) with 10% FCS. All the cells were cultured in a 37 °C incubator with 5% CO2 in the presence of 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 g/l sodium bicarbonate and 10 mM HEPES. TGF-β was purchased from Peprotech (100-21C).
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Serial deletions of GATA-2 were amplified by PCR, using a full-length cDNA encoding GATA-2 as template and the PCR products were cloned into plasmid vector pFLAG-CMV-2. The human full-length Smad4 (pcDNA3.1-Smad4 (his/myc)) and the reporter vectors including 3 × CAGA-Luc, PAI-Luc, and 3TP were kindly gifted from Dr. Qinong Ye (Beijing Institute of Biotechnology, Beijing, China).
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Cells were washed once in PBS, and lysed in 1 ml of lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), and centrifuged for 15 min at 12,000 rpm at 4 °C. The supernatant was transferred to a fresh tube, and immunoprecipitations were performed with anti-Flag affinity gel (A2220, Sigma) or anti-Myc antibody (Santa Cruz) followed by adsorption to protein A/G plus-agarose beads (sc-2003, Santa Cruz). After SDS-PAGE, the samples were transferred onto polyvinylidene difluoride membranes (Amersham life science) and probed with a variety of antibodies. For detecting interaction of endogenous GATA-2 with Smad4, K562 cells were lysed in 0.5 ml lysis buffer and immunoprecipitated with anti-Smad4 antibody or control serum (Santa Cruz). After extensive washing with the lysis buffer, the immunoprecipitates were resolved by SDS-PAGE, followed by Western blot analysis using the anti-GATA-2 antibody.
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To analyze the subcellular location of exogenous GATA-2 and Smad4, HepG2 cells were seeded in 6-well plates, cultured in DMEM supplemented with 10% fetal bovine serum and transfected with the pEGFP-N1- or pDsRED-N1-based vectors as indicated. Twenty four hours later, the cells were fixed for 30 min at room temperature with 4% paraformaldehyde in PBS and stained with Hoechst 33258. Confocal imaging was performed using Zeiss 510 META system. The green fluorescence was excited at 488 nm with 505–530 nm barrier filter and red fluorescence was simultaneously excited at 543 nm with 560 nm barrier filter.
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2.6. Lentiviral vector construction and production
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For construction of lentivirus-mediated GATA-2 overexpression, GATA-2 full length was cloned into the pBPLV vector to generate pBPLV-EDAG recombinant vector expressing GATA-2-GFP fusion protein. For construction of lentivirus-mediated RNA interference, two siRNA oligos against EDAG were synthesized in GenePharma Biotechnology, the sequences are as follows: si GATA-2-1: 5′-GGC TCG TTC CTG TTC AGA ATT-3′ and si GATA-2-2: 5′-GGA GGA GGA TTG TGF TGA TTT-3′. The siRNA sequences were cloned into a psicoR-GFP vector to generate siGATA-2 lentivirus. The siGATA-2 lentivirus expresses CMV promoter-driven GFP protein and U6 promoter-driven siRNA targeting GATA-2. A negative control siRNA was cloned into psicoR-GFP as a control. For production of lentivirus, HEK293T cells were cotransfected with transfer vectors pBPLV-GATA-2 or psicoR-GFP-si GATA-2 with the packaging vectors including pLP1, pLP2 and pLP-VSVG. Lentiviruses were harvested 72 h after transfection, passed through a 0.45-μm filter, and concentrated 100-fold by ultracentrifugation through 20% sucrose cushion (100,000 g for 90 min; 4 °C). Titers of viral stock were determined on K562 cells transduced by spinoculation (1000 g for 90 min; 30 °C) and analyzed by FACS 48 h later. GFP-positive cells were sorted using fluorescence-activated cell sorter (FACS; BD Biosciences).
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2.7. Colony formation assay
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Cells were transfected with indicated plasmids with Lipofectamine 2000 (Invitrogen), and then plated on 60-mm tissue culture dish (2 × 103 cells per well) and cultured in DMEM supplemented with 10% FBS. The cells were allowed to grow for 10 days in the presence of neomycin with medium changes every 2 days, after which they were washed, fixed with methanol, and stained with Giemsa (Sigma). Colonies 2-mm or greater in size were scored. All experiments were conducted three times.
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2.8. MTT assay
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1 × 103 cells were seeded in each well of 96-well plates and left overnight to adhere. Absorbance was determined by using CellTiter 96 Aqueous One Solution Reagent (Promega) according to the manufacturer's protocol at indicated time points. All experiments were conducted three times.
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For Western blotting, cells were lysed with M-PER® Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA). Then, Western blot analysis was performed according to standard procedures. Antibodies were used at the following concentrations: GATA-2 antiserum, 1: 1000; Smad4 (sc-47725, Santa Cruz), 1:1000; Myc (sc-40, Santa Cruz), 1:1000; Flag (F 3165, Sigma), 1:5000; GAPDH (sc-47778, Santa Cruz), 1:1000. Chemiluminescent detection was conducted using supersignal substrate (Pierce) according to the manufacturer's specifications.
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Evi-1-deficient P-Sp-derived cells showed a severely decreased colony forming capacity and in these cells the expression level of GATA-2 is up-regulated. Blocking of TGF-β signaling is also able to recover the hematopoietic defect of Evi-1-deficient P-Sp cells [16]. These findings suggest that Evi-1 promotes hematopoietic stem/progenitor expansion during embryogenesis through up-regulation of GATA-2 and inhibition of TGF-β signaling [16]. However, whether GATA-2 regulates signaling and the molecular mechanism remains unknown. In a study about building a global atlas of combinatorial transcriptional regulation in mouse and man, Smad4 was found to interact with GATA-2 [17]. In our recent study on the protein interaction network of the human liver, we also identified the interaction between Smad4 and GATA-2 [18]. Since Smad4 is the co-Smads of TGF-β signal pathway [19], it is easy to ask whether GATA-2 regulates TGF-β signal pathway. In the present study, we identified that GATA-2 interacts directly with Smad4, not with other Smad proteins such as Smad1,2, 3, 5 and Smad8, suggesting a specific interaction between GATA-2 and Smad4. GATA-2 negatively regulated TGF-β signaling and attenuates the growth inhibition induced by TGF-β. Furthermore, we found that GATA-2 could recruit HDAC3 to Smad4 and decreased the DNAbinding activity of Smad4. In a erythroleukemia cell line K562 cells, overexpression of GATA-2 inhibited the erythroid differentiation induced by TGF-β while knockdown of endogenous GATA-1 accelerated the TGF-β-induced erythroid differentiation. These results suggested a novel role of GATA-2 in regulating TGF-β signaling pathway.
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Total RNA was reverse-transcribed and amplified using reverse transcription and PCR kits, respectively (Promega Corp., Madison, WI, U.S.). Real-time RT-PCR was performed by Bio-Rad IQ5 (Bio-Rad, U.S.). The abundance of mRNA of each gene was normalized to GAPDH. The sequences of the primers are provided in Supplementary Table S1.
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Erythroid differentiation of K562 cells was assessed by the benzidine cytochemical test and the benzidine-positive cells represented more mature erythroid cells. Briefly, cells were washed twice with ice-cold PBS and then resuspended in ice-cold PBS. The benzidine solution (3 ml), which contained hydrogen peroxide (final concentration 0.0012%), was added and incubated for 10 min at room temperature. Benzidine-positive cells were quantitated under the microscope. At least 100 cells were counted in triplicate for each condition.
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All experiments were performed at least three times. Data were reported as means ± SD and the statistical significance was assessed by one-way analysis of variance followed by the Students–Newman– Keuls tests. A value of p ≤ 0.05 was considered to be significant.
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3.1. GATA-2 is a negative regulator of TGFβ signaling pathway
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We first investigated the effect of GATA-2 on the TGF-β signaling pathway. Cells were transiently transfected with the luciferase gene driven by the CAGA element (3 × CAGA-Luc) and GATA-2 expression vector in the presence or absence of TGF-β1. As shown in Fig. 1A, TGFβ1 induced the luciferase activity dramatically, reaching ~ 4.5fold at the concentration of 5 ng/ml. This increase was significantly reduced when GATA2 was cotransfected, in a manner dependent on the GATA2 dose. We then tested whether the similar repression was seen when R-Smads (Smad2, Smad3) or co-Smad (Smad4) proteins was overexpressed. The CAGA-Luc activity was highly enhanced when the R-Smads or co-Smads were cotransfected with CAGA-Luc. However, GATA2 dramatically repressed the transactivation by these Smads (Fig. 1B). We further investigated the effect of GATA-2 on Smad4mediated transcription in detail. GATA-2 inhibited Smad4-mediated transcription in a dose-dependent manner (Fig. 1C). Another GATA family member GATA-1 had no effect on the CAGA-Luc activity (Supplementary Figure S1), suggesting a specific effect of GATA-2 on TGF-β signaling pathway. It is well known that 3TP-Luc, a TGF-β1-responsive reporter plasmid carrying the plasminogen-activator inhibitor-1 promoter, is highly responsive to overexpression Smad2/3/4 and TGF-β in HepG2 cells. We used this system to evaluate the effect of GATA2 on transactivation by overexpressed Smad2/3/4 (Fig. 1D). The results clearly showed that co-transfection of GATA2 repressed the 3TP-Luc induced by Smad2/3/4 almost to the control level. Next, we examined whether GATA2 also repressed the transcriptional response induced by TGFβ1 stimulation. Just as Fig. 1E shows, GATA2 repressed the transcriptional activities induced by TGF-β1 stimulation. Real time PCR analysis
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3.2. GATA2 physically interacts with Smad4 and TGF-β1 treatment enhances the interaction between them
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Our previous study suggested that GATA-2 is a candidate binding protein of Smad4. To assess whether the interaction between GATA-2 and Smad4 is specific, we investigated the interaction between GATA2 and Smad proteins including Smad1, 2, 3, 4, 5 and Smad7 using yeast two hybrid. As Supplementary Figure S3 shows, GATA2 was detected to be a binding protein of Smad4 not other Smad proteins. To evaluate the interaction between GATA-2 and Smad4 in mammalian cells, cell lysates from HEK293T cells co-transfected with GATA-2FLAG and Myc epitope-tagged Smad4 expression vectors were subjected to immunoprecipitation with anti-Flag antibody. The efficiency of each transfection was monitored by GFP cotransfection and all of the transfections displayed similar efficiency (data not shown). As shown in Fig. 2A, Smad4 was specifically immunoprecipitated with GATA-2 by anti-Flag antibody. In contrast, no Smad4 protein was observed in FLAG antibody immunoprecipitates from cells transfected with Smad4-Myc and pCMV-FLAG. The interaction between GATA-2 and Smad4 was confirmed by reverse co-IP in HEK293T cells (Fig. 2B). The interaction between endogenous GATA-2 and Smad4 was further confirmed at physiological conditions in K562 cells. Immunoprecipitations were performed with anti-Smad4 antibody using normal mouse IgG as control. As shown in Fig. 2C, endogenous GATA2 can be immunoprecipitated by anti-Smad4 antibody. The possibility of ribosomal RNA or DNA mediating the binding was ruled out since the binding was not altered when RNase A and DNase were included throughout the cell lysis and IP. Given the observed physical interaction between overexpressed GATA-2 and Smad4 in HEK293T cells, we examined the intracellular location of the endogenous proteins in HepG2 cells in the presence or absence of TGF-β1. In the absence of TGF-β1, endogenous Smad4 mainly localizes in the cytoplasm, whereas GATA-2 is a nuclear diffuse protein, which was consistent with the previous studies (Supplementary Figure S4). After TGF-β1 treatment, Smad4 was induced to a remarkable nuclear translocation, which showed a complete co-localization with GATA-2 (Fig. 2D). Co-immunoprecipitation assay suggested that TGFβ1 treatment enhanced the interaction between GATA-2 and Smad4 (Fig. 2E). In K562 cells which express both GATA-2 and Smad4 endogenously, the same results were obtained (data not shown).
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3.3. Mapping the binding region of GATA-2 and Smad4
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Luciferase assays were carried out at 24 h posttransfection with the Dual-Luciferase Reporter Assay System (Promega). Transfection efficiencies were normalized using cotransfected plasmid pRL-TK measured by Renilla luciferase activity (Promega). Luciferase activity was measured 24 h later.
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also suggested that GATA-2 repressed the gene expression induced by TGF-β1 (Fig. 1F). Using lentivirus-mediated transfer, the full length coding sequence of GATA2 was stably expressed in human epithelial cell line HaCaT. The cells were treated with TGF-β1 and the proliferation was assessed by MTS analysis and colony formation assay. Just as shown in Fig. 1G–H, the growth of control HaCaT cells was significantly inhibited in the presence of TGF-β1, and GATA2 expressing HaCaT cells was resistant to the inhibition of TGF-β1. To investigate the effect of endogenous GATA-2 on TGF-β signaling, K562 cells were infected with lentivirus-based GATA-2 siRNA or universal scramble siRNA (siControl). Then the GFP positive cells were purified using FACS sorter and then transfected with CAGA-Luc reporter vector. As shown in Supplementary Figure S2, GATA-2 siRNA lentivirus decreased the endogenous GATA-2 protein level compared to the control cells and knockdown of endogenous GATA-2 led to increased activity of TGF-β signaling (Fig. 1I). Taken together, these results suggested that GATA2 inhibited the TGF-β1 signaling pathway.
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To determine which domain of GATA-2 is required for binding 290 Smad4, full-length GATA-2 and deletion mutants of GATA-2 were cloned 291 into vector pCMV-FLAG, expressing Flag-tagged proteins. HEK293T cells 292
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Fig. 1. GATA-2 negatively regulates TGF-β signaling pathway. (A) 293T cells were transfected with 0.2 μg of CAGA-Luc vector and different amounts of GATA-2 expression vector as indicated. Then 8 h later the cells were treated with or without 5 ng/ml TGF-β1 and 24 h later the luciferase activity was measured. (B) 293T cells were co-transfected with 0.2 μg of CAGA-Luc vector, 50 ng of the Smad protein expression vectors as indicated, and 200 ng of the expression vector for GATA2. The luciferase activity was measured 24 h later. (C) 293T cells were co-transfected with 0.2 μg CAGA-Luc, 50 ng Smad4 expression plasmid and different doses of GATA2 expression vector. Then cells were treated with or without 5 ng/ml TGF-β1 and the luciferase activity was measured 24 h later. (D) HepG2 cells were co-transfected with CAGA-Luc and the expression vectors as indicated. Then 24 h later cells were harvested for luciferase activity assay. (E) HepG2 cells were co-transfected with 0.2 μg p3TP-Lux vector and 200 ng GATA2 expression vector in the absence or presence of 5 ng/ml TGF-β1. Then the luciferase activity was measured 24 h later. (F) HaCaT cells were transfected with GATA-2 expression vector and then treated with or without TGF-β1 for 24 h. Total RNA was extracted for real-time PCR analysis and the values were normalized to GAPDH levels with the value obtained from untreated cells set as 1. (G) HaCaT infected with GATA-2 overexpression lentivirus or control lentivirus was treated with 5 ng/ml TGF-β1 and then cell proliferation was measured at the indicated time by MTS assay. The colony formation was measured 7 days later (H). (I) K562 cells were infected with lentivirus-based GATA-2 siRNA or universal scramble siRNA (siControl). Then the GFP positive cells were purified using FACS sorter and then transfected with CAGA-Luc reporter vector. Cells were treated with or without 5 ng/ml TGF-β1 and the luciferase activity was measured. Data are mean ± SD (n = 3). Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
were co-transfected with Myc tagged-Smad4 and GATA-2 deletion mutants or control vector. Immunoprecipitations were performed with anti-Flag antibody. As shown in Fig. 3A, GATA-2(N387) and GATA2(N270) can be co-immunoprecipated by Smad4, while no signal was detected in the immunoprecipitates from cells transfected with C-terminal deletion mutants of GATA-2 containing two zinc fingers. This result implicated that the GATA2 N-terminus domain (1-270aa) is required for Smad4 binding. To determine which region of Smad4 is required for GATA-2 binding, Myc-tagged full-length Smad4 and deletion mutants were co-transfected into HEK293T cells with GATA-2 expression vector. Immunoprecipitations were performed using anti-Myc antibody. As shown in Fig. 3B, all the Smad4 deletion mutants failed to interact
with GATA-2, suggesting that the full length of Smad4 protein is re- 306 quired for GATA-2 binding. 307
3.4. GATA-2 represses TGF-β signaling through Smad4
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To investigate whether GATA-2 repressed TGF-β signaling through binding to Smad4, the Smad4-deficient breast cancer cell line MDAMB-468 cells were co-transfected with GATA-2 expression vector and CAGA-Luc reporter vector. Then the luciferase activity was measured. As shown in Fig. 4A, GATA-2 failed to repress the reporter activity in MDA-MB-468 cells, implicating that GATA-2 repressed TGF-β signaling depending on Smad4 protein.
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Fig. 2. GATA2 physically interacts with Smad4. (A) 293T cells were co-transfected with the expression vectors as indicated. After 24 h, cells lysates were prepared, treated with RNase and DNase and subjected to immunoprecipitation with anti-Flag antibody (A) or anti-Myc antibody (B). The immunoprecipitates were analyzed with the indicated antibodies. (C) K562 cell lysates were prepared and immunoprecipitations were performed with anti-Smad4 antibody using normal mouse IgG as control in the presence of RNase A and DNase I. The immunoprecipitates were analyzed with anti-GATA-2 antibody and anti-Smad4 antibody. (D) HepG2 cells were co-transfected with GFP-Smad4 and RFP-GATA-2 vectors in the presence or absence of 5 ng/ml TGF-β1. Nucleus was stained with DAPI. The locations of Smad4 (green) and GATA2 (red) were observed through confocal imaging. (E) 293T cells were transfected with the expression vectors as indicated in the presence or absence of 5 ng/ml TGF-β1. Cell lysates were immunoprecipitated by anti-Flag antibody, and the immunoprecipitates were analyzed with the indicated antibodies.
Fig. 3. Mapping the binding region of GATA-2 and Smad4. (A) HEK293 cells were transiently co-transfected with the expression vectors as indicated. After 24 h, cell lysates were prepared and immunoprecipitations were performed with anti-Flag antibody. Immunoprecipitates were analyzed with anti-Flag antibody and anti-Myc antibody. The schematic illustration of GATA-2 and its truncated fragments and the relative binding of Smad4 were shown in the upper panel. (B) HEK293 cells were co-transfected with GATA-2 and the deletion mutants of Smad4 with Myc tag. After 24 h of transfection, cell lysates were prepared and immunoprecipitations were performed with anti-Flag antibody. Immunoprecipitates were analyzed with anti-Flag antibody and anti-Myc antibody.
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Fig. 4. GATA-2 represses TGF-β signaling through Smad4. (A) The Smad4-deficient breast cancer cell line MDA-MB-468 cells were co-transfected with CAGA-Luc vector and different doses of GATA2 expression vector as indicated. Then the luciferase activity was measured. (B) 293T cells were co-transfected with CAGA-Luc vector and full length or deletion mutants of GATA2. Then the cells were harvested and analyzed for luciferase activity. (C) 293T cells were co-transfected with CAGA-Luc vector, full length GATA2 expression vector and different doses of GATA-2 ZNF deletion mutant. Then the luciferase activity was measured. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
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To examine how GATA-2 regulates TGF-β signaling through Smad4, we determined the effect of GATA-2 on the phosphorylation of Smad2/3 and formation of the Smad2/3/4 complex induced by TGF-β. As shown in Fig. 5A–B, TGF-β induced a significant increase of Smad2/3 phosphorylation, and the Smad2/3/4 complex was enhanced. However, forced expression of GATA-2 did not affect the phosphorylation of Smad2/3 and the Smad2/3/4 complex formation. To test whether the effect of GATA-2 on Smad-transactivation is through direct competition with
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To define the full picture of the GATA2 domains that contribute to inhibition of TGF-β signaling, we employed a series of deletion mutants of GATA2 for specific regions and tested them for their repression activities. As shown in Fig. 4B, the deletion mutant GATA-2(1–387) severely represses the TGF-β signaling just as the wtGATA-2 did, and the C-terminal deletion mutants failed to repress TGF-β signaling. Interestingly, the deletion mutant GATA-2(N270) abolished the activity of repression although containing the binding domain with Smad4. This result indicated that the region between amino acids 270 and 387 of GATA2 containing two zinc fingers, although does not contribute to binding to Smad4, is required for repression of TGF-β signaling and some other co-repressor proteins might bind to this domain. To confirm this hypothesis, the deletion mutant containing two GATA-2 zinc finger (GATA-2(ZNF)) was co-transfected with GATA-2 expression vector and reporter vectors into HEK293T cells and the luciferase activity was measured. As Fig. 4C shows, GATA-2(ZNF) attenuated the inhibition effect of GATA-2 on TGF-β signaling activity in a dosedependent manner, suggesting that GATA-2(ZNF) might compete with full length GATA-2 to bind some repressors and released the effect of GATA-2.
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p300 for binding to Smads or a quenching of Smad transactivation by binding up of p300, the binding of p300 to Smad4 was determined. The result suggested that GATA-2 did not affect the binding between p300 and Smad4 (Fig. 5C). To determine whether GATA-2 altered the DNA binding activity of Smad proteins, EMSA was performed. As seen in Fig. 5D, incubation of the labeled Smad binding sites oligonucleotides with nuclear extract from Smad4-transfected HEK293T cells formed DNA–protein complex. However, when GATA-2 was co-transfected, the DNA-binding activity was decreased in a dose-dependent manner. This result suggested that GATA-2 decreased the DNA-binding activity of Smad proteins. To investigate how GATA-2 inhibited TGF-β signaling, histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) were used. As shown in Fig. 5E, TSA treatment attenuated the repression activity of GATA-2, while no obvious effect was observed with 5-Aza-dC treatment (Fig. 5F). Previous study reported that HDAC3 binds to the zinc finger domain of GATA-2 and plays key role in the transcriptional repression activity of GATA-2 [20]. Using IP analysis, we also found that HDAC3 can be recruited by GATA-2 on Smad4 and the GATA-2/Smad4/HDAC3 forms a complex in HEK293T cells (Supplementary Figure S5). These data suggested that GATA-2 negatively regulates TGF-β signaling through recruiting HDAC3.
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3.6. GATA-2 inhibited erythroid differentiation of K562 cells induced by 369 TGF-β signaling 370 Previously studies suggest that Activin A, a member of the TGF-β superfamily, accelerates the terminal erythroid differentiation assessed by up-regulation of hemoglobin expression in an erythroleukemia cell line K562 [21]. Because GATA-2 seems to repress TGF-β signaling, we tried to determine if GATA-2 affects TGF-β-dependant cell
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Fig. 5. GATA-2 decreased the DNA-binding activity of Smad4 and recruited histone deacetylase. (A) HaCaT cells were transfected with GATA-2 expression vector or control vector and then cells were treated with 5 ng/ml TGF-β1 for the indicated time. Lysates were prepared for Western blotting analysis with indicated antibodies. (B) 293T cells were co-transfected with the expression vectors as indicated in the presence or absence of 5 ng/ml TGF-β1. Two hours later cell lysates were immunoprecipitated with anti-Smad4 monoclonal antibody, and the immunoprecipitates were immunoblotted with indicated antibodies. (C) 293T cells were co-transfected with the expression vectors as indicated in the presence or absence of 5 ng/ml TGF-β1. Cell lysates were immunoprecipitated with anti-HA antibody, and the immunoprecipitates were then immunoblotted with antibodies indicated. (D) 293T cells were co-transfected with Myc-Smad4 and Flag-GATA2 in the presence or absence of 5 ng/ml TGF-β1 and the nuclear extracts were prepared for EMSA. Then 10 μg nuclear protein was combined with biotin-labeled SBE. The arrowhead pointed to the specifically shifted bands. For competition experiments, 100-fold excess of unlabeled oligonucleotides was added. (E) 293T cells were co-transfected with CAGA-Luc and 200 ng GATA2 expression vector or control vector. Then cells were treated with 100 ng/mL TSA or 40 μM 5-Aza-CdR (F) and 8 h later the luciferase activity was measured. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
differentiation. The lentiviruses expressing GATA-2 or control GFP were transduced into K562 cells and stable cell lines were constructed (Supplementary Figure S6). Then the cells were treated with Activin A and the hemoglobin production was measured by benzidine staining. In the control cells, Activin A treatment led to increased number of benzidine positive cells and hemoglobin production. However, GATA2 overexpression attenuates the erythroid differentiation induced by Activin A (Fig. 6A–B). Luciferase activity assay also confirmed that GATA-2 overexpression in K562 cells led to attenuated Activin A-induced TGF-β signaling pathway (Supplementary Figure S7). To confirm the effect of GATA2 on the TGF-β signaling pathway, we used lentivirus-mediated RNAi against GATA2 to knockdown endogenous GATA2 in K562 cells. As Fig. 6C–D shows, when GATA2 was
knockdown, the cells were more responsive to Activin A treatment, the percentage of benzidine positive cells and hemoglobin production were higher than the control cells, suggesting a negative role of GATA2 in the erythroid differentiation induced by TGF-β signaling pathway activation.
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Although a previous study on Evi-1 indicates an association between GATA-2 and TGFβ signal pathway [16], whether and how GATA-2 regulates TGFβ signal pathway remain unknown. Our present study provides direct evidence that GATA-2 negatively regulates TGFβ signal pathway through binding to Smad4 and then decreasing its DNA-
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binding activity. We found that GATA-2 reduced TGFβ-dependent transcription and cell growth inhibition. In Smad4-deficient cells, the repression activity on TGFβ signaling of GATA-2 was abolished. The N-terminal 270aa is required for GATA-2 to bind full length of Smad4, and the zinc finger domain of GATA-2 might recruit some co-repressors to decrease TGFβ signaling. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the formation of Smad2/3/4 complex, the DNA-binding activity of Smad4 was significantly reduced by GATA-2. Overexpression of GATA-2 in K562 cells inhibited the TGFβ-mediated erythroid differentiation and knockdown of GATA-2 accelerated the erythroid differentiation induced by TGFβ signaling activation. All these results indicated that GATA2 negatively regulate TGFβ signaling dependent on GATA-2/Smad4 interaction. Propagation of TGF-β signaling within the cell depends on a highly regulated combination of phosphorylation, complex formation, and nuclear translocation of the Smad proteins [22]. Many transcription factors and cofactors are known to be involved in the Smad signaling through directly associating with various Smad proteins. c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-β-dependent manner. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3
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Fig. 6. GATA-2 inhibited erythroid differentiation of K562 cells induced by TGF-β signaling. (A) K562 cells were infected with lentivirus-based expressing GATA-2 or control lentivirus and then GFP positive cells were purified by FACS sorter. Then the cells were treated with 50 ng/ml Activin A for the indicated time. Then the erythroid differentiation was investigated with benzidine staining. Total RNA was extracted from cells treated for 8 days and the expression level of β-hemoglobin (HBB) was analyzed by real-time PCR (B). (C) K562 cells were infected with lentivirus-based GATA-2 siRNA (siGATA-2-1, siGATA-2-2) or universal scramble siRNA (siControl). Cells were treated with 50 ng/ml Activin A for the indicated time. Then the erythroid differentiation was investigated with benzidine staining. Total RNA were extracted from cells treated for 8 days and the expression level of β-hemoglobin (HBB) was analyzed by real-time PCR (D). Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 or **p ≤ 0.001.
with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-β signaling [23]. Another oncogene v-ErbA was also found to associate with the Smad4, sequester Smad4 in the cytoplasm, and then antagonizing TGF-β-induced cell growth inhibition [24]. The TGF-β inducible protein Tsc-22 was reported to enhance TGF-β signaling by associating with Smad4 and then induce erythroid cell differentiation. Tsc-22 does not seem to affect subcellular translocation or phosphorylation of Smad proteins. Tsc-22 might enhance Smad activity by recruiting various transcriptional regulators including p300 and HDAC [25]. Furthermore, the homeoprotein DLX1 interacts with Smad4 and blocks a signaling pathway from Activin A in hematopoietic cells probably through sequestering transcriptional activator complex from the Smad3/Smad4 complex [26]. In our present study, we found that although GATA-2 significantly decreased the DNA-binding activity of Smad protein, GATA-2 did not affect the Smad2/3 phosphorylation, the Smad2/3/4 complex formation, the nuclear translocation of Smad4, and the binding between Smad4 and p300 in response to TGFβ, suggesting that GATA-2 inhibits TGFβ signaling through recruiting some co-repressors. Furthermore, we found that the regions containing 2 zinc finger (270aa–387aa) is essential for the repression activity of GATA-2, suggesting that this region might contribute to recruiting some co-repressors to suppress Smad4 activity. Very interestingly, a previous study suggests that HDAC3 interacts with GATA-2 and repressed
Please cite this article as: X.-M. Dong, et al., Cell. Signal. (2014), http://dx.doi.org/10.1016/j.cellsig.2014.01.028
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All authors declare no competing financial interests.
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Authors' contributions
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Xiao-Ming Dong, Rong-Hua Yin, Yang Yang, Zhi-Wei Feng, HongMei Ning, Lan Dong, Wei-Wei Zheng, Yu-Xin Jia,Yi-Nan Jiang, En-Dong Liu, Hui Chen, Yi-Qun Zhan, Miao Yu, and Chang-Hui Ge performed the experiments. Dong Li, Liu-Jun Tang and Jian Wang analyzed the data. Chang-Yan Li designed and performed experiments, and contributing to writing the manuscript. Xiao-Ming Yang designed the experiments and contributed to writing the manuscript.
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Acknowledgment
We thank Dr. Qinong Ye (Beijing Institute of Biotechnology, Beijing, 512 China) for providing the human full-length Smad4 (pcDNA3.1-Smad4 513 (his/myc)), 3 × CAGA-Luc, and 3TP-Luc plasmids. 514 515
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[1] N.P. Rodrigues, A.J. Tipping, Z. Wang, T. Enver, Int. J. Biochem. Cell Biol. 44 (2012) 457–460. [2] C. Vicente, A. Conchillo, M.A. Garcia-Sanchez, M.D. Odero, Crit. Rev. Oncol. Hematol. 82 (2012) 1–17. [3] C. Heyworth, K. Gale, M. Dexter, G. May, T. Enver, Genes Dev. 13 (1999) 1847–1860. [4] F.Y. Tsai, G. Keller, F.C. Kuo, M. Weiss, J. Chen, M. Rosenblatt, F.W. Alt, S.H. Orkin, Nature 371 (1994) 221–226. [5] K. Ohneda, M. Yamamoto, Acta Haematol. 108 (2002) 237–245. [6] F.Y. Tsai, S.H. Orkin, Blood 89 (1997) 3636–3643. [7] K. Kitajima, M. Masuhara, T. Era, T. Enver, T. Nakano, EMBO J. 21 (2002) 3060–3069. [8] K. Briegel, K.C. Lim, C. Plank, H. Beug, J.D. Engel, M. Zenke, Genes Dev. 7 (1993) 1097–1109. [9] C. Perry, H. Soreq, Eur. J. Biochem. 269 (2002) 3607–3618. [10] R. Ferreira, K. Ohneda, M. Yamamoto, S. Philipsen, Mol. Cell. Biol. 25 (2005) 1215–1227. [11] X. Fan, G. Valdimarsdottir, J. Larsson, A. Brun, M. Magnusson, S.E. Jacobsen, P. ten Dijke, S. Karlsson, J. Immunol. 168 (2002) 755–762. [12] M.C. Dickson, J.S. Martin, F.M. Cousins, A.B. Kulkarni, S. Karlsson, R.J. Akhurst, Development 121 (1995) 1845–1854. [13] Y. Zermati, B. Varet, O. Hermine, Exp. Hematol. 28 (2000) 256–266. [14] Y. Zermati, S. Fichelson, F. Valensi, J.M. Freyssinier, P. Rouyer-Fessard, E. Cramer, J. Guichard, B. Varet, O. Hermine, Exp. Hematol. 28 (2000) 885–894. [15] X. Bai, J. Kim, Z. Yang, M.J. Jurynec, T.E. Akie, J. Lee, J. LeBlanc, A. Sessa, H. Jiang, A. DiBiase, Y. Zhou, D.J. Grunwald, S. Lin, A.B. Cantor, S.H. Orkin, L.I. Zon, Cell 142 (2010) 133–143. [16] T. Sato, S. Goyama, E. Nitta, M. Takeshita, M. Yoshimi, M. Nakagawa, M. Kawazu, M. Ichikawa, M. Kurokawa, Cancer Sci. 99 (2008) 1407–1413. [17] T. Ravasi, H. Suzuki, C.V. Cannistraci, S. Katayama, V.B. Bajic, K. Tan, A. Akalin, S. Schmeier, M. Kanamori-Katayama, N. Bertin, P. Carninci, C.O. Daub, A.R. Forrest, J. Gough, S. Grimmond, J.H. Han, T. Hashimoto, W. Hide, O. Hofmann, A. Kamburov, M. Kaur, H. Kawaji, A. Kubosaki, T. Lassmann, E. van Nimwegen, C.R. MacPherson, C. Ogawa, A. Radovanovic, A. Schwartz, R.D. Teasdale, J. Tegner, B. Lenhard, S.A. Teichmann, T. Arakawa, N. Ninomiya, K. Murakami, M. Tagami, S. Fukuda, K. Imamura, C. Kai, R. Ishihara, Y. Kitazume, J. Kawai, D.A. Hume, T. Ideker, Y. Hayashizaki, Cell 140 (2010) 744–752. [18] J. Wang, K. Huo, L. Ma, L. Tang, D. Li, X. Huang, Y. Yuan, C. Li, W. Wang, W. Guan, H. Chen, C. Jin, J. Wei, W. Zhang, Y. Yang, Q. Liu, Y. Zhou, C. Zhang, Z. Wu, W. Xu, Y. Zhang, T. Liu, D. Yu, L. Chen, D. Zhu, X. Zhong, L. Kang, X. Gan, X. Yu, Q. Ma, J. Yan, L. Zhou, Z. Liu, Y. Zhu, T. Zhou, F. He, X. Yang, Mol. Syst. Biol. 7 (2011) 536. [19] S. Itoh, F. Itoh, M.J. Goumans, P. Ten Dijke, Eur. J. Biochem. 267 (2000) 6954–6967. [20] Y. Ozawa, M. Towatari, S. Tsuzuki, F. Hayakawa, T. Maeda, Y. Miyata, M. Tanimoto, H. Saito, Blood 98 (2001) 2116–2123. [21] Y. Fukuchi, M. Kizaki, K. Yamato, C. Kawamura, A. Umezawa, J. Hata, T. Nishihara, Y. Ikeda, Oncogene 20 (2001) 704–713. [22] J. Massague, Y.G. Chen, Genes Dev. 14 (2000) 627–644. [23] S. Akiyoshi, H. Inoue, J. Hanai, K. Kusanagi, N. Nemoto, K. Miyazono, M. Kawabata, J. Biol. Chem. 274 (1999) 35269–35277. [24] R.A. Erickson, X. Liu, Mol. Biol. Cell 20 (2009) 1509–1519. [25] S.J. Choi, J.H. Moon, Y.W. Ahn, J.H. Ahn, D.U. Kim, T.H. Han, Mol. Cell. Biochem. 271 (2005) 23–28. [26] S. Chiba, K. Takeshita, Y. Imai, K. Kumano, M. Kurokawa, S. Masuda, K. Shimizu, S. Nakamura, F.H. Ruddle, H. Hirai, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 15577–15582. [27] A. Moustakas, S. Souchelnytskyi, C.H. Heldin, J. Cell Sci. 114 (2001) 4359–4369. [28] N.O. Fortunel, A. Hatzfeld, J.A. Hatzfeld, Blood 96 (2000) 2022–2036. [29] H. Kamesaki, G.Y. Michaud, S.G. Irving, N. Suwabe, S. Kamesaki, M. Okuma, J. Cossman, Blood 87 (1996) 999–1005. [30] L. Attisano, J.L. Wrana, Science 296 (2002) 1646–1647. [31] L.M. Wakefield, A.B. Roberts, Curr. Opin. Genet. Dev. 12 (2002) 22–29. [32] J. Massague, Nat. Rev. Mol. Cell Biol. 1 (2000) 169–178. [33] M.S. Kumar, D.C. Hancock, M. Molina-Arcas, M. Steckel, P. East, M. Diefenbacher, E. Armenteros-Monterroso, F. Lassailly, N. Matthews, E. Nye, G. Stamp, A. Behrens, J. Downward, Cell 149 (2012) 642–655.
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This work was supported by Chinese National Natural Science 508 Foundation Projects (81070392, 81222005) and Special Funds for 509 Major State Basic Research of China (2012AA020206, 2013CB910800). 510
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GATA-2-mediated transcription, suggesting a potential role of GATA-2/ HDAC3 complex in gene repression [20]. The region containing whole 2 zinc fingers was required for HDAC3 binding [20]. Our result suggests that HDAC3 can be recruited to Smad4 by GATA-2 and a broad spectrum inhibitor of histone deacetylation transferase TSA attenuated the repression activity of GATA-2, suggesting an involvement of HDAC proteins. Whether HDAC3 is essential for GATA-2 to suppress Smad4 activity remains to be determined. Since Smad4 is the co-Smad protein in TGFβ signal pathway, it is easy to suppose that GATA-2 repressed the TGFβ signal pathway induce by various members of TGFβ superfamily such as TGFβ1, Activin A, and BMPs. In our present study, we found that GATA-2 inhibited TGFβ1induced transcription and Activin A-mediated erythroid differentiation. Whether GATA-2 regulates BMP-dependent TGFβ signaling pathway needs further investigation. In addition, the family of Smad proteins have MH1 and MH2 domains in common [27]. Our study suggests that GATA-2 binds to the full length of Smad4 protein but not any of the deletion mutants. Also we showed that GATA-2 specifically interacts with Smad4, but does not interact with other Smad proteins including Smad1, 2, 3, 5, and 7. Interestingly, we also investigated the effect of GATA-1 on TGFβ signaling pathway and no effect was observed. These data indicated that the negative regulation of TGFβ signaling pathway is specific for GATA-2, but not other GATA family member, and the interaction between GATA-2 and Smad4 is specific for Smad4, but not other Smad proteins. The low homology at the N-terminal between GATA-1 and GATA-2 protein might contribute to the difference. But how GATA-2 specifically interacts with Smad4 remains further investigation. TGFβ is one of the key regulators of erythropoiesis because it is involved in the control of both early and later stage of erythroid progenitor cell development [28]. GATA-2 is a negative regulator of erythropoiesis and makes major contributions to the proliferation of hematopoietic stem cells [29]. Our present study suggests that overexpression of GATA-2 inhibited TGFβ-induced erythroid differentiation and knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation in K562 cells. However, whether GATA-2/Smad4 interaction plays a role in erythroid differentiation in vivo needs further investigation. More importantly, the TGF-β signaling pathway is a major signaling that controls cell proliferation, differentiation, and tumor suppression [30–32]. GATA-2 negatively regulates TGF-β signaling pathway in HaCaT cells and attenuates the growth inhibition of TGF-β, indicating a potential role of GATA-2 in tumor development. In fact, a recent study suggests that the GATA-2 transcriptional network is requisite for Ras oncogene-driven non-small cell lung cancer and gata2 loss dramatically reduced tumor development [33]. Whether GATA-2/Smad4 interaction plays roles in tumor development remains an interesting question. Taken together, we provide direct evidence that GATA-2 is a negative regulator of TGFβ signaling pathway through binding to Smad4.
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Please cite this article as: X.-M. Dong, et al., Cell. Signal. (2014), http://dx.doi.org/10.1016/j.cellsig.2014.01.028
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