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HEMATOPOIESIS
From the Institut National de la Santé et de la
Recherche Médicale U362, Institut Gustave Roussy, Villejuif,
France; the Institut National de la Santé et de la Recherche
Médicale U363, Institut Cochin de Génétique
Moléculaire, Hôpital Cochin, Paris, France; and the
Department of Molecular Oncology, Genentech, San Francisco, CA.
Enforced expression of c-mpl in embryonic stem (ES)
cells inactivated for this gene results in protein expression
in all the ES cell progeny, producing cells that do not belong to the
megakaryocytic lineage and are responsive to PEG-rhuMGDF, a
truncated form of human thrombopoietin (TPO) conjugated to polyethylene
glycol. These include a primitive cell called BL-CFC, thought to
represent the equivalent of the hemangioblast, and all myeloid
progenitor cells. In this model, PEG-rhuMGDF was able to potentiate the
stimulating effects of other growth factors, including vascular
endothelial growth factor, on BL-CFC and a combination of cytokines on
the growth of granulocyte macrophage-colony-forming units. The
importance of the C-terminal domain of Mpl and of mitogen-activated
protein kinase (MAPK) activation in TPO-dependent megakaryocytic
differentiation has been well studied in vitro. Here, the role of this
domain and the involvement of MAPK in upstream and nonmegakaryocytic cells are examined by using 2 truncated mutants of Mpl ( Thrombopoietin (TPO), also termed Mpl ligand
(Mpl-L) or megakaryocyte growth and development factor (MGDF), is a
specific regulator of megakaryocytopoiesis.1-3 However,
TPO, in synergy with interleukin-3 (IL-3), Steel factor (KL),
and Flt-3 ligand (FL), has a pleiotropic role in
hematopoiesis,4-9 including regulation of the
hematopoietic stem cell compartment.10
Mpl belongs to the cytokine receptor superfamily.11-14 The
cytoplasmic domain of Mpl is 121 amino acids long, and there is no
recognizable kinase domain or enzymatic motif in this
region.13 On ligand binding, however, Mpl transmits
biochemical signals such as activation of the Janus family of protein
tyrosine kinases (JAKs), which in turn phosphorylate Mpl tyrosine
residues. This is followed by the activation of other target molecules,
including members of the latent transcription factor family named
signal transducers and activators of transcription (STATs),
phosphatidyl-inositol 3-kinase, Shc, mitogen-activated protein kinases
(MAPK), and proto-oncogenes such as Cbl and Vav.15-26
Various cell lines have been engineered to express murine or
human c-mpl on their surfaces, and they respond to TPO by
proliferating, differentiating, or both. Analysis of mutations in the
cytoplasmic domain of Mpl in these cell lines has shown that the
conserved membrane-proximal box1 and box2 domains are required for
TPO-induced phosphorylation of JAK2 and activation of STAT5 and for
TPO-induced megakaryocytic proliferation and
differentiation.15,16,23 In the C-terminal domain, the
tyrosine residue (Y) 112 is necessary for activation of the MAPK
pathway through Shc activation.27 Activation of the MAPK
pathway is required for TPO-induced megakaryocytic cell line
differentiation.15,16,24,25,28 In the multifactorial in
vivo context, mice lacking the C-terminal 60 amino acids of Mpl have
normal platelet and megakaryocyte numbers, such as the wild type, but
their megakaryocytes display a defective response to TPO in vitro and
in vivo.29 In addition, these megakaryocytes displayed a
low ploidy on TPO treatment,29 in agreement with the
decreased megakaryocyte ploidy in cultures containing MAPK inhibitors.30
MAPKs, also known as extracellular regulated kinases (ERK), are a class
of serine-threonine kinases that are activated by many cytokines. They
are key regulators of cell proliferation and differentiation in
numerous cell types.28,31,32 A common pathway leading from
cell surface receptors to ERKs involves p21ras, which
activates c-Raf protein kinase. c-Raf phosphorylates 2 MAPK kinases
(MEK1 and MEK2), which in turn phosphorylate ERK1 and ERK2
(p44MAPK and p42MAPK), respectively. On
activation, ERKs translocate to the nucleus and phosphorylate
transcription factors such as Elk-1.
Embryonic stem (ES) cells are a good model for studying the
consequences of genetic manipulations on hematopoiesis in vitro. They
are nontransformed cell lines in which targeted mutations can be
created readily, and they can differentiate in vitro to hematopoietic
cells. Several methods have been proposed for the generation
of hematopoietic cells from ES cells. In this work we used a 2-step
method that allows direct evaluation of the ability of ES cells to
generate hematopoietic progenitors33 and a
technique34 that gives access to a primitive cell called
blast colony-forming cells (BL-CFCs). BL-CFCs are able to form
blast cell colonies composed of hematopoietic and endothelial cells.
These cells may represent the hemangioblast, the long-suspected common
progenitor of the hematopoietic and endothelial
lineages.35 Thus, ES cell hematopoietic differentiation
allows us to study the commitment process of BL-CFCs to hematopoietic
and endothelial tissues in vitro and the mechanisms of regulation of
myeloid lineage differentiation and maturation.
In theory, enforced expression of genes in ES cells leads to protein
expression in all ES-derived cell types. In a previous paper,36 we show that wild-type ES cells do not respond to
PEG-rhuMGDF (a truncated form of human TPO conjugated to polyethylene
glycol), the generation of megakaryocytic progenitors and the formation of platelets excepted. Enforced expression of full-length
c-mpl in ES cells inactivated for c-mpl resulted
in PEG-rhuMGDF-dependent responses of ES cell progeny, namely, the
development of all myeloid progenitors and the maturation of
megakaryocytic and other myeloid lineages. Interestingly, blast cell
colony development from BL-CFCs, which normally depends strictly on
vascular endothelial growth factor (VEGF), also occurred in the
presence of PEG-rhuMGDF. Here, we showed that a synergistic effect of
PEG-rhuMGDF on the VEGF-dependent blast cell colony and on the growth
factor-dependent granulocyte macrophage progenitor development also
exists, and we investigated the signaling pathway involved in these
responses. Using Mpl mutants with truncated cytoplasmic domains and
specific inhibitors of the MAPK pathway, we examined the role of MAPK
activation in PEG-rhuMGDF-dependent development of ES cell-derived
hematopoietic cells, extending from the hemangioblast to myeloid mature
cells. We show that the MAPK pathway is required not only for
megakaryocytic and all other myeloid progenitor cell development but
also for BL-CFC development. In contrast, this pathway is not involved
in the synergistic effects of PEG-rhuMGDF with VEGF or a combination of cytokines.
Cells
Growth factors, antibodies, and reagents
Fluorescein isothiocyanate (FITC)-anti-CD41 (gpIIb) was obtained from PharMingen (San Diego, CA). Monoclonal antibody specific for the Flag epitope tag sequence (M1) was purchased from Eastman Kodak Company (New Haven, CT). The second-step reagent was FITC-conjugated goat F(ab')2 fragment, specific for mouse immunoglobulin (Ig)G (Silenius, Hawthorn, Australia). Various purified IgG was used as irrelevant control antibodies: anti-mouse IgG1 (PharMingen) and purified mouse IgG2b (DAKO, Glostrup, Denmark). Propidium iodide was from Sigma (Saint-Quentin Fallavier, France). Specific MEK inhibitors, PD98059 and U0126, were purchased from Promega France (Lyon, France). Stock solutions were prepared in dimethyl sulfoxide (DMSO; Sigma) at 20 mM for PD98059 and 10 mM for U0126. Plasmid constructions Full-length murine c-mpl (flmpl) and 2 mutant cDNAs, inserted into the retrovirus expression vector pBabepuro, have been previously described.23 Mutant 3 (3mpl) lacks
residues 576 to 599, and mutant 34 (34mpl) lacks residues 576 to
622 (Figure 1A). All constructs were
transferred to the EcoRI site of the expression vector
pEF-BOS,38 containing the promoter of the human elongation factor hEF-1 .
Embryonic stem cell electroporation and selection of transfected cells Mpl / ES cells were maintained in an
undifferentiated state and were electroporated as previously
described.36 Three days after electroporation, cells
expressing Flag were sorted by flow cytometry (FACS-Vantage; Becton
Dickinson) to be enriched in transfected cells as previously
described.36 A second round of sorting was performed 15 days later to select stable pools of transfected mpl/
ES cells. In some cases, a third round was necessary to obtain cell
populations with purity greater than 80%. Each type of transfectant (mpl/ flmpl, mpl/ 3mpl, and
mpl/ 34mpl) was expanded and frozen.
Embryonic stem cell differentiation To study the responses of myeloid progenitors to various cytokines, we used the 2-step method described by Keller et al.33 Embryoid bodies (EBs) were formed from ES cells, and their progenitor content was determined in the presence of various combinations of cytokines: 6 growth factors (6GF) (5 U/mL EPO + 5 U/mL KL + 1000 U/mL IL-1 + 50 U/mL IL-3 + 10 ng/mL
(nM) IL-6 + 10 ng/mL [nM] G-CSF) or 10 ng/mL (nM)
PEG-rhuMGDF or 6GF+PEG-rhuMGDF.36 Myeloid colonies
including megakaryocyte colony-forming units (CFU-MK), granulocyte
macrophage CFUs (CFU-GM), erythroid burst-forming units (BFU-E), and
mixed colonies (CFU-mix) were counted on day 7 of culture. Clones
larger than 50 cells were scored as colonies except for CFU-MK, for
which one unit consisted of at least a 3-megakaryocyte cluster.
We used the 3-step method described by Kennedy et al34 to study the development of BL-CFC. Three-day EBs (EBs3) were collected and plated as previously described with cytokines consisting of either 5 ng/mL (nM) VEGF or 10 ng/mL (nM) PEG-rhuMGDF or VEGF+PEG-rhuMGDF.36 Blast cell colonies were scored after 4 days of culture. Myeloid progenitors in day 4 blast cell colonies were assessed in the presence of 6GF. To study the role of MAPK in the formation of ES cell-derived hematopoietic cells, EBs3 or EBs6 cells were incubated with various concentrations of the MEK inhibitor PD98059 or U0126 or with DMSO for 1 hour before cell stimulation. Blast cell colony and progenitor assays were subsequently assessed. Maturation of hematopoietic cells To study the terminal differentiation of ES cell-derived myeloid progenitors in the presence of PEG-rhuMGDF, day 4 blast cell colonies were cultured as previously described.36 After 5 days of culture, platelet numbers were quantified in triplicate, and megakaryocyte DNA content was determined by flow cytometry.Flow cytometry analysis To quantify platelets produced in the cultures, cells from one well were collected and incubated for 20 minutes with FITC-anti-CD41, and the samples were adjusted to a final volume of 300 µL. The acquisition rate was 1 µL/s for 60 seconds. Events were gated based on normal murine blood platelets and collected using a log-scale forward scatter and side scatter. Samples were analyzed with a FACSort flow cytometer (Becton Dickinson).To examine the DNA content of megakaryocytes, cells were labeled with anti-FITC CD41 antibody in phosphate-buffered saline containing 5% fetal calf serum, 3 mM EDTA, 25 mM HEPES, 3.5% bovine serum albumin, and 8 µM prostaglandin 1 (Sigma), washed with the same buffer, and resuspended in 0.1% citrate sodium solution containing 50 µM propidium iodide and stored at 4°C for 18 hours. RNase (50 µg/mL [µM]) was added 10 minutes before analysis. Data were analyzed on a logarithmic scale by CellQuest software (Becton Dickinson). To purify hematopoietic cells contained in mpl Assay for MAPK activation The total EBs6 cell population or CD41+Flag+ double-positive EBs6-sorted cells were deprived of PEG-rhuMGDF for several hours in cytokine-free medium and then were stimulated with 100 ng/mL (nM) PEG-rhuMGDF for various times at 37°C. Whole-cell lysates were prepared by resuspending cell pellets in Laemmli sample buffer (Tris 62.5 mM, pH 6.8, sodium dodecyl sulfate [SDS] 2%, glycerol 10%) and boiling them for 10 minutes. Proteins were separated on SDS-12% polyacrylamide gels and were transferred to nitrocellulose membranes. ERK activity was measured by probing the membranes with an activation-specific antibody that recognizes the dual phosphorylated forms of p42 ERK2 and p44 ERK1 (Promega, Madison, WI). Total ERK amounts were determined by reprobing the same membranes with an antibody recognizing ERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was revealed with secondary antibodies coupled to horseradish peroxidase and the enhanced chemiluminescence system (Amersham).Statistical methods Data are presented as mean ± SEM. Student t test was used for statistical analysis.
Role of the C-terminal domain of Mpl in the response of embryonic stem cell-derived progenitors and BL-CFC to PEG-rhuMGDF Response of progenitors to PEG-rhuMGDF.
The first step of ES cell hematopoietic differentiation is the
formation of EBs. EBs6 contain all myeloid progenitors, identifiable by
clonogenic assays. To investigate the signaling pathway involved in the
response of ES cell-derived progenitors to PEG-rhuMGDF, we used ES
cells inactivated for c-mpl (mpl / ES cells in response to
PEG-rhuMGDF (Figure 2). Reintroduction of
flmpl into mpl/ ES cells restored this response. It
also allowed the differentiation of all types of myeloid progenitors (a
total of 345 ± 21 per 105 EBs6 cells), whereas the wild
type only gave rise to CFU-MK.36 EBs6 expressing 34mpl
generated few progenitors (61 ± 3 per 105 EBs6 cells)
(Figure 2), and these were significantly different from those generated
by mpl/. They included CFU-MK and nonmegakaryocytic
progenitors (BFU-E, CFU-mix). However, no CFU-GM developed.
Hematopoietic colonies were smaller than those derived from
mpl/ flmpl (not shown). Thus, EBs6 cells expressing
34mpl were impaired in their capacity to generate progenitors in
response to PEG-rhuMGDF. EBs6 cells expressing 3mpl gave rise to
progenitors (134 ± 8 per 105 EBs6 cells). Although fewer
than those obtained with flmpl, they included megakaryocytes and all
types of nonmegakaryocyte colonies including CFU-GM (Figure 2).
However, the 3mpl protein was expressed by a smaller proportion of
EBs6 cells than flmpl, suggesting that the hematopoietic activity of
the mutant 3 might have been underestimated. To overcome this
problem, we decided to sort the Flag+ hematopoietic
progenitors. Because all ES cell-derived myeloid progenitors express
the CD41 antigen (Mitjavila, manuscript submitted), we sorted
CD41+ Flag+ double-positive cells from
mpl/ 3mpl and mpl/ flmpl EBs6 cells
and then plated them for progenitor assays. Although the number of
progenitors was diminished after sorting because of the use of a less
efficient batch of methylcellulose in these experiments, there was no
difference between the progenitor contents of these 2 cell populations
(96 ± 2 and 105 ± 10 per 105 cells for 3mpl and
flmpl, respectively). The signals transmitted by the region deleted
from the mutant 3 (amino acids 71-94) do not appear to be required
for progenitor development.
Response of BL-CFC to PEG-rhuMGDF.
BL-CFC emerges in EBs after 3 days of differentiation. BL-CFC generates
blast cell colonies in which hematopoietic progenitors develop
concomitantly with endothelial cells.35 The growth of blast cell colonies is strictly dependent on the presence of
VEGF.34 We demonstrated36 that the ubiquitous
expression of flmpl allowed the growth of blast cell colonies in the
presence of PEG-rhuMGDF. These colonies had hematopoietic and
endothelial potential, similar to colonies grown with VEGF. The
Role of the C-terminal domain of Mpl in the terminal hematopoietic differentiation of embryonic stem cells In a previous paper,36 we show that mpl/ flmpl ES cells can develop into mature
megakaryocytic, erythroid, macrophagic, and granulocytic cell lineages
in serum-free liquid cultures initiated from blast cell colonies in the
presence of PEG-rhuMGDF. Here, we focused on consequences of the 3
and 34 deletions on megakaryocyte maturation. Figure
4A illustrates megakaryocytes that
developed from mpl/ flmpl, mpl/
3mpl, and mpl/ 34mpl ES cells in the presence of
PEG-rhuMGDF.
To examine ploidy distribution of megakaryocytes, cells were labeled
with anti-CD41, which specifically labels megakaryocytes, and with
propidium iodide to evaluate their DNA content. Like the wild type (not
shown), mpl Cultures initiated from mpl Role of the C-terminal domain of Mpl in the synergistic effect of PEG-rhuMGDF with VEGF or a combination of 6GF Enforced expression of flmpl in ES cells resulted in a potentialization by PEG-rhuMGDF of the stimulating effect of a mixture of 6GF on the growth of CFU-GM (Table 2). In contrast, no potentiation was observed with mpl/
3mpl, and 34mpl EBs6-derived CFU-GM (Table 2). Because the 3mpl protein was expressed by a smaller proportion of EBs cells than
fmpl and 34mpl, we decided to verify the absence of potentiation in
Flag+-sorted 3mpl EBs6 cells. No synergistic effect of
PEG-rhuMGDF with 6GF was observed: 52 ± 3, 61 ± 8, 133 ± 7
CFU-GM grew from 105 sorted 3 mpl cells cultured
with 6GF, PEG-rhuMGDF, or 6GF+PEG-rhuMGDF, respectively, (NS) versus
35 ± 2, 99 ± 10, and 274 ± 16 (P < .05) for
sorted flmpl cells grown in similar conditions, respectively). Therefore, the C-terminal region of Mpl, and the region deleted from
the mutant 3 were necessary for the synergistic effect of PEG-rhuMGDF with 6GF.
Similarly, the addition of PEG-rhuMGDF to VEGF in cultures of
mpl
PEG-rhuMGDF activates MAPK in hematopoietic cells derived from
mpl / flmpl,
mpl/ 3mpl, and mpl/ 34mpl ES
cells. EBs6 are composed of hematopoietic and nonhematopoietic cells.
To analyze ERK activation in the same proportion of
PEG-rhuMGDF-responding hematopoietic cells, EBs6 cells were sorted
into CD41+ and Flag+ cells.
CD41+Flag+ EBs6 populations derived from
mpl/ flmpl, 3mpl, and 34mpl ES cells were
stimulated with PEG-rhuMGDF for various times at 37°C, and ERK1-ERK2
activation was assessed in whole-cell lysates. PEG-rhuMGDF induced a
transient activation of ERK1 and ERK2 in mpl/ flmpl ES
cell-derived hematopoietic cells. The 3 deletion did not prevent
this activation, either in intensity or in duration (Figure
6). Phosphorylation of ERK1-ERK2 was
detected in hematopoietic cells derived from the mutant 34 after 15 minutes of PEG-rhuMGDF stimulation, though weak (Figure 6), but the
cells contained normal amounts of ERK1 and ERK2 proteins. Thus, ERK1
and ERK2 are activated by PEG-rhuMGDF in ES cell-derived hematopoietic
cells. These data suggest that most of the ERK activation can be
attributed to signaling arising from the carboxyl terminus.
MAPK pathway is involved in PEG-rhuMGDF-dependent growth of progenitors and blast cell colonies, but not in the synergy between PEG-rhuMGDF and VEGF The above results show that the34 deletion correlates with the
loss of MAPK activation. We investigated the role of this pathway in
PEG-rhuMGDF-induced development of progenitors and BL-CFCs and in the
synergistic effects of PEG-rhuMGDF with VEGF or with a mixture of 6GF.
Progenitors and blast cell colonies were assayed in the presence of
various concentrations of the specific MEK inhibitor, U0126 (Figure
7B). U0126 caused dose-dependent inhibition of the PEG-rhuMGDF-dependent growth of mpl/
flmpl and 3mpl blast cell colonies. The inhibitory effect was observed at a concentration of 2 µM (50% inhibition) but reached more than 90% inhibition at 20 µM. U0126 (20 µM) completely
inhibited ERK1-ERK2 phosphorylation as assessed by Western blot
analysis with anti-ERK-P antibody (Figure 7A). U0126 also inhibited
the PEG-rhuMGDF-dependent growth of progenitors derived from
mpl/ flmpl, 3mpl EBs6 cells, and the few progenitors
that developed with 34mpl (Figure 7B). Similar inhibition was
observed using another MEK inhibitor, PD98059 (not shown). Thus, the
development of both progenitors and BL-CFCs in response to PEG-rhuMGDF
requires the MAPK pathway.
In addition, 20 µM U0126 inhibited mpl
The importance of the carboxy-terminal domain of Mpl in
megakaryocyte differentiation has been studied in vitro using cell lines.15,23-25 Here, we analyzed the role of this domain
in response to PEG-rhuMGDF (a truncated form of human TPO conjugated to
polyethylene glycol) of various ES cell-derived hematopoietic cells
and the involvement of MAPK in PEG-rhuMGDF signaling. Our model was
based on the re-expression of the full-length c-mpl and the
expression of different mutants of c-mpl in ES cells
homozygously inactivated for this gene. We evaluated the consequences
of 2 deletions in the C-terminal domain of Mpl that affect the TPO
response of a cell line.23,25 One deletion ( The deletion of the 51 C-terminal amino acids of Mpl prevented the PEG-rhuMGDF-dependent development of blast cell colonies; however, it only partially inhibited progenitor formation. All types of myeloid colonies, except CFU-GM, grew, but their total number was very much lower than that from cells expressing the full-length Mpl (18%). This deletion did not impede the final maturation, as evidenced by platelet formation in vitro, but it caused reduced megakaryocyte polyploidization (8N versus 32N) in comparison with the full-length Mpl. Finally, it inhibited the synergistic effects of PEG-rhuMGDF with VEGF or a combination of growth factors. Therefore, in the absence of the C-terminal domain, the membrane-proximal region of Mpl allows a partial response of ES cell-derived hematopoietic cells to PEG-rhuMGDF. However, a complete biologic response requires the C-terminal domain. This suggests that the membrane-proximal domain is sufficient for CFU-MK and BFU-E differentiation, whereas the C-terminal domain is necessary for proliferation. In mice, the membrane-proximal region is sufficient to ensure megakaryocyte and platelet homeostasis.29 Signals from the distal and the proximal regions are necessary for a normal response of murine bone marrow megakaryocytes and platelets to exogenous TPO and recovery from myelosuppressive treatment.29 To discriminate between the role of the C-terminal 28 amino acids
(residues 94-121) and that of the internal amino acids (sequence 71-94)
in response to PEG-rhuMGDF of ES cell-derived cells, we analyzed the
consequences of The A small number of progenitors, consisting of BFU-E and CFU-MK, can differentiate from ES cells when the C-terminal domain of Mpl is deleted. One possibility is that the weak activation of ERK1/2 in the absence of the C-terminal domain of Mpl, also detected by other groups,29,30 contributes to this differentiation. Alternatively, pathways activated by the membrane-proximal domain of Mpl, probably including STAT5, protein kinase C, or an as yet undiscovered pathway, could be involved in differentiating signals.43,44 Interestingly, no CFU-GM developed in the absence of the C-terminal domain of Mpl. This suggests that signals that allow erythroid and megakaryocytic progenitor growth are not sufficient for that of granulocyte macrophage progenitors. Deletion of Mpl cytoplasmic residues 71 to 121, but not of 71 to 94, resulted in a reduced ploidy of ES cell-derived megakaryocytes without loss of CD41+ element production, suggesting that ERK1-ERK2 activation is involved in the ploidization of ES cell-derived megakaryocytes. This finding is consistent with previous data demonstrating that ploidy of TPO-dependent bone marrow megakaryocytes in culture is reduced by the presence of a MAPK inhibitor.30 The molecular mechanisms that regulate megakaryocyte ploidization are unknown. However, polyploidization, like cell proliferation, requires DNA replication. Thus, the effect of MAPK pathway activation on megakaryocyte polyploidization might be paralleled to its effect on cell proliferation in other cell lines. It could be the consequence of the up-regulation of cyclin D, which plays an important role in polyploidization, and the down-regulation of p27-p21.45 Deletion of residues 71 to 94 abolishes cell differentiation in the megakaryoblastic human cell line UT7.23 Loss of TPO-dependent megakaryocyte differentiation is associated with weaker and only transient ERK1-ERK2 activation compared with that of UT7 cells expressing full-length Mpl.25 Surprisingly, we found no abnormalities in ES cell-derived hematopoietic cell differentiation when this sequence was deleted and ERK1-ERK2 activation had kinetics of activation similar to that of full-length Mpl. The contribution of the amino acid sequence 71 to 94 of Mpl-to-Mpl signaling appears, therefore, to depend on the cellular context. In view of this, it may be preferable to use nontransformed cells to study the role of intracellular signals in cell proliferation and differentiation. Differences in Mpl signaling between species (murine versus human) or developmental stages (embryonic versus adult) could also explain this discrepancy. Although not required for BL-CFC, progenitor, or megakaryocyte formation, the amino acid sequence 71 to 94 was required for the synergistic effects of PEG-rhuMGDF, either on factor-dependent progenitor or on VEGF-dependent BL-CFC development. Little is known about the mechanisms of synergy between growth factors. ERK1-ERK2 or p38MAPK activation is responsible for the synergy between EPO and KL46 or between IL-12 and IL-2,47 respectively. We show here that ERK1-ERK2 activation does not contribute to the synergistic effect of PEG-rhuMGDF with other hematopoietic growth factors or VEGF. The synergy was not inhibited by the presence of specific MAPK inhibitors. At doses at which ERK activation is completely prevented, the synergy is not affected. Residues 71 to 94 may activate unknown signals necessary for the synergy. These signals could act directly or as primers of transduction pathways activated by 1 of the 2 receptors.48 Alternatively, because a molecular association between EPO-R and c-Kit has been reported to be responsible for the synergy between EPO and stem cell factor on BFU-E proliferation and differentiation,49 residues 71 to 94 might allow a similar association between Mpl and other hematopoietic receptors or Flk-1, the VEGF receptor. We analyzed the role of 2 regions of the Mpl C-terminal domain in a
model of nontransformed hematopoietic cells derived from ES cells in
vitro, in which the mpl gene is activated as
early as hemangioblast emergence. As summarized in Figure
8, we found that these regions mediate 2 different categories of signals, leading to different functions. MAPK
ERK1-ERK2 activation, mediated by the C-terminal 28 amino acids of
Mpl, supports PEG-rhuMGDF-dependent development of a cell considered
the equivalent of the hemangioblast (the BL-CFC), the production of
most progenitor cells, and megakaryocyte polyploidization. In contrast,
the synergistic effect of PEG-rhuMGDF with other growth factors
requires the amino acid sequence 71 to 94 but does not involve
ERK1-ERK2 activation. Signals emerging from the latter region and
responsible for growth factor synergy remain to be characterized. These
give evidence that the C-terminal domain of Mpl, mostly involved in
megakaryocyte differentiation,24,25,28,40-42 mediates
megakaryocyte proliferation. When ectopically expressed, this domain
mediates BFU-E proliferation, hemangioblast and CFU-GM differentiation
and proliferation.
We thank Kirin for providing PEG-rhuMGDF, Immunex for providing
IL-1
Submitted February 21, 2001; accepted September 27, 2001.
Supported by INSERM and by grants from the Association pour la Recherche contre le Cancer (contract no. 5377).
F.J.d.S. is employed by Genentech.
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: Francoise Sainteny, INSERM U362, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, 94805 Villejuif, France; e-mail: sainte{at}igr.fr.
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© 2002 by The American Society of Hematology.
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  H. Yao, B. Liu, X. Wang, Y. Lan, N. Hou, X. Yang, and N. Mao Identification of High Proliferative Potential Precursors with Hemangioblastic Activity in the Mouse Aorta-Gonad- Mesonephros Region Stem Cells, June 1, 2007; 25(6): 1423 - 1430. [Abstract] [Full Text] [PDF]
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 B. J. Lannutti and J. G. Drachman Lyn tyrosine kinase regulates thrombopoietin-induced proliferation of hematopoietic cell lines and primary megakaryocytic progenitors Blood, May 15, 2004; 103(10): 3736 - 3743. [Abstract] [Full Text] [PDF]
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L. C. Platanias Map kinase signaling pathways and hematologic malignancies Blood, June 15, 2003; 101(12): 4667 - 4679. [Abstract] [Full Text] [PDF]
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R. C.R. Perlingeiro, M. Kyba, S. Bodie, and G. Q. Daley A Role for Thrombopoietin in Hemangioblast Development Stem Cells, May 1, 2003; 21(3): 272 - 280. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
the amino acid sequence
71 to 94 appears to be required only for the synergistic action of
PEG-rhuMGDF; the last 28 amino acids seem to mediate all other
responses to PEG-rhuMGDF, including the development of hemangioblasts
to blast cell colonies, of myeloid progenitors in numbers comparable to
those formed after the binding of PEG-rhuMGDF to the full-length Mpl
and megakaryocyte polyploidization. However, because the deletion of
residues 94 to 121 was not tested directly, it is possible that
elimination of the C-terminus by itself would have less or greater
effect on the above findings.
