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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2002-03-0740.
HEMATOPOIESIS
From the Department of Pulmonary Diseases, University
Medical Center, Utrecht, The Netherlands.
Signal transducers and activators of transcription (STATs) have
been reported to play a critical role in the differentiation of several
myeloid cell lines, although the importance of STATs in the
differentiation of primary human hematopoietic cells remains to be
established. Terminal eosinophil differentiation is induced by
interleukin-5 (IL-5), which has also been demonstrated to activate STAT5. We have investigated whether STAT5 plays a critical role during
eosinophil differentiation using umbilical cord blood-derived CD34+ cells. In this ex vivo system, STAT5 expression and
activation are high early during differentiation, and STAT5 protein
expression is down-regulated during the final stages of eosinophil
differentiation. Retroviral transductions were performed to ectopically
express wild-type and dominant-negative STAT5a (STAT5a Eosinophils play an important role in immunity
against helminth infection. Parasite infection results in enhanced
eosinophil numbers and in migration toward and degranulation at the
site of parasite invasion. In vitro studies demonstrated that
eosinophils are capable of adhering to parasites in the presence of
different antibody isotypes (immunoglobulin E [IgE], IgG,
IgA)1,2 and complement components (C3b).3 At
the site of contact, eosinophils degranulate, resulting in damage to
the tegumental membranes and eventually in the death of
parasites.4 The toxic effect of eosinophilic granule
proteins, such as eosinophilic cationic protein (ECP), is complemented
by the generation of toxic oxygen metabolites.5 Besides
their role in immunity against parasitic infection, eosinophils are
thought to play an important role in the pathogenesis of allergic diseases, among them asthma and dermatitis. Eosinophils are recruited to the site of allergic inflammation by chemoattractants, such as C5a
and platelet activating factor (PAF).6,7 Release
of granular contents at the inflammatory locus can result in long-term tissue damage. For example, airway epithelium can be damaged during airway inflammation.8,9
Eosinophils are derived from pluripotent hematopoietic stem cells
(HSCs) in the bone marrow. These progenitors are defined as precursors
for all lineages of mature blood cells, and they are capable of
self-renewal. Such cells can be divided into long-term repopulating
hematopoietic stem cells (LT-HSCs) and short-term repopulating
hematopoietic stem cells (ST-HSCs). ST-HSCs can differentiate to
multipotent progenitor cells, which are capable of differentiation toward a subset of the hematopoietic lineage. These multipotent stem
cells include the common lymphoid precursor10 and the
common myeloid precursor.11 Common myeloid progenitor
cells, known as the granulocyte/erythrocyte/macrophage/megakaryocyte
colony-forming unit (CFU-GEMM), can differentiate toward the erythroid,
megakaryocytic, and myelomonocytic lineage. It has been demonstrated
that genes specific for erythroid, myeloid, or megakaryocytic lineages
are transcribed in the common myeloid progenitors before commitment to
a single lineage. Genes specific for differentiation toward other
lineages are down-regulated on commitment to a single
lineage.12 Myeloid differentiation is regulated by a
variety of cytokines, including erythropoietin (EPO),
granulocyte-colony-stimulating factor (G-CSF), thrombopoietin (TPO),
interleukin-3 (IL-3), granulocyte macrophage-colony-stimulating factor
(GM-CSF), macrophage-colony-stimulating factor (M-CSF), and IL-5. IL-3
and GM-CSF are cytokines that regulate proliferation and survival
during myeloid differentiation of various lineages, whereas EPO, TPO,
G-CSF, M-CSF, and IL-5 are required for the final maturation of
erythrocytes,13 megakaryocytes, platelets,14,15 neutrophils, monocytes,16 and
eosinophils,17,18 respectively.
Hematopoietic cytokines can activate several signal transduction
pathways, including the Ras/Raf/Erk,19-23
phosphatidylinositol 3 kinase (PI3K),24-26 and Janus
kinase/signal transducer and activator of transcription (JAK/STAT)
pathway.27,28 The JAK/STAT signal transduction pathway is
thought to play an important role in myeloid differentiation,29 and the signaling paradigm mediating
this pathway has been elucidated in detail.30 In short, on
ligand binding, cytokine receptor dimerization results in the
activation of members of the receptor-associated JAK family through
cross-phosphorylation. Subsequently, tyrosine phosphorylation of the
intracellular domains of the dimerized receptor occurs, enabling STAT
transcription factors to associate with the receptor. STATs are
subsequently phosphorylated by the receptor-associated JAKs. Homodimers
or heterodimers are formed, and, after translocation to the nucleus, transcription can be regulated through STAT-specific DNA-binding sites.31-34
It has been demonstrated that the stimulation of cells with IL-5, which
is required for terminal eosinophil differentiation, results in the
activation of STAT5.35,36 In this study, we have
investigated the role of STAT5 during IL-5-mediated eosinophil differentiation. Our data demonstrate that STAT5 is expressed throughout the eosinophil differentiation program of CD34+
progenitor cells. STAT5 expression and activation are high during early
differentiation, but STAT5 expression is down-regulated during terminal
eosinophil differentiation. To determine whether STAT5 plays a critical
role in the differentiation process, wild-type STAT5a,
dominant-negative STAT5a, and dominant-negative STAT5b were ectopically
expressed in CD34+ cells by retroviral transduction. Our
experiments demonstrated that STAT5a and STAT5b play an important role
in proliferation and maturation during eosinophil differentiation.
Ectopic expression of STAT5a was found to induce the expression of
Bcl-2, an antiapoptotic protein, and p21WAF/Cip1, a
cyclin-dependent kinase inhibitor, in differentiating eosinophils. These data demonstrate that STAT5 plays a critical role in regulating the differentiation of primary human hematopoietic cells toward eosinophils.
Isolation and culture of human CD34+ cells
Neutrophil differentiation was induced with the addition of SCF (50 ng/mL), FLT-3 (50 ng/mL) ligand, GM-CSF (0.1 nM), IL-3 (0.1 nM), and
G-CSF (30 ng/mL). After 6 days of culture, only G-CSF was added to
the cells.
Viral transduction of CD34+ cells
CD34+ cells were transduced in 24-well dishes precoated with 20 µg/cm2 recombinant human fibronectin fragment CH-296 (RetroNectin; Takara, Otsu, Japan) for 2 hours and with 2% bovine serum albumin (BSA) for 30 minutes. Transduction was performed by the addition of 0.5 mL viral supernatant to 0.5 mL medium containing 0.5 × 106 cells. Twenty-four hours after transduction, 0.7 mL medium was removed from the cells, and 0.5 fresh virus supernatant was added together with 0.5 mL fresh medium and cytokines (IL-3 and IL-5). The percentage of eGFP-positive living cells was determined every 3 or 4 days by fluorescence-activated cell sorter (FACS) analysis (FACS Vantage; Becton Dickinson, San Jose, CA). Histochemical staining of eosinophil and neutrophil precursors The percentage of eosinophil differentiation was determined by histochemical staining of the cells. The percentage of cells differentiating toward eosinophils was determined by Luxol Fast-Blue (LFB) staining (Avocado Research Chem, Heysham, United Kingdom), a dye that specifically stains eosinophil granules. Cytospins were prepared from 5 × 104 differentiated eosinophils. Slides were dried on silica gel for 24 hours before fixing them in dry acetone for 10 minutes. Slides were stained with 0.15% wt/vol LFB (Avocado Research) in urea-saturated ethanol for 2 hours.May-Grünwald-Giemsa staining was used to analyze differentiating eosinophils and neutrophils. Cytospins were prepared from 5 × 104 differentiating eosinophils and were fixed in methanol for 3 minutes. After fixation, cytospins were stained in a 50% eosin methylene blue solution (Sigma-Aldrich GmbH, Seelze, Germany) for 20 minutes and rinsed in water for 5 seconds, and the nuclei were counterstained with 10% Giemsa solution (Merck kGaA, Darmstadt, Germany) for 15 minutes. During eosinophil differentiation, different stages of maturation can be observed. Cells differentiate from blast cells to promyelocyte type 1, promyelocyte type 2, myelocyte, metamyelocyte, and, finally, mature eosinophils with segmented nuclei. These stages can be distinguished by cell size, ratio of cytoplasm to nucleus, presence of azurophilic granules, appearance of eosinophilic granules, and nucleus shape. Juvenile eosinophils were characterized as cells belonging to the stages of promyelocyte 2, myelocyte, metamyelocyte, and mature eosinophils. These cells were all observed to contain eosinophilic granules. Electrophoretic mobility shift assay Nuclear extracts were prepared from differentiating eosinophils as described previously.38 Oligonucleotides were labeled by filling in the cohesive ends with [ -32P] dCTP using
Klenow fragment of DNA polymerase 1. Nuclear extracts were incubated in
a final volume of 20 µL, containing 10 mM HEPES (N-2-hydroxylethypiperazine-N'-2-ethanesulfonic acid), pH 7.8, 50 mM
KCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 5 mM
MgCl2, 10% (vol/vol) glycerol, 5 mM dithiothreitol, 2 µg
poly(dI-dC), 20 µg BSA, and 1 ng 32P-labeled
oligonucleotide for 20 minutes at room temperature. Subsequently,
samples were subjected to electrophoresis for 3 hours on a 5%
nondenaturing polyacrylamide gel. Supershift analysis was performed by
preincubating 10 µg nuclear extract with 3 µg antibody against
STAT5, STAT3 (Transduction Laboratories, Lexington, KY), and STAT1
(Transduction Laboratories, Lexington, KY) for 2 hours on ice before
adding the binding buffer and the 32P-labeled oligonucleotide.
Western blot analysis Western blot analysis was performed using standard techniques. In short, for the detection of STAT5 expression and phosphorylation, differentiating eosinophils were lysed in Laemmli buffer (0.12 M Tris HCl, pH 6.8, 4% sodium dodecyl sulfate [SDS], 20% glycerol, 0.05 µg/µL bromophenol blue, and 35 mM -mercaptoethanol) and were
boiled for 5 minutes. Equal amounts of total lysate were analyzed by
8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were
transferred to Immobilon-P and incubated with blocking buffer
(Tris-buffered saline/Tween 20) containing 5% low-fat milk or BSA for
16 hours at 4°C before incubation with either an N-terminal STAT5
antibody (Santa Cruz Technology, Santa Cruz, CA), an antiphospho-STAT5 (Y695) antibody (Cell Signaling Technology, Beverly, CA), an antibody against -actin (Santa Cruz Technology), an antibody against Bcl-2 (Santa Cruz Technology), or an antibody against p21WAF/Cip1
(Santa Cruz Technology) for 2 hours in the same buffer. Subsequently, blots were incubated with peroxidase-conjugated secondary antibodies for 1 hour. Enhanced chemiluminescence (ECL) was used as a detection method according to the manufacturer's protocol (Amersham Pharmacia, Amersham, United Kingdom).
Measurement of apoptosis CD34+ cells were transduced with a retroviral vector (SFCMM-3) expressing a truncated nerve growth factor receptor (NGFR) as a surface marker. During eosinophil differentiation, the percentage of Annexin V-positive cells was determined. Cells were incubated for 20 minutes on ice with a primary mouse antibody against NGFR. After washing the cells with phosphate-buffered saline (PBS), the cells were simultaneously incubated with a phycoerythrin (PE)-conjugated goat-antimouse antibody and Annexin V-fluorescein isothiocyanate (FITC) in Annexin-binding buffer (Bender Medsystems, Austria) for 20 minutes on ice. Percentages of transduced and apoptotic cells were determined by FACS analysis. As a positive control, a cell-permeable tat-peptide consisting of the BH3 domain of the proapoptotic protein Bim (YGRKKRRQRRREIWIAQELRRIGDEFNAYY) was used. Cells were incubated for 1 hour with 40 µM peptide at 37°C.
Characterization of ex vivo eosinophil differentiation CD34+ progenitor cells, isolated from umbilical cord blood, were differentiated toward eosinophils in the presence of the cytokines IL-3 and IL-5, as described in "Materials and methods." The morphology of differentiating eosinophils was analyzed by May-Grünwald-Giemsa staining (Figure 1A). The percentage of juvenile eosinophils is shown in Figure 1B. After 7 days of differentiation, 20% of the cells were characterized as juvenile eosinophils, and most of the cells belonged to the promyelocyte 1 stage. After 17 days of culture, differentiation increased to 58% of juvenile eosinophils, of which most were mature myelocytes. During eosinophil differentiation, granules are formed. Final maturation of eosinophil differentiation was further investigated with LFB staining, which specifically stains eosinophil granules (Figure 1C). The percentage of LFB-positive cells increased from 15% after 10 days of differentiation to 35% after 17 days of differentiation (Figure 1D).
STAT5 expression and activation during eosinophil differentiation To investigate the regulation of STAT5a during eosinophil differentiation, protein lysates were prepared from CD34+ cells immediately after isolation from umbilical cord blood and during subsequent eosinophil differentiation. Equal amounts of protein were separated by SDS-PAGE, and Western blotting was performed with an N-terminal antibody against STAT5 or an antibody against tyrosine-phosphorylated STAT5 (Figure 2A). STAT5 was highly expressed in CD34+ cells as the nonphosphorylated, inactive form. However, STAT5 became phosphorylated on stimulation with IL-5. STAT5 expression and phosphorylation levels were high early during eosinophil differentiation, but protein expression was down-regulated after 14 to 17 days of differentiation. To demonstrate that STAT5 phosphorylation indeed correlates with STAT5 activation during eosinophil differentiation, nuclear extracts were prepared, and electrophoretic mobility shift assays (EMSAs) were performed using a-casein element
as a STAT5-binding site.39 Figure 2B shows that the
specific slower migrating (C1) DNA-protein complex, corresponding to
STAT5a (as demonstrated by supershift analysis with an antibody against
STAT5),40 decreased after 17 days of differentiation.
These experiments demonstrate that STAT5 is indeed expressed in
differentiating eosinophils but is down-regulated during terminal
maturation.
Retroviral transduction of CD34+ progenitor cells To investigate the role of STAT5a during eosinophil differentiation, a bicistronic retroviral DNA construct was used coexpressing eGFP. CD34+ cells, differentiated toward eosinophils for 2 days, were transduced with virus expressing either STAT5a, STAT5a 750, or empty vector. STAT5a750 is a
dominant-negative STAT5 mutant that can bind to DNA but is unable to
activate transcription.41 Expression of STAT5a and
STAT5a750 in transduced cells was confirmed by Western blotting of
protein lysates from transduced cells with a STAT5 antibody (Figure
3A). EMSA was also performed to analyze
the DNA binding of STAT5a and STAT5a750 in transduced cells (Figure
3B). After 7 or 14 days of differentiation, cells transduced with eGFP alone, STAT5a750, or STAT5a were starved of cytokines for 16 hours
before stimulation with IL-5 for 15 minutes. Nuclear extracts were
analyzed using a -casein element as a probe for STAT5 binding. Nuclear extracts prepared from transduced cells with STAT5a retrovirus showed increased DNA binding compared with cells transduced with eGFP.
Stimulation of cells transduced with STAT5a750 also resulted in
increased DNA binding to the -casein element. The DNA-protein complex C2 migrated faster than the DNA-protein complex C1 in cells
transduced with STAT5a (Figure 3B), clearly demonstrating that
retroviral transduction results in ectopic expression of STAT5a and its
inactive mutant.
Regulation of proliferation during eosinophil differentiation by STAT5 To determine whether STAT5 plays a critical role in regulating eosinophil differentiation, umbilical cord blood-derived cells were transduced with either eGFP, STAT5a, or STAT5a750. The percentage of
eGFP-positive cells represents the percentage of transduced cells and
was determined by FACS analysis (Figure
4A). Transduction efficiency of cells
transduced with eGFP was approximately 32%, whereas transduction of
cells with STAT5a or STAT5a750 resulted in average transduction
efficiency of approximately 25% and 21%, respectively. We observed an
increase in the percentage of eGFP-positive cells after transduction
with STAT5a (from 25% to 48%), indicating that STAT5a positively
influences proliferation during eosinophil differentiation compared
with cells transduced with eGFP alone. We also observed a decrease in
eGFP-positive cells after transduction with STAT5a750 compared with
cells transduced with eGFP alone. This demonstrates that STAT5a is both
necessary and sufficient for progenitor cell proliferation.
To determine whether STAT5 was indeed critical in the regulation of
proliferation during eosinophil differentiation, CD34+
cells were transduced with eGFP, STAT5a, or STAT5a To investigate whether the inhibition of proliferation in cells
ectopically expressing dominant-negative STAT5a resulted from enhanced
apoptosis, Annexin V staining was performed in transduced cells. It has
previously been described that eGFP tends to leak from apoptotic
cells42; in our experiments, no eGFP-positive apoptotic or
dead cells could be detected (results not shown). Therefore, an
alternative retroviral vector expressing a truncated NGFR as a surface
marker was used. The percentage of apoptotic cells during eosinophil
differentiation is very low, approximately 10%. No difference could be
observed in the percentage of Annexin V-positive cells between cells
transduced with empty vector alone and cells transduced with
STAT5a Regulation of eosinophil differentiation by STAT5 To investigate whether STAT5a also plays a role in eosinophil differentiation, cells were again transduced with eGFP, STAT5a, or STAT5a750. After 7, 10, 14, and 17 days of differentiation, eGFP-positive cells were sorted by FACS from the nontransduced cells
and cytospins prepared. The morphology of the differentiating eosinophils was analyzed by May-Grünwald-Giemsa staining as
described in "Materials and methods" (Figure
5A). The percentage of juvenile eosinophils is expressed in Figure 5B. Transduction of cells with eGFP
or STAT5a resulted in approximately 70% juvenile eosinophils after 17 days of differentiation, of which most were mature myelocytes or
metamyelocytes.
After the transduction of cells with STAT5a, approximately the same
percentages of juvenile eosinophils and cells transduced with eGFP were
observed. However, because cells transduced with STAT5a showed an
increase in proliferation, there was an increase in total numbers of
juvenile eosinophils. Transduction of cells with STAT5a Eosinophil differentiation was also analyzed by LFB staining of the
granules (Figure 5C). After fixation, cells were stained with LFB for 2 hours. After 10 days of differentiation, approximately 33% of the
cells transduced with eGFP were positive for LFB. This percentage
increased to 38% after 21 days of differentiation (Figure 5D).
Transduction of cells with STAT5a resulted in accelerated eosinophil
differentiation. After only 10 days of differentiation, more than 40%
of the cells were positive for LFB. Percentages of LFB-positive cells
increased to 52% after 17 days of differentiation. In contrast, cells
ectopically expressing STAT5a To determine whether STAT5a and STAT5b may have different functional
activities in eosinophil differentiation, cells were transduced with
eGFP or dominant-negative STAT5b (Figure 5E). Expression of
dominant-negative STAT5b in CD34+ cells resulted in a block
in eosinophil differentiation that was comparable to the inhibition in
differentiation caused by STAT5a Although STAT5 is thought to play a role in the differentiation of
neutrophils, this has only been demonstrated in the murine 32D cell
line.43 To determine whether STAT5a also plays a role in
neutrophil differentiation from human CD34+ cells, cells
were transduced with eGFP or STAT5a Regulation of STAT5 target genes during eosinophil differentiation Our results demonstrate that STAT5 plays an important role during eosinophil differentiation. To confirm the functionality of STAT5, the expression of several STAT5 target genes was evaluated. Protein lysates were prepared from differentiating eosinophils, and these were separated by SDS-PAGE (Figure 6A). Western blotting was performed with antibodies against Bcl-2 and p21WAF/Cip1, both previously described STAT5 target genes.44,45 Bcl-2 expression was high early during eosinophil differentiation, whereas expression was down-regulated during final maturation. In contrast, p21WAF/Cip1 expression was absent after 3 days of eosinophil differentiation and was up-regulated during final maturation.
Ectopic expression of STAT5a and STAT5a
In this study we have investigated the role of STAT5a during
eosinophil differentiation. Our experiments indicate that, during eosinophil differentiation of CD34+ umbilical cord blood
progenitors, STAT5a protein is differentially expressed and its
activity is regulated. Ectopic expression of STAT5a results in an
enhanced proliferation and an accelerated differentiation of umbilical
cord blood-derived CD34+ cells. Furthermore, ectopic
expression of STAT5a Although previous studies have suggested that STAT transcription factors may play an important role during myeloid differentiation, these studies were performed in myeloid leukemic cell lines, not in primary hematopoietic cells.43 We used an ex vivo differentiation system that resulted in the differentiation of a high percentage of juvenile eosinophils (Figure 1). Several studies have used similar ex vivo differentiation protocols for eosinophil differentiation from CD34+ progenitor cells derived from cord blood or peripheral blood.46-48 Recently, Hashida et al49 demonstrated that 80% of the cord blood-derived CD34+ cells in their study developed into mature eosinophils after 17 days of differentiation. However, they only distinguished between blast cells and mature eosinophils without mentioning the other stages of eosinophil differentiation. The same percentages of cells that resembled eosinophils, after 21 days of differentiation, were described by Velazquez et al.50 Our differentiation protocol resulted in approximately the same percentages of differentiated and juvenile eosinophils as these previous studies. This ex vivo system of eosinophil differentiation resulted in a high number of juvenile eosinophils that were positive for LFB and eosinophil peroxidase (data not shown), and it is an important tool for analyzing the role of transcription factors during early stages of the differentiation process. Retroviral transduction experiments were performed to ectopically
express either STAT5a or STAT5a A role for STAT5 in proliferation during myeloid differentiation has
been suggested because of the observation that the expression of
dominant-negative STAT5 results in an inhibition of proliferation in
IL-3-dependent cell lines.43,51 Bone marrow-derived
macrophages obtained from STAT5a( Although little is known about STAT5 target genes regulating differentiation, the activation of p21WAF1/Cip1 60 and p27kip1 cyclin-dependent kinase inhibitors have been demonstrated to be involved in megakaryocytic differentiation.61 Ectopic expression of p21WAF/Cip1 and p27kip1 in a human megakaryoblastic leukemia cell line resulted in the induction of megakaryocyte differentiation. Ectopic expression of p21WAF/Cip1 also resulted in the morphologic differentiation of Ba/F3 cells.59 We have demonstrated that p21WAF/Cip1 expression levels are up-regulated during eosinophil differentiation. Ectopic expression of STAT5a also resulted in enhanced expression of p21WAF/Cip1, whereas p21WAF/Cip1 expression is reduced in cells transduced with dominant-negative STAT5a. This suggests that transcriptional activation of p21WAF/Cip1 is involved in STAT5-dependent eosinophil differentiation, by inducing a cell-cycle arrest that might be necessary for the induction of terminal differentiation. In chickens, it has been demonstrated that CCAAT/enhancer
binding protein (C/EBP) The 2 STAT5 gene products, STAT5a and STAT5b, are highly
homologous and are thought to have overlapping functional
roles.70 Recently, it has been demonstrated that STAT5b
can be differentially regulated compared with STAT5a. Both
isoforms are phosphorylated by Src kinases, whereas only STAT5b
subsequently translocates to the nucleus in NIH 3T3 cells Furthermore,
it has been demonstrated that STAT5b may have unique transcriptional
functions. For example, STAT5a and STAT5b can regulate transcription of
the estrogen receptor- STAT5 expression and phosphorylation are high during the early stages
of eosinophil differentiation, whereas protein expression of STAT5 is
down-regulated at later stages. This indicates that STAT5 might play a
critical role, mainly during early eosinophil differentiation. Indeed,
the transduction of cells with STAT5a results in an acceleration of
differentiation but does not result in cells with more mature
morphology. To support this hypothesis, it has been recently suggested
that STAT5 plays a role in early hematopoiesis. In
STAT5a( This is the first study that clearly demonstrates that STAT5a plays a critical role in human eosinophil differentiation. The precise molecular mechanism underlying this process remains to be clarified. However, our data suggest that the transcriptional regulation of critical cell cycle and antiapoptotic genes is important in regulating differentiation fate.
We thank Dr G. Nolan for kindly providing us with the LZRS vector and the phoenix-ampho packaging cell line. We also would like to thank Dr H. Spits for providing us with the LZRS construct expressing dominant-negative STAT5b and M. Stijns for providing us with the SFCMM-3 vector and the antibody against NGFR.
Submitted March 11, 2002; accepted August 5, 2002.
Prepublished online as Blood First Edition Paper, August 15, 2002; DOI 10.1182/blood-2002-03-0740.
Supported by research grants from GlaxoSmithKline and by a grant from the Dutch Cancer Society (M.B.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Leo Koenderman, Department of Pulmonary Diseases, G03.550, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; e-mail: l.koenderman{at}hli.azu.nl.
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  J. M. Beekman, L. P. Verhagen, N. Geijsen, and P. J. Coffer Regulation of myelopoiesis through syntenin-mediated modulation of IL-5 receptor output Blood, October 29, 2009; 114(18): 3917 - 3927. [Abstract] [Full Text] [PDF]
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 C. R. Geest, M. Buitenhuis, E. Vellenga, and P. J. Coffer Ectopic expression of C/EBP{alpha} and ID1 is sufficient to restore defective neutrophil development in low-risk myelodysplasia Haematologica, August 1, 2009; 94(8): 1075 - 1084. [Abstract] [Full Text] [PDF]
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R. Bedi, J. Du, A. K. Sharma, I. Gomes, and S. J. Ackerman Human C/EBP-{epsilon} activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation Blood, January 8, 2009; 113(2): 317 - 327. [Abstract] [Full Text] [PDF]
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 A. T. J. Wierenga, E. Vellenga, and J. J. Schuringa Maximal STAT5-Induced Proliferation and Self-Renewal at Intermediate STAT5 Activity Levels Mol. Cell. Biol., November 1, 2008; 28(21): 6668 - 6680. [Abstract] [Full Text] [PDF]
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 L. H. Ulfman, V. M. Kamp, C. W. van Aalst, L. P. Verhagen, M. E. Sanders, K. A. Reedquist, M. Buitenhuis, and L. Koenderman Homeostatic Intracellular-Free Ca2+ Is Permissive for Rap1-Mediated Constitutive Activation of {alpha}4 Integrins on Eosinophils J. Immunol., April 15, 2008; 180(8): 5512 - 5519. [Abstract] [Full Text] [PDF]
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M. Buitenhuis, L. P. Verhagen, H. W. M. van Deutekom, A. Castor, S. Verploegen, L. Koenderman, S.-E. W. Jacobsen, and P. J. Coffer Protein kinase B (c-akt) regulates hematopoietic lineage choice decisions during myelopoiesis Blood, January 1, 2008; 111(1): 112 - 121. [Abstract] [Full Text] [PDF]
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 M. Buitenhuis, L. P. Verhagen, J. Cools, and P. J. Coffer Molecular Mechanisms Underlying FIP1L1-PDGFRA-Mediated Myeloproliferation Cancer Res., April 15, 2007; 67(8): 3759 - 3766. [Abstract] [Full Text] [PDF]
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 M. A. Meyn III, S. J. Schreiner, T. P. Dumitrescu, G. J. Nau, and T. E. Smithgall Src Family Kinase Activity Is Required for Murine Embryonic Stem Cell Growth and Differentiation Mol. Pharmacol., November 1, 2005; 68(5): 1320 - 1330. [Abstract] [Full Text] [PDF]
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 J. J. Schuringa, K. Y. Chung, G. Morrone, and M. A.S. Moore Constitutive Activation of STAT5A Promotes Human Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation J. Exp. Med., September 7, 2004; 200(5): 623 - 635. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
/
signals emanating from the erythropoietin receptor.
Eur J Biochem.
1997;249:637-647