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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2876-2883
Expression of a Functional N-Methyl-D-Aspartate-Type
Glutamate Receptor by Bone Marrow Megakaryocytes
By
Paul G. Genever,
David J.P. Wilkinson,
Amanda J. Patton,
Nicky M. Peet,
Ying Hong,
Anthony Mathur,
Jorge D. Erusalimsky, and
Tim M. Skerry
From the Department of Biology, University of York, York; and The
Wolfson Institute for Biomedical Research, University College London,
Cruciform Project Laboratories, Rayne Institute, London, UK.
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ABSTRACT |
Better understanding of hemostasis will be possible by the
identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that
belongs to a family of ionotropic glutamate receptors of the
N-methyl-D-aspartate (NMDA) subtype responsible for
synaptic neurotransmission in the central nervous system (CNS).
Northern blotting and reverse-transcriptase polymerase chain reaction
(RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type
subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal
megakaryoblastic cells. Immunohistochemistry and in vivo
autoradiographic binding of the NMDA receptor-specific antagonist
MK-801 confirmed that megakaryocytes expressed open channel-forming
NMDA receptors in vivo. Western blots indicated that megakaryocyte
NMDAR1 was either unglycosylated or only glycosylated to low levels,
and of identical size to CNS-type NMDAR1 after deglycosylation with
endoglycosidase F/peptide-N-glycosidase F. In functional
studies, we demonstrated that NMDA receptor activity was necessary for
phorbol myristate acetate (PMA)-induced differentiation of
megakaryoblastic cells; NMDA receptor blockade by specific antagonists
significantly inhibited PMA-mediated increases in cell size, CD41
expression, and adhesion of MEG-01 cells. These results provide
evidence for a novel pathway by which megakaryocytopoiesis and
platelet production may be regulated.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIESIS FOLLOWS a complex sequence
of events beginning with megakaryocyte differentiation from pluripotent
bone marrow stem cells and culminating with the release of platelets into the circulation from megakaryocyte cytoplasmic
fragments.1 The major regulator of this process is the
c-Mpl ligand thrombopoietin.2-4 In addition, a
variety of pleiotropic hematopoietic growth factors acting on different
cells of the megakaryocytic lineage promote the growth and maturation
of megakaryocytes.5 Despite these advances, the precise
cell-signaling events underlying the regulation of megakaryocytopoiesis
are incompletely understood.
In a recent study to investigate responses of bone cells to mechanical
load in vivo, we identified a glutamate/aspartate transporter (GLAST),
previously only detected in the central nervous system (CNS).6 GLAST performs an essential function during
glutamate-mediated synaptic neurotransmission by acting as a
high-affinity reuptake system to remove released glutamate from the
synaptic cleft, thus preventing overstimulation of postsynaptic
glutamate receptors. The presence of GLAST in bone led us to
hypothesize that glutamate signaling may also have a function outside
the CNS. To explore this hypothesis initially, we have investigated the
expression of glutamate receptor types in bone and bone marrow. One
cell type in bone marrow expressing
N-methyl-D-aspartate (NMDA)-type glutamate receptors
was the megakaryocyte, which suggests a role for glutamate signaling in
megakaryocyte maturation and platelet formation.
Glutamate is the predominant excitatory amino acid in the vertebrate
CNS, mediating synaptic neurotransmission through diverse ionotropic
and metabotropic receptors. Three subtypes of the ionotropic receptors
have been described:  -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and NMDA glutamate receptors, which are
classified according to their pharmacologic responses to these agonists.7 NMDA receptors have received particular
attention as principal participants in synaptic plasticity, which is
believed to underlie key neuronal events such as development, learning, and memory8-10; in vivo, they function as a heteropentamer
of NMDAR1 subunits with one of the four structurally related NMDAR2 subunits (A, B, C, or D).11 In addition, overstimulation of NMDA receptors, causing excessive Ca2+ influx, is thought
to promote neuronal degeneration and its clinical sequelae.12 The NMDA-type glutamate receptors we have
identified on megakaryocytes form open channels in vivo and have
functional activity in a human megakaryocyte-like cell line. These data
suggest that an auxiliary glutamate-mediated signaling pathway may be involved in the regulation of megakaryocytopoiesis.
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MATERIALS AND METHODS |
Cell culture.
Culture media and supplements were obtained from GIBCO-BRL (Paisley,
UK). MEG-01 cells were maintained in RPMI 1640 medium (as supplied
contains 140 µmol/L L-glutamate) supplemented with 100 U/mL
penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine, and 10% fetal calf serum at 37°C in 5% CO2/95% air
atmosphere. Differentiation was induced by culturing MEG-01 cells in
the presence of 10 7 mol/L phorbol myristate acetate
(PMA) for up to 3 days.13
Megakaryocytes were generated ex vivo from hematopoietic progenitors.
Briefly, CD34+ cells were isolated from umbilical cord
blood by a magnetic immunoselection technique (Milteny Biotec, Auburn,
CA) and cultured in Iscove's modified Dulbecco's medium supplemented
with minimum essential amino acids, 100 U/mL penicillin, 100 µg/mL
streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate,
2 mg/mL L-asparagine, 0.2% bovine serum albumin, 10% human
cord blood platelet-poor plasma, and 10 ng/mL thrombopoietin. After 14 days, megakaryocytic differentiation was assessed by flow cytometry as
previously described.14
Reverse-transcriptase polymerase chain reaction and Northern
blotting.
Total RNA was isolated from MEG-01 cells and bone marrow using Trizol
reagent (GIBCO-BRL). First-strand cDNA was synthesized using
Superscript RNase H reverse transcriptase (RT) (GIBCO-BRL). The
following polymerase chain reaction (PCR) primers were used: 5'-AATGACCCCAGGCTCAGAAAC-3' and
5'-TGAAGCCTCAAACTCCAGCAC-3', which amplify a product of 201 bp and correspond to position 2001 to 2222 of rat brain NMDAR1
(accession no. U08261), 5'-AGGTGTTCTATCAGCGTG-3' and
5'-TGTAGCTGTGCATGTCAG-3', which amplify a product of 619 bp and correspond to position 1570 to 2189 of rat brain NMDAR2D (accession no. L31612), and 5'-GATGTCTTCCAAGTATGCGGA-3' and
5'-GGGAATCTCCTTCTTGACCAG-3', which amplify a product of
667 bp and correspond to position 992 to 1658 of human NMDAR1
(accession no. L05666).
PCR was performed for 35 cycles of 94° for 30 seconds, 50°C for
30 seconds, and 72°C for 30 seconds. The PCR products were subcloned into pCR11 using the TA cloning kit (Invitrogen,
Groningen, The Netherlands) or into pGEM-T (Promega, Southampton,
UK), and sequenced using the ABI 377 DNA sequencer (Perkin
Elmer, Warrington, UK).
The cloned human NMDAR1 cDNA was used as a probe for Northern blotting
of RNA extracted from megakaryocytes derived from CD34+
progenitor cells using the technique described earlier. Northern blots
were performed using 2 µg total RNA extracted from cultures containing greater than 90% megakaryocytes.
Western blotting.
Brain homogenates and MEG-01 cells were lysed in 50 mmol/L Tris, 10%
glycerol, 1 mmol/L EDTA, 0.5 mmol/L dithiothreitol (DTT), 1% sodium
dodecyl sulfate (SDS), and separated by SDS-polyacrylamide gel
electrophoresis (PAGE). Western blotting was performed using a
monoclonal antibody against NMDAR1 (clone 54.1; Pharmingen, San Diego,
CA). This antibody identifies an extracellular peptide sequence that is
conserved among NMDAR1 splice variants. Deglycosylation was achieved by
preincubating samples with endoglycosidase
F/peptide-N-glycosidase F (Oxford Glycosystems, Abingdon, UK)
for 18 hours at 37°C.
Immunolocalization.
Adult rat ulnae and neonatal rat tibiae used for immunolocalization
studies were dissected out, dipped in 10% polyvinyl alcohol (PVA)
(Sigma, Poole, UK), and immediately frozen in chilled n-hexane ( 70°C). The tissues were mounted in 10% PVA on brass chucks
and 5- to 7-µm sections were cut using a Brights cryostat (Brights Instrument, Huntington, UK). Sections were collected on Vectorbonded slides (Vector Laboratories, Peterborough, UK) and stored at
35°C until use. Before immunolocalization, the sections were
fixed in 4% paraformaldehyde for 5 minutes and endogenous peroxidase activity depleted with 3% hydrogen peroxide (Sigma) for 30 minutes. A
further preincubation was performed with 10% normal horse serum (Vector Laboratories) for 30 minutes to block nonspecific antibody binding. The sections were incubated for 30 minutes with anti-NMDAR1 monoclonal antibody (Pharmingen; 1 µg/mL) followed by biotinylated horse anti-mouse secondary antibody (Vector Laboratories; 1:200 dilution) for 10 minutes and avidin-biotinylated-peroxidase reagent (ABC Elite, Vector Laboratories; 1:50 dilution) for 15 minutes. Peroxidase activity was visualized with 0.5 mg/mL 3, 3'-diaminobenzidine (Sigma) and 0.3% hydrogen peroxide as substrate.
All dilutions were made up in phosphate-buffered saline (PBS), pH 7.4, and incubations were performed at room temperature with three PBS
washes between each incubation. Negative controls received the same
concentrations of normal mouse IgGs (Vector Laboratories) in place of
primary antibody. Some sections were counterstained with hematoxylin
before mounting in glycerol/PBS. Similar immunolocalizations were
performed on blood and marrow smears from normal human subjects and on
cytospins of MEG-01 cells.
Acetylcholinesterase activity was used to identify rat megakaryocytes.
Unfixed sections were incubated in 0.1 mol/L acetate buffer, pH 6.0, containing 3 mmol/L copper sulfate, 5 mmol/L sodium citrate, 0.5 mmol/L
potassium ferricyanide, and 0.5 mg/mL acetylthiocholine iodide, for up
to 2 hours at room temperature in a humidified chamber. The slides were
then rinsed in buffer and fixed in 4% paraformaldehyde. For
colocalization with NMDAR1, acetylcholinesterase-stained sections were
blocked for 30 minutes with 10% goat serum (Vector Laboratories), then
incubated with the primary NMDAR1 antibody (Pharmingen; 1 µg/mL
overnight at 4°C). Antibody binding was detected by incubation with
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary
antibody (Sigma; 1:100 dilution) for 45 minutes at room temperature and
the mounted slides were viewed under UV light illumination.
For immunofluorescent localization of glutamate transporters on MEG-01
cells, cytospins were fixed in 4% paraformaldehyde for 5 minutes,
blocked with 10% goat serum for 30 minutes, and rabbit polyclonal
antibodies against GLAST, GLT-1, and EAAC1 (provided by Dr Jeffrey
Rothstein, Johns Hopkins University, Baltimore, MD) were applied to
sections (0.1 to 0.5 µg/mL, overnight, 4°C). Antibody binding was
identified using FITC-conjugated goat anti-rabbit secondary antibody
(Sigma; 1:50 dilution) for 45 minutes, mounted, and viewed under UV illumination.
Radioligand binding.
Male rats (50 to 70 g) were injected intracardially with 15 µg/kg (in
0.9% saline) of tritiated MK-801 ([3-3H]-MK-801; 23.9 Ci/mmol; Dupont NEN, Stevenage, UK), a specific noncompetitive NMDA
receptor antagonist. After 15 minutes, the animals were killed and
flushed with saline by intracardiac perfusion until the tissues were
blanched, to remove unbound ligand. To control for nonspecific
labeling, three rats were injected intracardially with a 60-fold excess
of cold MK-801 (1 mg/kg dizocilipine maleate; Research Biochemicals
International, Natick, MA) 15 minutes before administration of radiolabeled MK-801. Tissues were then rapidly removed, frozen, and sections cut as described previously.
Sections were dehydrated through graded alcohols, dipped in EM-1
emulsion (Amersham International, Little Chalfont, UK), and cooled on
an ice tray for 30 minutes. They were then air-dried in desiccated
light-tight boxes at room temperature for 4 hours, and finally exposed
at 4°C for up to 24 weeks. Some 3H-MK-801-labeled
tissue sections were subjected to the NMDAR1 immunoperoxidase
localization protocol described earlier, before dipping in emulsion.
Flow cytometry.
CD41 (GPIIb/IIIa) expression on MEG-01 cells was determined following
PMA treatment in the presence or absence of 100 µmol/L MK-801, using
flow cytometry and anti-human CD41 monoclonal antibody (clone 5B12;
Dako, High Wycombe, UK), which identifies glycoproteins IIb and IIIa.
MEG-01 cells (1 to 5 × 106 cells) were centrifuged,
washed in cold phenotyping buffer (PB; calcium and magnesium-containing
PBS with 0.1% bovine serum albumin), and blocked with PB containing 1 µg/mL normal rabbit IgGs for 10 minutes. Following centrifugation,
the cells were incubated with anti-human CD41 (1:100 dilution) in 100 µL PB for 30 minutes on ice. Following washing with PB, 50 µL goat
anti-mouse FITC-conjugated secondary antibody (Sigma; 1:100 dilution)
was added and the cells were incubated for a further 30 minutes on ice.
After a final wash with PB, the cells were resuspended in 1 mL PB for
analysis. FITC control samples were treated identically except
that they received the same concentration of normal mouse IgGs in place of primary antibody. Live cells were analyzed with a
Coulter XL flow cytometer (Coulter, Miami, FL) using
light-scatter parameters. The percentage of CD41+ cells was
evaluated according to an FITC gate set at 1% on samples stained with
the control antibody.
Similar experiments were repeated using an antibody that recognizes a
different epitope on the GPIIb/IIIa complex. FITC-conjugated monoclonal
antibody Y2/51 (Dako), which detects the megakaryocytic lineage-specific antigen CD61 (glycoprotein IIIa), was used as previously described.14 Antigen expression was determined
using a FACScan instrument and Lysis II software (Becton Dickinson, Mountain View, CA). Light-scatter parameters were used to exclude dead
cells. Relative antigen expression was estimated on live cells using
the median fluorescence intensity. The fluorescence of cells stained
with an FITC-conjugated isotype-matched control antibody was subtracted
from all determinations.
Adhesion assays.
Adhesion assays were performed as described by De Luca et
al15 with minor modifications. Briefly, MEG-01 and CMK
cells (2 × 105 cells/mL) were cultured in 96-well
plates with and without PMA (107 mol/L) for 72 hours
in the presence of varying concentrations (0 to 100 µmol/L) of MK-801
or the competitive glutamate receptor antagonist D-AP5
(D(-)-2-amino-5-phosphonopentanoic acid; Research Biochemicals International). Medium was then removed and the wells were
rinsed three times with PBS to remove nonadherent cells. Adherent cells
were fixed in 70% ethanol (15 minutes) and stained with 0.5% crystal
violet (25 minutes), followed by extensive washing with water to remove
unbound dye. Dye was eluted by the addition of 50% ethanol/0.1 mol/L
sodium citrate, pH 4.2. Absorbances were measured on a plate reader
(Dynatech MR5000; Dynatech, Billingshurst, UK) at 570 nm.
The methylthiotetrazole (MTT) assay was used to determine cell
viability.16 Briefly, parallel cultures were grown as
described earlier in phenol red-free RPMI 1640 growth medium. After 72 hours, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide; Thiazol blue; Sigma) solution was added directly to the wells to give a final concentration of 1 mg/mL and the plates were incubated for a further 3 hours. The cells were lysed with 0.05N HCl containing 10% SDS and absorbances read at 570 nm.
Statistical analysis.
Where indicated, significant differences were determined using
Student's t-test, as described in the text.
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RESULTS |
Expression of NMDA receptor subunits in ex vivo-generated
megakaryocytes and megakaryoblastic cells.
PCR of cDNA from rat marrow amplified single products for the NMDA
receptor subunits NMDAR1 and NMDAR2D of predicted size (Fig
1A and B). In agreement with these results,
amplification of NMDAR1 and NMDAR2D using cDNA from the human
megakaryoblastic cell line, MEG-01, identified single products of 667 bp and 619 bp, respectively (Fig 1C). RT-PCR did not identify
expression of NMDAR2A, B, or C in rat marrow or MEG-01 cells. All of
the PCR products showed 100% sequence homology with corresponding CNS-type NMDA subunits. NMDA receptor mRNA expression was confirmed on
Northern blots of total RNA derived from primary human megakaryocytes. As shown in Fig 1D, the NMDAR1 probe hybridized to an mRNA species of
approximately 4.4 kb, which matched the size of a previously identified
NMDAR1 transcript.17 Western blots of cell lysates from
MEG-01 cells showed the presence of major protein bands of approximately 100 kD corresponding to the size of deglycosylated CNS-type NMDAR1 (Fig 1E). Since NMDAR2D is the least characterized of
CNS NMDA-type subunits, and no anti-NMDAR2D antibodies yet exist
commercially, our immediate studies concentrated on investigating the
localization and expression of NMDAR1 in megakaryocytes.
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| Fig 1.
Expression of NMDA receptor subunits by megakaryocytes.
RT-PCR amplified products for the NMDAR1 (A) and NMDAR2D (B) receptor
subunit from rat bone marrow; NMDAR2A-C were identified in brain only
(B). (C) RT-PCR confirmed expression of NMDAR1 (NR1) and NMDAR2D (NR2D)
subunits by MEG-01 cells. (D) Northern blot analysis of total RNA (2 µg) from primary human megakaryocytes using an NMDAR1 probe, which
identified a 4.4-kb mRNA species. (E) Western blot analysis of NMDAR1
protein in rat cerebellum, MEG-01 cells (cultured in the absence or
presence of 107 mol/L PMA for 3 days) and
N-deglycosylated rat cerebellum. CNS-type NMDAR1 was of expected
size,17 116 kD, which was reduced to ~100 kD following
N-deglycosylation. Major MEG-01 NMDAR1 protein bands were detected at
~100 kD, matching N-deglycosylated CNS-type NMDAR1 size.
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Megakaryocytes express NMDAR1 protein in vivo.
Specific NMDAR1 immunoreactivity was observed on megakaryocytes in
sections of neonatal and adult rat bone (Fig
2A) and spleen (data not shown). Positive
staining was also detected on megakaryocytes in smears of mouse and
human marrow on human platelets and on cytospins of MEG-01 cells (Fig
2C through E). Colocalization studies showed that NMDAR1 staining was
present only on acetylcholinesterase-positive cells, and that all
acetylcholinesterase-positive megakaryocytes displayed NMDAR1 staining
regardless of stage of maturation (Fig 2F and G). Intense
immunostaining for the glutamate transporter GLAST was seen on MEG-01
cells (Fig 2H), although expression of the related transporters, GLT-1
and EAAC1, was not observed on these cells.
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| Fig 2.
Immunolocalization of NMDAR1 on megakaryocytes.
(A) Cryosection of rat bone indicating intense NMDAR1 immunoreactivity
(brown reaction product) on bone marrow megakaryocytes (arrows). (B)
Antibody control, hematoxylin counterstain. Positive immunostaining was
also identified on human megakaryocytes (C, arrow), human platelets (D,
arrows), and MEG-01 cells (E). (F and G) Colocalization studies showed
that cells displaying NMDAR1 immunofluorescence in rat bone marrow (F,
arrow) were acetylcholinesterase-positive (G, arrow). (H) MEG-01 cells
demonstrated strong immunostaining for the glutamate transporter,
GLAST. (I) MEG-01 antibody control. Original magnification: A and B,
×250; C, D, and E, ×1,000; F and G, ×500; H and I,
×200.
Fig 3. Binding of the specific NMDA channel
blocker, MK-801 to megakaryocytes in vivo. Autoradiography studies
localized 3H-MK-801, identified as numerous black silver
particles (A, arrows) to NMDAR1-positive (brown staining)
megakaryocytes in rat ulna cryosections. Ligand binding was displaced
by excess cold MK-801 (B). Original magnification × 1,000.
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Radioligand binding demonstrates open NMDA receptor channels.
MK-801 binds specifically to channel-forming NMDA receptors in the CNS.
As shown in Fig 3A, when animals were
injected with [3H]-MK-801, and binding was examined in
bone marrow sections, prominent radioactivity was detected on bone
marrow megakaryocytes, which colocalized with NMDAR1 antibody staining.
The number of developed silver grains present on megakaryocytes was
markedly reduced when the animals were pretreated with an excess of
cold MK-801 (Fig 3B).
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Fig 3.
Functional studies.
MEG-01 cells undergo further differentiation when treated with
PMA.13 As shown earlier, these cells were found to express NMDA receptor subunits by RT-PCR (Fig 1C) and Western blotting (Fig 1E)
and immunostaining (Fig 2E). Having demonstrated that megakaryocytes
bind MK-801 in vivo, a specific noncompetitive antagonist of the
CNS-type NMDA receptor, we investigated the effect of this in MEG-01 cells.
Flow cytometric analyses showed that 100 µmol/L MK-801 inhibited
PMA-mediated increases in cell size and CD41 (GPIIb/IIIa) expression.
Thus, in the presence of MK-801, mean forward scatter was significantly
reduced from 480 to 326 nm (P < .05), and CD41+
events from 34% to 20% (P < .005) (Fig
4 and Table
1). To confirm these observations
further, experiments were repeated using an alternative antibody
recognizing a different epitope on GPIIIa. These studies again
demonstrated that MK-801 caused a 30% to 55% inhibition of GPIIIa
expression in PMA-stimulated cultures of MEG-01 cells (Fig
5).
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| Fig 4.
Effect of the NMDA channel blocker, MK-801, on CD41
(GPIIb/IIIa) expression by MEG-01 cells. Charts show flow cytometric
analyses of CD41 expression by (A) untreated MEG-01 cells, (B) MEG-01
cells treated with PMA (107 mol/L), and (C) MEG-01 cells
treated with PMA (107 mol/L) and MK-801
(104 mol/L) for 3 days. Representative contour plots are
shown indicating forward-scatter (FS, cell size) against CD41-FITC
fluorescence (FL1). Events lying within the F gate indicate CD41
positivity. PMA-stimulated increases in cell size and CD41 expression
(compare A and B) are reduced in the presence of MK-801 (C). See Table
1.
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Table 1.
Effect of MK-801 (100 µmol/L) on PMA-Stimulated
Increases in Size and CD41 (GPIIb/IIIa) Expression of MEG-01 Cells
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| Fig 5.
Effect of MK-801 on GPIIIa expression in MEG-01 cells.
MEG-01 cells were incubated in the presence of the indicated compounds
for 3 days. Results expressed as percentage of the increase in the
median fluorescence intensity observed in the presence of PMA alone.
Addition of the PKC inhibitor GF109203X (3 µmol/L) was used as a
positive control and fluorescence intensity of samples not treated with
PMA was subtracted from values obtained in the presence of PMA. Results
indicated that 50 to 150 µmol/L MK-801 caused a 30% to 55%
inhibition of PMA-induced increase in GPIIIa expression.
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In addition, we investigated the effect of NMDA receptor blockade on
MEG-01 cell adhesion. It was found that PMA markedly increased MEG-01
adhesion, and that this effect was significantly inhibited (P < .005) by MK-801 in a dose-dependent manner (Fig 6A). In addition, another NMDA receptor
antagonist, D-AP5, which competitively blocks glutamate binding to
receptors, also caused a significant (P < .01) inhibition of
PMA-mediated adhesion (Fig 6B). MK-801 and D-AP5 did not affect cell
numbers or viability in MTT assays of parallel cultures (Fig 6C and D).
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| Fig 6.
Effect of NMDA receptor antagonists on MEG-01 adhesion.
MEG-01 cells were cultured in the absence ( ) or presence ( ) of
PMA (107 mol/L). (A) The noncompetitive antagonist,
MK-801, and (B) the competitive antagonist, D-AP5, caused significant
dose-dependent inhibition of PMA-stimulated adhesion. MTT assays of
parallel cultures indicated that MK-801 (C), and D-AP5 (D), had no
effect on cell numbers or viability. Values are means ± SEM (n = 4). *P < .05, **P < .01, ***P < .005 v controls (t-test).
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To rule out the possibility that the inhibition of PMA-induced adhesion
was a characteristic of this particular cell line, the effect of MK-801
was also investigated in another megakaryocytic cell line. Figure
7 shows that treatment of CMK cells with 1 to 100 µmol/L MK-801 caused a significant (P < .005)
dose-dependent inhibition of PMA-mediated cell attachment.
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| Fig 7.
Effect of the NMDA receptor antagonist, MK-801 on CMK
adhesion. Treatment of CMK cells with various concentrations of MK-801
in the absence () or presence () of PMA (107
mol/L) caused a significant dose-dependent inhibition of PMA-induced
adhesion. Values are means ± SEM (n = 4). *P < .05, ***P = .001 v controls (t-test).
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DISCUSSION |
Identification of additional regulatory signals governing megakaryocyte
maturation and platelet release is fundamental to our understanding of
thrombopoiesis. Megakaryocytic expression of an NMDA-type glutamate
receptor provides evidence for an additional level of control in this
regulatory hierarchy. Although NMDA receptors are concentrated at CNS
synaptic junctions, expression outside the CNS is not without
precedent. Similar functional receptors have been described in guinea
pig ileum, rat adrenal gland, islet cells of rat
pancreas,18-24 and bone cells25, indicating
that glutamate receptors may have a more widespread distribution,
including some nonneuronal peripheral tissues. Megakaryocytes are not
classical excitable cells, yet they interact directly with several
peripheral neurotransmitters. They express receptors, and display
storage and transporter mechanisms for serotonin
(5-HT)26-30 mimicking central serotonergic
neurons,31 and megakaryocytes interact with dopamine in a
similar manner.32,33 Receptors for vasoactive intestinal
peptide have been demonstrated on megakarocytes and platelets34 and the same cells express neuropeptide
Y.35 In addition, megakaryocytes synthesize
acetylcholinesterase,36,37 which may play a role in
megakaryocytopoiesis,38 although classically it is
responsible for terminating cholinergic neurotransmission at central
and peripheral synapses, by hydrolyzing acetylcholine. It is
noteworthy that the codistribution of acetylcholinesterase with glutamate receptors at noncholinergic synaptic junctions enhances glutamate-mediated signaling,39,40 possibly
through direct interaction with NMDA receptors,41 and such
synergy may also operate in megakaryocytes. Similarly, activation of
serotonin 5-HT2 receptors, which can stimulate growth and colony
formation in megakaryocytes,30 also enhances ion currents
through isolated NMDA receptor channels.42
Our findings may help to reinterpret the observation that platelets
express a high-affinity binding site for the NMDA receptor antagonist
phencyclidine.43,44 Results from those studies suggested that this binding site was not related to CNS NMDA receptors. In
contrast, in this work we have provided compelling evidence for
functional megakaryocyte NMDA receptors. Furthermore, we have observed
immunoreactivity for NMDAR1 on platelets.
The NMDA receptors expressed by megakaryocytes are open channel-forming
structures, as evidenced by their binding of MK-801, which cannot bind
to closed channels, confirming in vivo functionality. It is likely that
these receptor complexes are composed predominantly of NMDAR1-type
subunits, although our finding that NMDAR2D-type subunit transcripts
are also expressed suggests that native megakaryocyte NMDA receptors
form as heteromeric assemblies of NMDAR1/NMDAR2D subunits, consistent
with current theories regarding NMDA receptor assembly in central
neurones.11 An interesting observation is that the level of
glycosylation in megakaryocyte NMDA receptors is reduced or absent
compared with CNS-type, although the functional significance of this is
not entirely clear. It may be that the enhanced stability that
carbohydrate moieties would provide to NMDA receptors in the synaptic
membrane, where precise orientation and localization is essential, is
deemed unnecessary in megakaryocytes, which display evenly distributed
receptors across their cell surfaces, susceptible to multidirectional
agonist stimulation.
The finding that NMDA receptors are present on bone marrow
megakaryocytes is highly suggestive that glutamate-like signaling mechanisms are important for these cells. This view is reinforced by
our recent finding that in vivo, megakaryocytes exist in intimate contact with cells bearing the glutamate transporter proteins GLT-1 and
GLAST, which are found on mononuclear bone marrow cells and
osteoblasts, respectively.6 These transporters are
essential requirements for functional glutamate-mediated communication; at CNS synaptic junctions, they initiate the reuptake of released glutamate into nearby cells, terminating
neurotransmission.45 Furthermore, we have demonstrated
expression of NMDA-type glutamate receptors by osteoblasts and
osteoclasts,25 pointing to the existence of a multicellular
glutamate signaling pathway in bone.
Megakaryocyte-like MEG-01 cells also express NMDA receptors and GLAST,
suggesting that autocrine glutamate signaling pathways operate in these
cell cultures. In vitro studies with MEG-01 cells indicated that
functional NMDA receptors are necessary to allow these cells to
progress through PMA-induced differentiation. The differentiation-promoting effects of PMA are believed to be achieved through activation of protein kinase C (PKC).13,46,47 There is substantial evidence that NMDA receptor activity is regulated by
PKC,48-52 and that NMDA ion currents are positively
modulated by phorbol esters.40,51,53,54 It is possible
therefore, that PMA-induced PKC activation may stimulate NMDA receptor
activity in MEG-01 cells to promote their differentiation. This is
consistent with our finding that PMA-stimulated increases in cell size
and CD41 expression by MEG-01 cells were significantly reduced by blockade of NMDA receptors with the specific antagonist, MK-801 (Figs 4
and 5). Increased adhesiveness is an additional feature of PMA-induced
differentiation of megakaryoblastic cell lines and primary
megakaryocytes.55-60 The striking effect that MK-801 and
D-AP5 had on inhibiting PMA-stimulated MEG-01 adhesion suggests that
one of the primary actions of megakaryocyte NMDA receptors may lie in
regulating cell-cell and cell-matrix adhesion, both critical elements
of megakaryocyte and platelet function. As  1-integrins are likely
mediators of PMA-induced adhesion of erythroleukemia cell
lines,56 the relationship between megakaryocyte NMDA
receptor activation and the expression of these and other adhesion
molecules is currently being investigated.
This work demonstrates that megakaryocytes express ionotropic receptors
that have distinct similarities to NMDA-type glutamate receptors of the
CNS, although detailed pharmacokinetic studies have not yet been
performed. The strong implication that these receptors are involved in
megakaryocyte adhesion and differentiation stimulates further research
to determine the precise physiologic role of megakaryocyte NMDA receptors.
| |
ACKNOWLEDGMENT |
The authors thank Dr Jeffrey Rothstein (Johns Hopkins University,
Baltimore, MD) for kindly providing the antibodies to GLAST, GLT-1, and
EAAC1. We also thank Nigel Raven and Pamela Wiseman (York District
Hospital) for provision of human marrow smears.
| |
FOOTNOTES |
Submitted May 26, 1998; accepted December 17, 1998.
Supported by the Arthritis Research Campaign, Wellcome Trust, Medical
Research Council, and Smith and Nephew.
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 Paul G. Genever, PhD, Department of
Biology, PO Box 373, University of York, York YO10 5YW, UK; e-mail:
pg5{at}york.ac.uk.
| |
REFERENCES |
1.
Nurden P, Poujol C, Nurden AT:
The evolution of megakaryocytes to platelets.
Baillieres Clin Haematol
10:1, 1997[Medline]
[Order article via Infotrieve]
2.
de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang W-J, Oles KJ, Hultgren B, Solberg Jr LA, Goeddel DV, Eaton DL:
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.
Nature
369:533, 1994[Medline]
[Order article via Infotrieve]
3.
Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM, Bell LA, Spreacher CA, Blumberg H, Johnson R, Prunkard D, Ching AFT, Mathewes SL, Bailey MC, Forstrom JW, Buddle MM, Osborn SG, Evans SJ, Sheppard PO, Presnell SR, O'Hara PJ, Hagen FS, Roth GJ, Foster DC:
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369:565, 1994[Medline]
[Order article via Infotrieve]
4.
Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FS, Roth GJ, Papayannopoulou T, Foster DC:
Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.
Nature
369:568, 1994[Medline]
[Order article via Infotrieve]
5.
Wendling F, Han Z-C:
Positive and negative regulation of megakaryocytopoiesis.
Baillieres Clin Haematol
10:29, 1997[Medline]
[Order article via Infotrieve]
6.
Mason DJ, Suva LJ, Genever PG, Patton AJ, Steuckle S, Hillam RA, Skerry TM:
Mechanically regulated expression of a neural glutamate transporter in bone: A role for excitatory amino acids as osteotropic agents?
Bone
20:199, 1997[Medline]
[Order article via Infotrieve]
7.
Hollman M, Heinemann S:
Cloned glutamate receptors.
Ann Rev Neurosci
17:31, 1994[Medline]
[Order article via Infotrieve]
8.
Collingridge GL, Singer W:
Excitatory amino acid receptors and synaptic plasticity.
Trends Pharmacol Sci
11:290, 1990[Medline]
[Order article via Infotrieve]
9.
Bliss TV, Collingridge GL:
A synaptic model of memory: Long-term potentiation in the hippocampus.
Nature
361:31, 1993[Medline]
[Order article via Infotrieve]
10.
Collingridge GL, Bliss TV:
Memories of NMDA receptors and LTP.
Trends Neurosci
18:54, 1995[Medline]
[Order article via Infotrieve]
11.
Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, Seeburg PH:
Heteromeric NMDA receptors: Molecular and functional distinction of subtypes.
Science
256:1217, 1992[Abstract/Free Full Text]
12.
Choi DW:
Glutamate neurotoxicity and diseases of the nervous system.
Neuron
1:623, 1988[Medline]
[Order article via Infotrieve]
13.
Ogura M, Morishima Y, Okumura M, Hotta T, Takamoto S, Ohno R, Hirabayashi N, Nagura H, Saito H:
Functional and morphological differentiation induction of a megakaryoblastic leukemia cell line (Meg-01s) by phorbol esters.
Blood
72:49, 1988[Abstract/Free Full Text]
14.
Hong Y, Martin JF, Vainchenker W, Erusalimsky JD:
Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells.
Blood
87:123, 1996[Abstract/Free Full Text]
15.
De Luca M, Tamura RN, Kajiji S, Bondanza S, Rossino P, Cancedda R, Marchisio PC, Quaranta V:
Polarized integrin mediates human keratinocyte adhesion to basal lamina.
Proc Natl Acad Sci USA
87:6888, 1990[Abstract/Free Full Text]
16.
Mossman T:
Rapid colorimetric assay for cellular growth and application to proliferation and cytotoxicity assays.
J Immunol Methods
65:55, 1983[Medline]
[Order article via Infotrieve]
17.
Moriyoshi K, Masayuki M, Ishii T, Shigemoto R, Mizuno N, Nakanishi S:
Molecular cloning and characterisation of the NMDA receptor.
Nature
354:31, 1991[Medline]
[Order article via Infotrieve]
18.
Shannon HE, Sawyer BD:
Glutamate receptors of the N-methyl-D-aspartate subtype in the myenteric plexus of the guinea pig ileum.
J Pharmacol Exp Ther
251:518, 1989[Abstract/Free Full Text]
19.
Yoneda Y, Ogita K:
Enhancement of [3H]glutamate binding by N-methyl-D-aspartic acid in rat adrenal.
Brain Res
406:24, 1987[Medline]
[Order article via Infotrieve]
20.
Inagaki N, Kuromi H, Gonoi T, Okamoto Y, Ishida H, Seino Y, Kaneko T, Iwanaga T, Seino S:
Expression and role of ionotropic glutamate receptors in pancreatic islet cells.
FASEB J
9:686, 1995[Abstract]
21.
Bertrand G, Gross R, Puech R, Loubatieres-Mariani MM, Bockaert J:
Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas.
Br J Pharmacol
106:354, 1992[Medline]
[Order article via Infotrieve]
22.
Gonoi T, Mizuno N, Inagaki N, Kuromi H, Seino Y, Miyazaki J, Seino S:
Functional neuronal ionotropic glutamate receptors are expressed in the non-neuronal cell line MIN6.
J Biol Chem
269:16989, 1994[Abstract/Free Full Text]
23.
Weaver CD, Yao TL, Powers AC, Verdoorn TA:
Differential expression of glutamate receptor subtypes in rat pancreatic islets.
J Biol Chem
271:12977, 1996[Abstract/Free Full Text]
24.
Molnar E, Varadi A, McIlhinney RAJ, Ashcroft SJH:
Identification of functional ionotropic glutamate receptor proteins in pancreatic -cells and in islets of Langerhans.
FEBS Lett
371:253, 1995[Medline]
[Order article via Infotrieve]
25.
Patton AJ, Genever PG, Birch MA, Suva LJ, Skerry TM:
Expression of an N-Methyl-D-Aspartate-type receptor by human and rat osteoblasts and osteoclasts suggests a novel glutamate signaling pathway in bone.
Bone
6:645, 1998
26.
White JG:
Serotonin storage organelles in human megakaryocytes.
Am J Pathol
63:403, 1971[Medline]
[Order article via Infotrieve]
27.
Tranzer JP, da Prada M, Pletscher A:
Storage of 5-hydroxytryptamine in megakaryocytes.
J Cell Biol
52:191, 1972[Free Full Text]
28.
Fedorko ME:
The functional capacity of guinea pig megakaryocytes. I. Uptake of 3H-serotonin by megakaryocytes and their physiologic and morphologic response to stimuli for the platelet release reaction.
Lab Invest
36:310, 1977[Medline]
[Order article via Infotrieve]
29.
Rudnick G, Fishkes H, Nelson PJ, Schuldiner S:
Evidence for two distinct serotonin transport systems in platelets.
J Biol Chem
255:3638, 1980[Abstract/Free Full Text]
30.
Yang M, Srikiatkhachorn A, Anthony M, Chong BH:
Serotonin stimulates megakaryocytopoiesis via the 5-HT2 receptor.
Blood Coagul Fibrinolysis
7:127, 1996[Medline]
[Order article via Infotrieve]
31.
Pletscher A, Laubscher A, Graf M, Saner A:
Blood platelets as models for central 5-hydroxytryptaminergic neurons.
Ann Biol Clin Paris
37:35, 1979[Medline]
[Order article via Infotrieve]
32.
Daimon T, Kawai K, Uchida K:
Location of sites of dopamine storage in megakaryocytes by autoradiography.
Histochemistry
93:263, 1990[Medline]
[Order article via Infotrieve]
33.
Dean B, McAdam AJ, Sundram S, Pavey G, Harrison LC, Copolov DL:
Identification of a dopamine-binding protein on the membrane of the human platelet.
Biochem J
287:45, 1992
34.
Park SK, Olson TA, Ercal N, Summers M, O'Dorisio MS:
Characterisation of vasoactive intestinal peptide receptors on human megakaryocytes and platelets.
Blood
87:4629, 1996[Abstract/Free Full Text]
35.
Ericsson A, Hemsen A, Lundberg JM, Persson H:
Detection of neuropeptide Y-like immunoreactivity and messenger RNA in rat platelets: The effects of vinblastine, reserpine and dexamethasone on NPY expression in blood cells.
Exp Cell Res
192:604, 1991[Medline]
[Order article via Infotrieve]
36.
Paulus JM, Maigne J, Keyhani E:
Mouse megakaryocytes secrete acetylcholinesterase.
Blood
58:1100, 1981[Abstract/Free Full Text]
37.
Lev-Lehman E, Deutsch V, Eldor A, Soreq H:
Immature human megakaryocytes produce nuclear-associated acetylcholinesterase.
Blood
89:3644, 1997[Abstract/Free Full Text]
38.
Soreq H, Patinkin D, Lev-Lehman E, Grifman M, Ginzberg D, Eckstein F, Zakut H:
Antisense oligonucleotide inhibition of acetylcholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosis ex vivo.
Proc Natl Acad Sci USA
91:7907, 1994[Abstract/Free Full Text]
39.
Appleyard ME, Vercher J-L, Greenfield SA:
Release of acetylcholinesterase from the guinea pig cerebellum in vivo.
Neuroscience
25:133, 1988[Medline]
[Order article via Infotrieve]
40.
Appleyard ME, Jahnsen H:
Actions of acetylcholinesterase in the guinea pig cerebellar cortex in vitro.
Neuroscience
47:291, 1992[Medline]
[Order article via Infotrieve]
41.
Webb CP, Nedergaard S, Giles K, Greenfield SA:
Involvement of the NMDA receptor in the non-cholinergic action of acetylcholinesterase in Guinea-pig substantia nigra pars compacta neurons.
Eur J Neurosci
8:837, 1996[Medline]
[Order article via Infotrieve]
42.
Blank T, Zwart R, Nijholt I, Spiess J:
Serotonin 5-HT2 receptor activation potentiates N-methyl-D-aspartate receptor-mediated ion currents by a protein kinase C-dependent mechanism.
J Neurosci Res
45:153, 1996[Medline]
[Order article via Infotrieve]
43.
Jamieson GA, Agrawal AK, Greco NJ, Tenner TE, Jones GD, Rice KC, Jacobson AE, White JG, Tandon NN:
Phencyclidine binds to platelets with high affinity and specifically inhibits their activation by adrenaline.
Biochem J
285:35, 1992
44.
Raulli RE, Jackson B, Tandon N, Mattson M, Rice K, Jamieson GA:
Phencyclidine inhibits epinephrine-stimulated platelet aggregation independently of high affinity N-methyl-D-aspartate (NMDA)-type glutamate receptors.
Biochem Biophys Acta
1224:175, 1994[Medline]
[Order article via Infotrieve]
45.
Kanner BI:
Glutamate transporters from brain.
FEBS Lett
1:95, 1993
46.
Zauli G, Bassini A, Catani L, Gibellini D, Celeghini C, Borgatti P, Caramelli E, Guidotti L, Capitani S:
PMA-stimulated megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level.
Br J Haematol
92:530, 1996[Medline]
[Order article via Infotrieve]
47.
Ballen KK, Ritchie AJ, Murphy C, Handin RI, Ewenstein BM:
Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line.
Exp Hematol
24:1501, 1996[Medline]
[Order article via Infotrieve]
48.
Chen L, Huang L-YM:
Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation.
Nature
356:521, 1992[Medline]
[Order article via Infotrieve]
49.
Ben-Ari Y, Aniksztejn L, Bregestovski P:
Protein kinase C modulation of NMDA currents: An important link for LTP induction.
Trends Neurosci
15:333, 1992[Medline]
[Order article via Infotrieve]
50.
Tingley WG, Roche KW, Thompson AK, Huganir RL:
Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain.
Nature
364:70, 1993[Medline]
[Order article via Infotrieve]
51.
Koga T, Sakai N, Tanaka C, Saito N:
Presynaptic and Ca(2+)-independent PKC subspecies modulates NMDAR1 current.
Neuroreport
7:477, 1996[Medline]
[Order article via Infotrieve]
52.
Leonard AS, Hell JW:
Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites.
J Biol Chem
272:12107, 1997[Abstract/Free Full Text]
53.
Gerber G, Kangrga I, Ryu PD, Larew JS, Randic M:
Multiple effects of phorbol esters in the rat spinal dorsal horn.
J Neurosci
9:3606, 1989[Abstract]
54.
Yamazaki M, Mori H, Araki K, Mori KJ, Mishina M:
Cloning, expression and modulation of a mouse NMDA receptor subunit.
FEBS Lett
300:39, 1992[Medline]
[Order article via Infotrieve]
55.
Jarvinen M, Ylanne J, Vartio T, Virtanen I:
Tumor promoter and fibronectin induce actin stress fibres and focal adhesion sites in spreading human erythroleukemia (HEL) cells.
Eur J Cell Biol
44:238, 1987[Medline]
[Order article via Infotrieve]
56.
Molla A, Berthier R, Chapel A, Schweitzer A, Andrieux A:
Beta 1 integrins mediate adherent phenotype of human erythroblastic cell lines after phorbol 12-myristate 13-acetate induction.
Biochem J
309:491, 1995
57.
Leven RM:
Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading.
J Cell Physiol
163:597, 1995[Medline]
[Order article via Infotrieve]
58.
Murate T, Saga S, Hamaguchi M, Asano H, Ito T, Watanabe T, Adachi K, Koizumi KT, Yoshida S, Saito H, Hotta T:
Herbimicin A inhibits phorbol ester-induced morphological changes, adhesion, and megakaryocytis differentiation of leukemia cell line, MEG-01.
Proc Soc Exp Biol Med
209:270, 1995[Medline]
[Order article via Infotrieve]
59.
Steiner M, Li W, Ciaramella JM, Anagnostou A, Sigounas G:
DL-alpha-tocopherol, a potent inhibitor of phorbol ester induced shape change of erythro- and megakaryoblastic leukemia cells.
J Cell Physiol
172:351, 1997[Medline]
[Order article via Infotrieve]
60.
Herrera R, Hubbell S, Decker S, Petruzzelli L:
A role for the MEK/MAPK pathway in PMA-induced cell cycle arrest: Modulation of megakaryocytic differentiation of K562 cells.
Exp Cell Res
238:407, 1998[Medline]
[Order article via Infotrieve]
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ABSTRACT
INTRODUCTION