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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1273-1282
Thrombopoietin-Induced Activation of the Mitogen-Activated Protein
Kinase (MAPK) Pathway in Normal Megakaryocytes: Role in Endomitosis
By
Ponlapat Rojnuckarin,
Jonathan G. Drachman, and
Kenneth Kaushansky
From the Division of Hematology, University of Washington, Seattle,
WA.
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ABSTRACT |
Thrombopoietin (TPO) plays a critical role in megakaryocyte
proliferation and differentiation. Using various cultured cell lines,
several recent studies have implicated the mitogen-activated protein
kinase (MAPK) pathway in megakaryocyte differentiation. In the study
reported here, we examined the role played by thrombopoietin-induced MAPK activity in a cytokine-dependent cell line (BAF3/Mpl) and in
primary murine megakaryocytes. In both systems, extracellular signal-regulated protein kinase (ERK) 1 and 2 MAPK phosphorylation was
rapidly induced by TPO stimulation. To identify the Mpl domain responsible for MAPK activation, BAF3 cells expressing truncated forms
of the Mpl receptor were studied. Phosphorylation of ERKs did not
require elements of the cytoplasmic signaling domain distal to Box 2 and was not dependent on phosphorylation of the adapter protein Shc.
ERK activation in murine megakaryocytes was maximal at 10 minutes and
was markedly decreased over the subsequent 3 hours. Next, the
physiologic consequences of MAPK inhibition were studied. Using the
MAPK kinase (MEK) inhibitor, PD 98059, blockade of MAPK activity
substantially reduced TPO-dependent proliferation in BAF3/Mpl cells and
markedly decreased mean megakaryocyte ploidy in cultures. To exclude an
indirect effect of MAPK inhibition on stromal cells in whole bone
marrow, CD41+ cells were selected and then cultured in
TPO. The number of polyploid megakaryocytes derived from the
CD41-selected cells was also significantly reduced by MEK inhibition,
as was their geometric mean ploidy. These studies show an important
role for MAPK in TPO-induced endomitosis and underscore the value of
primary cells when studying the physiologic effects of signaling pathways.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIETIN (TPO), fulfills most, if
not all, of the properties predicted for the primary regulator of
platelet production. Both in vitro and in vivo experiments have
demonstrated that TPO is sufficient for full megakaryocyte
development.1-3 Binding of the hormone to its receptor,
Mpl, results in activation of a variety of signaling molecules,
including components of the JAK/STAT pathway4 and the
Shc/Ras/MAPK pathway.5,6 Most of the studies to date have
been conducted in cell lines that either normally express the Mpl
receptor or have been engineered to express it on the cell surface.
Activation of the JAK/STAT pathway has also been demonstrated both in
normal megakaryocytes7 and in platelets.8-10
However, the activation of MAPK pathway has never been reported in
primary megakaryocytes.
Extrapolation of data derived from cell lines to normal physiologic
conditions is difficult for several reasons. First, the expression of
specific signaling molecules may differ between cell lines and primary
cells. Second, signaling molecules in distinct cell types may be
compartmentalized by both organelles and scaffolding proteins into
different subcellular locations, potentially altering the cross-talk
between pathways. Third, it is likely that transformed cell lines
develop anomalous signaling pathways to promote their proliferation and
survival, pathways that differ significantly from primary cells.
Although much has been learned about the signaling pathways triggered
by the binding of TPO to its receptor on cell lines, the physiologic
role of each pathway in megakaryocyte proliferation and differentiation
is still largely unknown. Truncated thrombopoietin receptors that fail
to activate JAK2 are also unable to support proliferation of
BAF3/Mpl11 and UT-712 cell lines in the
presence of TPO. This suggests that the JAK/STAT pathway may contribute to TPO-induced proliferation. Distinct signaling pathways may control
different cellular processes. For example, JAK/STAT may be responsible
for proliferation and MAPK pathway may function for differentiation.
Alternatively, various pathways may have complex interactions that lead
to an integrated response or that may be redundant.
Mitogen-activated protein kinases are serine/threonine kinases that are
highly conserved in all eukaryotic cells from yeast to humans. At least
6 signaling cascades classified as MAPK pathways have been
identified13 and have been found to mediate cell
proliferation, survival, apoptosis, and/or differentiation, depending
on the cell line used. For instance, MAPK induces proliferation in NIH 3T3 cells, a murine fibroblastic cell line, but induces differentiation in PC12 cells, a neural pheochromocytoma cell line.14 Two
of the classical MAPKs, extracellular signal-regulated protein kinase 1 (ERK1) and ERK2, have been shown to be involved in TPO signaling. A
study in the erythroleukemia cell line, K562, has shown that constitutively active mutants of MEK or phorbol esters can cause megakaryocyte-like differentiation, including cell enlargement, inhibition of cell growth, and expression of platelet-specific glycoprotein IIb/IIIa on cell surfaces.15 In addition, a
constitutively active form of ERK has been shown to induce the
expression of megakaryocyte-specific surface markers and morphological
changes in CMK cells, a human megakaryoblastic leukemia cell line. The MEK inhibitor, PD 98059, was found to block these
effects.16 Although taken as evidence that MEK and MAPK are
involved in normal megakaryocyte development, megakaryocytic
differentiation was induced by stem cell factor in this system, which
cannot promote differentiation in normal megakaryocyte progenitor
cells. Recently, similar results were observed in UT7-Mpl cell line
stimulated by thrombopoietin,17 although these cells fail
to differentiate into fully mature megakaryocytes. Like all transformed
or immortal cell lines, it is possible that UT-7 cells use aberrant
pathways that are not present in normal cells. Alternatively, these
pathways in cell lines may lead to differentiation by nonphysiologic
mechanisms. For example, endomitosis in the HEL cell line is reportedly
due to the absence of cdc2.18 In contrast, endomitosis in
MegT cells is believed to result from the absence of
cyclinB.19 Both of these proteins are present in primary
endomitotic megakaryocytes.20,21
To define the physiologic role of the MAPK pathway in megakaryocytes,
we used murine bone marrow cells cultured in TPO. We found that ERK1
and ERK2 were rapidly but only transiently activated in normal murine
megakaryocytes after exposure to TPO, despite the continued presence of
the hormone. Inhibition of the MAPK pathway significantly reduced
megakaryocyte polyploidization in both whole bone marrow cultures and
CD41-selected cell cultures. These results point to the importance of
the MEK/ERKs pathway in a specific aspect of megakaryocyte development,
endomitosis, and argue persuasively for the use of primary cells to
dissect the role of various intracellular signals on cellular differentiation.
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MATERIALS AND METHODS |
Cell line and growth conditions.
BAF3/Mpl cells obtained from Zymogenetics, Inc (Seattle,
WA)11,22 were maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) with 10% heat-inactivated fetal calf
serum (HyClone, Logan, UT), 2 mmol/L L-glutamine, 100 U/L penicillin,
100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B (BioWhittaker)
supplemented with murine interleukin-3 (mIL-3; 0.2% vol/vol
conditioned culture medium from baby hamster kidneys cells engineered
to constitutively secrete mIL-3). BAF3 cell lines expressing truncated
forms of Mpl were previously described11 and were
maintained in an identical fashion.
Purification of murine megakaryocytes.
BDF-1 mice (Jackson Labs, Bar Harbor, ME) were subcutaneously injected
with pure, recombinant human TPO for 5 days (2 µg/d; Zymogenetics,
Inc) and killed, and bone marrow cells were flushed from femurs and
tibias into serum-free Iscove's modified Dulbecco's medium
(IMDM) supplemented with 1% Nutridoma (Boerhinger
Mannheim, Indianapolis, IN) with penicillin, streptomycin, and
L-glutamine. Cells were incubated (37°C, 5% CO2) with
recombinant murine TPO (5% vol/vol conditioned medium, ~37.5 ng/mL)
for 72 hours. Mature megakaryocytes were purified over a discontinuous
bovine serum albumin (BSA; Sigma, St Louis, MO) density gradient
(0%/1.5%/3.0% BSA).7 Ninety percent of cells that
settled to the bottom in 30 minutes were mature megakaryocytes by
immunostaining. Additionally, some of mature megakaryocytes remained
attached to the plastic syringe and could be recovered by gentle washing.
Stimulation of cells.
Cells were starved in serum-free, growth factor-free medium (16 hours
for BAF3/Mpl cells and 7 hours for murine megakaryocytes). PD 98059 (New England Biolab, Beverly, MA) was dissolved in dimethyl sulfoxide
(DMSO) and added to cells 20 minutes before TPO stimulation at final
concentrations between 20 and 100 µmol/L. An equal volume of DMSO was
used as a negative control. In some experiments, 100 nmol/L Wortmannin
(Sigma), a PI3K inhibitor, was also used. After preincubation, cells
were stimulated by TPO (2% vol/vol; conditioned medium, ~14 ng/mL)
for 10 minutes or for the indicated duration. In some experiments,
fetal calf serum (10% vol/vol), recombinant murine stromal
cell-derived factor 1 (SDF-1; 100 ng/mL; a gift from N. Yamamoto, Tokyo Medical and Dental University, Tokyo, Japan) or murine stem cell factor (SCF; 100 ng/mL; Kirin
Pharmaceutical, Gunma, Japan) was added. The cells were then mixed with
ice-cold phosphate-buffered saline (PBS) and a cell lysate was prepared by Triton-X 100 solubilization as previously described.4
A BAF3/Mpl cell proliferation assay was performed as previously
described.11 Briefly, cells were washed twice with
cytokine-free media and then grown in various concentrations of murine
TPO-conditioned supernatant (0.01%, 0.1%, 1%, and 10% vol:vol; 1%
is ~7 ng/mL) or murine IL-3-conditioned medium at the maximal
proliferation dose. After 36 hours of incubation,
3,4,5-dimethylthiazole-2-yl-2,5-diphenyl tetrazolium bromide (MTT;
Sigma) was added (final concentration, 1 mg/mL) and
incubation was continued for 5 hours. Cells were then lysed and
absorbance at 570 to 630 nm was determined using an enzyme-linked
immunosorbent assay (ELISA) plate reader. Each data point
was performed in triplicate. Proliferation was expressed as a
percentage of maximal mIL-3-induced growth.
Western blot analysis.
The protein concentrations of BAF3/Mpl cell lysate were measured using
Protein/DC Assay (Bio-Rad, Hercules, CA) to assure equal loading
between lanes. For megakaryocytes, equal numbers of purified cells were
used to generate each sample. Lysates were denatured by boiling for 5 minutes in loading buffer as described by Laemmli23 and
separated on 10% polyacrylamide gels. Proteins were then transferred
to nitrocellulose membranes (Schleicher & Schuell, Keane, NH) and
blocked for 16 hours in Tris-buffered saline with 0.05% Tween 20 (TBST) and 3% BSA. Rabbit polyclonal antibody specific for doubly
phosphorylated MAPK was purchased from Promega (Madison, WI). Rabbit
polyclonal ERK 2 antibody was obtained from Santa Cruz Biotech (Santa
Cruz, CA). Phosphotyrosine antibody (4G10) and Shc antibody were
purchased from Upstate Biotechnology, Inc (Lake Placid, NY). Each
antibody was diluted in blocking buffer at the concentration
recommended by each supplier and incubated with the blot at room
temperature for 2 hours. After four 5-minute washes in
TBST, the nitrocellulose blots were gently rocked (30 to 60 minutes at
room temperature) in solution containing blocking buffer and the
detecting antibody: goat antirabbit antibody (MAPKs and Shc) or goat
antimouse antibody (4G10) coupled to horseradish peroxidase (Bio-Rad)
at a final dilution of 1:5,000. The membrane was then washed 4 times in
TBST (5 minutes each), incubated with chemiluminescent reagents (Santa
Cruz Biotech), and exposed to film. In some cases, the antibodies were
stripped off the blot by washing with 62.5 mmol/L Tris (pH 6.8), 2%
sodium dodecyl sulfate, and 100 mmol/L  -mercaptoethanol at 50°C
for 30 minutes. Subsequently, the blots were reblocked and reprobed
with new primary and secondary antibodies.
Whole marrow culture.
BDF1 mice were killed, and the marrow was flushed from femurs and
tibias. The cell suspension was then filtered through 2 layers of
gauze, pelleted, and resuspended in red blood cell lysis buffer (140 mmol/L NH4Cl in 17 mmol/L Tris-HCl, pH 7.2). Cells were
washed and incubated in IMDM/1%
Nutridoma/penicillin/streptomycin/L-glutamine supplemented with 5%
TPO-conditioned medium (approximate concentration, 35 ng/mL). PD 98059 at final concentrations of 20, 50, or 100 µmol/L was added. An equal
volume of diluent (DMSO) was added to control cultures. Megakaryocyte
cultures were then incubated for 72 hours at 37°C in a 5%
CO2-containing fully humidified atmosphere. Cells were
observed by inverted light microscopy, small aliquots were stained with
trypan blue and counted for total viable cells by hemocytometer, and
cytospins were prepared on BSA-coated slides. Slides were then
Wright-stained to assess megakaryocytic morphology and stained for
acetylcholinesterase activity as a marker of murine megakaryocyte
differentiation. The acetylcholinesterase stain was performed according
to the method of Jackson.24 The remaining cells were
subjected to immunofluorescence staining and analyzed by flow cytometry.
Immunofluorescence and flow cytometric analysis.
Ten milliliters of megakaryocyte suspension culture was layered over 1 mL of 3% BSA in CATCH buffer (0.36% sodium citrate, 2 mmol/L
theophylline, and 1 mmol/L adenosine in calcium-free, magnesium-free
Hank's Balanced Salt Solution), and the cells were pelleted at
200g. Cells were resuspended in 100 µL of 3% BSA in CATCH
buffer. Antimouse Fc receptor antibody (Pharmingen, San Diego, CA) was
added at a final concentration of 10 µg/mL to block nonspecific Fc
binding and incubated on ice for 30 minutes. Fluorescein isothiocyanate
(FITC)-conjugated antimurine CD41 or its isotype control
(Pharmingen) was then added at 1:50 dilution and incubated on ice for 1 hour. One milliliter of 3% BSA in CATCH buffer was added, the
suspension was layered over 5% BSA in CATCH, and the cells were
pelleted at 200g. Cells were then resuspended in propidium iodide (PI) buffer containing 0.1% sodium citrate, 0.1% Triton X-100,
50 µg/mL PI, and 6% RNAse. Cells were then analyzed by two-colored
flow cytometry. FITC-CD41+ cells (megakaryocytes) were
gated to determined DNA content (ploidy) by PI stain and the
distribution of cell size (forward scattering). Ploidy analysis is
displayed semilogarithmically.
CD41-selected cell culture.
Bone marrow cells from BDF1 mice were separated by density gradient
centrifugation. Five milliliters of cell suspension was layered on 3 mL
of Optiprep (Nycomed, Oslo, Norway) at a specific gravity
1.080 and centrifuged at 400g for 20 minutes at
room temperature. Low-density cells at the interface were then
collected, washed twice, and resuspended in PBS with 5% fetal calf
serum at 108 cells/mL. FITC-conjugated anti-CD41 antibody
was then added at 0.35 µg/106 cells and incubated on ice
for 45 minutes. Cells were then washed twice and incubated with 1:10
dilution of microbeads coated with anti-FITC antibody (Miltenyi
Biotech, Auburn, CA) in PBS/0.5% BSA/2 mmol/L EDTA at 6°C to
12°C for 15 minutes and washed once. Labeled cells were then
passed through a high gradient magnetic column, MidiMACS
(Miltelyi Biotech). The positive fraction retained in the column after
3 washes was then eluted and cultured in 1% Nutridoma and 5%
TPO-conditioned medium (approximate concentration, 35 ng/mL) with or
without PD 98059. After 84 hours of culture, cells were then stained
with anti-CD41-FITC and PI using the methods described above.
Approximately 30% of cells after culture were CD41+ megakaryocytes.
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RESULTS |
ERKs are activated by TPO in BAF3/Mpl cells and mediate TPO-induced
proliferation.
BAF3/Mpl cells were incubated in serum-free, growth factor-free
medium for 16 hours to allow maximal dephosphorylation of cellular proteins. Cultures were then stimulated by TPO for 10 minutes
in the presence or absence of the specific MEK inhibitor, PD 98059, and
cell lysates were prepared and subjected to Western blot analysis. When
probed with an antibody specific for the
double-phosphorylated, activated form of ERKs
(Fig 1), the TPO-stimulated cell lysate contained two prominent bands at 44 and 42 kD that were absent in
unstimulated cells. These 44- and 42-kD bands were independently verified to represent activated ERK1 and ERK2, respectively, by blotting with an ERK1/2-specific antibody. Addition of the MEK inhibitor PD 98059 20 minutes before TPO stimulation substantially decreased ERK phosphorylation. The blot was stripped and reprobed with
ERK2-specific antibody to demonstrate equal loading of each lane (see
lower panel, Fig 1). It is noteworthy that concentrations of PD 98059 as high as 100 µmol/L could not completely inhibit TPO-induced MAPK
activation in the BAF3/Mpl cell line, the maximal concentration
achievable because of limited inhibitor solubility. These results
suggest that an MEK-independent MAPK activation pathway may exist in
BAF3 cells. The phosphoinositol-3-kinase (PI3K) pathway has been
previously reported to induce MAPK phosphorylation without MEK
activation.25 Consistent with this hypothesis, the combination of PD 98059 and Wortmannin to TPO-stimulated BAF3/Mpl cells
at the concentration of 100 µmol/L and 100 nmol/L, respectively, resulted in more inhibition of MAPK phosphorylation than either inhibitor alone (data not shown). This result suggests that maximal TPO-induced ERK activation in this cell line may be dependent on both
MEK- and PI3K-induced pathways.
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| Fig 1.
TPO-induced MAPK phosphorylation in BAF3/Mpl cells.
BAF3/Mpl cells were cultured in serum-free, cytokine-free media for 16 hours. Twenty minutes before stimulation, PD 98059 (PD) at a final
concentration of either 50 or 100 µmol/L, was added. Cells were then
stimulated by the addition of 14 ng/mL murine TPO for 10 minutes,
lysed, and subjected to Western blot analysis. The blot was probed with
anti-double-phosphorylated ERKs/MAPK antibody (upper
panel), showing 2 specific bands corresponding to ERK1 (44 kD) and ERK2
(42 kD). ERK phosphorylation could be partially inhibited by PD 98059. The blot was then stripped and reprobed with anti-ERK2 antibody (lower
panel) to assure equal loading in all lanes.
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Next, we determined the functional consequences of MAPK activation in
BAF3/Mpl cells. Using an MTT reduction assay, we found that
proliferation of BAF3/Mpl cells grown in TPO was decreased by PD 98059 in a dose-dependent manner (Fig 2). This
suggests that ERK activation contributes to TPO-induced proliferation
in BAF3/Mpl cells. In contrast, PD 98059 has no significant effect on
IL-3-induced proliferation of BAF3/Mpl cells in 3 separate experiments. Correlated with these data, IL-3 induced only minor ERK
phosphorylation in BAF3/Mpl cells compared with TPO (data not shown).
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| Fig 2.
The effect of MEK inhibition on TPO-induced proliferation
of BAF3/Mpl cells. BAF3/Mpl cells were cultured in the presence of
various concentrations of recombinant murine TPO. The cultures also
contained PD 98059 (dissolved in DMSO) at final concentrations of 50 µmol/L ( ) or 100 µmol/L (X), whereas the control cultures
contained a similar volume of DMSO ( ). The number of living cells
was determined after 36-hour cultures using the MTT method described in
Materials and Methods. The TPO-induced proliferation is presented as
the percentage of maximal IL-3-induced proliferation. The abscissa is
displayed in a logarithmic scale. The results represent the mean
(±SD) of triplicate determination in a single representative
experiment. This experiment has been performed 3 times with similar
results.
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MAPK phosphorylation by TPO does not depend on Shc phosphorylation.
Previously, we used truncated forms of the Mpl cytoplasmic domain to
identify subdomains involved in the activation of signaling proteins in
response to TPO.11 A similar approach was taken to map the
region of Mpl responsible for MAPK phosphorylation. We found that
truncation of the 10 carboxy-terminal residues (T-111, missing the
primary sites of Mpl tyrosine phosphorylation) resulted in a moderate
reduction in detectable phospho-MAPK (Fig
3A). Thrombopoietin stimulation in BaF3/Mpl cells with further
truncation such that only 69 cytoplasmic amino acids of the cytoplasmic
domain remain (T-69, including box1 and box2) still caused definite
MAPK phosphorylation. In contrast, TPO does not induce significant
tyrosine phosphorylation of the adapter protein Shc (Fig 3B) in cells
expressing either of the truncated receptors. This difference is
significant because Shc, which binds to the distal portion of the
phosphorylated Mpl cytoplasmic domain, was believed to be a major route
to Ras-Raf-MEK-MAPK activation. Therefore, the significant ERK
phosphorylation in these cells (~70% and 60% for T-111 and T-69,
respectively, compared with full-length Mpl as measured by
densitometry) suggests that a part of TPO-induced ERK activation is Shc
independent.
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| Fig 3.
MAPK phosphorylation is not fully dependent on Shc
phosphorylation. Cell lysates were prepared either before ( ) or
after (+) stimulation with exogenous TPO (14 ng/mL for 10 minutes).
Parental BAF3 cells were used as well as clones engineered to express
the full-length murine Mpl receptor (mMpl) or mutated receptors, which
were truncated after either 111 or 69 cytoplasmic amino acids (T-111
and T-69, respectively).11 (A) One hundred micrograms of
each lysate was evaluated by Western blotting and probed to detect
double-phosphorylated ERK1 and ERK2. The blot was
stripped and reprobed with ERK2 antibody to confirm equal loading in
all lanes. (B) Shc was immunoprecipitated from 1 mg of each cell
lysate. The immunoprecipitated protein was evaluated by Western
blotting and probed with a phosphotyrosine-specific antibody (4G10).
The blot was stripped and reprobed to verify the presence of Shc in all
lanes.
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ERK is activated by TPO in purified murine megakaryocytes.
To determine whether the MAPKs are also activated by TPO in primary
cells, mature murine megakaryocytes were expanded in vitro by
incubation of whole bone marrow cells in serum-free media containing exogenous TPO. The large cells were purified using a unit gravity sedimentation column. The resulting cells (~200,000 cells/mouse) contained approximately 90% mature megakaryocytes by immunostaining. Two hundred thousand megakaryocytes were starved in serum-free, cytokine-free conditions for 7 hours. The short period (compared with
BAF3 cells) was chosen because primary megakaryocytes undergo apoptosis
more rapidly than do transformed cells when deprived of serum and
growth factors (data not shown). Megakaryocytes were then stimulated by
TPO in the presence or absence of the MEK inhibitor (PD 98059), lysed,
and subjected to Western blot analysis. Like the results in BAF3/Mpl
cells, TPO induced marked ERK phosphorylation in normal murine
megakaryocytes (Fig 4). But, in contrast to
the transformed BAF3/Mpl cells, this effect was completely inhibited by
the MEK inhibitor alone at a relatively low concentration of PD 98059 (20 µmol/L). Some investigators have proposed that the protein kinase
C pathway plays a major role in MAPK activation. However, we found that
ERK phosphorylation was only partially inhibited by Bis-indolylmalemide
I, a specific protein kinase C (PKC) inhibitor (data not
shown).
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| Fig 4.
TPO-induced MAPK phosphorylation in purified murine
megakaryocytes. Bone marrow cells were harvested from mice preinjected
with 2 µg/d human TPO for 5 days, grown in serum-free medium with 35 ng/mL murine TPO for 3 days, and then purified by an albumin
density-gradient column. Purified megakaryocytes were incubated in a
serum-free, cytokine-free medium for 7 hours and stimulated with 14 ng/mL murine TPO for 10 minutes. Twenty minutes before stimulation, PD
98059 (PD) at 20 µmol/L or 50 µmol/L was added. The cell lysates
were size-fractionated, transferred to nitrocellulose, and probed with
anti-double-phosphorylated ERK antibody (upper panel).
The blot was then stripped and reprobed with anti-ERK2 antibody (lower
panel) to assure equal loading in all lanes.
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MAPK activation in megakaryocytes is transient.
Evidence from other cell systems suggests that the duration of MAPK
activation plays an important role in determining the cellular
response; transient MAPK activation (minutes to hours) is typical of
proliferation, whereas prolonged MAPK activation (lasting many hours or
several days) is characteristic of differentiation.14 To
evaluate the duration of ERK activation, purified murine megakaryocytes were starved for 7 hours and then stimulated by TPO for 10 minutes, 1 hour, and 3 hours. ERK phosphorylation was greatest at 10 minutes and
then progressively decreased at the later time points
(Fig 5A), becoming nearly undetectable by 3 hours. This result differs from a previous study in the UT-7 cell lines
that suggested that sustained ERK activation was required for cellular
differentiation.17
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| Fig 5.
Time course of TPO-induced MAPK phosphorylation. Bone
marrow cells were prepared and stimulated as described in the legend to
Fig 4. Cell lysates were subjected to Western blot analysis and probed
with anti-double-phosphorylated ERK antibody (upper
panels) and then stripped and reprobed with anti-ERK2 antibody (lower
panels). The cultures contained (A) 14 ng/mL TPO alone or (B) TPO plus
10% (vol:vol) fetal calf serum or TPO plus 100 ng/mL SDF-1. The
TPO-inducible bands found just above ERK1 were sometimes seen. They
were not cross-reactive with anti-ERK1 antibody as the blot was
stripped and reprobed. The origin of these bands is still unclear.
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We hypothesized that other growth factors or cytokines might synergize
with TPO to produce sustained ERK activation in primary megakaryocytes
and thereby fall in line with the conclusions of others.14
To evaluate this possibility, we studied the effect of fetal calf
serum, SDF-1, and murine SCF on MAPK activation when used in
combination with TPO. Each of these agents has been shown to act in
synergy with TPO to augment megakaryocyte
development.2,26,27 Murine megakaryocytes were starved and
stimulated with TPO for 10 and 90 minutes with or without murine 100 ng/mL SCF, 10% fetal calf serum, or 100 ng/mL SDF-1. Neither fetal
calf serum nor SDF-1 could prolong MAPK responses (Fig 5B), although
the intensity of the double-phosphorylated ERK activation
was significantly enhanced by both serum and SDF-1, demonstrating that
converging signaling pathways from other receptors can enhance MAPK
response to TPO, but do not prolong it. Murine SCF could neither
enhance nor prolong MAPK phosphorylation in megakaryocytes (data not shown).
MAPK pathway contributes to megakaryocyte endomitosis.
To identify the functional effects of the MAPK pathway in
megakaryocytes, murine marrow cells were cultured in serum-free media
with TPO for 3 days. PD 98059 or diluent (DMSO) was added at the
beginning of culture. The low amount of DMSO used in the culture had no
adverse effects on bone marrow cells as compared with culture without
DMSO. Nonadherent cells were stained by trypan blue and viable cells
were counted using a hemocytometer. Total cell numbers were lower in
the inhibitor group than in the control group (see table in Fig 7).
This effect was most obvious on nonhematopoietic, adherent cells (bone
marrow stromal cells). As seen by inverted microscopy, the numbers of
adherent cells were greatly reduced at higher doses of PD 98059. At 50 µmol/L, the adherent cells were virtually absent, suggesting that
MAPK pathway is essential for survival of these cells. The percentage
of megakaryocytes in each culture were determined by staining cells
with FITC-tagged antibody to CD41, a megakaryocyte-specific marker, and
analyzing by flow cytometry. The total number of megakaryocytes was
calculated as the total number of cells multiplied by the percentage of
CD41+ cells. In multiple experiments, megakaryocyte number
in whole bone marrow culture was not significantly affected by
inhibition of MEK. We next studied the effect of blocking the MAPK
pathway on several aspects of megakaryocyte differentiation. Cytospins were prepared and the slides were Wright-stained for morphologic analysis in 2 separate experiments. Megakaryocytes, with or without inhibitor, display similar morphology by light microscopy
(Fig 6A and B). They could increase in size
and nuclear lobulation, and some cells in both groups developed pink
intracytoplasmic granules. Surface acetylcholinesterase activity,
another marker of murine megakaryocyte differentiation, appeared
similar in both the inhibitor and control groups in 2 separate
experiments (Fig 6C and D). These results were observed at PD 98059 concentrations of 20 and 50 µmol/L. Therefore, we did not detect
nonspecific toxicity of the MEK inhibitor on megakaryocytes in culture
within this dose range.
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| Fig 6.
MEK inhibition has no obvious effect on murine
megakaryocyte morphology and acetylcholinesterase activity in whole
marrow cultures. Murine bone marrow cells were harvested and split into
2 flasks. Both were grown in serum-free media with 35 ng/mL murine TPO
for 3 days. One culture contained 50 µmol/L PD 98059 and the other
contained an equal amount of DMSO. Cytospin preparations were then
performed and stained with Giemsa ([A] DMSO; [B] PD 98059) or
acetylcholinesterase activity (brown staining; [C] DMSO; [D] PD
98059).
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As megakaryocytes mature, they undergo DNA replication without
cytokinesis. The DNA content increases from 2 N to 4 N, 8 N, 16 N, up to 128 N in some cells. This process, termed
endomitosis, is unique to normal megakaryocytic
differentiation.28 To evaluate the DNA content of
individual megakaryocytes, we used flow cytometry with CD41 to
distinguish megakaryocytes from other lineages in liquid culture, and
after cell permeabilization, with PI staining to evaluate DNA content.
As shown in Fig 7, treatment of
megakaryocyte cultures for 3 days with PD 98059 greatly
reduced the proportion of cells developing 32 N and 64 N DNA content.
In contrast, when murine marrow cells were grown in the same
conditions, except with Bis-Indolylmalemide I (BIM), a specific PKC
inhibitor, the cells developed their full polyploid potential (data not
shown).
View larger version (46K):
[in this window]
[in a new window]
| Fig 7.
The effect of MEK inhibition on megakaryocyte
endomitosis. Murine bone marrow cells were cultured as described in the
legend to Fig 6. Cells were then stained with FITC-conjugated
anti-CD41, solubilized, and stained with PI. In the left-hand panels,
the abscissa represents log FITC intensity and the ordinate represents
cell size, as determined by forward light scatter. Megakaryocytes,
identified by CD41 positivity, were gated to analyze for DNA content by
PI staining (right-hand panels). The dot plot of cells stained with the
isotype control for anti CD41-FITC is also shown ([A] isotype). The
log intensity of PI is on the abscissa and the numbers of cells are on
the ordinate. The bars mark the DNA contents of 2N, 4N, 8N, 16N, 32N,
64N, and 128N, respectively. Cells grown in DMSO (A) are compared with
cells grown in PD 98059 (B). These data are the representative results
from 3 separate experiments. Cell numbers after 72-hour cultures are
shown in the table (C). The total numbers of viable cells were
determined using a hemocytometer. The percentage of CD41+
cells in each population was determined by immunofluorescent staining
and flow cytometry. Megakaryocyte numbers were calculated as follows:
total viable cells multiplied by the percentage of CD41+
cells. The results represent the mean of 3 to 4 experiments.
|
|
The interpretation of these data is still limited. Whole bone marrow
cultures contain accessory cells that can produce growth factors and
cytokines. Inhibition of MAPK decreased the numbers of these cells
considerably and may have indirectly affected megakaryocyte maturation.
To address this problem, bone marrow cells were selected for CD41
expression using a high gradient magnetic system (MiDiMACS) before
culture in TPO. Consistent with the results in whole bone marrow cell
cultures, the proportion of polyploid cells was substantially lower in
the presence of PD 98059 (Fig 8). Data in
these partially purified cells suggest that MEK inhibition directly
affects megakaryocyte development. However, in contrast to the result
in whole bone marrow cultures, the total number of cells and
megakaryocytes decreased significantly in purified cell cultures
containing PD 98059. Moreover, cells became smaller than the control
culture as assessed by Giemsa staining.
View larger version (21K):
[in this window]
[in a new window]
| Fig 8.
The effect of MEK inhibition on megakaryocyte endomitosis
in CD41-selected cell culture: Analysis of polyploid cells. Murine bone
marrow cells were harvested and CD41+ cells were selected
using a high gradient magnetic system (MiDiMACS). Purified cells were
grown in serum-free media containing 35 ng/mL murine TPO for 84 hours.
In 1 culture, PD 98059 at final concentration of 25 µmol/L was added
initially and a similar dose was added 24 hours later (B). An equal
volume of DMSO was added in the control culture. Cells were then
stained with FITC-conjugated anti-CD41, solubilized, and stained with
PI before being analyzed using flow cytometry. Only viable cells,
defined by forward and side light scattering characteristics, were
analyzed for fluorescent intensity. After culture, there was an 80%
reduction in the total number of CD41+ cells in the
presence of the inhibitor compared with control. However, equal number
of cells were counted in each group. The histograms show the ploidy
distribution of cells with DNA contents of 4N or higher. The
consecutive peaks represent the frequencies of cells with DNA contents,
as indicated by bars. These data are the representative results from 3 separate experiments.
|
|
| |
DISCUSSION |
The cloning of TPO has opened a new era of research into the molecular
mechanisms of megakaryocyte development. Intracellular changes after
ligand-receptor binding have been extensively studied in the past
several years. Various immortalized cell lines expressing the TPO
receptor have provided models for signal transduction experiments,
because the large number of cells necessary for biochemical studies can
be obtained. We have demonstrated in this study that MAPKs (ERK1 and
ERK2) are activated in the BAF3/Mpl cell lines. Like other studies of
the effects of MAPKs, our results with the MEK inhibitor, PD 98059, demonstrated an inhibitory effect on BAF3/Mpl cell proliferation.
Therefore, the MAPK kinase pathway contributes to proliferation of
BAF3/Mpl cells, but the effects on cellular maturation could not be
evaluated, because these cells do not differentiate in response to TPO.
Interestingly, PD 98059 could not totally block MAPK activation in this
cell line. It is possible that there may be other MEK-independent
pathways that are able to directly activate ERK, such as the PI3K
pathway. The inhibitor, PD 98059, selectively inhibits MEK1 relative to
MEK2.29 Therefore, an alternative explanation is that MEK2
may be more critical for ERK phosphorylation in BAF3 cells.
Consequently, MEK1 inhibition alone by this compound cannot completely
block ERK phosphorylation. In 1 series of experiments, the PI3K
inhibitor, Wortmannin, when added to PD 98059, completely blocked ERK1
and ERK2 phosphorylation. However, additional experiments will be required to confirm the role of PI3K in ERK activation in BAF3 cells. A
concern about this type of experiment is the specificity of a chemical
inhibitor. PD 98059 may be able to interfere with biochemical reactions
in cells other than the MAPK pathway. We have transfected BAF3/Mpl
cells with a hyperactive ERK2 construct (a generous gift from Dr Terry
Vik [Wells Center for Pediatric Research, Indianapolis,
IN] and colleagues16) and found that these cells had
higher proliferative response to TPO compared with cells transfected
with a control vector. This result confirms that the ERK pathway is
involved in TPO-induced proliferation. Although the use of cell lines
is convenient, extrapolation to signaling in normal cells is not always
reliable, because BAF3/Mpl may have acquired an aberrant pathway for
unlimited growth.
As a result of these limitations, we expanded our studies of MAPK
signaling to primary cell cultures, a physiologically relevant model
for studying hematopoiesis. In the present study, 2 of the MAPKs, ERK1
and especially ERK2, have been found to be reproducibly phosphorylated
after 10 minutes of TPO stimulation in murine megakaryocytes. We also
found that MAPK phosphorylation wanes over a 3-hour time course,
despite the continued presence of TPO, suggesting a role for an as-yet
unidentified phosphatase. Addition of either fetal calf serum or SDF-1
increased the intensity of MAPK phosphorylation but could not prolong
the response. Studies in other cell systems suggest that the duration
of ERK activation affects the cellular response. A study in a
pheochromocytoma cell line, PC12, has shown that transient ERK
activation by epidermal growth factor (EGF) resulted in
cell proliferation, but sustained ERK activation by nerve growth factor
(NGF) resulted in differentiation.14 In PC12
cells engineered to express the platelet-derived growth factor (PDGF) receptor, PDGF induced sustained activation of ERK
and differentiation.30 This may be the result of different
rates of receptor downregulation, because the EGF receptor is
downregulated more rapidly than the NGF receptor14 or
differential activation of cellular phosphatases. Interestingly, PC12
overexpressing the EGF receptor can also cause differentiation,
suggesting that the number of surface receptors determines the duration
of responses.31 Nevertheless, this differential response
may be dependent on some unknown property of this transformed cell line
and may not represent the actual phenotype of primary neuronal cells. A
recent study has shown that thrombopoietin induces sustained ERK
activation in UT7/Mpl cells and results in cellular
differentiation.17 This cell line was engineered to
overexpress the Mpl receptor (possibly greater than Mpl expression in
normal megakaryocytes that express ~1,900 Mpl receptors per
cell32). Therefore, sustained MAPK response in cell lines
might be an artifact attributable to the overexpression of the Mpl
receptor. Alternatively, mature and immature megakaryocytes may respond
to TPO differently. Our density gradient purification system enriched
primarily mature megakaryocytes that may no longer require or may no
longer generate a prolonged MAPK signal. Therefore, the kinetics of the
ERK response in immature megakaryocytes or their progenitors may
require further investigation. Unfortunately, at present, primary
megakaryocyte progenitors cannot be obtained in sufficient quantities
for biochemical analysis.
The pathway from the Mpl receptor to MAPK phosphorylation/activation
has yet to be fully defined. Much evidence has been gathered suggesting
that the Shc adapter protein interacts with cytokine receptors via its
phosphotyrosine binding domain, and upon phosphorylation, Shc can form
a molecular bridge leading to Ras activation (association of GRB2/SOS
complex). Previous reports suggested that the loss of Shc activity
blocked TPO-induced partial differentiation of WEHI-3B and M1 cell
lines.12 However, Porteu et al33 reported that
deletion of Mpl cytoplasmic residues 71-94 prevented MAPK activation
despite retaining the Shc recruitment and activation site. Our results
clearly demonstrate that neither Shc phosphorylation nor residues 71-94 are absolutely required for TPO-induced MAPK phosphorylation in
BAF3/Mpl cells (Fig 3). Such discrepant results further point out
concerns over too great a reliance on conclusions derived from
transformed cell lines. The Shc-independent pathway of ERK activation
has to be further determined, but in our view, should be studied in
primary cells.
In our culture system, bone marrow cells were grown in TPO-containing
serum-free media for 3 to 4 days before the numbers and various aspects
of differentiation of megakaryocytes were evaluated. Because of the
short duration of culture, the roles of MAPK in very primitive cells
cannot be evaluated. The stages of development we studied are the steps
from committed megakaryocyte precursors to fully mature megakaryocytes.
As shown in Fig 7C, the number of CD41+ megakaryocytes at
the end of whole marrow cultures was not significantly affected by MAPK
inhibition, whereas total cell numbers decreased. This indicates that
MAPK is involved in proliferation of other murine marrow cells but is
less critical for megakaryocytes, at least within the complex cellular
environment of a whole marrow culture. Nevertheless, at 20 µmol/L PD
98059, there was clear inhibition of megakaryocyte endomitosis. At this
dose, MAPK phosphorylation was markedly inhibited in megakaryocytes
(Fig 3). Megakaryocyte morphologies, as observed by
Giemsa staining and acetylcholinesterase activity, were all comparable
between control cells and those grown in PD 98059, suggesting that the
effect on endomitosis is not a nonspecific toxicity of PD 98059. However, whole marrow cultures also contain stromal cells that could
possibly produce cytokines affecting megakaryocyte development. The
inhibitor PD 98059 eliminated these stromal cells from the cultures.
Thus, it was possible that the effects of the inhibitor on
megakaryocytes were indirect. To address this issue, we purified
CD41+ marrow cells that were highly enriched in
megakaryocyte progenitors. The addition of PD 98059 to these cultures
again led to substantial blockade of megakaryocyte endomitosis.
However, unlike the whole bone marrow cells, culture of CD41-selected
cells in PD 98059 significantly reduced the number and size of
CD41+ cells present after 84 hours of culture. Although we
cannot fully explain the difference between the 2 culture systems, it
is possible that stromal cells in whole bone marrow can elaborate
factors to support megakaryocyte growth compensating for the MEK
inhibition. However, these factors in whole marrow could not compensate
for the inhibition of the endomitotic process, suggesting that MAPK is
critical for full polyploidization.
Polyploidy is widely believed to be essential for full megakaryocyte
development, especially for maximal platelet production. An in vivo
experiment has demonstrated that TPO is essential for full ploidy
development.34 We propose that this action of TPO is
mediated, at least in part, via the ERK-MAPK pathway. However, the
mechanisms of MAPK-mediated megakaryocyte endomitosis remain unclear.
MAPK has been shown to increase cyclin D1 expression in
fibroblasts.35 Moreover, overexpression of cyclin D1 in
megakaryocytic cell lines has been shown to enhance phorbol
ester-induced differentiation.36 However, recent studies
showed that cyclin D3 was prominently expressed in megakaryocytes and
necessary for megakaryocyte differentiation,37,38 because
antisense oligonucleotides to cyclin D3 inhibit megakaryocyte endomitosis in murine bone marrow culture system.37 In
addition, cyclin D3 overexpression in vivo by a transgenic mouse model
resulted in an increase in megakaryocyte number, size, and
ploidy.38 TPO has been shown to increase cyclin D3
expression in megakaryocytes in vivo.38 Whether this
increase is influenced by MAPK activation remains to be determined.
In conclusion, we found that the MAPK pathway is transiently activated
by TPO in normal murine megakaryocytes, reflecting the effects in an
Mpl bearing leukemic cell line. Although they have been shown to affect
the proliferation of Mpl-expressing cell lines or to stimulate various
aspects of cellular differentiation in other cell lines, we have found
that ERK1 and ERK2 play a very important role in megakaryocyte
development, supporting endomitosis. The target of MAPK responsible for
coupling to the cell cycle changes necessary for the uncoupling of DNA
synthesis and mitosis, the precise molecular pathways coupling the Mpl
receptor and MAPK activation, and the extent to which other members of
the MAPK family or other signaling pathways contribute to megakaryocyte differentiation remain active areas for further investigation.
| |
ACKNOWLEDGMENT |
The authors thank Norma Fox and Colleen O'Rork for technical
assistance; Chong Kim and Kathryn Allen for assistance with flow cytometry; and Catherine Carow and Yoshitaka Miyakawa for their thoughtful discussions. Furthermore, we thank Zymogenetics (BAF3/Mpl cells), Kirin Pharmaceuticals (purified TPO and SCF), and Naoki Yamamoto (SDF-1) for generously providing important reagents.
| |
FOOTNOTES |
Submitted September 29, 1998; accepted April 21, 1999.
P.R. and J.G.D. contributed equally to this work.
Supported by National Institutes of Health (NIH) Grant No. R01DK49855
(to K.K.); by Chulalongkorn University, Thailand (to P.R.); and by NIH
Grant No. K08HL03498 and an ASH Fellow Scholar Award (to J.G.D.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kenneth Kaushansky, MD, Division of
Hematology, University of Washington Medical Center, Seattle, WA 98195.
| |
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TOP
ABSTRACT
INTRODUCTION