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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1601-1613
Megakaryocyte Growth and Development Factor-Induced Proliferation
and Differentiation Are Regulated by the Mitogen-Activated Protein
Kinase Pathway in Primitive Cord Blood Hematopoietic Progenitors
By
Serge Fichelson,
Jean-Marc Freyssinier,
Françoise Picard,
Michaela Fontenay-Roupie,
Martine Guesnu,
Mustapha Cherai,
Sylvie Gisselbrecht, and
Françoise Porteu
From the Laboratoire d'Hématopoièse, Site
Transfusionnel, the Laboratoire d'Hématologie, and the Institut
Cochin de Génétique Moléculaire (ICGM), Insitut
National de la Santé et de la Recherche Médicale (INSERM
U363), Hôpital Cochin, Université René Descartes,
Paris, France.
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ABSTRACT |
In several erythroleukemia cell lines, activation of
mitogen-activated protein kinases (MAPK) by phorbol esters or
megakaryocyte growth and development factor (MGDF) is required for
induction of megakaryocytic phenotype and growth arrest. To support
this model, we have examined the effect of a specific inhibitor of this
pathway (PD98059) on human CD34+ hematopoietic
progenitors isolated from cord blood (CB), induced to differentiate
along the megakaryocytic lineage in liquid cultures supplemented with
rhuMGDF. RhuMGDF induced a sustained activation of MAPK in
megakaryocytes and this activation was completely inhibited in the
presence of low concentrations of PD98059 (6 to 10 µmol/L). At this
concentration, PD98059 induced an increase in cell proliferation, resulting in accumulation of viable cells and a prolongation of the
life time of the cultures. This increase correlated with an increase in
DNA synthesis rather than with a reduction in apoptosis. This effect
was combined with developmental changes indicative of delayed
megakaryocytic differentiation: (1) PD98059-treated cells tended to
retain markers of immature progenitors as shown by the increased
proportion of both CD34+ and
CD41+CD34+ cells. (2) PD98059-treated
cultures were greatly enriched in immature blasts cells. (3) PD98059
increased megakaryocytic progenitors able to form colonies in semisolid
assays. Thus, the MAPK pathway, although not required for megakaryocyte
formation, seems to be involved in the transition from proliferation to
maturation in megakaryocytes. Inhibition of MAPK activation also led to
an increase in the number and size of erythroid colonies without
affecting granulocyte/macrophage progenitor numbers suggesting that, in addition to the megakaryocytic lineage, the MAPK pathway could play a
role in erythroid lineage differentiation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS is an ordered and tightly
regulated process of proliferation and differentiation leading to the
generation of mature blood cells from a small number of totipotent stem
cells. The regulatory system governing megakaryocytopoiesis and
platelet production follows this general scheme, involving the
committment of mutipotential progenitors to megakaryocyte precursors
and final maturation leading to the formation of platelets. In their
development, megakaryoblasts undergo a series of transformations such
as expression of lineage-specific antigens, several rounds of
endomitotic replication resulting in large polyploid cells, and the
development of an extensive system of demarcation membranes, which
delimits platelet territories.1 Both these proliferative
and maturative steps have been shown to be controlled in vitro and in
vivo by the c-Mpl receptor and its cognate ligand thrombopoietin (TPO),
also known as megakaryocyte growth and development factor
(MGDF).2-6 In addition to its effects on megakaryopoiesis
and thrombopoiesis, MGDF supports the survival and proliferation of
human and murine primitive hematopoietic cells.7,8
Cells in which the balance between proliferation and differentiation is
altered are more prone to leukemogenesis. Thus, it is of considerable
interest to determine the molecular mechanisms controlling this choice.
Because growth factors play a central role in these processes, great
efforts have been made at elucidating the signal transduction pathways
linking receptors at the cell surface to gene transcription in the
nucleus. The classical mitogen-activated protein kinase (MAPK) (also
known as extracellular-regulated kinase, [ERK]) pathway is the major
of these mechanisms. ERKs are activated by most growth factors and have
been shown to be a key regulator of both proliferation and
differentiation in different cell types. Constitutive activation of
MAPK is sufficient to transform fibroblasts, while inhibition of this
pathway blocks Ras-mediated cell transformation and growth
factor-induced mitogenesis.9,10 However, recent observations have also underscored the essential role of this pathway
in cell differentiation.10-13 In hematopoietic cells, the MAPK pathway has been shown to control the differentiation of immature
thymocytes downstream of the T-cell receptors14,15 and to
regulate early development of B cells.16
ERK are serine/threonine kinases whose activation itself involves the
dual phosphorylation of two crucial threonine and tyrosine residues.
This phosphorylation occurs on sequential activation of p21Ras and the
protein kinases Raf and MAPK kinases, MEK 1 and MEK2 (reviewed in
Dhanasekaran et al17). Once activated, ERKs phosphorylate a
variety of cytosolic proteins including other kinases such as
pp90Rsk18 and phospholipase A2.19 On
activation, ERKs also translocate to the nucleus,13,20
where they phosphorylate transcription factors such as Elk-1, leading
to the transcription of c-Fos.17,21
Mpl is a member of the cytokine receptor superfamily, which includes
the receptors for erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), and most interleukins.2 A number of receptors of this family, including Mpl, promote both proliferation and differentiation signals when they are introduced in
various cell lines.22-27 We have previously shown that
these functions are separately controlled by distinct regions of Mpl cytoplasmic domain.22 This scheme applies also to other
cytokine receptors.24-27 ERK activation has been reported
for most of these receptors and has been shown either necessary or
dispensable for proliferation.28 Correlations between the
loss of maturation effect on mutation of the intracellular domain of
the receptors and ERK activation have been described in some instances.
The MAPK pathway was shown to be dispensable for GM-CSF-induced
myeloid differentiation of the WT19 cell line.27 By
contrast, we have previously shown that MGDF induces a sustained
activation of ERK in UT7 cells in which we introduced Mpl (UT7-Mpl) and
that this prolonged activation is required for MGDF-induced increase in megakaryocytic-specific antigens, CD41 and CD42b.29
The importance of the MAPK pathway in megakaryocytic differentiation
was corroborated in several erythroleukemia cell lines where increase
in CD41 can be induced on introduction of constitutively activated
mutants of MEK.30-32 Interestingly, this same pathway was
found to repress erythroid differentiation, suggesting that MAPK might
balance the commitment to megakaryocyte versus erythroid cell lineage
from a common ancestor. However, bipotent cell lines can neither mimic
differentiation and proliferative capacities of hematopoietic
progenitors nor reflect all stages of megakaryocytic differentiation.
In addition, 1 study has reported the existence of significant
differences in MGDF-induced signaling between megakaryocytes and
established cell lines.33 Likewise, sustained activation of
the MAPK pathway has been shown to lead to exactly opposite effects on
proliferation in primary and transformed fibroblasts.34 This prompted us to analyze in the present study the effects of a
specific MEK inhibitor, PD98059,35 on freshly isolated
primary human CD34+ hematopoietic progenitor cells induced
to differentiate along the megakaryocytic lineage in liquid cultures
supplemented with rhuMGDF.
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MATERIALS AND METHODS |
Growth factors.
Pegylated-human recombinant megakaryocyte growth and differentiation
factor (PEG-rhu-MGDF) was a generous gift from Amgen (Thousand Oaks,
CA). Human erythopoietin (specific activity, 120,000 U/mg) was provided
by Dr M. Brandt (Boehringer, Mannheim, Germany). Recombinant stem cell
factor (SCF) was from Amgen and interleukin-3 (IL-3),
IL-6, GM-CSF were obtained from Dr Shimosaka (Kirin Brewery Co, Tokyo, Japan).
Isolation of CD34+progenitors from cord blood.
Umbilical cord blood (CB) units from normal full-term deliveries were
obtained, after informed consent of the mothers, from the Obstetrics
Unit of Hôpital Saint-Vincent de Paul, Paris, France, and
collected in placental blood collection bags (Maco Pharma, Tourcoing,
France) by the Centre d'Hémobiologie Périnatal. The mean
CB total volume collected was 85 mL. CB units were diluted with 50 mL
phosphate-buffered saline (PBS; GIBCO-BRL, Life Technologies, Cergy-Pontoise, France) containing 0.8% bovine serum albumine (BSA;
StemCell Technologies, TEBU, Le Perray-en-Yvelines, France), then
submitted to Ficoll density gradient (Lymphocyte separation medium;
Eurobio, Les Ulis, France). Low-density cells were recovered and
enriched for CD34+ cells by 2 cycles of positive selection
using anti-CD34 antibody (QBEND/10) and magnetic cell sorting on
Midi-MACS then Mini-MACS columns (CD34 isolation kit; Miltenyi Biotech,
TEBU). CD34+-enriched cells were numbered and viability was
determined by trypan blue exclusion.
The purity of the CD34-selected cells was determined after each
isolation by fluorescence-activated cell sorting (FACS) analysis using
a monoclonal antibody directed against CD34 directly conjugated to
phycoerythrin. These preparations usually averaged about 85% to 90%
CD34+ cells.
Liquid suspension cultures.
To avoid interference of serum, purified CD34+ cells (4 × 104 to 105/mL) were cultured in
serum-free Iscove's modified Dulbecco's medium (IMDM) (GIBCO-BRL)
supplemented with 15% of a commercial mixture BSA, insulin, and
transferrin (BIT 9500; StemCell Technologies), and 100 ng/mL
PEG-rhuMGDF in the presence of various concentrations (3 to 20 µmol/L) PD9805935 diluted in dimethyl sulfoxide
(DMSO) or the equivalent volumes of DMSO alone as
controls. Every 3 days, viable cells were scored by Trypan blue dye
exclusion and a fraction of them was harvested for further analysis
(see below). The remaining cells were amplified with fresh serum-free
IMDM containing PEG-rhuMGDF and either 6 µmol/L MEK inhibitor or
DMSO. Cell density was reajusted to 1 to 4 × 105
cells/mL.
Cell labeling and flow cytometry analysis.
Immunophenotyping was essentially performed as described.22
Briefly, CD34+ cells were washed twice in PBA (PBS
containig 1% BSA and 0.2% sodium azide) and incubated for 30 minutes
at 4°C with 1 mg of normal goat aggregated IgG (Sigma, St Louis,
MO) per mL to saturate Fc receptors. After 1 wash in PBA, cells were
incubated with fluorescein isothiocyanate (FITC)- or
phycoerythrin-labeled (R-PE) mouse antibodies directed against the
following antigens: CD34, CD41, CD61 GpA (Immunotech, Marseille,
France) or CD42b (Dako, Glostrup, Denmark), applied at the recommended
concentrations. Control stainings were performed with isotype-matched
control antibodies. Incubation was performed for 30 minutes on ice.
After staining, the cells were washed twice and resuspended in PBA
containing 1% formaldehyde. Fluorescence was analyzed on an ELITE flow
cytometer (Coulter Corp, Miami, FL). The percentage of positive cells
and the mean fluorescence intensity (MFI), a measure of the relative
density of the antigen on the surface of stained cells, were determined for each sample. In some experiments, CB hematopoietic progenitors were
stained simultaneously with R-PE-anti-CD34 and either FITC-anti-CD41 or FITC-anti-CD42b antibodies, or FITC-anti-CD34 and R-PE-anti-CD41. Spillover of green fluorescence into the red fluorescence was compensated electronically for each experiment by using single-stained samples.
To determine the frequency of apoptotic cells within the cultures,
cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) and incubated on ice with FITC-labeled annexin V at
the recommended concentration (Coulter) for 10 minutes. FACS analysis was performed immediately.
DNA synthesis.
DNA synthesis was measured by thymidine incorporation as follows:
CD3+ progenitor cultures performed in the presence or
absence of MEK inhibitor were harvested at regular intervals (between 6 and 15 days) and triplicate samples of 2 × 104 cells
were plated directly in 96-well plates. A total of 2 µCi [3H]thymidine was then added and the amount of
radioactivity incorporated in cells was determined 15 hours later.
Quantification of clonogenic progenitors in semisolid culture
assays.
The presence of erythroid (burst-forming unit-erythroid [BFU-E]),
colony-forming unit granulocyte/macrophage (CFU-GM), and colony-forming
unit-megakaryocytic (CFU-MK) progenitors was evaluated every 3 to 4 days along the culture. For erythroid and GM progenitors, previously described methylcellulose assays were used36:
aliquots of cells, which had been cultured in the presence or absence
or PD98059 were seeded at 500 to 5,000 cells/mL depending on the day of
culture, in StemGEM 1d methylcellulose medium. This medium contains
Epo, IL-3, IL-6, SCF, G-CSF, GM-CSF, IL-11 and allows the growth of all
types of erythroid and myeloid progenitors (StemBio Research, CNRS,
Villejuif, France). Cultures were performed in duplicate and incubated
in fully humidified atmosphere with 5% CO2 in air at
37°C. Colonies were scored after 14 days. Megakaryocyte progenitors
were cultured using serum-free fibrin clot, as described.6 Briefly, cells (1 × 105/mL) were suspended in IMDM
suplemented with the BIT 9500 mixture, 20 ng/mL PEG-rhuMGDF, and 50 ng/mL SCF. Fibrin clot formation was obtained by adding 1 mg/mL plasma
fibrinogen, 0.01 mol/L  -aminocaproic acid, and 7 IU/mL
thrombin (Thrombiprest; Diagnostica Stago, Asnières, France). Cultures were incubated for 12 days at 37°C
in a 5% CO2 95% air humidified incubator. Colonies were
quantified by indirect immunophosphatase alkaline labeling after
staining with anti-CD61 monoclonal antibody (Y2-51; Dako). Dishes were
scanned completely under an inverted microscope at 40× or
100× magnification. Colonies are defined as 1 or more
CD61+ megakaryocyte.
Assays for MAPK activation.
ERK activity was measured by immunoblotting of whole-cell extracts with
an activation-specific polyclonal antibody, which recognizes the dual
phosphorylated forms of p42 ERK2 and p44 ERK1 (Promega Corp, Madison,
WI). Total ERK amounts were determined by reprobing the same membranes
with an anti-ERK1 antibody recognizing both ERK1 and ERK2 (SC-93; Santa
Cruz Biotechnology Inc, Santa Cruz, CA). Whole-cell lysates were
prepared as described29 from 7- to 11-day cultures of
CD34+ cells. Cells were either lysed directly or washed 3 times and deprived of rhuMGDF by incubation for 5 hours in
cytokine-free medium before stimulation. Stimulation was performed for
30 minutes and 1 hour at 37°C with 100 ng/mL rhuMGDF, in the
presence or absence of 6 µmol/L PD98059 or the equivalent amount of
DMSO as control.
Morphological studies.
Cells were collected every 2 or 3 days of culture and concentrated on
glass slides by cytocentrifugation. Cell morphology was examined after
May-Grünwald-Giemsa (MGG) staining.
Statistical analysis.
Statistical differences in cell expansion and apoptosis between the
subfractions of cells grown in the presence or absence of PD98059 were
analyzed using Fischer-Snedecor test applied to analysis of variance (ANOVA).
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RESULTS |
MGDF-mediated MAPK activation in primary megakakaryocytic cells derived
from CB CD34+ progenitors.
MGDF has been shown to drive unilineage differentiation along the
megakaryocytic pathway and to lead to almost pure megakaryocytes in
suspension cultures.1,3-6 To determine whether MAPK pathway activation could be of relevance in MGDF-mediated functions in megakaryocytes, we first analyzed whether rhuMGDF was able to trigger
MAPK activation in physiological conditions, ie, primary normal
megakaryocytes. CD34+ cells freshly isolated from human
umbilical CB were cultured in suspension in serum-free medium
containing only rhuMGDF as growth factor (as described in Materials and
Methods). After 10 days, about 85% of the cells were positive for the
megakaryocytic-specific antigen CD41 (see below). No phosphorylated ERK
could be detected when lysates from these cells were directly prepared
and loaded on gel (data not shown). However, rhuMGDF was able to
trigger MAPK activation in CD41+ cells starved of cytokine
by a 5-hour incubation in rhuMGDF-free medium before stimulation
(Fig 1A). In some experiments, depending on
the donor sample, a unique band corresponding to phosphorylated ERK2
was observed (see Fig 1B). This was probably due to the far greater
abundance of ERK2 than ERK1 in cells from these donnors, as shown by
immublotting with an antibody recognizing both active and inactive ERK1
and ERK2 (Fig 1A and B). Active ERK2 was still detected after 120 minutes of stimulation, indicating that rhuMGDF induced a sustained
MAPK activation in primary megakaryocytes. This activation, however,
decayed more rapidly than that previously reported in the UT7-Mpl
megakaryoblastic cell line stimulated with MGDF.29 This
might be due to some downregulation of ERKs occuring during the several
days of preculture of CD34+ cells in the presence of high
concentrations of MGDF.
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| Fig 1.
MAPK activation in CB-derived megakaryocytes. (A) Kinetic
of ERK activation in day 7 and day 10 cells derived from purified
CD34+ cells grown in the presence of rhuMGDF. Cells were
deprived of growth factor by incubation for 5 hours in cytokine-free
medium and stimulated for the indicated times with 100 ng/mL rhuMGDF.
(B) Inhibition of rhuMGDF-induced ERK activation by PD98059. Cells were
grown for 10 days with rhuMGDF, starved, and stimulated for 60 minutes
at 37°C with 100 ng/mL of rhuMGDF in the presence of 6 µmol/L
PD98059 (+) or the equivalent amount of DMSO as control ( ). ERK
activity was detected by immunoblotting whole-cell lysates with
antiphospho ERK antibody. The amount of sample loaded in each lane was
verified by immunoblotting the same membranes with an antibody
recognizing both active and inactive ERK1 and ERK2.
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The MEK inhibitor increases rhuMGDF-induced proliferation of
CD34+ cells.
The results described with K56230,31 and UT7-Mpl cell lines
(unpublished data, March 1998) have suggested that
the MAPK pathway could play a role in cell fate decision towards
erythroid or megakaryocytic lineages. To explore this
hypothesis, the MEK inhibitor was added to CB-derived
CD34+ progenitors cultured in the presence of
rhuMGDF. In a first set of experiments, we determined the optimal
concentration of PD98059 that can be used in this system. Based on the
dose (10 µmol/L), which inhibits rhuMGDF-induced differentiation in
the UT7-Mpl model,29 concentrations of PD98059 ranging from
3 to 20 µmol/L were tested on CB CD34+ cell cultures (not
shown). The concentration of 6 µmol/L was found both to fully inhibit
MAPK activation triggered by rhuMGDF in this system (Fig 1B) and to be
devoid of toxicity. This concentration was used in all of the following studies.
As shown in Fig 2A, the cultures treated
with the MEK inhibitor always contained more viable cells than control
cultures, after day 10. In both cultures, expansion was observed until
around day 12. After this time point, the number of viable cells in
control cultures declined quickly and a massive cell death occured
usually around day 15; at day 15 to 18, all of the cells were dead. By contrast, in PD98059-treated samples, although proliferation reached a
maximum around day 12, the total number of viable cells remained almost
constant or declined slowly until day 18. Eventually, all PD98059-treated cells were dead by day 22. Thus, the difference in
viable cell numbers between the 2 cultures culminated at the end of the
cultures where PD98059-treated samples contained up to 13-fold more
cells at day 17 than controls. The relative expansions of the cells
after 17 days of culture with rhuMGDF in cultures containing PD98059
(58-fold ± 20-fold, mean ± standard deviation [SD], n = 7) or
the corresponding volume of solvent (10-fold ± 5-fold, mean ± SD, n = 8) were significantly different (P < .01).
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| Fig 2.
Relative expansion of CD34+ cells in the
presence ( ,  ) or absence ( ,  ) of 6 µmol/L PD98059 induced
by MGDF (A) or a mixture of SCF + IL-3 +IL-6 (B). The data have
been normalized to the number of purified CD34+ cells
seeded on day 0. The results presented are those from a representative
experiment of 8 (A) or 3 (B) performed.
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Thus, the presence of the MEK inhibitor resulted in prolongation of the
cultures in the presence of MGDF. This negative effect of the MAPK
pathway on the proliferation seemed specific for rhuMGDF responses.
Indeed, in cultures of CB CD34+ cells performed in the
presence of a mixture of either SCF + IL-3 + IL-6, the same
concentration of PD98059 inhibited cell proliferation (Fig 2B). The MEK
inhibitor has no effect on cell growth induced by a mixture of G-CSF
and FLT3-ligand (data not shown).
To determine whether the difference in absolute viable cell numbers
observed between PD98059-treated and control cultures was due to a
blockage of apoptosis, cells were stained at various time intervals
with annexin V coupled to FITC. Figure 3
shows that although still expanding, day 7 cultures already contained a
significant number of apoptotic cells, and this proportion increased gradually during the culture. In controls, consistent with the drop in
viable cell numbers, at day 16 to 17, almost all of the cells have
entered the apoptotic process (Fig 3A). Although significantly (P < .05) inferior to that of control, the frequency of
apoptotic cells was quite high in PD98059-treated cultures reaching
about 70% at day 16 to 17 (Fig 3A). This results in a higher absolute number of apoptotic cells in cultures containing the inhibitor (Fig
3B). From these data, the rate of apoptosis of the cells in 2 days
(between days 10 and 12 or 12 and 14) was estimated by the following
ratio: (absolute number of new apoptotic cells generated in between
days 10 to 12 [or 12 to 14])/total number of viable cells on day 10 [or 12])×100. We found that about 30% of the cells were dying
during these 2-day periods in both control and PD98059-treated
cultures. These results suggest that inhibition of apoptosis is
probably not the mechanism responsible for the increased proliferation
of PD98059-treated cells.
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| Fig 3.
Appearance of apoptotic cells in the cultures as measured
by annexin V staining. CD34+ cells were grown with
rhuMGDF in the absence or presence of 6 µmol/L PD98059 and stained
with annexin V-FITC at the indicated days. The percentage of annexin
V-positive cells was determined by FACS analysis. (A) Frequency of
apoptotic cells in control cultures () and cultures containing the
inhibitor ( ). The results represent mean ± SD from 4 independent
experiments. (B) Total number of apoptotic cells in the presence ()
or absence () of PD98059. The results from a representative
experiment are shown.
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We therefore assessed whether PD98059 could affect the capacity of the
cells to enter S phase. For that, DNA synthesis was tested by measuring
incorporation of [3H]thymidine into the cells at regular
intervals. The kinetic profile of incorporation was exactly inverse of
that of apoptosis: in the presence or absence of inhibitor,
incorporation was maximun around day 8 and declined gradually
thereafter (Fig 4). However, DNA synthesis
was greatly increased in the presence of the MEK inhibitor, reaching up
to 4-fold. By days 12 to 15, [3H]thymidine incorporation
was still significant in PD98059-treated cells, while it had reached
background levels in control cells.
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| Fig 4.
Effect of the MEK inhibitor on DNA synthesis capacity of
CD34+ cells grown with rhuMGDF. Cells from cultures
performed in the absence () or presence () of 6 µmol/L PD98059
were harvested at the indicated days and DNA synthesis was assessed by
measuring [3H]thymidine incorporation. Results are mean ± SD of triplicate determinations from a representative experiment of
3 performed. The relative increase in [3H]thymidine
incorporation in PD98059-treated cells as compared with control
DMSO-treated cells is indicated below the bars for each time point.
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Globally, these data suggest that the augmentation cell numbers in
cultures treated with the MEK inhibitor results mainly from an
increased mitosis capacity of the cells. Thus in CD34+
progenitors, the MAPK pathway functions as a negative regulator of
rhuMDGF-induced mitogenesis.
Phenotypic analysis of cultured progenitors in the presence or
absence of MEK inhibitor.
Previous studies have shown that during differentiation of
megakaryocytic progenitors, the proliferative capacity of progenitors decreases as a continuum, as cell surface phenotype change from CD34+CD41+/CD61+ cells to
CD34 CD41+/CD61+
cells.37,38 Therefore, we analyzed whether the increased
proliferation in PD98059-treated cultures could be due to the presence
of more immature progenitors, and in greater numbers, than in control cultures. Surface expression of CD34 and of the megakaryocytic markers
CD41, CD61, and CD42b were analyzed at regular time intervals in
cultures treated or not with PD98059.
As shown in Table 1 and
Fig 5, and as previously
described,37,38 CD41 appeared early in the culture, while
CD42b was expressed later during megakaryocytic differentiation. By
contrast with what had been observed with cell lines, no major
difference in expression of these markers was observed between cultures
containing PD98059 or control. The mean fluorescence intensity of
anti-CD41 staining was only slightly decreased (by 21% ± 10% at
day 10, mean ± SD, n = 5) in cells treated with the inhibitor.
However, the 2 cultures clearly differed by the surface expression of
CD34 (Fig 5 and Table 1): both the proportion of CD34+
cells and the level of expression of CD34 on the positive cells were
consistently increased in PD98059-treated cultures (58% ± 26% and
51% ±15% of increase in CD34+ cell number and MFI,
respectively, at day 7, mean ± SD, n = 5). This result suggested
that inhibition of the MAPK pathway in this system delayed
rhuMGDF-induced megakaryocytic differentiation.
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| Fig 5.
Effect of the MEK inhibitor on CD34 and CD41 cell surface
expression. Representative histograms from 1 of at least 4 experiments
performed with blood from different donors are shown for each marker.
Cells from control cultures (filled histograms) or cultures containing
6 µmol/L PD98059 (open histograms) were harvested at the indicated
days and stained separately with either anti-CD34 antibody coupled to
R-PE or anti-CD41 antibody coupled to FITC. In some experiments,
labeling was performed with FITC-anti-CD34 and R-PE-anti-CD41
antibodies and similar results were obtained. For each marker, the
percentage of positive cells (or the MFI for CD41 at days 10 and 13)
are indicated: gray labeling, controls; black labeling, PD98059-treated
samples.
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To further assess this possibility, cells were stained simultaneously
with anti-CD34 and anti-CD41 antibodies. As shown in Figs 6A and 7,
as for CD34+ cells, the frequency of doubly stained
CD34+CD41+ cells was greatly increased (71% ± 14% of increase, mean ± SD, n = 5) in cultures containing
the MEK inhibitor, as compared with control cultures. By contrast the
number of CD34+CD41 cells remained
almost unchanged (Fig 6A, 16% ± 12% of increase, mean ± SD, n = 5). Figure 6B shows that the relative proportion of double-stained
CD34+CD41+ cells among the CD41+
cells was greatly augmented by the MEK inhibitor in 5 of 5 independent experiments. On average, 30% of day 7-CD41+ cells
expressed CD34 in control cultures, while this proportion averaged 58%
in the presence of PD98059. After 11 days of culture, the expression of
CD34 dropped, but the same phenomenon was still observed (Fig 6B).
Likewise, the proportion of double-positive CD34+CD42b+ cells was greatly enriched after
treatment with the MEK inhibitor (44% v 75% of day 7-CD42b
cells also expressing CD34 were found in control and PD98059-treated
cultures, respectively).
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| Fig 6.
Increased frequency of double-stained
CD34+CD41+ cells in PD98059-treated
cultures. Cells from cultures containing or not 6 µmol/L PD98059 were
harvested at different time intervals and stained simultaneously with
R-PE-anti-CD34 and FITC-anti-CD41 (or FITC-anti-CD34 and
R-PE-anti-CD41) antibodies. (A) FACS dot plot from a representative
experiment on day 7 cells. (B) Percent of CD34+ cells
among the CD41+ cell population after 7 and 11 days of
culture in the absence () or in the presence (+) of PD98059 in 5 independent experiments.
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| Fig 7.
Dose-response analysis of PD98059 effect on the presence
of CD34+, CD41+, and
CD41+CD34+ cells in MGDF-induced cultures.
CD34+ cells were grown with rhuMGDF in the presence of
the indicated concentrations of PD98059 diluted in DMSO or the
equivalent volumes of DMSO alone. After 7 days, cells were labeled with
FITC-anti-CD41 and R-PE-anti-CD34 antibodies either separately or
simultaneously. Results are expressed as a percent of the frequency of
positives cells for each marker obtained with control cells.
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As shown in Fig 7, maximum increases in the numbers CD34+
and CD34+CD41+ cells were obtained with a
concentration of 6 to 10 µmol/L of PD98059.
Taken together, these results suggest that the megakaryocytic CD41 or
CD42b positive cells present in the cultures containing the MEK
inhibitor are in a more immature differentiation state than their
control counterparts.
Enhanced megakaryocytic and erythroid colony formation in the
presence of the MEK inhibitor.
The presence of progenitors was evaluated all along the culture by the
capacity of the cells to derive clonogenic colonies in semisolid
assays. Cells grown with rhuMGDF in the presence or absence of MEK
inhibitor, were harvested after various days in culture, and were
plated in the absence of inhibitor with a cocktail of cytokines
allowing the growth of either erythroid and GM or MK colonies. Colonies
were counted 12 to 14 days later.
MK colonies were derived from cells, which had received the MEK
inhibitor during the liquid cultures and in greater number than in
controls (Fig 8A). Both the relative
(number of colonies/1,000 cells) and the absolute number of colonies
present in the cultures were increased in the presence of PD98059 (Fig
8A and B). This increase was consistently observed in 5 of 5 independent experiments. The median number of MK colonies/1,000 cells
formed with cells cultured 12 days without and with PD98059 was
respectively, 1.9 (range, 0.4 to 4) and 3.9 (range, 1.1 to 7). The
increased clonogenic capacity of PD98059-treated cells confirms that
activation of the MAPK pathway negatively regulates proliferation of
megakaryocytic progenitors.
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| Fig 8.
Evolution of erythroid (Ery), megakaryocytic (MK), and GM
clonogenic progenitors in MGDF-induced cultures. Clonogenic progenitors
in cultures performed in the absence or the presence of 6 µmol/L
PD98059 were quantified at the indicated days by semisolid culture
assays as described in Materials and Methods. (A) Total number of
colonies formed with control () or PD98059-treated cultures ().
The results from a representative experiment, where determinations were
done in duplicate, are shown. (B) Percent of increase in relative ( )
or total () colony numbers obtained with PD98059-treated cells as
compared with control cells. Results are mean ± SD of at least 4 independent experiments for each type of colony.
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|
More surprisingly, a similar consistent increase in erythroid colonies
was also found (Fig 8A and B). There was a wide interindividual variation, but in every case (6 of 6 independent experiments) inhibition of the MAPK pathway resulted in an augmentation of the
number of erythroid colonies: median 5.1 (range, 0.8 to 10) and 10.4 (range, 2.3 to 28) colonies/1,000 cells for control and PD98059-treated
cells, respectively, after 10 days of culture. Moreover, the average
size of the erythroid colonies was larger in the presence of PD98059,
as compared with controls (data not shown), suggesting the presence of
more immature erythroid progenitors in these cultures.
By contrast, inhibition of MAPK activation did not significantly alter
the formation of GM colonies (Fig 8A and B): the median number of GM
colonies/1,000 cells was 11 (range, 0.25 to 30) and 14 (range, 0.5 to
31) in the absence or presence of PD98059, respectively. To further
examine whether the MAPK pathway could influence the formation of
colonies other than erythroid or megakaryocytic, CD34+
cells were cultured with G-CSF and Flt-3 ligand, conditions allowing exclusively the development of granulocytic progenitors. In these conditions, no difference was observed in the number of colonies developed between cultures containing the PD98059 or control (data not shown).
Morphological examination of cells from liquid cultures.
Cells from both PD98059-treated cultures or untreated controls were
examined at days 7, 10, 13, and 17 of culture by MGG staining. Analysis
was performed on cytocentrifugation products of 3 independent experiments. Whereas day 7 cells from both PD98059-treated or control
cultures displayed a similar immature blastic morphology (not shown),
striking differences appeared from day 10 (Fig 9A and D). At day 13, control cultures
contained a majority of mature micromegakaryocytes (more than 90%) and
only 6% of blast cells (Fig 9E and F). By contrast, MEK-treated
cultures showed a slower differentiation with the presence, at day 13, of 55% of blast cells and only 45% of mature megakaryocytes and
micromegakaryocytes (Fig 9B and C). In these cultures, the presence of
undifferentiated blast cells was observed until day 17 (not shown).
Examination of cells at the end of the culture (from day 17) showed
drastic differences between PD98059-treated cells and controls, in
agreement with the proliferation curves shown Fig 2: a mixture of
immature and mature living cells was maintained in the presence of the MEK inhibitor, whereas massive cell necrosis was observed in controls (not shown).
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| Fig 9.
Morphologic analysis of cells from liquid cultures. MGG
staining of cytocentrifugated cells from PD98059-treated (A to C) or
control (D to F) cultures, at day 10 (A and D) and day 13 (B, C, E, and
F); original magnification × 100.
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On the other hand, PD98059-treated cultures contained much more
megakaryoblastic cells of larger size and with a polylobulated nucleus
than controls: the number of polyploid cells averaged 10% and 30% in
control and PD98059-treated cultures, respectively. These results are
in agreement with previous studies39,40 showing that
CB-derived megakaryocytes exhibit a high proliferative capacity without
maturation to hyperploid cells. Among the polyploid cells, differences
in maturation clearly appeared between the 2 cultures. Indeed, more
than half of the polyploid cells present in PD98059-treated cultures
had a blastic immature morphology, while more than 95% of polyploid
cells from control cultures were mature megakaryocytes. Measurement of
cell DNA content after propidium iodide staining confirmed that
megakaryocytes from control cultures have a low ploidy with most of the
cells retaining 2N and 4N ploidy status throughout the culture period
(data not shown). No major difference in the frequency of polyploid
cells between control cultures and those containing PD98059 could be
detected by FACS analysis. The discrepancy between these data and those
obtained from morphological examination is probably due to the
relatively low number of polyploid cells even in PD98059-treated
cultures and to the high heterogenity in the ploidy class among these
cells, so that the number of cells in a given ploidy class might be too
low to be detected by FACS analysis.
| |
DISCUSSION |
Investigation on the function of the MAPK pathway has been greatly
aided recently by the discovery of inhibitors acting specifically on
MEK1 and 2 and preventing efficiently ERK activation when added to
intact cells.35 Such inhibitors are now widely used to
study the role of ERK in various cell lines or primary cells. Here, we
show that the MEK inhibitor can be added to long-term cultures of
normal human hematopoietic CD34+ progenitors grown in the
presence of MGDF, without displaying toxicity. A low concentration of
PD98059 (6 to 10 µmol/L) was found to fully inhibit ERK activation
induced by MGDF. At this concentration, PD98059 increased MGDF-induced
progenitor proliferation, while delaying their megakaryocytic
differentiation. Previous studies have shown that at concentrations up
to 50 µmol/L, PD98059 inhibits the MEK kinases without affecting a
variety of other kinase activities, including the related JNK and p38
MAPK pathways in different cellular models.35,41 Very
recently, however, PD98059 has been shown to act as a direct inhibitor
of cyclooxygenases.42 Indomethacin, at a dose currently
used to inhibit both cyclooxygenase-1 and -2,42 had no
effect on MGDF-induced proliferation of CD34+ progenitors
(data not shown), ruling out that inhibition of cyclooxygenase might be
responsible for the observed effect of PD98059. Previous studies in
cell lines have shown that PD98059 and dominant-negative mutant of MEK
have a similar inhibitory effect on megakaryocytic differentiation.30,31 In addition, a very recent study has reported that the megakaryocytic-specific enhancer of the  2 integrin gene is directly controlled by MAPKs,43 further suggesting
a role of the MAPK pathway in megakaryocytic differentiation. These data support the results described here and suggest that the delay in
megakaryocytic differentiation induced by PD98059 is most probably related to its ability to inhibit MAPK activation, although we cannot
definitively eliminate an effect of PD98059 on unknown processes.
One of the most surprising findings of this study is the increase in
proliferation of the cells in response to MGDF on treatment with the
MEK inhibitor. This effect seems to be mainly due to an increase in DNA
synthesis rather than to a reduction in the proportion of cells in
apoptosis. This phenotype is at odds with the well-admitted role played
by the MAPK signaling modules in mitogenesis induced by growth factors
and in the aberrant cell proliferation associated with neoplastic
transformation in mammals.9,10,17 However, an
antiproliferative role of the MAPK pathway is not unprecedented.
Several studies have reported that activation of the MAPK pathway by
growth factors or overexpression of constitutively activated forms of
kinases of this pathway can lead to cell-cycle arrest, senescence, or
even apoptosis.34,44-47 In a given cellular context, the
ultimate cellular response to activation of the MAPK pathway has been
shown to depend on the strength and duration of the signal: transient
or acute activation may contribute to cell-cycle progression, whereas a
chronic high activity may result in cell-cycle
arrest.34,45-48 MGDF was found to induce sustained ERK
activation both in erythromegakaryocytic leukemia cell
lines29 and here in normal megakaryocytes derived from CB
progenitors. ERK activation has been reported for most members of the
cytokine receptors and works on hematopoietic cell lines or normal
progenitors have documented the requirement for this pathway in
proliferation27,28,49-52 and/or survival52,53
in response to EPO, GM-CSF, IL-3, or G-CSF. However, the sustained or
transient nature of the MAPK activity was not reported in these models.
Interestingly, the pro-proliferative effects of the MEK
inhibitor were found only in cultures of CB progenitors performed in
the presence of MGDF: PD98059 was found to inhibit cell proliferation
in cultures containing a mixture of IL-3, SCF, and IL-6, and it had no
effect on cell numbers in cultures grown with G-CSF plus Flt3-ligand.
Likewise, in the UT7-Mpl cell line, PD98059 inhibits cell growth in the
presence of either GM-CSF or EPO, while it enhances MGDF-induced DNA
synthesis29 (and unpublished data, December
1996). In this system, both GM-CSF and EPO can induce
MAPK activation, but this activation is more transient and less intense
than that triggered by MGDF.29,54 Thus, the use of
the MAPK pathway to transduce events other than proliferation
or prevention of apoptosis seems to be specific for Mpl and does
not reflect a general feature of cytokine receptor signaling.
Very recently, it has been shown that the ultimate cellular response of
constitutive activation of the MAPK cascade, cell-cycle arrest or
transformation, is dependent on the integrity of the cell-cycle
machinery signal, ie, that it can be opposite whether the context is a
normal or an immortalized cell.34 The increase in
MGDF-induced thymidine incorporation and long-term cell growth induced
by the MEK inhibitor in both normal hematopoietic progenitors and
leukemic cell lines indicates that the antiproliferative function of
the MAPK pathway in cells of the megakaryocytic lineage is not
specifically linked to a normal cellular context.
Apart from the MAPK pathway, MGDF has been shown to trigger
phosphorylation and activation of many transducing molecules including members of the JAK and STAT family, phosphatidylinositol 3-kinase (PI3-K) or the ERK-related JNK and p38 MAPK.33,55-59 A
recent study60 has shown that dominant negative forms of
STAT5, but not those of STAT1 or STAT3, were able to reduce
MGDF-induced proliferation of F-36-P cells expressing Mpl, suggesting
that STAT5 could be involved, at least partially, in mitogenesis
induced by MGDF. An inhibition of similar magnitude was also detected on expression of a dominant negative mutant of Ras in these
cells.60 Among the Ras effectors, PI3-K is a good canditate
that might participate in MGDF mitogenic response, as this kinase has
recently been shown to couple growth factor receptors to the cell-cycle machinery and antiapoptotic signals.46,61 Further studies
will be required to determine whether 1 or several of these signaling cascades are essential for MGDF-induced proliferation and/or survival of megakaryocytes.
In most cell lines, cell-cycle arrest is combined with
differentiation.22-24,30,31,47,48 Likewise, it has been
reported that megakaryocytic progenitor cells represent a continuum of cells from high to low proliferative capacities associated with changes
in the surface phenotype.37,38 Notably, a decrease in
progenitor proliferation has been described on the loss of the CD34
marker of immature progenitors, ie, on transition from the
CD34+CD61+ to the
CD34CD61+ phenotype.37
Accordingly, the increase in proliferation of the cells observed in
cultures containing the MEK inhibitor was combined with several
developmental changes indicative of a delay in the differentiation
process. First, although the MEK inhibitor mostly did not affect the
expression of megakaryocytic markers CD41 or CD42b, PD98059-treated
cells tended to retain markers of immature progenitors, as shown by the
increased proportion of both CD34+ and double-positive
CD41+CD34+ cells in cultures containing the MEK
inhibitor, as compared with control cultures. Second, morphologic
studies clearly showed that the cultures containing the MEK inhibitor
were greatly enriched in immature blast cells even after extended
culture (days 17 to 21), while control cultures presented a majority of
mature megakaryocytes around day 13. This analysis also showed an
increase in polyploid cells of large size in PD98059-treated cells.
Although surprising, this observation fits well with a previous study
showing that cytoplasmic and nuclear maturation are dissociated, and
that polyploid cells can be found among immature megakaryocytic
precursors: despite their more immature state, the
CD34+CD41+ and
CD34+CD42b+ cell populations were found to
contain a higher proportion of polyploid cells than the
CD34CD41+ and
CD34CD42b+ populations,
respectively.37 Thus, this suggests that the expression of
CD34 is related to the ability of megakaryocytic precursors to
accomplish DNA synthesis, either mitosis or endomitosis. The higher
polyploidization capacity of CD34+CD41+ cells
may be explained by the presence, among the
CD34CD41+ cells of micromegakaryocytes,
ie, mature megakaryocytes, which have no endomitotic capacity.
Supporting this possibility, we observed a majority of mature small
megakaryocytes in control cultures. These results are in agreement with
the low ploidy of megakaryocytes derived ex vivo from CB, even in the
presence of MGDF.39,40 Finally, the delayed maturation of
megakaryocytes induced by the MEK inhibitor was also supported by the
increased megakaryocytic colony formation with cells derived from
cultures treated with PD98059. Thus, the MAPK pathway, although not
required for megakaryoblast formation, may regulate the transition from proliferation to maturation in this lineage
Consistently higher numbers of erythroid colonies of large size were
formed with PD98059-treated cells, indicating the presence of erythroid
progenitors both more numerous and more immature in these cultures than
in control cultures. This result suggests that the MEK inhibitor could
affect the differentiation of erythroid progenitors generated in the
cultures and/or the differentiation of a bipotent erythromegakaryocytic
progenitor.62 The absence of a consistent alteration in GM
colony numbers in the presence of PD98059 supports this possibility.
Our experiments did not allow direct assessment of erythroid
differentiation. However, because erythroid differentiation cannot be
studied in the presence EPO alone and requires cytokine mixtures, the
involvement of the MAPK pathway in erythroid lineage differentiation
might not be easily detected in such a culture system.
During T-cell development, activation of the ERK pathway has been shown
to influence commitment by favoring the differentiation into the CD4
versus the CD8 lineage.63 Studies performed with bipotent
erythromegakaryocytic cell lines have shown that activation of MAPK
pathway in K562 cells, while increasing megakaryocytic-specific antigens, suppresses expression of hemoglobin, a key marker of erythroid differentiation.30 Conversely, inhibition of MAPK activity, even at the basal level, results in increased expression of
globins and prevents phorbol myristate acetate
(PMA)-induced downregulation of erythroid markers in K562
cells. In UT7 cells, increased surface expression of GpA was induced
even in the presence of GM-CSF on inhibition of basal ERK activity (F. Porteu, unpublished data, March 1998). Thus, it was
suggested that MAPK might also control erythroid versus megakaryocytic
lineage commitment. The present study does not support this hypothesis.
Indeed, first, PD98059 did not prevent the commitment towards the
megakaryocytic lineage. Second, no cells expressing the erythroid
marker, GpA, were generated in the cultures grown with TPO in the
absence of EPO.
The best characterized of MAPK targets are members of the ETS and AP-1
families of transcription factors. Phosphorylation has been shown to
play a critical role in modulating activity of transcription
factors.64 ERKs also phosphorylate and regulate the
activities of transcription factors of the STAT family65 and of various cytosolic molecules involved in signal transduction such
as kinases18,19 or effectors of apoptosis.66
Further studies are currently underway to characterize ERK-specific
targets in megakaryocytes.
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ACKNOWLEDGMENT |
We thank Amgen for providing PEG-rhuMGDF and SCF. We also greatfully
aknowledge Dr I. Dusanter-Fourt for critical review of the manuscript
and I. Bouchaert (ICGM) for help in cytometry analysis.
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FOOTNOTES |
Submitted December 31, 1998; accepted M |
TOP
ABSTRACT
INTRODUCTION