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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3847-3855
Collagen Mediates Changes in Intracellular Calcium in Primary Mouse
Megakaryocytes Through syk-Dependent and -Independent Pathways
By
Stephen J. Briddon,
Steven K. Melford,
Martin Turner,
Victor Tybulewicz, and
Steve P. Watson
From the Department of Pharmacology, University of Oxford, Oxford,
UK; and the National Institute for Medical Research, London, UK.
 |
ABSTRACT |
We have characterized changes in [Ca2+]i
in primary mouse megakaryocytes in response to fibrillar collagen and
in response to cross-linking of the collagen receptor, the integrin
 2 1. The response to collagen was markedly
different from that seen to a triple helical collagen-related peptide
(CRP), which signals via the tyrosine kinases p59fyn and
p72syk. This peptide binds to the collagen receptor
glycoprotein VI (GPVI), but not to the integrin
21. Collagen elicited a sustained increase in [Ca2+]i composed primarily of
influx of extracellular Ca2+ with some Ca2+
release from internal stores. In contrast to CRP, this response was
only partially (~30%) inhibited by the src-family kinase inhibitor PP1 (10 µmol/L) or by microinjection of the tandem SH2 domains of
p72syk. Collagen also caused an increase in
[Ca2+]i in megakaryocytes deficient in
either p59fyn or p72syk, although the response
was reduced by approximately 40% in both cases: Cross-linking of the
2 integrin increased [Ca2+]i
in these cells exclusively via Ca2+ influx. This response
was reduced by approximately 50% after PP1 pretreatment, but was
significantly increased in fyn-deficient megakaryocytes. Collagen
therefore increases [Ca2+]i in mouse
megakaryocytes via multiple receptors, including GPVI, which causes
Ca2+ mobilization, and 21,
which stimulates a substantial influx of extracellular
Ca2+.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
COLLAGENS ARE IMPORTANT and widespread
extracellular matrix proteins. In blood vessels, type I and type III
collagen form the subendothelial matrix, where they play an important
role in the maintenance of vascular integrity. After damage to the blood vessel lining, platelets adhere to these collagens and are subsequently activated, leading to the formation of a hemostatic plug.
Platelets express several receptors for collagen, including the
integrin  21,1 glycoprotein IV
(GPIV),2 and glycoprotein VI (GPVI),3 and other
proteins of 65 kD and 85-90 kD and GPIIb/IIIa.4,5 These
receptors appear to be variously responsible for adhesion to and
activation of platelets by collagen. A two-step, two-site model of
collagen action has been proposed involving initial adhesion to
21 followed by subsequent binding to an
activatory receptor, which mounting evidence suggests is
GPVI.6 The signaling pathway that is thought to be
activated downstream of GPVI in human platelets involves its
association with the Fc receptor  chain (FcR chain), which is
better known as a component of receptors for IgG and IgE.7
After stimulation of platelets by collagen or a collagen-related peptide (CRP), the FcR chain is phosphorylated on tyrosine
residues, probably within a conserved cytoplasmic domain known as the
immune receptor tyrosine-based activation motif or ITAM.8
ITAM phosphorylation allows subsequent binding and activation of the
tyrosine kinase p72syk via its SH2 domains, leading to
activation of phospholipase C2 (PLC2), an increase
in intracellular calcium [Ca2+]i, and
activation of protein kinase C.9,10 This pathway is essential for platelet secretion and aggregation by collagen, because
platelets from mice deficient in either p72syk or the FcR
chain are not activated when stimulated with fibrillar collagen.11
Less is known of the signaling capacity of other platelet collagen
receptors. The integrin 21 is essential
for efficient platelet adhesion to collagen, particularly under
conditions of high shear.1,12 However, its role in
intracellular signaling is less clear. Keely and Parise13
have shown that 21 is necessary for full
collagen-induced activation and phosphorylation of p72syk
and PLC2, whereas Kamiguti et al14 have also suggested a
role for 21 in p72syk
phosphorylation. In pancreatic acinar cells,
21 has been linked to increases in
[Ca2+]i,15 and a number of other
reports have shown calcium elevation after cross-linking of other
integrins such as v3.16
Platelet GPIV is known to bind collagen and is associated with the
src-family kinases p59fyn, p53/56lyn, and
p62yes, suggesting that it may be capable of transducing
intracellular signals.17
Less is known regarding the signaling pathways activated by collagen in
other cell types and in particular the platelet precursor cell, the
megakaryocyte. Megakaryocytes are large polyploid bone marrow cells
that mature in the bone marrow, before releasing platelets into the
bloodstream. At full maturity, they express many of the cell surface
proteins that are expressed on platelets, including the fibrinogen
receptor GPIIb/IIIa (2b3), the receptor for von Willebrand's factor, GPIb, and
21.18,19 They also respond to
classic platelet agonists such as thrombin and ADP and to
CRP. CRP consists of a glycine-proline-hydroxyproline
repeat sequence, cross-linked via cysteine residues in its C- and
N-terminals, and binds to GPVI, but not
21.20,21 In megakaryocytes,
it elicits an increase in [Ca2+]i in these
cells through a tyrosine kinase-dependent pathway that is thought to be
the same as that found in platelets, with crucial roles for
p72syk and the src-family kinase
p59fyn.22
The presence of other collagen receptors prompted us to investigate the
nature of the response in megakaryocytes to collagen itself and, after
cross-linking of one of its receptors, the
21 integrin. This showed differences in
the signaling pathways activated by collagen in megakaryocytes and
platelets. Collagen can activate at least two pathways in
megakaryocytes leading to the mobilization of calcium from
intracellular stores and the influx of extracellular calcium. The
evidence suggests a role for both GPVI and the integrin 21 in the response to collagen and may
indicate a difference in the role of collagen signaling in the two cell types.
| |
MATERIALS AND METHODS |
Materials.
Collagen fibers (predominantly type I) derived from equine Achilles
tendon were obtained from Nycomed (Munich, Germany). CRP [GCP*(GPP*)10GCP*G,
single letter amino acid code; P* = hydroxyproline] was
provided by Dr M. Barnes (Strangeways Laboratories, Cambridge, UK) and
cross-linked as described.20 Armenian hamster antimouse
CD49b (integrin 2 subunit) and mouse antihamster IgG known to recognize the anti-2 monoclonal antibody (MoAb)
were obtained from Pharmingen (Cambridge Bioscience, Cambridge, UK). PP1 was provided by Dr J. Hanke (Pfizer, Groton, CT).
Bovine thrombin, apyrase, staurosporine, and poly-L-lysine (70,000 molecular weight [MW]) were from Sigma (Poole, Dorset,
UK). FURA-2 and calibration standards were from Molecular Probes
(Eugene, OR). A recombinant protein containing
glutathione-S-transferase (GST) fusion protein linked to the tandem SH2
domains of p72syk was expressed in Escherichia coli
and purified as previously described.8 GST was then removed
by thrombin cleavage, and thrombin was removed by passage over Hi-Trap
heparin columns (Pharmacia, Uppsala, Sweden). All other
reagents were from previously reported sources.22
Protein-deficient mice.
BALB/c radiation chimeric mice reconstituted with syk-deficient fetal
liver were generated as previously described.23
Fyn-deficient mice on a C57BL/6 background were obtained from Jackson
Laboratories (Bar Harbor, ME).
Preparation of mouse megakaryocytes.
Mouse megakaryocytes were prepared as previously
described.22 Briefly, 4- to 8-week-old BALB/c or
protein-deficient mice were killed by cervical dislocation and the
femurs were removed. Bone marrow was flushed out gently in a
calcium/magnesium-free Hanks' buffer containing 0.4% wt/vol bovine
serum albumin (BSA) and 0.2 U/mL apyrase using a 23-gauge needle. Cells
were collected by centrifugation (200g for 8 minutes) in the
presence of 1 µg/mL prostacyclin. For experimentation, cells were
resuspended in a modified Hanks' buffer, containing low calcium (200 µmol/L) that prolonged cell survival (143 mmol/L NaCl, 5.6 mmol/L
KCl, 2 mmol/L MgCl2, 10 mmol/L HEPES, 10 mmol/L glucose,
0.2 mmol/L CaCl2, and 0.4% BSA, pH 7.2). Cells were
maintained at 37°C in this buffer until experimentation.
Single-cell microinjection and calcium imaging.
Cell suspension (50 µL) was plated onto poly-L-lysine (70,000 MW)-coated coverslips and allowed to settle for 3 minutes. Excess suspension was removed and modified Hanks' buffer was added as bathing
medium. Unless stated otherwise, experiments were performed with
[Ca2+]e = 200 µmol/L and at room
temperature (22°C ± 2°C). Cells were viewed on an inverted
Axiovert 110 microscope (Zeiss, Herts, UK), and stage
III/IV megakaryocytes were identified on the basis of size and
morphology. We have previously confirmed their identity as
megakaryocytes22 through specific staining with the 1C2 Ab, which is specific to mouse megakaryocytes.24
Microinjection of FURA-2 and recombinant protein using glass
capillaries was performed using an Eppendorf micromanipulator 5170 and
microinjector 5242 (Merk Ltd, Dorset, UK). FURA-2 pentapotassium salt
was dissolved in a standard intracellular buffer and was present in the
injection needle at a concentration of 2.5 mmol/L, resulting in an
estimated intracellular concentration of 20 to 200 µmol/L.
Recombinant fusion protein containing the tandem SH2 domains of
p72syk was present in the injection needle at 135 µg/mL,
giving an estimated final concentration of 0.5 µmol/L. Single-cell
digital imaging was performed using Ionvision software (Improvision,
Warwick, UK). Fluorescence video images were captured at excitation
wavelengths of 340 and 380 nm, with emission at 510 nm. Calculation of
[Ca2+]i from the 340:380 ratio was performed
by the use of a previously established calibration curve using standard
solutions of various free Ca2+ concentrations applying a
viscosity correction factor25 and using the appropriate
kd for the FURA-2/Ca2+ interaction at this
temperature. Analysis was performed using Ionvision software for the
Macintosh (Improvision).
Cell stimulation and
21
cross-linking.
Generally, agonists were dissolved or diluted in modified Hanks'
buffer and added directly to the bathing medium. Staurosporine and PP1
were added 3 minutes before stimulation and
[Ca2+]e was modified 2 minutes before any
further addition. For 21 cross-linking
studies, cells were plated onto poly-L-lysine-coated slips and
incubated with hamster anti-2 MoAb (10 µg/mL) for 5 minutes. Excess antibody was removed, bathing medium was added, and a
suitable cell was micro-injected with FURA-2. Cross-linking was
initiated by the addition of mouse antihamster IgG (20 µg/mL).
Data analysis and statistics.
Data are presented as both the peak value of
[Ca2+]i after agonist addition and the
increase in [Ca2+]i above the basal level,
taken immediately before agonist addition. Data are presented as the
mean ± standard error (SE) mean for the number of
values indicated, with each condition tested on cells from a minimum of
3 different mice. Normality of data was tested using a one-group
 2 analysis. Differences were analyzed by either an
unpaired Student's t-test or, where the sample size was small,
by Mann-Whitney U-test. In each case, P < .05 was the minimum
value taken to indicate statistical significance.
| |
RESULTS |
Characterization of collagen-mediated changes in
[Ca2+]i.
The addition of fibrillar type I collagen (100 µg/mL) to murine
megakaryocytes in the presence of 200 µmol/L extracellular calcium
([Ca2+]e) caused a steady increase in
[Ca2+]i from a basal of 66 ± 7 nmol/L to
a peak of 217 ± 19 nmol/L after a delay of approximately 30 to 60 seconds, leading to a sustained plateau (mean ± SE mean, n = 10;
increase over basal = 151 ± 26 nmol/L;
Figs 1A and
2A). This plateau generally persisted for
at least 10 minutes after the addition of collagen, decreasing slightly
in this time. A similar profile of changes in
[Ca2+]i were seen in megakaryocytes isolated
from human bone marrow (unpublished observations). The
increase in [Ca2+]i seen after collagen
stimulation was highly dependent on the level of
[Ca2+]e, suggesting a prominent role for
calcium influx in this response (Fig 1A). The addition of 10 mmol/L
EGTA caused a small decrease in basal [Ca2+]i
(57 ± 21 nmol/L, n = 6). Collagen elicited a small but significant increase in [Ca2+]i in the presence of EGTA,
with a mean peak [Ca2+]i value of 100 ± 24 nmol/L (n = 6), giving an increase over basal of 44 ± 12 nmol/L
(P < .01 v basal). This indicates that collagen is
capable of mobilizing calcium from intracellular stores in mouse
megakaryocytes. The peak calcium level in the presence of 10 mmol/L
EGTA was significantly reduced compared with that seen in 200 µmol/L
[Ca2+]e (P < .05), indicating that
a significant component of the response was due to influx of
[Ca2+]e. This was further demonstrated by
increasing the [Ca2+]e to 1.2 mmol/L, which
caused a further elevation in the response to collagen, with increases
of greater than 500 nmol/L above basal seen in all cells tested (peak
[Ca2+]i = 695 ± 150 nmol/L, n = 6;
P < .01 v peak value in 200 µmol/L [Ca2+]e; Fig 1A). The addition of 1.2 mmol/L
[Ca2+]e caused no significant increase in the
resting level of [Ca2+]i in these cells (108 ± 37 nmol/L, n = 6).
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| Fig 1.
Collagen-mediated changes in
[Ca2+]i in megakaryocytes from wild-type
and syk  / mice. Megakaryocytes from either (A) wild-type Balb/c
mice or (B) radiation chimeric Balb/c mice repopulated with syk /
fetal liver were isolated from bone marrow as described. After plating
onto poly-L-lysine-coated coverslips, stage III/IV megakaryocytes were
identified and injected with FURA-2 and then stimulated with collagen
(100 µg/mL) as indicated. [Ca2+]e levels
were adjusted 2 minutes before the addition of agonist. FURA-2
fluorescence was measured as described and
[Ca2+]i was determined from a precalculated
calibration curve. Representative traces in (A) and (B) are each taken
from cells from a single animal, but a representative of at least 6 (A)
or 3 (B) cells from a minimum of three mice.
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| Fig 2.
The role of Syk- and Src-family kinases in collagen- and
CRP-mediated calcium increases in mouse megakaryocytes. Single mouse
megakaryocytes from Balb/c bone marrow were microinjected with FURA-2,
and the changes in [Ca2+]i were determined
in response to (A) 100 µg/mL collagen or (B) 2 µg/mL CRP. The
response to collagen (100 µg/mL) was then determined (C) after
comicroinjection of a recombinant fusion protein containing the tandem
SH2 domains of syk; (D) in syk-deficient megakaryocytes; (E) in the
presence of the Src-family kinase inhibitor, PP1 (10 µmol/L for 3 minutes); or (F) in fyn-deficient megakaryocytes. Each trace is
representative of 4 to 10 cells from a minimum of three different mice.
In each case, [Ca2+]e was fixed at 200 µmol/L.
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We have previously characterized the calcium-mobilizing capacity of CRP
in these cells. This triple-helical peptide, consisting of
glycine-proline-hydroxyproline repeats, is a potent agonist at the GPVI
collagen receptor expressed in human platelets, but does not bind to
the integrin 21.10,21 The
addition of CRP (2 µg/mL) to mouse megakaryocytes causes a sharp
increase in [Ca2+]i followed by a second,
sustained increase, which returns to basal levels approximately 200 seconds after the addition of agonist (Fig 2B). Neither the peak in
[Ca2+]i seen after addition of CRP in these
cells nor the profile of the response was significantly changed by
removing [Ca2+]e (data not shown). Thus, in
contrast to collagen, CRP appears to act predominantly by mobilizing
Ca2+ from intracellular stores.
The role of syk- and src-family kinases in the collagen-mediated
changes in [Ca2+]i.
Initial results suggested that, in megakaryocytes, in addition to GPVI,
collagen activates other cell membrane receptors. Further evidence for
this was obtained from work investigating the role of syk- and
src-family kinases in this change in [Ca2+]i.
Initially, we investigated the role of p72syk in the
Ca2+ response to collagen in these cells (Fig 2 and
Table 1). Our previous work with CRP
demonstrated that the pathway by which this agonist increased
[Ca2+]i in mouse megakaryocytes was entirely
dependent on the tyrosine kinase p72syk.22 This
was shown partly by microinjection of a recombinant protein composed of
the tandem SH2 domains of p72syk. The inhibition is
believed to be through the binding of the recombinant protein to the
phosphorylated ITAM motif on the FcR chain, which then prevents
further signal propagation via this pathway. Injection of this protein
therefore allows us to investigate the role of the GPVI/FcR chain
pathway in the collagen response, in addition to other
syk-SH2-dependent signaling pathways. In agreement with our previous
study, microinjection of the SH2 domains of p72syk protein
did not significantly affect the resting calcium levels in the cells
(56 ± 7 nmol/L, n = 10; Table 1). However, the addition of collagen
in the presence of this protein caused a large increase in
[Ca2+]i levels, although the peak value of
188 ± 11 nmol/L gave a mean increase above basal that was
significantly reduced compared with control cells (133 ± 11 nmol/L,
n = 10; P < .05; Table 1 and Fig 2C). To further investigate
the role of p72syk in collagen-mediated calcium increases,
radiation chimeric mice, repopulated with syk / fetal
liver, were produced as previously described.23 Syk
/ megakaryocytes isolated from these animals had a
similar basal [Ca2+]i to control cells (73 ± 6 nmol/L, n = 6). Collagen caused a significant increase in
[Ca2+]i in syk / cells, with a
peak value of 179 ± 10 nmol/L and an increase above basal levels of
114 ± 10 nmol/L (P < .01 v basal). This is
approximately 40% lower than in control cells (P < .05; Table 1 and Fig 2D). The increase in [Ca2+]i
mediated by collagen in the syk / megakaryocytes was,
like the response in control cells, highly dependent on the
concentration of [Ca2+]e, indicating that
collagen could initiate calcium influx independently of
p72syk (Fig 1B). Interestingly, there was a small but
significant increase in [Ca2+]i in syk
/ megakaryocytes (P < .05) in the presence of
10 mmol/L EGTA, suggesting the existence of a minor syk-independent
pathway for collagen-mediated mobilization of
[Ca2+]i from intracellular stores.
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Table 1.
Collagen-Mediated Changes in
[Ca2+]i in Mouse Megakaryocytes in the
Presence of Tyrosine Kinase Inhibitors and in Kinase-Deficient
Megakaryocytes
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We then investigated the role of Src-family kinases in the
collagen-mediated increases in [Ca2+]i,
because the response to CRP in mouse megakaryocytes is mediated predominantly via the src-family kinase p59fyn.
Pretreatment of mouse megakaryocytes with the Src-family kinase inhibitor PP126 (10 µmol/L) affected neither the resting
[Ca2+]i (67 ± 13 nmol/L, n = 9) nor the
peak in [Ca2+]i after collagen stimulation
(201 ± 18 nmol/L), although the increase above resting levels was
significantly reduced (136 ± 8 nmol/L, n = 9; P < .05;
Table 1 and Fig 2E). However, interestingly, the duration of the
collagen response was reduced in the presence of PP1, suggesting a role
for a Src-family kinase in the maintenance of calcium influx. Because
PP1 may not be specific to p59fyn at this concentration, we
also studied the collagen-mediated change in
[Ca2+]i in megakaryocytes from fyn-deficient
mice. In these cells, collagen elicited a smaller increase in
[Ca2+]i to that seen in control cells, but
this failed to reach significance (peak
[Ca2+]i = 183 ± 26 nmol/L, rise above
basal = 106 ± 27 nmol/L, n = 4; Fig 2F).
To test the overall kinase-dependency of the collagen response, we used
the general kinase inhibitor, staurosporine, which is known to prevent
CRP-mediated calcium changes in these cells and also functional
responses to collagen in human platelets. Preincubation of mouse
megakaryocytes with staurosporine (3 µmol/L for 5 minutes) did not
significantly affect either the resting level of
[Ca2+]i (74 ± 19 nmol/L, n = 8) or the
change in [Ca2+]i seen after the addition of
100 µg/mL collagen (peak [Ca2+]i = 224 ± 33 nmol/L; increase above basal = 155 ± 37 nmol/L; data not shown). This result strongly suggests that collagen can increase [Ca2+]i independently of tyrosine
phosphorylation in these cells.
The role of
21 in
collagen-mediated changes in [Ca2+]i in mouse
megakaryocytes.
Taken together, these results show that the collagen-mediated increase
in [Ca2+]i in mouse megakaryocytes is
partially mediated by the syk-dependent pathway previously described
for CRP in these cells. This implies the existence of a second pathway
of calcium elevation. Of the proposed receptors for collagen other than
GPVI, the integrin 21 is the best
characterized, and its presence on megakaryocytes has been
described.19 We therefore investigated whether this integrin was capable of causing a change in
[Ca2+]i in mouse megakaryocytes using a
previously characterized MoAb to the 2 integrin subunit,
which is known to block the adhesion of T cells to collagen
gels.27 This was used in preference to anti-1 subunit antibodies to avoid coincident
cross-linking of, for instance, v1 or
51 integrins.
Mouse megakaryocytes were coated with the antimouse 2
MoAb (10 µg/mL) for 5 minutes and the excess was removed. A suitable cell was microinjected with FURA-2 and the primary antibody was cross-linked by the addition of the antihamster IgG secondary antibody
(20 µg/mL). Cross-linking of 2-subunits caused a small but significant increase in [Ca2+]i in the
presence of 200 µmol/L [Ca2+]e after a
delay of 20 to 30 seconds (increase above basal = 95 ± 8 nmol/L, n = 8; Fig 3C). The response was smaller but
similar in profile to that seen with collagen, consisting of an initial slow increase in [Ca2+]i followed by a
prolonged plateau. The addition of primary antibody (10 µg/mL) or
secondary antibody (20 µg/mL) alone to the incubation medium caused
no significant change in cytosolic calcium levels (Fig 3A and B). The
response seen after cross-linking with the anti-2 Ab was
dependent on [Ca2+]e. With
[Ca2+]e at 1.2 mmol/L, cross-linking caused a
significantly larger and prolonged response than in 200 µmol/L
calcium (increase above basal = 279 ± 44 nmol/L, n = 11; P < .05; Fig 3D). In the presence of 10 mmol/L EGTA, clustering of
2 subunits did not cause any significant increase in
[Ca2+]i (Fig 3D). The changes in
[Ca2+]i caused by cross-linking of the
2 subunit appear, therefore, to be mediated entirely by
influx of [Ca2+]e.
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| Fig 3.
The effect of cross-linking the 2-integrin
subunit of mouse megakaryocytes on [Ca2+]i
levels. (A) After plating of a bone marrow suspension onto
poly-L-lysine-coated coverslips, cells were coated with
anti-2 MoAb for 5 minutes and then the excess was
removed. Extracellular medium ([Ca2+]e = 200 µmol/L) was added and a suitable cell was microinjected with
FURA-2. The change in [Ca2+]i in response
to the addition of antihamster secondary Ab (20 µg/mL) as indicated
was determined. (B) After FURA-2 injection of a suitable cell,
anti-2 MoAb was added (10 µg/mL) and the change in
[Ca2+]i was recorded. In (C), similar
experiments were performed after the addition of secondary Ab alone.
(D) The effect of increasing the [Ca2+]e to
1.2 mmol/L or chelating extracellular Ca2+ with EGTA (10 mmol/L) on the subsequent response to 2-subunit
cross-linking was determined. [Ca2+]e was
altered 2 minutes before the addition of the secondary Ab. (E) After
coating with primary antibody, the effect of pretreatment of cells with
PP1 (10 µmol/L for 3 minutes) on subsequent 2-subunit
cross-linking was determined, and in (F) 2-subunit
cross-linking was performed in fyn-deficient megakaryocytes. Unless
otherwise indicated, experiments were performed in
[Ca2+]e equal to 200 µmol/L. Each trace
is representative of 6 to 12 cells from a minimum of three different
mice.
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Because the influx of [Ca2+]e after collagen
activation was partially sensitive to PP1 treatment, we investigated
the role of src-family kinases in the response to cross-linking of the
integrin 2-subunit. Preincubation with the src-family
kinase inhibitor PP1 before the addition of the cross-linking antibody
caused an approximate 50% decrease in the increase above resting
[Ca2+]i with
[Ca2+]e at 1.2 mmol/L (137 ± 31 nmol/L,
n = 12). This was also the case when
[Ca2+]e was at 200 µmol/L (Fig 3E). To
further dissect the role of src-family kinases in this calcium influx
pathway, these experiments were also performed in megakaryocytes from
fyn / mice. Surprisingly, in 200 µmol/L
[Ca2+]e, cross-linking of 2
subunits in these cells produced a significantly greater increase in
[Ca2+]i than in control cells (202 ± 37 nmol/L, n = 11; Fig 3F).
| |
DISCUSSION |
The data presented in this study provide, to our knowledge, the first
description of the changes in [Ca2+]i
mediated by collagen in primary megakaryocytes. The study also provides
evidence for the activation of at least two collagen receptors in these
cells, which can influence [Ca2+]i via
multiple pathways. We have delineated roles for (1) a
collagen-stimulated mobilization of calcium from intracellular stores,
dependent on p72syk and probably mediated by
GPVI,22 and (2) a collagen-mediated influx of extracellular
calcium, which is p72syk- and
p59fyn-independent and may be mediated by
21. These results demonstrate the complex
nature of collagen signaling in these cells and highlight an important
difference between signaling by this agonist in megakaryocytes and in
platelets, in which the increase in [Ca2+]i
mediated by collagen is mediated entirely by a tyrosine
kinase-dependent pathway.9,28 We believe that this
difference may reflect the differing physiological roles of the
response to collagen in the two cell types.
Our previous studies using the synthetic collagen agonist, CRP, have
demonstrated that a calcium-mobilizing pathway similar to that
activated by GPVI/FcR chain in platelets exists in mouse megakaryocytes.22 The tyrosine kinases p59fyn
and p72syk play crucial roles in this pathway, which is
presumed to culminate in the activation of PLC2 and the generation
of the calcium-mobilizing messenger, inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3]. The response to CRP is virtually
abolished in mice megakaryocytes deficient in either p72syk
or p59fyn. This response consists almost entirely of
calcium release from intracellular stores, because it is not
significantly altered in the presence of EGTA. In contrast to CRP, the
collagen response was highly dependent on the level of extracellular
calcium, indicating that a large component of the response is mediated
by calcium influx. This suggests that the
p59fyn/p72syk pathway was not the only pathway
leading to an increase in [Ca2+]i. Moreover,
whereas collagen undoubtedly releases stored calcium in these cells,
because there was still a response in the presence of EGTA, at
physiologically relevant concentrations of
[Ca2+]e, influx comprises the greater part of
the collagen response.
Evidence for the separation of the mechanisms underlying entry and
mobilization of calcium by collagen came from the use of tyrosine
kinase inhibitors and protein-deficient megakaryocytes. Collagen caused
a significant increase in [Ca2+]i in the
presence of the general kinase inhibitor staurosporine, in the presence
of the src-family kinase inhibitor PP1, and after microinjection of a
protein containing the tandem SH2 domains of
p72syk. Each of these treatments is known to
block the response to CRP in these cells.22 Staurosporine
and PP1 have also been shown to block the calcium mobilizing responses
of collagen and CRP in human platelets.28,29 After
microinjection of the tandem SH2 domains of p72syk, there
was approximately 30% reduction in the peak response to collagen. This
protein is thought to act by binding to phosphorylated FcR chain
ITAM and preventing downstream signaling. These results suggest that
the FcR chain pathway is responsible for only part of the collagen
response. This was further supported by the persistence of a collagen
response in syk-deficient megakaryocytes, whereas CRP-mediated calcium
mobilization is absent. Importantly, collagen could also mediate
substantial calcium influx in syk-deficient megakaryocytes, showing
that calcium influx can be mediated independently of this kinase. The
maintenance of calcium influx stimulated by collagen was partly
affected by PP1, reflecting a role for a Src-family kinase in this
response. A role for this kinase family in calcium influx has been
demonstrated in HEL cells,30 whereas in platelets the
presence of a tyrosine kinase-dependent route of calcium influx has
been described.31
Because a component of the collagen response is dependent on the
tyrosine kinase p72syk and is affected in duration by PP1,
it is noteworthy that the general kinase inhibitor, staurosporine, does
not have the same effect. It is possible that collagen influences the
calcium levels in megakaryocytes through both positive and negative
kinase-dependent mechanisms. One could then envisage a situation in
which a general kinase inhibitor such as staurosporine would affect
both stimulatory and inhibitory pathways. For example, staurosporine
inhibits serine/threonine kinases such as protein kinase C (PKC), which
can directly affect capacitative calcium influx, or calcium extrusion
mechanisms.32-34
Cross-linking of the integrin 2 in mouse megakaryocytes
demonstrated that clustering of the integrin
21 could also stimulate calcium influx.
The integrins are a family of heterodimeric cell surface molecules that
interact with a variety of extracellular matrix components such as
collagens, fibronectin, and vitronectin.35 The integrin
21 is an important receptor for collagen
that is present on platelets, megakaryocytes, and a variety of other
cell types. Whereas the signaling functions of
21 are not well defined in platelets,
there is evidence that binding of collagen to this receptor is
necessary for full activation of p72syk and
PLC213,14 and may mediate collagen activation of
p60c-src.36 In pancreatic acinar cells, the
increase in [Ca2+]i caused by collagen
appears to be mediated predominantly by 21.15,37 The calcium
signaling capacity of integrins in other cells has also been
demonstrated, with contributions from tyrosine kinase-dependent and
-independent pathways.16 For example, both soluble
fibronectin and vitronectin mediate an increase in
[Ca2+]i in human endothelial
cells,38 whereas the v3
integrin in rat osteoclasts is responsible for calcium increases to
vitronectin in this cell line.39 Integrins also regulate or
induce protein tyrosine phosphorylation in many cell
types.35 This seems to be predominantly linked to the
formation of focal adhesions, but can lead to phosphorylation or
activation of a variety of other proteins, including ZAP-70, PLC1,
and Src-family kinases.37,40,41
Cross-linking of the integrin 2 subunit caused an
increase in [Ca2+]i in mouse megakaryocytes.
This response appears to be mediated exclusively by the influx of
extracellular calcium and is maintained at a lower level in the
presence of PP1 but potentiated in fyn-deficient cells. It is therefore
possible that differing src-family kinases play stimulatory and
inhibitory roles in 21 signaling in
megakaryocytes. The pathway mediating the component of the response to
2-subunit cross-linking that is independent of
src-family kinases is unknown, although there is some evidence to
suggest that integrins can directly activate calcium influx channels
independently of kinases.16
A major finding of this study is the difference in calcium signaling
pathways activated by collagen in megakaryocytes compared with those
activated in platelets. In platelets, collagen mediates aggregation and
dense granule release via a tyrosine kinase-dependent pathway, which
can be inhibited by staurosporine and PP1.10,21,28 This is
the predominant pathway in these cells, because mice platelets lacking
p72syk or the FcR chain fail to undergo aggregation or
secretion in response to collagen.11 However, in
megakaryocytes, it appears that calcium influx, probably mediated by
21, is the predominant pathway. This may
reflect differences in the level of expression or the signaling
pathways coupled to the integrin 21
between megakaryocytes and platelets. This is likely to reflect
important differences in the physiological role of collagen in the two
cell types. For example, whereas collagen, CRP, and thrombin induce similar responses in the platelet, there are notable differences in
megakaryocyte morphology after stimulation by the three agonists. We
have sometimes observed a shape change or blebbing response of
megakaryocytes in response to CRP (30% of cells) or thrombin (60% of
cells; unpublished observations), which others have
speculated is related to proplatelet formation.42 However,
the addition of collagen did not cause blebbing, suggesting that the
adhesion molecule overrides the response to cross-linking of GPVI,
thereby protecting against premature proplatelet shedding. Collagen may also perform other functions in the megakaryocyte that have little significance to platelet function. We speculate, for example, that
collagen may play a role in the migration and subsequent adhesion of
mature megakaryocytes to the blood vessels, leading to protusion into
the circulation.19 It is notable that integrin-mediated Ca2+ fluxes have been shown to regulate adhesion and
migration in other cells.39,43,44
In conclusion, we have demonstrated the complex nature of the
collagen-induced changes in [Ca2+]i in
primary megakaryocytes. Collagen causes both calcium mobilization from
intracellular stores and the influx of calcium from the extracellular solution. Although multiple pathways are involved in this response, mobilization from intracellular stores is mediated predominantly via a
syk-dependent pathway, which may be via the GPVI pathway previously
described in these cells. In contrast, calcium influx occurs via a
predominantly syk-independent pathway, which is at least partly
mediated by 21.
| |
ACKNOWLEDGMENT |
The authors thank Drs M. Barnes and R. Farndale for the CRP, J. Hanke
for the gift of PP1, and Dr Che-Leung Law for the Syk fusion construct.
| |
FOOTNOTES |
Submitted August 27, 1998; accepted February 1, 1999.
Supported by The Wellcome Trust and the British Heart Foundation.
S.P.W. is a BHF Senior Research Fellow.
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 Steve P. Watson, PhD,
Department of Pharmacology, University of Oxford, Mansfield Road,
Oxford, OX1 3QT, UK; e-mail: steve.watson{at}pharmacology.ox.ac.uk.
| |
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TOP
ABSTRACT
INTRODUCTION