This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wierenga, A. T. J.
Right arrow Articles by Schuringa, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wierenga, A. T. J.
Right arrow Articles by Schuringa, J. J.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, November 2008, p. 6668-6680, Vol. 28, No. 21
0270-7306/08/$08.00+0     doi:10.1128/MCB.01025-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Maximal STAT5-Induced Proliferation and Self-Renewal at Intermediate STAT5 Activity Levels{triangledown} ,{dagger}

Albertus T. J. Wierenga, Edo Vellenga, and Jan Jacob Schuringa*

University Medical Center Groningen, University of Groningen, Department of Hematology, Hanzeplein 1, 9700 RB Groningen, The Netherlands

Received 30 June 2008/ Returned for modification 11 August 2008/ Accepted 29 August 2008


arrow
ABSTRACT
 
The level of transcription factor activity critically regulates cell fate decisions, such as hematopoietic stem cell (HSC) self-renewal and differentiation. We introduced STAT5A transcriptional activity into human HSCs/progenitor cells in a dose-dependent manner by overexpression of a tamoxifen-inducible STAT5A(1*6)-estrogen receptor fusion protein. Induction of STAT5A activity in CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis, which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased cobblestone area-forming cell and long-term-culture-initiating cell frequencies, which were predominantly elevated at intermediate STAT5A activity levels but not at high STAT5A levels. Self-renewal of progenitors was addressed by serial replating of CFU, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. By extensive gene expression profiling we could identify gene expression patterns of STAT5 target genes that predominantly associated with a self-renewal and long-term expansion phenotype versus those that identified a predominant differentiation phenotype.


arrow
INTRODUCTION
 
STAT5 (signal transducer and activator of transcription 5) is widely expressed throughout the hematopoietic system, in both stem and progenitor cells as well as in committed erythroid, myeloid, and lymphoid cells (8, 39, 49). Indeed, it is not surprising that STAT5 can be activated by a wide variety of cytokines and growth factors (23, 49). These include those that act on the most immature human hematopoietic stem compartment, such as the FLT3 ligand (69), stem cell factor (43), and thrombopoietin (37), as well as those that act on erythroid cells (erythropoietin) (38), lymphoid cells (interleukin 7 [IL-7] and IL-2) (53) or myeloid cells (IL-3, IL-5, granulocyte/macrophage-colony stimulating factor [GM-CSF], and CSF-1) (49). Loss-of-function and gain-of-function experiments have revealed critical roles for STAT5 in several of these cell types. STAT5AB{Delta}N/{Delta}N mice, which express an N-terminally truncated form of STAT5, have been used to assess stem cell function in the absence of wild-type (wt) STAT5 signaling. These mice were characterized by normal hematopoietic stem cell (HSC) numbers, and stem cells isolated from the bone marrow or fetal liver were capable of engrafting irradiated recipients (9), yet the competitive repopulating capacity of STAT5AB{Delta}N/{Delta}N HSCs was severely impaired (5, 6, 9, 55-57). The underlying mechanisms are not fully elucidated yet, but it has been observed that the responsiveness of STAT5AB{Delta}N/{Delta}N HSCs to early-acting cytokines such as IL-3 and stem cell factor is reduced, while the sensitivity to 5-fluorouracil is enhanced (6). More recently, mice completely devoid of STAT5 expression were developed as well, and data have indicated that STAT5 is required for HSC, lymphocyte, and erythrocyte development. (20, 29, 70). We have observed that downmodulation of STAT5 expression impairs long-term self-renewal and maintenance of human stem and progenitor cells (46). Conversely, it has been shown that introduction of activating mutants of STAT5 in hematopoietic stem and progenitor cells enhances long-term self-renewal (26, 50). Within the erythroid compartment, STAT5 fulfils an important antiapoptotic role by upregulating Bcl-Xl (35, 58, 59, 70), although a more direct role in initiating erythroid commitment might exist as well (17, 50, 51). Although in STAT5–/– mice myelopoiesis appears to be relatively unaffected (29), it is likely that in myeloid cells many of the signals initiated by, e.g., IL-3 and GM-CSF are, at least in part, mediated by STAT5 (2, 33). STAT5 is also involved in eosinophil differentiation of human cord blood (CB) CD34+ cells in response to IL-5 (7). Also, lymphoid development is severely impaired in STAT5–/– mice (21). These studies have highlighted the fact that that STAT5 signaling is involved at various stages throughout hematopoietic development. However, little is known about the kinetics of cytokine-induced STAT5 signaling and the manner in which STAT5 responds to lineage-restricted cytokine exposure. Also, it is currently unclear whether similar levels of STAT5 are required for the various functions of this transcription factor within the hematopoietic system.

Besides having a role in normal hematopoiesis, elevated STAT signaling has been associated with leukemic transformation as well. Overexpression of a STAT5A S710F mutant (cSF5F) in murine bone marrow resulted in the development of multilineage leukemias upon transplantation into lethally irradiated wild-type or nonirradiated Rag2–/– mice (32). The underlying mechanisms might involve STAT5 tetramer formation (32), but cytoplasmic STAT5 functions have recently been described as well, whereby STAT5 forms a complex with PI-3K and Gab2, resulting in activation of the Akt/PKB pathway (19). Other mechanisms by which STAT5 can contribute to the transformation process are the induction of genomic instability and DNA damage (30), e.g., by upregulation of reactive oxygen species (44). Constitutive activation of STAT5 has been observed in the vast majority of acute myeloid leukemia (AML) cases (4, 16, 60). This may be attributed to the activating mutations in upstream kinases such as FLT3, c-KIT, and JAK2 (8), or it may be due to autocrine growth factor production (4). We recently demonstrated that in primary AML CD34+ cells, downmodulation of STAT5 expression by lentiviral RNA interference impairs long-term expansion of leukemic stem/progenitor cells (46). In murine cells, constitutive activation of STAT5 is sufficient to induce leukemia-like disease (32, 52). Elevated STAT5 signaling is also involved in the pathogenesis of other hematological malignancies, including chronic myeloid leukemia induced by BCR-ABL (10, 24, 48, 54, 65) or polycythemia vera induced by activated JAK2 V617F (28, 63, 67).

Little information is available regarding mechanisms that are involved in STAT5-induced transformation. Recent reports highlight the importance of transcription factor dosage that is associated with malignant transformation. Examples include PU.1, which is highly leukemic in murine models when expressed at 20% of normal endogenous levels, while complete loss or heterozygous dosage is not leukemic (42). Furthermore, low levels of C/EBP{alpha} are associated with leukemic transformation of myeloid lineages, while increased expression results in precursor B acute lymphoblastic leukemia (12). On the basis of these findings, we analyzed the effects of STAT5 on HSC self-renewal and differentiation in a dose-dependent manner. We observed that at a low to intermediate STAT5 activity, maximal effects on self-renewal of stem and progenitor cells were obtained in conjunction with long-term expansion. High STAT5 activity levels resulted in a predominant erythroid commitment with impaired myeloid development. Gene expression profiling over a range of STAT5 activities in human CB CD34+ cells revealed subsets of genes that were associated with the self-renewal and long-term expansion phenotype. This subset contained (besides known growth-promoting genes such as PIM1 and the gene encoding cyclin D2) a large set of genes encoding membrane and membrane-associated proteins.


arrow
MATERIALS AND METHODS
 
Cell culture and retroviral transductions. CD34+ cells were derived from neonatal cord blood from healthy full-term pregnancies from the obstetrics departments of the Martini Hospital and University Medical Center in Groningen (UMCG), Groningen, The Netherlands, after informed consent had been obtained. The protocol was approved by the Medical Ethical Committee of the UMCG. For all retroviral transduction experiments, the murine stem cell virus (MSCV) retroviral expression vector was used, which contained an encephalomyelocarditis virus-derived internal ribosomal entry site (IRES2) in front of the truncated neural growth factor receptor (MiNR1 vector). The STAT5A(1*6) retroviral vector was described previously (50, 64). STAT5A(1*6)-ER was constructed by fusing the STAT5(1*6) cDNA, from which the stop codon had been removed by PCR-mediated mutagenesis, to the cDNA of the ligand binding domain of the murine estrogen receptor (ER) in pBlueScript (kindly provided by Hergen Spits, Department of Cell Biology and Histology, University of Amsterdam, Amsterdam, The Netherlands). The resulting cDNA was cloned into the EcoRI site of pMSCV-iNGFR. pMSCV-STAT5A(S710F) was made by replacing the BamHI/NsiI fragment from pMSCV-STAT5(1*6) with the same fragment from pMSCV-STAT5A(wt). STAT5A(S710F)-ER and STAT5A(wt)-ER were cloned similarly to pMSCV-STAT5(1*6), using cDNA from the corresponding pMSCV vectors. Details of the cloning and primer sequences are available upon request. pMSCV-STAT5B-ER was made by cloning the STAT5B-ER cDNA (kindly provided by Hergen Spits, Department of Cell Biology and Histology, University of Amsterdam) from pLZRS into the EcoRI site of the pMSCV-iNGFR vector. Stable PG13 high-titer retroviral producer cell lines were generated, and CB CD34+ cells were transduced in three consecutive rounds as described previously (50, 64).

Stromal coculture and CFC, CAFC, LTC-IC, and second CFC assays. MS5 cocultures were performed as described previously. Briefly, MS5 stromal cells were preplated on T25 flasks in minimal essential medium alpha supplemented with 10% horse serum (Sigma, Zwijndrecht, The Netherlands), penicillin, streptomycin, and 200 mM glutamine, and cells were grown to full confluence. A total of 3 x 104 transduced CB CD34+ cells were plated on MS5 in LTC medium (minimal essential medium alpha supplemented with heat-inactivated 12.5% fetal calf serum, heat-inactivated 12.5% horse serum [Sigma, Zwijndrecht, The Netherlands], penicillin, streptomycin, 200 mM glutamine, 57.2 µM β-mercaptoethanol [Sigma], and 1 µM hydrocortisone [Sigma]). Half of the cultures was removed weekly and was replaced with fresh LTC medium. Harvested cells were counted and analyzed by fluorescence-activated cell sorting (FACS) and by analyzing progenitor content (colony-forming cells [CFCs]) as indicated below. CFC assays were performed as described previously. Briefly, CFC assays were performed in MethoCult H4230 (StemCell Technologies, Grenoble, France) supplemented with 20 ng/ml IL-3, 20 ng/ml IL-6, 20 ng/ml G-CSF, 20 ng/ml c-KIT ligand, and 6 U/ml erythropoietin. A total of 103 to 104 cells were plated in methylcellulose per plate in duplicate. For secondary CFC assays, cells were harvested from methylcellulose after 2 weeks, washed three times with phosphate-buffered saline, and replated in new methylcellulose for another 2 weeks, after which secondary CFCs were enumerated. Cobblestone area-forming cell (CAFC) and long-term-culture-initiating cell (LTC-IC) assays were performed by plating transduced CB CD34+ cells in limiting dilutions in the range of 6 to 1,458 cells per well on MS5 stromal cells in 96-well plates in LTC medium. Cultures were fed weekly with new medium, and after 5 weeks, methylcellulose was added to the wells. Two weeks later, wells containing CFCs were scored as positive. CAFC assays were performed similarly to LTC-IC assays, but CAFCs were counted at day 10 and at week 5 by microscopic evaluation of cocultures, and phase dark colonies underneath the stroma were enumerated.

mRNA analysis. Total RNA was isolated using an RNeasy kit from Qiagen (Venlo, The Netherlands) according to the manufacturer's recommendations. For real-time reverse transcriptase PCR, cDNA was prepared by reverse transcribing the total RNA of 0.15 x 106 sorted cells using Moloney murine leukemia virus reverse transcriptase (Fermentas GmbH, St. Leon-Roth, Germany) according to the manufacturer's instructions. cDNA was diluted 20-fold, and 3 µl was then real-time amplified using iQ SYBR Green Supermix (Bio-Rad, Veenendaal, The Netherlands) in a MyIQ thermocycler (Bio-Rad) and quantified using MyIQ software (Bio-Rad). Hypoxanthine phosphoribosyltransferase, RPL27, and RPL30 expression levels (14) were used to normalize samples and to calculate relative expression levels. Primers and conditions are given in Table S1 in the supplemental material. Genome-wide expression analysis was performed on Illumina (Illumina, Inc., San Diego, CA) BeadChip arrays (Sentrix Human-6; 46,000-probe sets). Typically, 0.5 to 1 µg of mRNA combined from three independent transduction experiments was used for labeling reactions and hybridization with the arrays according to the manufacturer's instructions. Data were analyzed using the BeadStudio v3 gene expression module (Illumina, Inc.) and Genespring (Agilent, Amstelveen, The Netherlands).

Flow cytometry analysis. All antibodies were obtained from Becton Dickinson (Alphen a/d Rijn, The Netherlands). Cells were incubated with antibodies at 4°C for 30 min. All FACS analyses were performed on a FACSCalibur (Becton Dickinson) analyzer, and data were analyzed using WinList 3D (Topsham). Cells were sorted on a MoFlo (DakoCytomation, Carpinteria, CA).

EMSA. Nuclear extracts of retrovirally transduced KG1a cells were made according to the miniscale procedure. Electrophoretic mobility shift assays (EMSAs) were performed by incubating 5 µg of nuclear extract with 5'-IRDye700-labeled double-stranded oligonucleotides for 30 min at room temperature. Binding reactions were run on nondenaturing 4% acrylamide gels in 1x Tris-borate-EDTA, and the gels were scanned using an Odyssey infrared scanner (Li-Cor Biosciences, Lincoln, NE). To check for specificity of the reactions, a 50-fold molar excess of unlabeled oligonucleotide (either self or nonself) was added to the binding reaction mixtures. For supershift analysis, 1 µl of STAT5-specific antibody (C17; Santa Cruz Biotech, Santa Cruz, CA) was added simultaneously with the probe.

Immunoblotting, histochemistry, and cytospins. Nuclear extracts were obtained as described above. Laemmli sample buffer was added to the samples, and samples were boiled for 5 min prior to separation on 12% sodium dodecyl sulfate-acrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Etten Leur, The Netherlands) by semidry electroblotting. Membranes were blocked in Odyssey blocking buffer prior to incubation with antibodies. Binding of antibodies was detected by incubating with Alexa680- or IRDye800-labeled secondary antibodies (Invitrogen, Breda, The Netherlands) and scanning of the membrane on an Odyssey infrared scanner (Li-Cor Biosciences, Lincoln, NE). Antibodies against STAT5 (C17) were obtained from Santa Cruz (Santa Cruz Biotech, Santa Cruz, CA), and antibodies against tyrosine-phosphorylated STAT5 (pY694 and C11C5) were obtained from Cell Signaling Technologies (Leiden, The Netherlands) and were used at dilutions of 1:1,000. May-Grünwald Giemsa staining was used to analyze cytospins.

Luciferase assays. For transactivation studies, 293T cells were transiently transfected in 12-well plates with the appropriate constructs using Fugene6 (Roche, Basel, Switzerland) according to the manufacturer's instructions. Cells were transfected with a luciferase vector containing three STAT5-binding sites from the beta-casein promoter (3xGAS-luciferase), together with a pDM-LacZ vector as an internal control for transfection efficiency. At 24 h after transfection, cells were stimulated for 24 h with 0 to 500 nM 4-hydroxytamoxifen (4-OHT) (Sigma) to induce STAT5 activation, and cell extracts were made using Luciferase lysis buffer (Promega, Leiden, The Netherlands). Luciferase expression was measured according to the manufacturer's protocol (Promega, Leiden, The Netherlands). β-Galactosidase expression was measured to correct for differences in transfection efficiency.


arrow
RESULTS
 
Dose-dependent induction of STAT5A activity in human CD34+ cells. In order to generate a system in which STAT5 transactivity levels could be introduced into primary human CD34+ cells in a dose-dependent manner, we constructed a fusion protein of the STAT5A(1*6) mutant with the modified ligand-binding domain of the ER that is responsive to 4-OHT. STAT5A(1*6)-ER was inserted into the retroviral MiNR1 vector (schematically depicted in Fig. 1 A). Dose-dependent STAT5A inducibility was tested first in 293T human kidney and KG1a cells and later in transduced CB-derived CD34+ cells. Western blotting of nuclear extracts of transduced KG1a cells revealed that STAT5 tyrosine 694 phosphorylation and nuclear translocation could be induced in a dose-dependent manner upon treatment of cells with increasing concentrations of 4-OHT (Fig. 1B). Specific STAT5A DNA binding was determined by EMSAs and supershift experiments (Fig. 1C), which could also be induced in a dose-dependent manner by increasing concentrations of 4-OHT (Fig. 1D). STAT5A transactivation was determined by transient transfections of 293T cells with the STAT5A(1*6)-ER fusion together with a 3xGAS-luciferase reporter, and these studies also revealed a dose-dependent induction of STAT5A transactivation upon treatment with increasing concentrations of 4-OHT (Fig. 1E). To study the effects of STAT5 transactivation on the expression of endogenous target genes, CB CD34+ cells were retrovirally transduced with STAT5A(1*6)-ER, and RNA was isolated from cells that were treated with increasing concentrations of 4-OHT for 24 h. Illumina microarray analysis as well as quantitative PCR (Q-PCR) analysis revealed that STAT5 target genes were induced in a dose-dependent manner, and an example for SOCS2 expression is shown in Fig. 1F.


Figure 1
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 1. Dose-dependent induction of STAT5 activity using 4-OHT-inducible STAT5A(1*6)-ER fusion proteins. (A) Schematic representation of the retroviral vectors used in this study. (B) Western blot of nuclear extracts of KG1a cells transduced with MiNR1 control or STAT5A(1*6)-ER vectors. Cells were stimulated with 4-OHT in concentrations from 0 to 500 nM for 24 h, and lysates were probed using antibodies against STAT5 and tyrosine 694-phosphorylated STAT5. (C) EMSA using nuclear extracts of MinR1 and STAT5A(1*6)-ER-transduced KG1a cells using a STAT5-specific probe from the beta-casein promoter. Cells were stimulated with 500 nM 4-OHT for 24 h or left unstimulated, and nuclear extracts were prepared. Supershift experiments were performed using antibodies against STAT5. (D) EMSA experiment as described for panel C, except that cells were stimulated with various concentrations of 4-OHT. (E) 293T cells were transiently transfected with MiNR1 or STAT5A(1*6)-ER vectors together with a luciferase reporter containing three STAT5 binding sites in the promoter. Cells were stimulated with various concentrations of 4-OHT for 24 h, and cell lysates were prepared and analyzed for luciferase activity. (F) CB CD34+ cells were transduced with STAT5A(1*6)-ER and stimulated with 4-OHT for 24 h, and RNA was isolated. Q-PCR analysis was performed using primers against SOCS2. Q-PCR data were compared to results obtained from gene expression profiling using Illumina BeadChip arrays. (G) CB CD34+ cells were stimulated with a cocktail of cytokines (KFT) in a dose-dependent manner. Total cell lysates were prepared, and Western blotting was performed using antibodies against STAT5 and tyrosine 694-phosphorylated STAT5. (H) CB CD34+ cells were transduced with wt STAT5A-ER and stimulated with 10 ng/ml IL-3, 400 nM 4-OHT, or both. Total cell lysates were prepared, and Western blotting was performed using antibodies against STAT5 and tyrosine 694-phosphorylated STAT5. (I) CB CD34+ cells were stimulated with increasing concentrations of KFT as for panel G or transduced with STAT5A(1*6)-ER or wt STAT5A-ER, after which cells were stimulated with 4-OHT as for panel B (for wt STAT5A-ER, 0, 3.2, 16, 80, 400, and 2,000 nM 4-OHT was used). RNA was isolated, and Q-PCRs were performed using primers against CISH.

Finally, we wished to determine how the induced STAT5A(1*6)-ER transactivity levels compare to normal endogenous STAT5 activity levels induced by cytokines as well as to wt STAT5A-ER activity levels. CB CD34+ cells were stimulated with the cytokines c-KIT ligand, FLT3 ligand, and thrombopoietin (KFT) in increasing concentrations, and STAT5 tyrosine 694 phosphorylation was determined by Western blotting. As shown in Fig. 1B and G, 4-OHT-induced STAT5A(1*6) tyrosine phosphorylation was within a similar range and never exceeded endogenous STAT5 phosphorylation levels induced by cytokines. Furthermore, endogenous IL-3-induced STAT5 signaling was compared to wt STAT5A-ER signaling in CB CD34+ cells. IL-3-induced STAT5A tyrosine phosphorylation was comparable to wt STAT5A-ER tyrosine phosphorylation induced by 4-OHT (Fig. 1H). Moreover, similar observations were made in Q-PCR analyses for STAT5 target genes, and an example is shown in Fig. 1I in which cytokine-induced cytokine-inducible SH2-containing protein (CISH) expression was within a range similar to that of STAT5A(1*6)-ER- and wt STAT5A-ER-induced CISH expression, whereby somewhat higher 4-OHT concentrations were required to activate the wt STAT5A-ER molecules.

The proliferative advantage of CD34+ cells is most pronounced at intermediate STAT5A activity levels. CB CD34+ cells were transduced with MiNR1 empty vectors or STAT5A(1*6)-ER vectors, and cocultures on MS5 bone marrow stroma were initiated. 4-OHT was added to the cocultures in concentrations from 0 to 500 nM. Cultures were demidepopulated weekly, and expansion of suspension cells was monitored. Control MiNR1 cultures expanded about 15- to 20-fold over a period of 3 weeks, which was not affected by addition of 4-OHT to the cultures (Fig. 2A). Activation of STAT5A resulted in a proliferative advantage, as we have observed before (50), but surprisingly, the maximum degree of expansion of over 80-fold within 3 weeks was not reached in the groups with the highest STAT5 activity levels (Fig. 2A). Rather, transduced CD34+ cells treated with 20 nM 4-OHT, resulting in intermediate STAT5 activity levels, displayed the maximal proliferative advantage (Fig. 2A). In order to determine whether the effects on proliferation were specific for the STAT5A(1*6)-ER mutant, we repeated the coculture experiments with STAT5A S710F-ER and wild-type STAT5A-ER (Fig. 2B and C) as well as with STAT5B-ER (data not shown), and basically similar results were observed, albeit with somewhat different kinetics. STAT5A(S710F)-ER resulted in maximal long-term expansion at 20 nM (Fig. 2B), at which the maximal number of CAFCs was observed. While at 500 nM a strong expansion was observed at week 1, this expansion was transient; all cells differentiated toward an erythroid fate, and no long-term cultures could be maintained after 3 weeks of MS5 coculture. wt STAT5A-ER-induced proliferation was maximal at 400 nM 4-OHT and did not increase further upon further activation of STAT5 (Fig. 2C).


Figure 2
View larger version (40K):
[in this window]
[in a new window]

  		FIG. 2. Maximal proliferative advantage of CB CD34+ cells at intermediate STAT5A activity levels while differentiation is most affected at high STAT5A transactivation levels. CB CD34+ cells were transduced with MiNR1 control or with STAT5A(1*6)-ER (A) or STAT5A(S710F)-ER (B) vectors and cocultures were initiated on MS5 bone marrow stromal cells in the presence of 4-OHT. Cultures were demidepopulated weekly, and cumulative cell counts of a representative experiment (out of three) are presented. (C) Cells were treated as described for panel A, and cocultures were initiated on MS5 bone marrow stromal cells in the presence of 4-OHT. (D) Cells treated as described for panel A were harvested by weekly demidepopulation and analyzed by FACS for expression of myeloid (CD14, CD15) and erythroid (GPA) antigens. Data from a representative week 2 experiment are shown. (E) Cells treated as described for panel A were harvested at week 2 and analyzed by cytospin and May-Grunwald-Giemsa staining. (F and G) Cells treated as described for panels B and C were harvested by weekly demidepopulation and analyzed by FACS for expression of myeloid (CD14, CD15) and erythroid (GPA) antigens. Data from representative week 2 experiments are shown.

Impaired myelopoiesis and induction of erythropoiesis at high STAT5A transactivation levels. Hematopoietic differentiation was monitored in suspension cells from MS5 cocultures by FACS and morphological analysis of centrifuged cells by MGG staining. As observed previously (50), STAT5A activation impairs myelopoiesis, as demonstrated by a reduction in cells that express the monocytic and granulocytic antigens CD14 and CD15 (Fig. 2D), while erythroid differentiation was induced, as demonstrated by an increase in cells expressing glycophorin A (Fig. 2D), CD36, and CD71bright (data not shown). Analysis of centrifuged cells from suspensions at week 2 yielded similar results, with an impairment of myelopoiesis and an induction of erythropoiesis upon STAT5 activation (Fig. 2E). Importantly, maximum inhibition of myelopoiesis was observed at the highest STAT5 activity levels (Fig. 2D). These data contrast our observations related to the proliferative capacity of cells, which was maximal at intermediate STAT5 activity levels. Maximal erythroid differentiation and impaired myeloid differentiation were also achieved at the highest activity levels of STAT5A S710F-ER (Fig. 2F), wt STAT5A-ER (Fig. 2G), and STAT5B-ER (data not shown).

Self-renewal of progenitors at intermediate STAT5A activity levels. To address the self-renewal capacity of hematopoietic progenitors, CB CD34+ cells were transduced with MiNR1 controls vectors or STAT5A(1*6)-ER, and 103 cells were plated in methylcellulose in duplicate in the absence or presence of increasing concentrations of 4-OHT. As depicted in Fig. 3A, increasing STAT5 activity levels resulted in a dose-dependent reduction in CFCs. No apoptotic cells were observed, suggesting that reduced proliferation or differentiation beyond the progenitor stage was involved rather than programmed cell death (data not shown). After 2 weeks, CFC colonies were harvested and replated in fresh methylcellulose. Only cells that were cultured with low concentrations of 4-OHT were capable of giving rise to secondary CFCs (Fig. 3B, right). Secondary colonies were exclusively of myeloid origin, while erythroid colonies lacked replating capacity. Thus, low to intermediate STAT5 activity levels could confer self-renewal properties to myeloid progenitors, but self-renewal of erythroid progenitors was not observed.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 3. (A) Self-renewal of progenitors at intermediate STAT5A activity levels. CB CD34+ cells were transduced with MiNR1 control or STAT5A(1*6)-ER vectors and plated in methylcellulose in the presence of 4-OHT. Progenitor numbers were scored 2 weeks later. (B) After scoring, cells were harvested, washed with phosphate-buffered saline, and replated in new methylcellulose to initiate second CFC assays, which were scored 2 weeks after plating (n = 3).

CAFC and LTC-IC frequencies are increased at intermediate but not high STAT5A activity levels. To determine the effects of dose-dependent STAT5 signaling on the human HSC population, CAFC and LTC-IC assays were performed. Previous studies have shown that induction of STAT5A activity facilitates the interaction of human CD34+ cells with the bone marrow stroma. These data indicated that activation of STAT5 induces HSCs into a hyperactive state with the capacity to form CAFCs underneath bone marrow stromal cells that retain the capacity for long-term ex vivo self-renewal (50). First we determined the formation of CAFCs at week 2 in sorted transduced cells that were plated in limiting dilution in 96-well plates on MS5 bone marrow stromal cells. Control MiNR1 cells normally do not form CAFCs until week 5 in these cocultures, and indeed no CAFCs were observed in MiNR1 control cells at week 2 (Fig. 4A). In contrast, upon induction of STAT5A activity, early CAFCs appeared within 1 week after plating and continued to grow for over 5 weeks (Fig. 4A and B). Importantly, the highest CAFC frequencies were observed at intermediate STAT5A activity levels (20 nM 4-OHT), and CAFC frequencies were significantly lower at high STAT5A activity levels (500 nM 4-OHT). Representative photographs of MS5 cocultures at week 2 are shown in Fig. 4B. LTC-IC frequencies were determined in limiting dilution in 96-well plates, and in line with our CAFC data at week 2, LTC-IC frequencies were significantly enhanced upon induction of STAT5A activity, which were maximal at intermediate but not high STAT5A activity levels (results of two representative experiments are shown in Fig. 4C). Similar results were obtained in experiments in which CB CD34+ cells were transduced with wt STAT5A-ER vectors, whereby maximal LTC-IC frequencies were observed at intermediate STAT5 activity levels (Fig. 4E).


Figure 4
View larger version (45K):
[in this window]
[in a new window]

  		FIG. 4. CAFC and LTC-IC frequencies are increased at intermediate, but not high, STAT5A activity levels. (A and B) CB CD34+ cells were transduced with MiNR1 control or STAT5A(1*6)-ER vectors, and cocultures were initiated on MS5 bone marrow stromal cells at limiting dilutions on 96-well plates in the presence of 4-OHT. Early CAs were enumerated at week 2 (n = 3). (C and D) Stem cell frequencies were determined in LTC-IC assays at limiting dilutions in 96-well plates on MS5 stroma by plating MiNR1- or STAT5A(1*6)-ER-transduced cells at concentrations of 6 to 1,458 cells per well. Cultures were fed weekly with new medium; at week 5, methylcellulose was added, and LTC-IC frequencies were determined 2 weeks later. Data from two representative experiments are shown. (Left) LTC-IC data over a range of plated cell densities without 4-OHT or in the presence of 500 nM 4-OHT. (Right) LTC-IC frequencies over a range of 4-OHT concentrations. (E) Stem cell frequencies were determined in LTC-IC assays similar to those used for panels C and D but with wt STAT5A-ER-transduced cells.

Genome-wide changes in gene expression induced by dose-dependent STAT5 signaling. In order to correlate global changes in gene expression with levels of STAT5A transactivity, Illumina BeadChip array analysis were performed with STAT5A(1*6)-ER-transduced CD34+ cells that had been stimulated with increasing concentrations of 4-OHT (0 to 500 nM) for 24 h. mRNA was isolated from transduced and MoFlo-sorted cells and used to hybridize human Illumina BeadChip arrays that contained 46,000 probe sets. STAT5 expression levels, as well as the number and location of retroviral integration sites, were identical in all groups, as one batch of CD34+ cells was divided into six groups after transduction and MoFlo sorting that then received a certain dose of 4-OHT. Data was first analyzed by identifying significant changes in gene expression that were induced at intermediate STAT5A activity levels (20 nM versus 0 nM 4-OHT; change of >2-fold; P < 0.05). Thus, 437 changes in gene expression were identified (Fig. 5; also, see Table S2 in the supplemental material). Of the 437 genes with changes, 220 were upregulated and 217 were downregulated (Fig. 5; also, see Table S2 in the supplemental material). A number of the changes in gene expression were verified by independent Q-PCR analysis, and we observed that the Illumina BeadChip array analyses were in good agreement with data obtained by Q-PCR (26 of 29 changes identified by Illumina BeadChip arrays were confirmed by Q-PCR [see Fig. S1 in the supplemental material]). To determine whether the identified target genes were indeed potential STAT5 target genes or whether they were specific for the STAT5A(1*6)-ER mutant, we also performed gene expression studies on CB CD34+ cells transduced with wt STAT5A-ER vectors that had been stimulated with 0 or 500 nM 4-OHT. Relative changes are presented in Table S2 and Fig. S1 in the supplemental material; we observed that the expression of the vast majority of identified genes was affected by both STAT5A(1*6)-ER and wt STAT5A-ER in a similar manner, although the changes were typically somewhat lower in the STAT5A-ER-transduced cells.


Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 5. Genome-wide changes in gene expression induced by dose-dependent STAT5 signaling. CB CD34+ cells were transduced with MiNR1 control or STAT5A(1*6)-ER vectors, and cells were stimulated with 4-OHT for 24 h. RNA was isolated, and genome-wide gene expression patterns were determined on Illumina Sentrix Human-6 BeadChip arrays. The data are gene expression changes in the 20 nM versus 0 nM groups (more than twofold up- or downregulation). Of the 437 genes with changes, 217 were downregulated and 220 were upregulated. The upregulated genes were further subdivided into genes that reached their maximal expression levels at 20 nM (500 nM = 20 nM, ±20%; 138 changes) and genes that were further upregulated in response to further increasing STAT5 activity levels (500 nM > 20 nM, at least 1.2-fold; 60 changes). Gene lists are provided in the supplemental material. A number of gene expression changes were verified by Q-PCR analysis (all Q-PCR analyses are shown in Fig. S1 in the supplemental material). Gene expression changes were annotated according to cellular component, and significant gene ontology (see http://www.geneontology.org/GO.annotation.shtml) cellular component annotations are presented in pie charts.

As we observed different phenotypes at 20 nM (predominant long-term growth and self-renewal) and 500 nM (predominant erythroid differentiation, schematically represented in Fig. 6), we analyzed our datasets in order to determine which differences in gene expression between these groups might exist. We noted that within the upregulated genes, two subgroups could be identified. In one subgroup, maximal gene expression induced by STAT5 was reached at 20 nM 4-OHT ([STAT5]int group, defined as follows: expression at 20 nM > expression at 0 nM [>2-fold] and expression at 500 nM = expression at 20 nM [±20%]). This group contained 138 changes (see Table S3 in the supplemental material). A second subgroup contained genes that responded in a more linear fashion upon increasing STAT5 activity (linear up group, defined as follows: expression at 20 nM > expression at 0 nM [>2-fold] and expression at 500 nM > expression at 20 nM [>1.2-fold]). This group contained 60 changes (see Table S4 in the supplemental material). The [STAT5]int group contained genes such as PIM1, GATA2, IGFBP4 (encoding insulin growth factor binding protein 4), IGFBP5, CISH, CALB2 (encoding calbindin 2), IL3Ra (encoding IL-3 receptor {alpha}), ITGA2 (encoding integrin {alpha}2) and MUC1. The oncogene MAF was also upregulated maximally at intermediate STAT5 activity levels. The linear up group contained genes such as SOCS2 (encoding suppressor of cytokine signaling 2) and RGS1 (encoding regulator of G-protein signaling 1), as well as various novel potential STAT5 target genes, including members of the CCL chemokine family (CCL2, -3, -5, and -22). Q-PCR analysis also revealed that Bcl2 and p21 are induced by STAT5 in a linear fashion (Fig. 5). A total of 217 genes were found to be downregulated by induction of STAT5A activity in CD34+ cells (expression at 20 nM < expression at 0 nM 4-OHT [>2-fold]), and this group included CXCR4, ELA2 (encoding neutrophil elastase), and C/EBP{alpha}, genes that we previously identified as being downmodulated via STAT5A (50, 64), as well as various novel STAT5-regulated genes, including GJA4 (encoding gap junction protein alpha 4) and ARHGAP21 (encoding Rho GTPase activating protein 21) (Fig. 5; also, see Table S2 in the supplemental material).


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 6. Proposed model for dose-dependent STAT5 signaling in human CD34+ cells. Intermediate STAT5 activity levels induce maximal self-renewal and long-term proliferative advantage. High STAT5 activity levels predominantly associate with an induction of erythropoiesis and impaired myelopoiesis. Intermediate STAT5 levels are sufficient to induce maximal levels of growth-promoting oncogenes, such as PIM1 and MAF, or low levels of C/EBP{alpha}, as well as maximal levels of membrane-associated genes, such as MUC1, that might mediate the enhanced interaction of human HSCs/progenitor cells with their bone marrow microenvironment. This pattern of gene expression drives long-term expansion and self-renewal. Upon further increases in STAT5 activity, only specific subsets of STAT5 target genes are further enhanced, and these include p21CIP and members of the chemokine family. These negatively affect cell cycle progression and expansion, resulting in a shift toward a differentiation phenotype along the erythroid lineage.

We annotated the groups of genes according to biological process, cellular component, and molecular function. It was striking that genes within the [STAT5]int group were strongly enriched for the membrane (P = 0.0152), integral-to-membrane (P = 0.00548), and intrinsic-to-membrane (P = 0.00563) categories (Table 1; Fig. 5). Genes within this group are listed and further annotated in Table S3 in the supplemental material. These categories were absent from the linear up group, which was most strongly enriched in genes belonging to the extracellular-space (P = 4.80E-05) category (Table 1; Fig. 5). The downmodulated group was enriched for genes in the plasma membrane categories (P = 0.000586) and also contained genes in the actin cytoskeleton (P = 0.00574) and cell junction (P = 0.00941) categories, among others (Table 1).


View this table:
[in this window]
[in a new window]

  		TABLE 1. GO annotations related to gene expression changes induced by STAT5A(1*6)-ERa


arrow
DISCUSSION
 
The balance between HSC self-renewal and differentiation needs to be tightly controlled, as a shift toward differentiation might exhaust the stem cell pool, while a shift toward self-renewal might mark the onset of leukemic transformation. A number of transcription factors have been proposed to be critically involved in governing stem cell fate and lineage commitment, and these include HOX transcription factors (1), c-Myc (34), Notch1 (61), β-catenin (41), C/EBP{alpha} (68), PU.1 (25), and STAT5 (26, 29, 50). It is therefore no surprise that dysregulation of these transcription factors can contribute to the development of leukemias. Over the past few years, it has become increasingly evident that the dose of transcription factor activity is critical in determining cellular fates, whereby low versus high levels of activity might associate with very different phenotypes. Here, we report that maximal self-renewal and long-term expansion is induced in human stem/progenitor cells by relatively low STAT5 transcriptional activity levels, while high STAT5 activity levels predominantly result in erythroid commitment without long-term growth.

Maybe the clearest example to date that illustrates transcription factor dosage is PU.1. This transcription factor performs a variety of functions within the hematopoietic system, including maintenance of the stem cell pool, the generation of myeloid and lymphoid progenitors, and monocytic differentiation (25). While a 50% reduction in PU.1 expression did not result in severe phenotypes, a reduction to 20% of normal PU.1 levels was shown to be highly leukemogenic (42). Complete deletion of the PU.1 locus did not induce leukemia, and these data convincingly indicate that the levels of PU.1 are highly important in determining the phenotype. Other examples include c-Myc, which is thought to be expressed at very low levels in resting HSCs but whose expression increases upon HSC activation and is high in proliferating progenitors (34). Enforced overexpression of c-Myc in HSCs resulted in a quick loss of stem cells, presumably due to the induction of differentiation. Notch1, when expressed at high levels in neural stem cells leads to growth arrest, while low levels of the active form promote proliferation (18). In human CD34+ cells, overexpression of the active form of Notch1 results in opposing phenotypes, ranging from the induction of apoptosis (11) to stem cell expansion (61), and it is plausible that the dose of active Notch1 is critical in determining the phenotype. Activation of β-catenin by WNT3A was suggested to enhance stem cell self-renewal (41), although a loss of HSC repopulation and a block in multilineage differentiation was reported by others (27, 45), which might be explained by the different dosages of β-catenin activity that were introduced.

These examples indicate that the level of transcription factor activity that is introduced within the hematopoietic stem/progenitor compartment strongly influences the decision whether to self-renew, proliferate, or differentiate. In the present study we used a model system in which the transcription factor activity of STAT5 could be tightly controlled by making use of 4-OHT-inducible chimeras of the modified ER ligand binding domain fused to STAT5. Even though this 30-kDa domain is relatively large and we therefore cannot exclude the possibility that target gene expression might be altered by, for instance, changing STAT5 tetramer formation (32), we have observed induction of the previously described STAT5 target genes with our ER fusion molecules. Thus, this approach allowed us to perform gene dosage experiments in transduced human CD34+ stem/progenitor cells in relation to self-renewal, long-term expansion, and differentiation. Our studies revealed that low to intermediate STAT5 activity, but not high levels of STAT5 activity, are required to induce optimal self-renewal and long-term expansion on the stroma. This intermediate STAT5 activity was associated with cobblestone area (CA) formation underneath the bone marrow stroma within a few days after plating, and these CAs contain self-renewing cells, as demonstrated by their serial replating capacity (31, 50, 51). In fact, this interaction with the bone marrow stroma was required for self-renewal, as no long-term cultures could be established in cytokine-driven liquid cultures using cells transduced with either activated STAT5 or FLT3-ITD (13, 50). Genome-wide gene expression analysis revealed that at intermediate STAT5 activity levels, this group was significantly enriched in genes encoding membrane proteins. These include membrane proteins that have been associated with adhesion, such as protocadherin 21, CEECAM1, and MUC1. While loss-of-function experiments are still required to determine whether these membrane proteins are indeed involved in self-renewal and long-term growth by mediating the interaction between the bone marrow microenvironment and HSCs expressing STAT5, we have already performed studies focusing on the role in MUC1 in normal and leukemic CD34+ cells. These studies indeed indicate that normal CD34+ cells can be further subfractionated into MUC1+ cells to enrich for stem and progenitor cells, that overexpression of MUC1 is sufficient to elevate stem cell self-renewal of human CD34+ cells, and finally that MUC1 expression is increased in CD34+ cells from AML patients in the majority of cases (15). Besides an enrichment for adhesion molecules in the group of genes that is upregulated by intermediate STAT5 activity levels, we found that the group of downmodulated genes is also enriched for membrane-associated genes. Together, these data argue that STAT5 might strongly alter the membrane composition of stem/progenitor cells, such that interactions with the niche that allow long-term self-renewal are facilitated. This would be in line with our observations that cells expressing activated STAT5 cannot be maintained long-term outside of a bone marrow microenvironment.

Besides membrane-associated genes, we also identified a number of growth-promoting oncogenes as STAT5 targets. These include the gene encoding the serine/threonine kinase PIM1, which was initially identified as a Myc cofactor that predisposes to lymphomagenesis (62). Recently, it was shown that Myc recruits PIM1 to target genes to phosphorylate the serine 10 residue of histone H3 on the nucleosome, thereby contributing to transcriptional activation (71). PIM1 has previously been identified as a STAT5 target and can induce cytokine-independent growth of cell lines (35). The MAF gene was also identified as a STAT5 target. In multiple myeloma, the MAF oncogene not only stimulates cell cycle progression but also improves the interaction between tumor and stromal cells (22). C/EBP{alpha} expression was reduced by STAT5, as we observed previously (64), and in a variety of cases, low expression levels or impaired signaling of C/EBP{alpha} has been associated with enhanced self-renewal and leukemic transformation (3, 36, 40, 47, 68). Importantly, the effects of STAT5 on these oncogenes, as well as on the cluster of genes that was enriched for genes encoding membrane proteins, were all maximally induced or repressed at intermediate STAT5 activity levels and did not significantly change upon further increases in STAT5 activity. However, the phenotype did change from a predominant growth and self-renewal phenotype at intermediate STAT5 transactivity levels to a predominant differentiation phenotype at high STAT5 levels. The group of target genes at high STAT5 activity levels was significantly enriched for those encoding secreted extracellular factors. Within this group, we identified the genes for the chemokine family members CCL2, -3, -5, and -22 as novel STAT5 targets, and several potential STAT5 binding sites were found in their 5' promoter sequences (unpublished observations). It has been reported that several of these chemokines have growth-inhibitory effects on murine progenitor cells, as determined by colony assays in methylcellulose (66). These genes respond in a linear fashion in response to STAT5 activity and reach their highest expression levels al maximal STAT5 activity. The same is true for the cell cycle inhibitor p21CIP, and it is plausible that these growth-inhibitory genes might shift the balance from proliferation toward differentiation at the highest STAT5 transactivation levels.

In conclusion, we propose a model which is summarized in Fig. 6. Intermediate STAT5 activity levels are sufficient to induce maximal levels of growth-promoting proto-oncogenes such as PIM1 and MAF, or low levels of C/EBP{alpha}, as well as maximal levels of membrane-associated genes, such as the MUC1 gene, that might mediate the enhanced interaction between human HSCs/progenitor cells with their bone marrow microenvironment. This pattern of gene expression drives long-term expansion and self-renewal. Upon further increases in STAT5 activity, only specific subsets of STAT5 target genes are further enhanced, and these include genes for p21CIP and members of the chemokine family. This set of genes negatively affects cell cycle progression and expansion, resulting in a shift toward a differentiation phenotype along the erythroid lineage. On the basis of this model, we predict that relatively low, but not high, levels of STAT5 transcription factor activity favor leukemic transformation, and it will be challenging to perform these gene-dosage studies in an in vivo setting to confirm these hypotheses. Also, it will be intriguing to decipher why genes respond so differently to increasing levels of STAT5.


arrow
ACKNOWLEDGMENTS
 
This work was supported by KWF grant RUG 2003-2316 to E.V. and an NWO-VENI 2004 grant to J.J.S.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: University Medical Center Groningen, University of Groningen, Department of Hematology, Hanzeplein 1, 9700 RB Groningen, The Netherlands. Phone: 31-50-3619391. Fax: 31-50-3614862. E-mail: j.schuringa{at}int.umcg.nl Back

{triangledown} Published ahead of print on 8 September 2008. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


arrow
REFERENCES
 
    1
  1. Argiropoulos, B., and R. K. Humphries. 2007. Hox genes in hematopoiesis and leukemogenesis. Oncogene 26:6766-6776.[CrossRef][Medline]
    2. 2
  2. Azam, M., H. Erdjument-Bromage, B. L. Kreider, M. Xia, F. Quelle, R. Basu, C. Saris, P. Tempst, J. N. Ihle, and C. Schindler. 1995. Interleukin-3 signals through multiple isoforms of Stat5. EMBO J. 14:1402-1411.[Medline]
    3. 3
  3. Barjesteh van Waalwijk van Doorn-Khosrovani, S., C. Erpelink, J. Meijer, S. van Oosterhoud, W. L. van Putten, P. J. Valk, H. Berna Beverloo, D. G. Tenen, B. Lowenberg, and R. Delwel. 2003. Biallelic mutations in the CEBPA gene and low CEBPA expression levels as prognostic markers in intermediate-risk AML. Hematol. J. 4:31-40.[CrossRef][Medline]
    4. 4
  4. Birkenkamp, K. U., M. Geugien, H. H. Lemmink, W. Kruijer, and E. Vellenga. 2001. Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts. Leukemia 15:1923-1931.[Medline]
    5. 5
  5. Bradley, H. L., C. Couldrey, and K. D. Bunting. 2004. Hematopoietic-repopulating defects from STAT5-deficient bone marrow are not fully accounted for by loss of thrombopoietin responsiveness. Blood 103:2965-2972.[Abstract/Free Full Text]
    6. 6
  6. Bradley, H. L., T. S. Hawley, and K. D. Bunting. 2002. Cell intrinsic defects in cytokine responsiveness of STAT5-deficient hematopoietic stem cells. Blood 100:3983-3989.[Abstract/Free Full Text]
    7. 7
  7. Buitenhuis, M., B. Baltus, J. W. Lammers, P. J. Coffer, and L. Koenderman. 2003. Signal transducer and activator of transcription 5a (STAT5a) is required for eosinophil differentiation of human cord blood-derived CD34+ cells. Blood 101:134-142.[Abstract/Free Full Text]
    8. 8
  8. Bunting, K. D. 2007. STAT5 signaling in normal and pathologic hematopoiesis. Front. Biosci. 12:2807-2820.[CrossRef][Medline]
    9. 9
  9. Bunting, K. D., H. L. Bradley, T. S. Hawley, R. Moriggl, B. P. Sorrentino, and J. N. Ihle. 2002. Reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice lacking expression of STAT5. Blood 99:479-487.[Abstract/Free Full Text]
    10. 10
  10. Carlesso, N., D. A. Frank, and J. D. Griffin. 1996. Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J. Exp. Med. 183:811-820.[Abstract/Free Full Text]
    11. 11
  11. Chadwick, N., M. C. Nostro, M. Baron, R. Mottram, G. Brady, and A. M. Buckle. 2007. Notch signaling induces apoptosis in primary human CD34+ hematopoietic progenitor cells. Stem Cells 25:203-210.[CrossRef][Medline]
    12. 12
  12. Chapiro, E., L. Russell, I. Radford-Weiss, C. Bastard, M. Lessard, S. Struski, H. Cave, S. Fert-Ferrer, C. Barin, O. Maarek, V. la-Valle, J. C. Strefford, R. Berger, C. J. Harrison, O. A. Bernard, and F. Nguyen-Khac. 2006. Overexpression of CEBPA resulting from the translocation t(14;19)(q32;q13) of human precursor B acute lymphoblastic leukemia. Blood 108:3560-3563.[Abstract/Free Full Text]
    13. 13
  13. Chung, K. Y., G. Morrone, J. J. Schuringa, B. Wong, D. C. Dorn, and M. A. Moore. 2005. Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis. Blood 105:77-84.[Abstract/Free Full Text]
    14. 14
  14. de Jonge, H. J., R. S. Fehrmann, E. S. de Bont, R. M. Hofstra, F. Gerbens, W. A. Kamps, E. G. de Vries, A. G. van der Zee, G. J. te Meerman, and A. ter Elst. 2007. Evidence based selection of housekeeping genes. PLoS One 2:e898.[CrossRef][Medline]
    15. 15
  15. Fatrai, S., H. Schepers, H. Tadema, S. Daenen, E. Vellenga, and J. J. Schuringa. Mucin1 expression is enriched in the human stem cell fraction of cord blood and is up-regulated in majority of the AML cases. Exp. Hematol., in press.
    16. 16
  16. Gouilleux-Gruart, V., F. Gouilleux, C. Desaint, J. F. Claisse, J. C. Capiod, J. Delobel, R. Weber-Nordt, I. Dusanter-Fourt, F. Dreyfus, B. Groner, and L. Prin. 1996. STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients. Blood 87:1692-1697.[Abstract/Free Full Text]
    17. 17
  17. Grebien, F., M. A. Kerenyi, B. Kovacic, T. Kolbe, V. Becker, H. Dolznig, K. Pfeffer, U. Klingmuller, M. Muller, H. Beug, E. W. Mullner, and R. Moriggl. 2008. Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2. Blood 111:4511-4522.[Abstract/Free Full Text]
    18. 18
  18. Guentchev, M., and R. D. McKay. 2006. Notch controls proliferation and differentiation of stem cells in a dose-dependent manner. Eur. J. Neurosci. 23:2289-2296.[CrossRef][Medline]
    19. 19
  19. Harir, N., C. Pecquet, M. Kerenyi, K. Sonneck, B. Kovacic, R. Nyga, M. Brevet, I. Dhennin, V. Gouilleux-Gruart, H. Beug, P. Valent, K. Lassoued, R. Moriggl, and F. Gouilleux. 2007. Constitutive activation of Stat5 promotes its cytoplasmic localization and association with PI3-kinase in myeloid leukemias. Blood 109:1678-1686.[Abstract/Free Full Text]
    20. 20
  20. Hennighausen, L., and G. W. Robinson. 2008. Interpretation of cytokine signaling through the transcription factors STAT5A and STAT5B. Genes Dev. 22:711-721.[Abstract/Free Full Text]
    21. 21
  21. Hoelbl, A., B. Kovacic, M. A. Kerenyi, O. Simma, W. Warsch, Y. Cui, H. Beug, L. Hennighausen, R. Moriggl, and V. Sexl. 2006. Clarifying the role of Stat5 in lymphoid development and Abelson-induced transformation. Blood 107:4898-4906.[Abstract/Free Full Text]
    22. 22
  22. Hurt, E. M., A. Wiestner, A. Rosenwald, A. L. Shaffer, E. Campo, T. Grogan, P. L. Bergsagel, W. M. Kuehl, and L. M. Staudt. 2004. Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma. Cancer Cell 5:191-199.[CrossRef][Medline]
    23. 23
  23. Ihle, J. N. 2001. The Stat family in cytokine signaling. Curr. Opin. Cell Biol. 13:211-217.[CrossRef][Medline]
    24. 24
  24. Ilaria, R. L., Jr., and R. A. Van Etten. 1996. P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members. J. Biol. Chem. 271:31704-31710.[Abstract/Free Full Text]
    25. 25
  25. Iwasaki, H., C. Somoza, H. Shigematsu, E. A. Duprez, J. Iwasaki-Arai, S. Mizuno, Y. Arinobu, K. Geary, P. Zhang, T. Dayaram, M. L. Fenyus, S. Elf, S. Chan, P. Kastner, C. S. Huettner, R. Murray, D. G. Tenen, and K. Akashi. 2005. Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation. Blood 106:1590-1600.[Abstract/Free Full Text]
    26. 26
  26. Kato, Y., A. Iwama, Y. Tadokoro, K. Shimoda, M. Minoguchi, S. Akira, M. Tanaka, A. Miyajima, T. Kitamura, and H. Nakauchi. 2005. Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis. J. Exp. Med. 202:169-179.[Abstract/Free Full Text]
    27. 27
  27. Kirstetter, P., K. Anderson, B. T. Porse, S. E. Jacobsen, and C. Nerlov. 2006. Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block. Nat. Immunol. 7:1048-1056.[CrossRef][Medline]
    28. 28
  28. Lacout, C., D. F. Pisani, M. Tulliez, F. M. Gachelin, W. Vainchenker, and J. L. Villeval. 2006. JAK2V617F expression in murine hematopoietic cells leads to MPD mimicking human PV with secondary myelofibrosis. Blood 108:1652-1660.[Abstract/Free Full Text]
    29. 29
  29. Li, G., Z. Wang, Y. Zhang, Z. Kang, E. Haviernikova, Y. Cui, L. Hennighausen, R. Moriggl, D. Wang, W. Tse, and K. D. Bunting. 2007. STAT5 requires the N-domain to maintain hematopoietic stem cell repopulating function and appropriate lymphoid-myeloid lineage output. Exp. Hematol. 35:1684-1694.[CrossRef][Medline]
    30. 30
  30. Mallette, F. A., M. F. Gaumont-Leclerc, and G. Ferbeyre. 2007. The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence. Genes Dev. 21:43-48.[Abstract/Free Full Text]
    31. 31
  31. Moore, M. A., D. C. Dorn, J. J. Schuringa, K. Y. Chung, and G. Morrone. 2007. Constitutive activation of Flt3 and STAT5A enhances self-renewal and alters differentiation of hematopoietic stem cells. Exp. Hematol. 35:105-116.[CrossRef][Medline]
    32. 32
  32. Moriggl, R., V. Sexl, L. Kenner, C. Duntsch, K. Stangl, S. Gingras, A. Hoffmeyer, A. Bauer, R. Piekorz, D. Wang, K. D. Bunting, E. F. Wagner, K. Sonneck, P. Valent, J. N. Ihle, and H. Beug. 2005. Stat5 tetramer formation is associated with leukemogenesis. Cancer Cell 7:87-99.[CrossRef][Medline]
    33. 33
  33. Mui, A. L., H. Wakao, A. M. O'Farrell, N. Harada, and A. Miyajima. 1995. Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J. 14:1166-1175.[Medline]
    34. 34
  34. Murphy, M. J., A. Wilson, and A. Trumpp. 2005. More than just proliferation: Myc function in stem cells. Trends Cell Biol. 15:128-137.[CrossRef][Medline]
    35. 35
  35. Nosaka, T., T. Kawashima, K. Misawa, K. Ikuta, A. L. Mui, and T. Kitamura. 1999. STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells. EMBO J. 18:4754-4765.[CrossRef][Medline]
    36. 36
  36. Pabst, T., B. U. Mueller, P. Zhang, H. S. Radomska, S. Narravula, S. Schnittger, G. Behre, W. Hiddemann, and D. G. Tenen. 2001. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBP{alpha}), in acute myeloid leukemia. Nat. Genet. 27:263-270.[CrossRef][Medline]
    37. 37
  37. Pallard, C., F. Gouilleux, L. Benit, L. Cocault, M. Souyri, D. Levy, B. Groner, S. Gisselbrecht, and I. Dusanter-Fourt. 1995. Thrombopoietin activates a STAT5-like factor in hematopoietic cells. EMBO J. 14:2847-2856.[Medline]
    38. 38
  38. Pallard, C., F. Gouilleux, M. Charon, B. Groner, S. Gisselbrecht, and I. Dusanter-Fourt. 1995. Interleukin-3, erythropoietin, and prolactin activate a STAT5-like factor in lymphoid cells. J. Biol. Chem. 270:15942-15945.[Abstract/Free Full Text]
    39. 39
  39. Paukku, K., and O. Silvennoinen. 2004. STATs as critical mediators of signal transduction and transcription: lessons learned from STAT5. Cytokine Growth Factor Rev. 15:435-455.[CrossRef][Medline]
    40. 40
  40. Radomska, H. S., D. S. Basseres, R. Zheng, P. Zhang, T. Dayaram, Y. Yamamoto, D. W. Sternberg, N. Lokker, N. A. Giese, S. K. Bohlander, S. Schnittger, M. H. Delmotte, R. J. Davis, D. Small, W. Hiddemann, D. G. Gilliland, and D. G. Tenen. 2006. Block of C/EBP alpha function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations. J. Exp. Med. 203:371-381.[Abstract/Free Full Text]
    41. 41
  41. Reya, T., A. W. Duncan, L. Ailles, J. Domen, D. C. Scherer, K. Willert, L. Hintz, R. Nusse, and I. L. Weissman. 2003. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423:409-414.[CrossRef][Medline]
    42. 42
  42. Rosenbauer, F., K. Wagner, J. L. Kutok, H. Iwasaki, M. M. Le Beau, Y. Okuno, K. Akashi, S. Fiering, and D. G. Tenen. 2004. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nat. Genet. 36:624-630.[CrossRef][Medline]
    43. 43
  43. Ryan, J. J., H. Huang, L. J. McReynolds, C. Shelburne, J. Hu-Li, T. F. Huff, and W. E. Paul. 1997. Stem cell factor activates STAT-5 DNA binding in IL-3-derived bone marrow mast cells. Exp. Hematol. 25:357-362.[Medline]
    44. 44
  44. Sallmyr, A., J. Fan, K. Datta, K. T. Kim, D. Grosu, P. Shapiro, D. Small, and F. Rassool. 2008. Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML. Blood 111:3173-3182.[Abstract/Free Full Text]
    45. 45
  45. Scheller, M., J. Huelsken, F. Rosenbauer, M. M. Taketo, W. Birchmeier, D. G. Tenen, and A. Leutz. 2006. Hematopoietic stem cell and multilineage defects generated by constitutive beta-catenin activation. Nat. Immunol. 7:1037-1047.[CrossRef][Medline]
    46. 46
  46. Schepers, H., D. van Gosliga, A. T. Wierenga, B. J. Eggen, J. J. Schuringa, and E. Vellenga. 2007. STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells. Blood 110:2880-2888.[Abstract/Free Full Text]
    47. 47
  47. Schepers, H., A. T. Wierenga, D. van Gosliga, B. J. Eggen, E. Vellenga, and J. J. Schuringa. 2007. Reintroduction of C/EBP{alpha} in leukemic CD34+ stem/progenitor cells impairs self-renewal and partially restores myelopoiesis. Blood 110:1317-1325.[Abstract/Free Full Text]
    48. 48
  48. Scherr, M., A. Chaturvedi, K. Battmer, I. Dallmann, B. Schultheis, A. Ganser, and M. Eder. 2006. Enhanced sensitivity to inhibition of SHP2, STAT5, and Gab2 expression in chronic myeloid leukemia (CML). Blood 107:3279-3287.[Abstract/Free Full Text]
    49. 49
  49. Schindler, C., and J. E. Darnell, Jr. 1995. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621-651.[Medline]
    50. 50
  50. Schuringa, J. J., K. Y. Chung, G. Morrone, and M. A. Moore. 2004. Constitutive activation of STAT5A promotes human hematopoietic stem cell self-renewal and erythroid differentiation. J. Exp. Med. 200:623-635.[Abstract/Free Full Text]
    51. 51
  51. Schuringa, J. J., K. Wu, G. Morrone, and M. A. Moore. 2004. Enforced activation of STAT5A facilitates the generation of embryonic stem-derived hematopoietic stem cells that contribute to hematopoiesis in vivo. Stem Cells 22:1191-1204.[CrossRef][Medline]
    52. 52
  52. Schwaller, J., E. Parganas, D. Wang, D. Cain, J. C. Aster, I. R. Williams, C. K. Lee, R. Gerthner, T. Kitamura, J. Frantsve, E. Anastasiadou, M. L. Loh, D. E. Levy, J. N. Ihle, and D. G. Gilliland. 2000. Stat5 is essential for the myelo- and lymphoproliferative disease induced by TEL/JAK2. Mol. Cell 6:693-704.[CrossRef][Medline]
    53. 53
  53. Sexl, V., R. Piekorz, R. Moriggl, J. Rohrer, M. P. Brown, K. D. Bunting, K. Rothammer, M. F. Roussel, and J. N. Ihle. 2000. Stat5a/b contribute to interleukin 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced transformation are independent of stat5. Blood 96:2277-2283.[Abstract/Free Full Text]
    54. 54
  54. Shuai, K., J. Halpern, J. ten Hoeve, X. Rao, and C. L. Sawyers. 1996. Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene 13:247-254.[Medline]
    55. 55
  55. Snow, J. W., N. Abraham, M. C. Ma, N. W. Abbey, B. Herndier, and M. A. Goldsmith. 2002. STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells. Blood 99:95-101.[Abstract/Free Full Text]
    56. 56
  56. Snow, J. W., N. Abraham, M. C. Ma, S. K. Bronson, and M. A. Goldsmith. 2003. Transgenic bcl-2 is not sufficient to rescue all hematolymphoid defects in STAT5A/5B-deficient mice. Exp. Hematol. 31:1253-1258.[CrossRef][Medline]
    57. 57
  57. Snow, J. W., N. Abraham, M. C. Ma, and M. A. Goldsmith. 2003. Bone marrow transplant completely rescues hematolymphoid defects in STAT5A/5B-deficient mice. Exp. Hematol. 31:1247-1252.[CrossRef][Medline]
    58. 58
  58. Socolovsky, M., A. E. Fallon, S. Wang, C. Brugnara, and H. F. Lodish. 1999. Fetal anemia and apoptosis of red cell progenitors in Stat5a–/–5b–/– mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 98:181-191.[CrossRef][Medline]
    59. 59
  59. Socolovsky, M., H. Nam, M. D. Fleming, V. H. Haase, C. Brugnara, and H. F. Lodish. 2001. Ineffective erythropoiesis in Stat5a–/–5b–/– mice due to decreased survival of early erythroblasts. Blood 98:3261-3273.[Abstract/Free Full Text]
    60. 60
  60. Spiekermann, K., M. Pau, R. Schwab, K. Schmieja, S. Franzrahe, and W. Hiddemann. 2002. Constitutive activation of STAT3 and STAT5 is induced by leukemic fusion proteins with protein tyrosine kinase activity and is sufficient for transformation of hematopoietic precursor cells. Exp. Hematol. 30:262-271.[CrossRef][Medline]
    61. 61
  61. Suzuki, T., and S. Chiba. 2005. Notch signaling in hematopoietic stem cells. Int. J. Hematol. 82:285-294.[CrossRef][Medline]
    62. 62
  62. van Lohuizen, M., S. Verbeek, P. Krimpenfort, J. Domen, C. Saris, T. Radaszkiewicz, and A. Berns. 1989. Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell 56:673-682.[CrossRef][Medline]
    63. 63
  63. Wernig, G., T. Mercher, R. Okabe, R. L. Levine, B. H. Lee, and D. G. Gilliland. 2006. Expression of Jak2V617F causes a polycythemia vera-like disease with associated myelofibrosis in a murine bone marrow transplant model. Blood 107:4274-4281.[Abstract/Free Full Text]
    64. 64
  64. Wierenga, A. T., H. Schepers, M. A. Moore, E. Vellenga, and J. J. Schuringa. 2006. STAT5-induced self-renewal and impaired myelopoiesis of human hematopoietic stem/progenitor cells involves down-modulation of C/EBP{alpha}. Blood 107:4326-4333.[Abstract/Free Full Text]
    65. 65
  65. Ye, D., N. Wolff, L. Li, S. Zhang, and R. L. Ilaria, Jr. 2006. STAT5 signaling is required for the efficient induction and maintenance of CML in mice. Blood 107:4917-4925.[Abstract/Free Full Text]
    66. 66
  66. Youn, B. S., C. Mantel, and H. E. Broxmeyer. 2000. Chemokines, chemokine receptors and hematopoiesis. Immunol. Rev. 177:150-174.[CrossRef][Medline]
    67. 67
  67. Zaleskas, V. M., D. S. Krause, K. Lazarides, N. Patel, Y. Hu, S. Li, and R. A. Van Etten. 2006. Molecular pathogenesis and therapy of polycythemia induced in mice by JAK2 V617F. PLoS One 1:e18.[CrossRef]
    68. 68
  68. Zhang, P., J. Iwasaki-Arai, H. Iwasaki, M. L. Fenyus, T. Dayaram, B. M. Owens, H. Shigematsu, E. Levantini, C. S. Huettner, J. A. Lekstrom-Himes, K. Akashi, and D. G. Tenen. 2004. Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP{alpha}. Immunity 21:853-863.[CrossRef][Medline]
    69. 69
  69. Zhang, S., S. Fukuda, Y. Lee, G. Hangoc, S. Cooper, R. Spolski, W. J. Leonard, and H. E. Broxmeyer. 2000. Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling. J. Exp. Med. 192:719-728.[Abstract/Free Full Text]
    70. 70
  70. Zhu, B. M., S. K. McLaughlin, R. Na, J. Liu, Y. Cui, C. Martin, A. Kimura, G. W. Robinson, N. C. Andrews, and L. Hennighausen. 2008. Hematopoietic-specific Stat5-null mice display microcytic hypochromic anemia associated with reduced transferrin receptor gene expression. Blood 112:2071-2080.[Abstract/Free Full Text]
    71. 71
  71. Zippo, A., A. De Robertis, R. Serafini, and S. Oliviero. 2007. PIM1-dependent phosphorylation of histone H3 at serine 10 is required for Myc-dependent transcriptional activation and oncogenic transformation. Nat. Cell Biol. 9:932-944.[CrossRef][Medline]

Molecular and Cellular Biology, November 2008, p. 6668-6680, Vol. 28, No. 21
0270-7306/08/$08.00+0     doi:10.1128/MCB.01025-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Huang, X., Sakamoto, H., Ogawa, M. (2009). Thrombopoietin controls proliferation of embryonic multipotent hematopoietic progenitors. GENES CELLS 14: 851-860 [Abstract] [Full Text]  
  • Wang, Z., Li, G., Tse, W., Bunting, K. D. (2009). Conditional deletion of STAT5 in adult mouse hematopoietic stem cells causes loss of quiescence and permits efficient nonablative stem cell replacement. Blood 113: 4856-4865 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wierenga, A. T. J.
Right arrow Articles by Schuringa, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wierenga, A. T. J.
Right arrow Articles by Schuringa, J. J.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»