JBC Origene Your Gene Company

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M413461200 on January 25, 2005

J. Biol. Chem., Vol. 280, Issue 13, 13163-13170, April 1, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/13/13163    most recent
M413461200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ezoe, S.
Right arrow Articles by Kanakura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ezoe, S.
Right arrow Articles by Kanakura, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

GATA Transcription Factors Inhibit Cytokine-dependent Growth and Survival of a Hematopoietic Cell Line through the Inhibition of STAT3 Activity*

Sachiko Ezoe{ddagger}, Itaru Matsumura{ddagger}§, Karin Gale¶, Yusuke Satoh{ddagger}, Jun Ishikawa{ddagger}, Masao Mizuki{ddagger}, Satoru Takahashi||, Naoko Minegishi**, Koichi Nakajima{ddagger}{ddagger}, Masayuki Yamamoto§§, Tariq Enver¶¶, and Yuzuru Kanakura{ddagger}

From the {ddagger}Department of Hematology/Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan, the Section of Gene Function and Regulation, Chester Beatty Laboratories, Institute of Cancer Research, Fulham Road, London SW3 6JB, United Kingdom, the ||Division of Anatomy and Embryology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan, the **Department of Molecular Diagnostics, Tohoku University School of Medicine, 2-1 Seiryotyo Aobaku, Sendai 980-8575, Japan, the {ddagger}{ddagger}Department of Immunology, Osaka City University Graduate School of Medicine,1-4-3, Asahityo, Abenoku, Osaka 545-8585, Japan, the §§Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Institute, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-0006, Japan, and the ¶¶MRC Molecular Haematology Unit, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom

Received for publication, November 30, 2004 , and in revised form, January 19, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although GATA-1 and GATA-2 were shown to be essential for the development of hematopoietic cells by gene targeting experiments, they were also reported to inhibit the growth of hematopoietic cells. Therefore, in this study, we examined the effects of GATA-1 and GATA-2 on cytokine signals. A tamoxifen-inducible form of GATA-1 (GATA-1/ERT) showed a minor inhibitory effect on interleukin-3 (IL-3)-dependent growth of an IL-3-dependent cell line Ba/F3. On the other hand, it drastically inhibited TPO-dependent growth and gp130-mediated growth/survival of Ba/F3. Similarly, an estradiol-inducible form of GATA-2 (GATA-2/ER) disrupted thrombopoietin (TPO)-dependent growth and gp130-mediated growth/survival of Ba/F3. As for this mechanism, we found that both GATA-1 and GATA-2 directly bound to STAT3 both in vitro and in vivo and inhibited its DNA-binding activity in gel shift assays and chromatin immunoprecipitation assays, whereas they hardly affected STAT5 activity. In addition, endogenous GATA-1 was found to interact with STAT3 in normal megakaryocytes, suggesting that GATA-1 may inhibit STAT3 activity in normal hematopoietic cells. Furthermore, we found that GATA-1 suppressed STAT3 activity through its N-zinc finger domain. Together, these results suggest that, besides the roles as transcription factors, GATA family proteins modulate cytokine signals through protein-protein interactions, thereby regulating the growth and survival of hematopoietic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GATA family transcription factors are composed of six members (GATA-1–6) and play indispensable roles in the development of diverse cell types according to their unique tissue distribution (1). These factors bind to a consensus DNA sequence called a GATA motif (A/T)GATA(A/G) in the regulatory region of their target genes through two highly conserved zinc finger domains, both of which include the configuration Cys-X2-Cys-X17-Cys-X2-Cys. The carboxyl (C)1 finger is absolutely required for the DNA binding, whereas the amino (N) finger stabilizes the DNA-binding and confers full specificity (2). Among GATA family members, at least three GATA factors (GATA-1, GATA-2, and GATA-3) are critically involved in different aspects of hematopoiesis.

GATA-1, the first cloned member of this family, is expressed at high levels in erythroid cells, mast cells, and megakaryocytes and at lower levels in hematopoietic stem/progenitor cells. GATA-1 is considered to be essential for primitive and definitive erythropoiesis, because GATA-1-null cells did not contribute to erythropoiesis in chimeric mice generated from embryonic stem cells lacking GATA-1 (3). In addition, in vitro experiments showed that GATA-1-null embryonic stem cells are unable to differentiate into mature erythroid cells due to apoptosis (4, 5). Furthermore, lineage-selective GATA-1 knock-down mice exhibited striking thrombocytopenia as well as severe anemia (6). These results imply that GATA-1 plays an essential role not only in erythropoiesis but also in megakaryopoiesis and thrombopoiesis. However, the increased proliferation of megakaryocytes was also observed in the spleen of lineage-selective GATA-1 knock-down mice (6). Similarly, heterozygous mutant mice chimeric for GATA-1 gene displayed marked splenomegaly due to the massive accumulation of proerythroblasts and megakaryocytes (7). Moreover, primary megakaryocytes prepared from GATA-1-deficient mice exhibited hyperproliferation in liquid cultures (8). These lines of evidence suggest that GATA-1 might suppress the growth of erythroid and megakaryocytic cells regardless of its critical role in their development.

GATA-2 is highly expressed in pluripotent hematopoietic stem cells and moderately in early stage of erythroid cells, mast cells, and megakaryocytes (9). 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 (10). Furthermore, the in vitro experiment using GATA-2-/- embryonic stem cells showed that GATA-2 was necessary for proliferation/survival of early hematopoietic cells (11). However, as was the case with GATA-1, it was also reported that retrovirus-transferred wild-type (WT) GATA-2 inhibited the growth of normal hematopoietic progenitor cells (12). Moreover, we found that an estrogen-inducible chimeric form of GATA-2, GATA-2/estrogen receptor (ER), suppressed cytokine-dependent growth of normal hematopoietic progenitor cells in response to estradiol (ED) (13, 14). Therefore, at present, the functional properties of GATA-1 and GATA-2 in the growth and survival of hematopoietic cells is controversial, and, of course, its mechanism remains unknown.

Hematopoiesis is regulated by a number of hematopoietic growth factors, including IL-3, IL-5, IL-6, GM-CSF, G-CSF, erythropoietin, TPO, stem cell factor, and fms-like tyrosine kinase 3 ligand. These growth factors exert their effects through activation of various intracellular signaling cascades such as Janus family of protein tyrosine kinases (JAKs)/signal transducers and activators of transcription (STATs), Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt pathways. Among these signaling pathways, the STAT family of transcription factors plays a pivotal role in the regulation of growth and survival of hematopoietic cells (15). For example, STAT3 regulates cell growth through the transcriptional regulation of cyclin D1 and c-myc (16, 17) and mediates the anti-apoptotic signal from gp130 via the induction of Bcl-2 (18). Also, STAT5 mediates cell growth through the induction of cyclin D1 and pim-1 (19, 20) and prevents apoptosis by inducing Bcl-XL and Bcl-2 (21, 22).

In an attempt to clarify the roles of GATA transcription factors in the growth and survival of hematopoietic cells, in this study, we examined their effects on cytokine signals. Here we found that GATA-1 and GATA-2 inhibit STAT3 activity through the direct protein-protein interactions, thereby inhibiting cytokine-dependent growth and survival of hematopoietic cells. These results suggest that, in addition to the roles as transcription factors, GATA transcription factors would exert various effects through protein-protein interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—The antibodies (Abs) against GATA-1 (N6) and STAT3 (H190) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), the anti-HA Ab (12CA5) was from Roche Molecular Biochemicals (Indianapolis, IN), and an anti-FLAG Ab (M2) was from Sigma.

Plasmid Constructs and cDNAs—Expression vectors for GATA-1/ERT and GATA-2/ER were described previously (13). Expression vectors for WT GATA-1, GATA-2, and GATA-3 were generated by subcloning these cDNAs into pcDNA3 (Invitrogen). Various types of GATA-1 mutants were constructed by the PCR method and subcloned into pcDNA3. Expression vectors encoding FLAG-tagged WT and constitutively active STAT3 (STAT3-C) were kindly supplied by Dr. J. E. Darnell, Jr. (Rockefeller University, New York, NY) (16). An expression vector for constitutively active STAT5A (1*6-STAT5A) was provided by Dr. T. Kitamura (University of Tokyo, Tokyo, Japan) (23). The cDNAs of SOCS1, CIS, and OSM were provided by Dr. A. Yoshimura (Kyushu University, Fukuoka, Japan).

Cell Lines and Cultures—A murine IL-3-dependent cell line Ba/F3 was cultured in RPMI (Nakarai Tesq, Kyoto, Japan) supplemented with 10% fetal bovine serum (Flow, North Ryde, Australia) and 1 ng/ml IL-3. NIH3T3, 293T, and C2C12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

Preparation of Stable Transformants from Ba/F3—We stably introduced various expression vectors into Ba/F3 by electroporation (250 V and 960 microfarads) (Bio-Rad). The transfected clones were selected by the culture with G418 (1.2 mg/ml), puromycin (1 µg/ml), hygromycin (0.5 mg/ml), and/or blasticidin S hydrochloride (30 µg/ml) according to the transfected plasmid.

Flow Cytometry—DNA content of the cultured cells was quantitated by staining with propidium iodide and analyzed on FACSort (BD Biosciences) (24). Cell cycle analysis was performed with the program Modfit LT2.0 (BD Biosciences).

Northern Blot Analysis—The methods for the isolation of total cellular RNA and Northern blot were described previously (24).

Coimmunoprecipitation Analysis and Immunoblotting—293T cells were transfected with various expression vectors by the calcium phosphate coprecipitation method (25). After 12 h, the cells were washed and cultured for 24 h. The isolation of total cellular lysate, immunoprecipitation, gel electrophoresis, and immunoblotting were performed according to the procedures described previously (25). Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (PerkinElmer Life Sciences, Boston, MA).

Luciferase Assays—Luciferase assays were performed with a Dual-Luciferase Reporter System (Promega, Madison, WI) as previously described (19). In short, NIH3T3 and C2C12 cells (2 x 105 cells seeded in 60-mm dish) were transfected with the indicated effector genes along with 2 µg of a reporter gene for STAT3 (4 x APRE-Luc), STAT5 (3 x {beta}-Cas-Luc) (26), or GATA-1 (pGL3M{alpha}P-Luc) (25), and 10 ng of pRL-TK, an expression vector for Renilla luciferase, by the calcium phosphate coprecipitation method. After 12 h, the cells were washed and serum-deprived for 24 h. Then, the cells were lysed in lysis buffer supplied by the manufacturer, followed by the measurement of the firefly and Renilla luciferase activities on a luminometer LB96P (Berthold Japan, Tokyo, Japan). The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the Renilla luciferase activities. The experiments were performed in triplicate, and similar results were obtained from at least three independent experiments.

Electrophoretic Mobility Shift Assays—The isolation of nuclear extract and electrophoretic mobility shift assay were performed according to the procedures described previously (27). The sequences of the probes were as follows: 5'-GCGTGATTTCCCCGAAATGATGAGGCA-3' for STAT3 (17); 5'-CTCGTGGCGTTCTTGGAAATGCGCCC-3' for STAT5 (19), recognition sequences are underlined.

Chromatin Immunoprecipitation Assays—Chromatin immunoprecipitation assays were performed with a chromatin immunoprecipitation assay kit (Upstate, Lake Placid, NY) according to the manufacture's instructions. Briefly, cells (3 x 106 per sample) were fixed with 1% formaldehyde at 37 °C for 10 min. After the isolation of nuclear extract, chromatin was sonicated to shear DNA to lengths between 200 and 1000 bp by the sonifier. STAT3·DNA binding complexes were immunoprecipitated with an anti-STAT3 Ab or control rabbit IgG. The immunoprecipitated DNA was eluted and subjected to the PCR reactions using AmpliTaq Gold (PerkinElmer Life Sciences), in the following thermocycling conditions: 94 °C for 10 min, 35 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 30 s, followed by 72 °C for 10 min. The sequences of the primer set in the JunB promoter are as follows: sense, 5'-TGGCCGCTGTTTACAAGGACAC-3'; antisense, 5'-GCTTTTCTCTCCCTCCGTGGTA-3'. PCR products were electrophoresed on agarose gels and visualized by Syber Green (Cambrex, East Rutherford, NJ) staining.

GST Pull-down Assays—The production and purification of GST-STAT3 fusion proteins were performed according to the procedures as previously reported (28). 35S-Labeled GATA-1 was prepared by a TNT reticulocyte lysate system (Promega). For each binding reaction, 20 µg of GST fusion protein bound to glutathione-Sepharose beads was incubated with 50 µg of TNT reaction solution at 4 °C for 1 h. The binding complexes were separated by gel electrophoresis and subjected to autoradiography.

Isolation of Murine Bone Marrow Lin- Cells and Their Culture with TPO—Murine bone marrow mononuclear cells were isolated by density gradient centrifugation, and Lin- cells were purified by MACS (Miltenyl Biotec, Bergisch Gladbach, Germany) (14). The purified Lin- cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 30 ng/ml TPO for 6 days. After this culture, about 80% of the cultured cells showed polyploid morphology compatible with megakaryocytes and were positive for CD41 (data not shown).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of GATA-1 on Cytokine-dependent Growth and Survival of Ba/F3—To examine the effects of GATA-1 on cytokine-dependent growth of hematopoietic cells in a ligand-inducible system, in this study, we utilized GATA-1/ERT, a chimera consisting of full-length GATA-1 and the mutated ligand-binding domain of estrogen receptor. This chimeric GATA-1 reveals GATA-1 activity in response to tamoxifen (4-HT) as previously reported (13). At first, we stably introduced GATA-1/ERT into a murine IL-3-dependent proB cell line Ba/F3; this clone was designated Ba/F3/G1ERT. Under the culture without 4-HT, Ba/F3/G1ERT proliferated in response to IL-3 with the growth curve similar to that of parental Ba/F3 (Fig. 1A, left panel; the growth curve of parental Ba/F3 is not shown). Although the 4-HT treatment hardly influenced the growth of the mock-transfected Ba/F3 (Ba/F3/Mock) (Fig. 1A, right panel), 4-HT-activated GATA-1 suppressed the growth of Ba/F3/G1ERT by 20% (Fig. 1A, left panel). In agreement with this result, the 4-HT treatment reduced the proportion of proliferating cells in S-G2/M phase from 60% to 47% at 72 h in DNA content analysis (Fig. 1D).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 1.
Effects of GATA-1 on cytokine-dependent growth and survival of murine IL-3-dependent cell lines. A, Ba/F3/G1ERT and Ba/F3/Mock cells were seeded at a cell density of 100/µl and cultured with 1 ng/ml IL-3 in the presence or absence of 1 µM 4-HT. The total number of viable cells was counted by trypan blue dye exclusion method at the time indicated. The results are shown as mean ± S.D. of triplicate cultures. B and C, Ba/F3/c-Mpl/G1ERT, Ba/F3/gp130/G1ERT, Ba/F3/c-Mpl/G2ER, and Ba/F3/gp130/G2ER clones were cultured with the corresponding cytokine (TPO (30 ng/ml) or gp130L (30 ng/ml)) in the presence or absence of 1 µM 4-HT or ED as indicated. The growth curves of these clones were indicated. D, after the 72-h culture with IL-3, TPO, or gp130L, the DNA content of the cultured cells was examined by propidium iodide staining. Apo, apoptotic fraction.

 
To further examine the effects of GATA-1 on cytokine-dependent growth of Ba/F3, we introduced the thrombopoietin (TPO) receptor c-Mpl and a chimeric receptor G-CSFR/gp130 (consisting of the extracellular domain of G-CSF receptor and the cytoplasmic domain of gp130) (17, 18) into Ba/F3/G1ERT and Ba/F3/Mock; these clones were designated Ba/F3/c-Mpl/G1ERT and Ba/F3/gp130/G1ERT, Ba/F3/c-Mpl/Mock, and Ba/F3/gp130/Mock, respectively. As shown in Fig. 1B, Ba/F3/c-Mpl/G1ERT and Ba/F3/gp130/G1ERT proliferated in response to TPO and gp130 ligand (gp130L) (in this paper, we denote G-CSF as gp130L when it acts on G-CSFR/gp130), respectively. When these clones were treated with 4-HT for 4 days, GATA-1 suppressed TPO-dependent growth by 70%, and inhibited gp130L-dependent growth completely (changes in the proportion of S-G2/M phase before and after the 72-h 4-HT treatment in DNA content analysis under the culture with TPO, 57% to 20%; gp130L, 43% to 22% (Fig. 1D). Furthermore, the 4-HT treatment induced apoptosis in 39% of Ba/F3/gp130/G1ERT cells cultured with gp130L (apoptotic cells were detected as a sub-diploid fraction in DNA content analysis, Fig. 1D). In contrast, the 4-HT treatment neither influenced TPO-dependent growth/survival of Ba/F3/c-Mpl/Mock nor gp130L-depedent growth/survival of Ba/F3/gp130/Mock (data not shown). Together, these results suggest that GATA-1 can more efficiently affect signaling molecules of TPO and gp130L than those of IL-3.

Next, we analyzed whether GATA-2 inhibits TPO- or gp130-dependent growth and survival of Ba/F3 cells as well as GATA-1. We introduced GATA-2/ER (a chimera consisting of full-length GATA-2 and the estrogen receptor, which reveals GATA-2 activity in response to ED (13)) or a Mock vector into Ba/F3 clones each expressing c-Mpl and G-CSFR/gp130; these clones were designated as Ba/F3/c-Mpl/G2ER, Ba/F3/gp130/G2ER, Ba/F3/c-Mpl/Mock, and Ba/F3/gp130/Mock, respectively. The ED treatment did not influence TPO-dependent growth/survival of Ba/F3/c-Mpl/Mock or gp130L-dependent growth/survival of Ba/F3/gp130/Mock (data not shown). In contrast, ED-activated GATA-2 inhibited both TPO- and gp130L-dependent growth of these clones almost completely (Fig. 1C) (changes in the proportion of S-G2/M phase before and after the 72-h ED treatment: Ba/F3/c-Mpl/G2ER cultured with TPO, 53% to 4%; Ba/F3/gp130/G2ER cultured with gp130L, 40% to 3%) (Fig. 1D). Furthermore, the ED treatment induced severe apoptosis in 52% of Ba/F3/gp130/G2ER cells under the culture with gp130L (Fig. 1D).

Effects of GATA Transcription Factors on STAT Activity— STATs have been reported to mediate cytokine-dependent growth and survival of hematopoietic cells (15, 17–19). Particularly, STAT3 was shown to be crucial for gp130L-depednent growth and survival of Ba/F3 cells (17, 18). To evaluate the effects of GATA-1 and GATA-2 on STAT3 and STAT5 activities, we examined the expression their target molecules during the 4-HT or ED treatment in Ba/F3/gp130/G1ERT, Ba/F3/gp130/G2ER, and Ba/F3/gp130/Mock by Northern blot analysis. As shown in Fig. 2A, time course analyses demonstrated that both 4-HT treatment, and ED treatment suppressed the expression of target molecules for STAT3 (c-myc, JunB, and SOCS1) to an undetectable level under the culture with gp130L in Ba/F3/gp130/G1ERT and Ba/F3/gp130/G2ER, whereas these treatments did not affect their expression in Ba/F3/gp130/Mock (data not shown). In contrast, neither the 4-HT treatment nor ED treatment influenced the expression of target molecules for STAT5 (CIS and Pim-1) under the culture with IL-3 in Ba/F3/gp130/G1ERT, Ba/F3/gp130/G2ER (Fig. 2A) and Ba/F3/gp130/Mock (data not shown). After deprivation of gp130L for 12 h, we stimulated these clones with gp130L or IL-3 for 30 min and investigated induction levels of these target molecules. As shown in Fig. 2B, upper left panel, the induction of target molecules for STAT3 (Jun B, SOCS1, and Tis11) by gp130L was severely inhibited by the 2-h pretreatment with 4-HT in Ba/F3/gp130/G1ERT but not in Ba/F3/gp130/Mock. Also, the ED pretreatment suppressed their induction by gp130L in Ba/F3/gp130/G2ER but not in Ba/F3/gp130/Mock (Fig. 2B, lower left panel). In contrast, the induction of target molecules for STAT5 (CIS, OSM, and Pim-1) by IL-3 was hardly affected by these pretreatments in Ba/F3/gp130/G1ERT and Ba/F3/gp130/G2ER (Fig. 2B, right panels), and Ba/F3/gp130/Mock (data not shown). To further analyze the effects of GATA transcription factors on STAT activities, we performed luciferase assays with reporter genes for STAT3 (4 x APRE-Luc) and STAT5 (3 x {beta}-Cas-Luc) (26) in NIH3T3 cells. As shown in Fig. 2C, WT GATA-1, GATA-2, and GATA-3 inhibited STAT3-C (constitutively active STAT3)-induced 4 x APRE-Luc activities with similar efficiencies. By contrast, none of GATA-1, GATA-2, or GATA-3 affected 1*6-STAT5A (constitutively active STAT5A)-induced 3 x {beta}-Cas-Luc activities. These results suggest that GATA transcription factors would specifically suppress activity of STAT3 but not of STAT5.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 2.
Effect of GATA transcription factors on STAT3 and STAT5 activities. A, Ba/F3/gp130/G1ERT and Ba/F3/gp130/G2ER cells cultured with gp130L or IL-3 were treated with 1 µM 4-HT or ED and subjected to Northern blot analysis at the time indicated. B, Ba/F3/gp130/G1ERT, Ba/F3/gp130/G2ER, and Ba/F3/gp130/Mock cells maintained with gp130L were deprived of gp130L for 12 h, then stimulated with gp130L or IL-3 for 30 min with or without the 2-h pretreatment with 4-HT or ED, and subjected to Northern blot analysis. C, NIH3T3 cells were transfected with 2 µg of the expression vector for constitutively active STAT3 (STAT3-C) or STAT5 (1*6-STAT5), 8 µg of the expression vector for GATA-1, GATA-2, or GATA-3, 1.0 µg of an appropriate reporter gene (4 x APRE-Luc for STAT3 or 3 x {beta}-Cas-Luc for STAT5) and 10 ng of pRL-CMV-Rluc by the calcium phosphate coprecipitation method. After 12-h cultures, the cells were washed, serum-starved for 24 h, and then lysed in the lysis buffer, followed by the measurement of the firefly and the Renilla luciferase activities. The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the Renilla luciferase activities. The results are shown as the mean ± S.D. of triplicate experiments.

 
GATA-1 Inhibits DNA-binding Activity of STAT3 but Not of STAT5—To clarify the mechanism through which GATA-1 and GATA-2 inhibit STAT3 activity, we examined the effects of GATA-1 and GATA-2 on gp130L-induced tyrosine phosphorylation of STAT3. However, neither 4-HT-activated GATA-1 nor ED-activated GATA-2 influenced gp130L-induced tyrosine phosphorylation of STAT3 in Ba/F3/gp130/G1ERT and Ba/F3/gp130/G2ER (data not shown). So, we next examined the effects of GATA-1 and GATA-2 on DNA-binding activity of STAT3 by electrophoretic mobility shift assay. After deprivation of gp130L for 12 h, we stimulated Ba/F3/gp130/G1ERT and Ba/F3/gp130/Mock with gp130L or IL-3 for 30 min with or without the 4-HT pretreatment. Without the pretreatment, the nuclear extract isolated after the stimulation with gp130L bound to the probe for STAT3 (Fig. 3A, upper left panel, lane 2). This complex was abolished by the WT competitor but not by the mutated (MT) competitor, and was supershifted by the anti-STAT3 Ab, indicating that this complex was formed from STAT3 (Fig. 3A, upper left panel, lanes 3–5). The 4-HT pretreatment drastically suppressed DNA-binding activity of STAT3 in Ba/F3/gp130/G1ERT (Fig. 3A, upper left panel, lane 2 versus lane 7), whereas the 4-HT pretreatment by itself showed no effect on its DNA binding in Ba/F3/gp130/Mock (Fig. 3A, lower left panel). In contrast, IL-3-induced DNA-binding activity of STAT5 was scarcely influenced by the 4-HT pretreatment in Ba/F3/gp130/G1ERT (Fig. 3A, upper right panel, lane 2 versus lane 7) and Ba/F3/gp130/Mock (Fig. 3A, lower right panel). Like the 4-HT pretreatment, the ED pretreatment severely inhibited gp130L-induced DNA-binding activity of STAT3 in Ba/F3/gp130/GT2 (Fig. 3B, upper left panel), whereas the ED pretreatment by itself did not affect its DNA binding in Ba/F3/gp130/Mock (Fig. 3B, lower left panel). Also, the ED treatment hardly influenced IL-3-induced DNA-binding activity of STAT5 in Ba/F3/gp130/G2ER (Fig. 3B, upper right panel) and Ba/F3/gp130/Mock (Fig. 3B, lower right panel). Together, these results imply that both GATA-1 and GATA-2 would inhibit DNA-binding activity of STAT3 but not of STAT5.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 3.
Effects of GATA-1 and GATA-2 on DNA-binding activities of STAT3 and STAT5. Ba/F3/gp130/G1ERT, Ba/F3/gp130/G2ER, and Ba/F3/gp130/Mock cells maintained with gp130L were deprived of gp130L for 12 h and then stimulated with gp130L or IL-3 for 30 min with or without the 2-h pretreatment with 4-HT or ED, followed by the isolation of the nuclear extract. A and B, the nuclear extract was incubated with the end-labeled probe for STAT3 and STAT5 in the binding buffer, electrophoresed on 4% polyacrylamide gel, dried, and subjected to autoradiography. In competition assays, a 200-fold molar excess of unlabeled wild-type (WT) or mutant (MT) competitor oligonucleotide was added to the binding mixture. In supershift assays, nuclear extracts were preincubated with 1 µg of the indicated Ab prior to the binding reaction. SS, supershifted complex. C, after the isolation of nuclear extract, chromatin was sonicated to shear DNA. Sheared genomic DNA was used as a positive control (Input). Then, STAT3·DNA binding complexes were immunoprecipitated with the anti-STAT3 Ab or control rabbit IgG. The immunoprecipitated DNA was eluted and subjected to PCR. PCR primers were designed to amplify -454/-186 of the JunB promoter.

 
To further investigate the effects of GATA-1 on DNA-binding activity of STAT3 in the chromatin structure, we performed the chromatin immunoprecipitation analysis on the STAT3-binding site in the 5' region of Jun-B gene. After deprivation of gp130L for 12 h, Ba/F3/gp130/G1ERT and Ba/F3/gp130/Mock cells were stimulated with gp130L for 30 min with or without the 4-HT pretreatment. As shown in Fig. 3C, the STAT3-binding site was amplified by PCR in all of the sheared DNA samples (top panel, indicated as "Input"). Also, this region was amplified by PCR from the anti-STAT3 immunoprecipitate obtained from 4-HT-untreated, gp130L-stimulated cells (middle panel, lane 2), indicating that STAT3 activated by gp130L bound to this element. However, 4-HT pretreatment suppressed this binding severely (middle panel, lane 4). In contrast, the 4-HT pretreatment by itself did not influence this binding in Ba/F3/gp130/Mock (middle panel, lane 6 versus lane 8). In agreement with the results of the electrophoretic mobility shift assay, these findings indicate that GATA-1 also inhibits DNA-binding activity of STAT3 in the chromatin structure.

Both GATA-1 and GATA-2 Directly Bind to STAT3 in Vitro and in Vivo—To clarify the mechanism through which GATA-1 and GATA-2 inhibit DNA-binding activity of STAT3, we analyzed their bindings to STAT3. For this purpose, we transfected HA-tagged GATA-1, GATA-2, and/or FLAG-tagged STAT3 together with G-CSFR/gp130 into 293T cells and performed the coimmunoprecipitation analysis. After the stimulation with gp130L for 30 min, we prepared total cellular lysate and immunoprecipitated GATA-1 with the anti-HA Ab. As shown in Fig. 4A, the immunoblot with the anti-FLAG Ab revealed that STAT3 was coimmunoprecipitated with GATA-1 only when both molecules were cotransfected (top panel, lane 4). Also, GATA-1 was coimmunoprecipitated with STAT3 by the anti-FLAG Ab (third panel, lane 4). Similarly, we found that GATA-2 bound to STAT3 in 293T cells (fifth and seventh panels, lane 4).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 4.
GATA-1 binds to STAT3 in vitro and in vivo. A, 293T cells were transfected with HA-tagged GATA-1, HA-tagged GATA-2, and/or FLAG-tagged STAT3 as indicated. After 36 h, total cellular lysate was isolated and subjected to the coimmunoprecipitation analysis with the indicated Abs. B, the in vitro bindings GATA-1-STAT3 and GATA-2-STAT3 were examined by GST-pull-down assays. 35S-Labeled GATA-1 and GATA-2 were incubated with several types of GST-STAT3, and the binding complexes were separated by gel electrophoresis and subjected to autoradiography. C, the in vivo binding between GATA-1 and STAT3 was examined by the coimmunoprecipitation method using normal megakaryocytes that developed from murine bone marrow stem/progenitor cells after 5-day cultures with TPO. 10% of total lysate was loaded as input, and the remaining sample was divided into three aliquots and immunoprecipitated with the anti-GATA-1 antibody, the anti-STAT3 antibody, and anti-actin antibody, respectively.

 
Next, we investigated whether GATA-1 and GATA-2 directly binds to STAT3 in vitro. Because we could not purify the GST fusion protein containing the full portion of STAT3 due to the formation of inclusion body, we generated several GST-STAT3 fusion proteins each consisting of the fragment as follows: GST-STAT3 (amino acids 1–154), GST-STAT3 (103–377), and GST-STAT3 (320–590). After verifying the quality and quantity of the fusion proteins by the Coomassie Brilliant Blue staining (data not shown), we analyzed their binding to in vitro translated GATA-1 and GATA-2. As shown in Fig. 4B, 35S-labeled GATA-1 and GATA-2 bound to GST-STAT3 (320–590), but not to GST alone, GST-STAT3 (1–154) or GST-STAT3 (103–377). Because the DNA-binding domain of STAT3 resides in around amino acids 400–500, it was speculated that GATA-1 and GATA-2 may inhibit DNA-binding activity of STAT3 through the direct interactions.

To further confirm the relevance of this finding in normal hematopoietic cells, we examined the binding between GATA-1 and STAT3 in murine megakaryocytes that developed from bone marrow stem/progenitor cells after the culture with TPO. As shown in Fig. 4C, both STAT3 and GATA-1 were detected by immunoblot analysis on the total cell lysate prepared from normal megakaryocytes (both panels, lane 4). Also, as was seen in 293T cells, STAT3 was coimmunoprecipitated with GATA-1 (left panel, lane 2), and vice versa (right panel, lane 3). In addition, we confirmed that neither the anti-STAT3 Ab recognizes GATA-1 (left panel, lane 2) nor the anti-GATA-1 Ab recognizes STAT3 (right panel, lane 3). Also, we verified that neither STAT3 nor GATA-1 was immunoprecipitated by the anti-actin Ab (both panels, lane 1). From these results, it was assumed that the binding between GATA-1 and STAT3 in normal megakaryocytes is not attributable to the nonspecific reactions.

GATA-1 Inhibits STAT3 Activity through Its N-zinc Finger— Next, we tried to determine which domain of GATA-1 inhibits STAT3 activity. As shown in Fig. 5A, STAT3-C-induced 4 x APRE-Luc activities were inhibited by WT, del N90, del N180, del C80, and {Delta}CZF but not by del NZF, del BZF, del C115, {Delta}NZF, or {Delta}CENT, indicating that the N-zinc finger domain is necessary for GATA-1 to inhibit STAT3 activity. However, because del C115 also could not inhibit STAT3 activity, the C-terminal domain around amino acids 80–115 was also supposed to play some role in this inhibition. In agreement with this finding, the in vitro binding assay showed that mutants of GATA-1 lacking the N-zinc finger did not bind to GST-STAT3 (320–590) (Fig. 5B). Also, the in vivo binding assay using 293T cells showed that GATA-1 mutants lacking the N-zinc finger did not bind to STAT3 (Fig. 5C). These results suggest that GATA-1 inhibits STAT3 activity by binding to STAT3 through its N-zinc finger domain. Furthermore, we found that del C115, which could not inhibit STAT3 activity, did not bind to STAT3 in vivo despite its in vitro binding to GST-STAT3 (Fig. 5B, lane 5 versus Fig. 5C, lane 6), suggesting that the C-terminal domain around amino acids 80–115 would be necessary for the in vivo binding between GATA-1 and STAT3.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 5.
GATA-1 inhibits STAT3 activity though its N-zinc finger domain. A, to determine which domain of GATA-1 inhibits STAT3 activity, NIH3T3 cells were transfected with 2 µg of STAT3-C, 8 µg of the indicated GATA-1 mutant, 1.0 µg of a reporter gene (4 x APRE-Luc), and 10 ng of pRL-CMV-Rluc by the calcium phosphate coprecipitation method. After 12-h culture, the cells were washed, serum-starved for 24 h, and then lysed, followed by the measurement of the firefly and the Renilla luciferase activities. The results are shown as the mean ± S.D. of triplicate experiments. TAD, transactivating domain. B, GST-STAT3 (320–590) was incubated with 35S-labeled GATA-1 mutants, and the binding complexes were separated by gel electrophoresis and subjected to autoradiography. C, 293T cells were transfected with G-CSFR/gp130, HA-tagged GATA-1 mutant, and/or FLAG-tagged STAT3 as indicated. After 36 h, cells were stimulated with gp130L for 30 min, and total cellular lysate was isolated from each sample and subjected to the coimmunoprecipitation analysis with the indicated Abs. D, Ba/F3/gp130/WT GATA-1, Ba/F3/gp130/{Delta}NZF GATA-1, Ba/F3/gp130/{Delta}CZF GATA-1, and Ba/F3/gp130/Mock cells were seeded at a cell density of 100/µl and cultured with 30 ng/ml gp130L. The total number of viable cells was counted by trypan blue dye exclusion method at the time indicated. The results are shown as mean ± S.D. of triplicate cultures. E, Ba/F3/gp130/WT GATA-1, Ba/F3/gp130/{Delta}NZF GATA-1, Ba/F3/gp130/{Delta}CZF GATA-1, and Ba/F3/gp130/Mock cells maintained with IL-3 were deprived of IL-3 for 12 h, then stimulated with gp130L for 30 min, and subjected to Northern blot analysis.

 
Next, we examined the effects of deletion mutants in the in vivo system. For this purpose, we introduced WT GATA-1, {Delta}NZF GATA-1, and {Delta}CZF GATA-1 into Ba/F3/gp130, and selected several clones under the culture with IL-3, in which each transgene was effectively expressed; these clones were designated Ba/F3/gp130/WT GATA-1, Ba/F3/gp130/{Delta}NZF GATA-1, and Ba/F3/gp130/{Delta}CZF GATA-1, respectively. As shown in Fig. 5A, WT GATA-1 and {Delta}CZF GATA-1 can inhibit STAT3 activity, whereas {Delta}NZF GATA-1 cannot. These clones proliferated with the similar growth curves under the culture with IL-3 (data not shown). When cultured with gp130L, Ba/F3/gp130/WT GATA-1, and Ba/F3/gp130/{Delta}CZF GATA-1 cells scarcely proliferated (Fig. 5D), and a part of the cultured cells led to apoptosis (data not shown). In contrast, Ba/F3/gp130/{Delta}NZF GATA-1 was able to grow in response to gp130L, whereas its growth was reduced by 40% compared with that of Ba/F3/gp130/Mock (Fig. 5D). In addition, we found that the induction of JunB by gp130L was apparently inhibited in Ba/F3/gp130/WT GATA-1 and Ba/F3/gp130/{Delta}CZF GATA-1, whereas it was retained in Ba/F3/gp130/{Delta}NZF GATA-1 (Fig. 5E). The similar growth characteristics and JunB induction pattern were observed at least in three other clones of each transfectant. Together, these results suggest that GATA-1 would inhibit STAT3 activity through the N-zinc finger domain, thereby impeding gp130L-dependent growth/survival of Ba/F3 cells. However, it was to be noted that {Delta}NZF GATA still inhibited gp130L-dependent growth of Ba/F3 cells about 40%, whereas it did not inhibit STAT3 activity. This result implied that GATA-1 would disrupt an additional gp130-mediated growth signaling pathway besides STAT3.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In previous studies, we reported that GATA-2/ER suppresses the growth of normal hematopoietic stem/progenitor cells (13, 14). As for this mechanism, we found that GATA-2 down-regulates the expression of c-myc mRNA, which was assumed to be primarily responsible for GATA-2-induced growth suppression. Because the expression of c-myc mRNA was reported to be transcriptionally regulated by cytokine-induced STAT3 and Ras/MAPK activities (17, 29), in the present study, we examined the effects of GATA transcription factors on STAT3 activity. As a result, we found that GATA transcription factors (at least GATA-1, GATA-2, and GATA-3) inhibit STAT3 activity through the direct protein-protein interaction. In addition, we found that GATA-1 utilizes its N-zinc finger to suppress STAT3 activity. Considering the fact that GATA transcription factors utilize both zinc fingers as DNA-binding domains (2), these domains seem to harbor a number of functions, because they also associate with various nuclear factors besides STAT3. For example, the N-zinc finger of GATA-1 interacts with FOG, a critical cofactor for GATA-1 (30, 31) and c-Myb that inhibits its activity (32). The C-zinc finger of GATA-1 forms complexes with p300/CREB-binding protein and the Sp1/EKLF heterodimer, both of which augment GATA-1 activity (33–35). Moreover, GATA-1 binds to PU.1 through the C-zinc finger (25, 36–38). In this interaction, GATA-1 and PU.1 were shown to antagonize each other. These lines of evidence together with our data suggest that, in addition to the roles as transcription factors, GATA transcription factors would exhibit various biological effects by modulating the function of nuclear factors through the direct protein-protein interactions. Also, it was supposed that the activities and roles of GATA transcription factors in cell growth, differentiation, and survival would be considerably different according to the expression profiles of these interacting factors.

In the current study, we showed that ectopically expressed GATA-1/ERT severely suppressed TPO- and gp130-dependent growth of Ba/F3 cells. In addition, because hyperproliferation of megakaryocytes and proerythroblasts were observed in GATA-1 knock-down mice (6–8), it was assumed that endogenous GATA-1 also suppresses the growth of GATA-1-positive cells, at least partly by inhibiting STAT3 activity. Supporting this hypothesis, the binding between GATA-1 and STAT3 was detected in normal megakaryocytes. Then, the next question is whether STAT3 activities are completely suppressed (or to what degree they are suppressed) in GATA-1-positive cells. As for this problem, STAT3 activated by erythropoietin or TPO was shown to still function in GATA-1-positive erythroid and megakaryocytic cells (39, 40). Taken together, these results indicate that appropriately regulated (i.e. suppressed by GATA-1) STAT3 activities would be required for normal development of GATA-1-positive erythroid and megakaryocytic cells.

As was the case with GATA-1 knock-down mice that revealed the myclodysplastic syndrome-like phenotype (6, 7), the germ line mutation of GATA-1 in the N-zinc finger was reported to cause X-linked dyserythropoietic anemia and macrothrombocytopenia (41, 42). In affected individuals, erythrocytes in the peripheral blood were abnormal in size and shape (poikilocytosis and anisocytosis). The bone marrow was packed with excessive large, multinucleated erythroid precursors and with numerous small, dysplastic megakaryocytes. Although these phenotypes were believed to be caused by the defect in transcriptional activities of GATA-1, our result raises a possibility that some phenotype such as hyperproliferation of erythrocytes and megakaryocytes might be caused by unrestricted STAT3 activities due to the GATA-1 mutation in the N-zinc finger.

Among GATA and STAT family members, in the present study, we focused on the effects of GATA-1 and GATA-2 on STAT3 activity. However, it is possible that other members of GATA and STAT interact with and regulate each other. Especially, it will be interesting to examine the functional relationship between STAT3 and GATA-4/GATA-6 in cardiomyocytes, because both molecules play key roles in their development (43, 44). Also, it is attractive to explore the interaction between STAT4/STAT6 and GATA-3 in CD4+ Th lymphocytes, because the functional balance among these molecules was supposed to determine the differentiation toward Th1 or Th2 lymphocytes (45–49).

In summary, we demonstrated here that, in addition to the roles as transcription factors, GATA family proteins modulate cytokine signals through protein-protein interactions, thereby regulating the growth and survival of hematopoietic cells.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Tel.: 81-6-6879-3871; Fax: 81-6-6879-3879; E-mail: matumura{at}bldon.med.osaka-u.ac.jp.

1 The abbreviations used are: C-finger, carboxyl finger; N-finger, amino finger; WT, wild-type; ER, estrogen receptor; ED, estradiol; IL-3, interleukin-3; GM-CSF, granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; TPO, thrombopoietin; STAT, signal transducers and activators of transcription; JAK, Janus tyrosine kinase; MAPK, mitogen-activated protein kinase; Ab, antibody; GST, glutathione S-transferase; 4-HT, tamoxifen; HA, hemagglutinin; del, deletion mutant. Back


   ACKNOWLEDGMENTS
 
We thank Drs. A. Yoshimura, T. Kitamura, and J. E. Darnell, Jr. for providing the plasmids.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Weiss, M. J., and Orkin, S. H. (1995) Exp. Hematol. 23, 99-107[Medline] [Order article via Infotrieve]
  2. Yang, H.-Y., and Evans, T. (1992) Mol. Cell. Biol. 12, 4562-4570[Abstract/Free Full Text]
  3. Penvy, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S. F., D'Agani, V., Orkin, S. H., and Costantini, F. (1991) Nature 349, 257-260[CrossRef][Medline] [Order article via Infotrieve]
  4. Weiss, M. J., and Orkin, S. H. (1994) Genes Dev. 8, 1184-1197[Abstract/Free Full Text]
  5. Weiss, M. J., and Orkin, S. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9623-9627[Abstract/Free Full Text]
  6. Schivdasani, R. A., Fujiwara, Y., McDevitt, M., and Orkin, S. H. (1997) EMBO J. 16, 3965-3973[CrossRef][Medline] [Order article via Infotrieve]
  7. Takahashi, S., Komeno, T., Suwabe, N., Yoh, K., Nakajima, O., Nishimura, S., Kuroha, T., Nagasawa, T., and Yamamoto, M. (1998) Blood 92, 434-442[Abstract/Free Full Text]
  8. Vyas, P., Ault, K., Jackson, C. W., Orkin, S. H., and Shivdasani, R. A. (1999) Blood 93, 2867-2875[Abstract/Free Full Text]
  9. Orlic, D., Anderson, S., Biesecker, L. G., Sorrentino, B. P., and Bodine, D. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4601-4605[Abstract/Free Full Text]
  10. Tsai, F.-Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W., and Orkin., S. H. (1994) Nature 371, 221-226[CrossRef][Medline] [Order article via Infotrieve]
  11. Tsai, F.-Y., and Orkin, S. H. (1997) Blood 89, 3636-3643[Abstract/Free Full Text]
  12. Persons, D. A., Allay, J. A., Allay, E. R., Ashmun, R. A., Orlic, D., and Jane, S. M. (1999) Blood 93, 488-499[Abstract/Free Full Text]
  13. Heyworth, C., Gale, K., Dexter, M., May, G., and Enver, T. (1999) Genes Dev. 13, 1847-1860[Abstract/Free Full Text]
  14. Ezoe, S., Matsumura, I., Nakata, S., Gale, K., Ishihara, K., Minegishi, N., Machii, T., Kitamura, T., Yamamoto, M., Enver, T., and Kanakura, Y. (2002) Blood 100, 3512-3520[Abstract/Free Full Text]
  15. Levy, D. E., and Darnell., J. E., Jr. (2002) Nat. Rev. Mol. Cell. Biol. 3, 651-662[CrossRef][Medline] [Order article via Infotrieve]
  16. Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G., Zhao, Y., Pestell, R. G., Albanese, C., and Darnell, J. E., Jr. (1999) Cell 98, 295-303[CrossRef][Medline] [Order article via Infotrieve]
  17. Kiuchi, N., Nakajima, K., Ichiba, M., Fukada, T., Narimatsu, M., Mizuno, K., Hibi, M., and Hirano, T. (1999) J. Exp. Med. 189, 63-73[Abstract/Free Full Text]
  18. Fukada, T., Hibi, M., Yamanaka, Y., Takahashi-Tezuka, M., Fujitani, Y., Yamaguchi, T., Nakajima, K., and Hirano, T. (1996) Immunity 5, 449-460[CrossRef][Medline] [Order article via Infotrieve]
  19. Matsumura, I., Kitamura, T., Wakao, H., Tanaka, H., Hashimoto, K., Albanese, C., Downward, J., Pestell, R. G., and Kanakura, Y. (1999) EMBO J. 18, 1367-1377[CrossRef][Medline] [Order article via Infotrieve]
  20. Shirogane, T., Fukada, T., Muller, J. M., Shima, D. T., Hibi, M., and Hirano, T. (1999) Immunity 11, 709-719[CrossRef][Medline] [Order article via Infotrieve]
  21. Socolovsky, M., Fallon, A. E., Wang, S., Brugnara, C., and Lodish, H. F. (1999) Cell 98, 181-191[CrossRef][Medline] [Order article via Infotrieve]
  22. Lord, J. D., McIntosh, B. C., Greenberg, P. D., and Nelson, B. H. (2000) J. Immunol. 164, 2533-2541[Abstract/Free Full Text]
  23. Onishi, M., Nosaka, T., Misawa, K., Mui, A. L., Gorman, D., McMahon, M., Miyajima, A., and Kitamura, T. (1998) Mol. Cell. Biol. 18, 3871-3879[Abstract/Free Full Text]
  24. Matsumura, I., Ishikawa, J., Nakajima, K., Oritani, K., Tomiyama, Y., Miyagawa, J., Kato, T., Miyazaki, H., Matsuzawa, Y., and Kanakura, Y. (1997) Mol. Cell. Biol. 17, 2933-2943[Abstract]
  25. Matsumura, I., Kawasaki, A., Tanaka, H., Sonoyama, J., Ezoe, S., Minegishi, N., Nakajima, K., Yamamoto, M., and Kanakura, Y. (2000) Blood 96, 2440-2450[Abstract/Free Full Text]
  26. Matsumura, I., Nakajima, K., Wakao, H., Hattori, S., Hashimoto, K., Sugahara, H., Kato, T., Miyazaki, H., Hirano, T., and Kanakura, Y. (1998) Mol. Cell. Biol. 18, 4282-4290[Abstract/Free Full Text]
  27. Tanaka, H., Matsumura, I., Ezoe, S., Satoh, Y., Sakamaki, T., Albanese, C., Machii, T., Pestell, R. G., and Kanakura, Y. (2002) Mol. Cell 9, 1017-1029[CrossRef][Medline] [Order article via Infotrieve]
  28. Kawasaki, A., Matsumura, I., Kataoka, Y., Takigawa, E., Nakajima, K., and Kanakura, Y. (2003) Blood 101, 3668-3673[Abstract/Free Full Text]
  29. Kerkhoff, E., Houben, R., Loffler, S., Troppmair, J., Lee, J. E., and Rapp, U. R. (1998) Oncogene 16, 211-216[CrossRef][Medline] [Order article via Infotrieve]
  30. Tsang, A. P., Visvader, J. E., Turner, C. A., Fujiwara, Y., Weiss, M. J., Crossley, M., and Orkin, S. H. (1997) Cell 90, 109-119[CrossRef][Medline] [Order article via Infotrieve]
  31. Fox, A. H., Liew, C., Holmes, M., Kowalski, K., Mackay, J., and Crossley, M. (1999) EMBO J. 17, 2812-2822[CrossRef]
  32. Takahashi, T., Suwabe, N., Dai, P., Yamamoto, M., Ishii, S., and Nakano, T. (2000) Oncogene 19, 134-140[CrossRef][Medline] [Order article via Infotrieve]
  33. Merika, M., and Orkin, S. H. (1995) Mol. Cell. Biol. 15, 2437-2447[Abstract]
  34. Boyes, J., Byfield, P., Nakatani, Y., and Ogryzko, V. (1998) Nature 396, 594-598[CrossRef][Medline] [Order article via Infotrieve]
  35. Blobel, G. A., Nakajima, T., Eckner, R., Montminy, M., and Orkin, S. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2061-2066[Abstract/Free Full Text]
  36. Rekhtman, N., Radparvar, F., Evans, T., and Skoultchi, A. I. (1999) Genes Dev. 13, 1398-1411[Abstract/Free Full Text]
  37. Zhang, P., Behre, G., Pan, J., Iwama, A., Wara-Aswapati, N., Radomska, H. S., Auron, P. E., Tenen, D. G., and Sun, Z. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8705-8710[Abstract/Free Full Text]
  38. Nerlov, C., Querfurth, E., Kulessa, H., and Graf, T. (2000) Blood 95, 2543-2551[Abstract/Free Full Text]
  39. Kirito, K., Nakajima, K., Watanabe, T., Uchida, M., Tanaka, M., Ozawa, K., and Komatsu, N. (2002) Blood 99, 102-110[Abstract/Free Full Text]
  40. Kirito, K., Osawa, M., Morita, H., Shimizu, R., Yamamoto, M., Oda, A., Fujita, H., Tanaka, M., Nakajima, K., Miura, Y., Ozawa, K., and Komatsu, N. (2002) Blood 99, 3220-3227[Abstract/Free Full Text]
  41. Nichols, K. E., Crispino, J. D., Poncz, M., White, J. G., Orkin, S. H., Maris, J. M., and Weiss, M. J. (2000) Nat. Genet. 24, 266-270[CrossRef][Medline] [Order article via Infotrieve]
  42. Freson, K., Devriendt, K., Matthijs, G., Van Hoof, A., De Vos, R., Thys, C., Minner, K., Hoylaerts, M. F., Vermylen, J., and Van Geet, C. (2001) Blood 98, 85-92[Abstract/Free Full Text]
  43. Mascareno, E., Dhar, M., and Siddiqui, M. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5590-5594[Abstract/Free Full Text]
  44. Liang, Q., De Windt, L. J., Witt, S. A., Kimball, T. R., Markham, B. E., and Molkentin, J. D. (2001) J. Biol. Chem. 276, 30245-30253[Abstract/Free Full Text]
  45. Thierfelder, W. E., van Deursen, J. M., Yamamoto, K., Tripp, R. A., Sarawar, S. R., Carson, R. T., Sangster, M. Y., Vignali, D. A., Doherty, P. C., Grosveld, G. C., and Ihle, J. N. (1996) Nature 382, 171-174[CrossRef][Medline] [Order article via Infotrieve]
  46. Kaplan, M. H., Sun, Y. L., Hoey, T., and Grusby, M. J. (1996) Nature 382, 174-177[CrossRef][Medline] [Order article via Infotrieve]
  47. Takeda, K., Tanaka, T., Shi, W., Matsumoto, M., Minami, M., Kashiwamura, S., Nakanishi, K., Yoshida, N., Kishimoto, T., and Akira, S. (1996) Nature 380, 627-630[CrossRef][Medline] [Order article via Infotrieve]
  48. Shimoda, K., van Deursen, J., Sangster, M. Y., Sarawar, S. R., Carson, R. T., Tripp, R. A., Chu, C., Quelle, F. W., Nosaka, T., Vignali, D. A., Doherty, P. C., Grosveld, G., Paul, W. E., and Ihle, J. N. (1996) Nature 380, 630-633[CrossRef][Medline] [Order article via Infotrieve]
  49. Farrar, J. D., Ouyang, W., Lohning, M., Assenmacher, M., Radbruch, A., Kanagawa, O., and Murphy, K. M. (2001) J. Exp. Med. 193, 643-650[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
P. Minoo, L. Hu, Y. Xing, N. L. Zhu, H. Chen, M. Li, Z. Borok, and C. Li
Physical and Functional Interactions between Homeodomain NKX2.1 and Winged Helix/Forkhead FOXA1 in Lung Epithelial Cells
Mol. Cell. Biol., March 15, 2007; 27(6): 2155 - 2165.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
N. Onai, A. Obata-Onai, R. Tussiwand, A. Lanzavecchia, and M. G. Manz
Activation of the Flt3 signal transduction cascade rescues and enhances type I interferon-producing and dendritic cell development
J. Exp. Med., January 23, 2006; 203(1): 227 - 238.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
C. Kuhl, A. Atzberger, F. Iborra, B. Nieswandt, C. Porcher, and P. Vyas
GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1
Mol. Cell. Biol., October 1, 2005; 25(19): 8592 - 8606.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/13/13163    most recent
M413461200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow