From the Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom
Received for publication, October 10, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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Extracellular signal-regulated kinases
(ERKs), are common participants in a broad variety of signal
transduction pathways. Several studies have demonstrated the presence
of ERKs in human platelets and their activation by the physiological
agonist thrombin. Here we report the involvement of the ERK cascade in
store-mediated Ca2+ entry in human platelets.
Treatment of
dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-loaded platelets with thapsigargin to deplete the
intracellular Ca2+ stores resulted in a time- and
concentration-dependent activation of ERK1 and ERK2.
Incubation with either U0126 or PD 184352, specific inhibitors of
mitogen-activated protein kinase kinase (MEK), prevented thapsigargin-induced ERK activation. Furthermore, U0126 and PD 184352 reduced Ca2+ entry stimulated by thapsigargin or thrombin,
in a concentration-dependent manner. The role of ERK in
store-mediated Ca2+ entry was found to be independent of
phosphatidylinositol 3- and 4-kinases, the tyrosine kinase pathway, and
actin polymerization but sensitive to treatment with inhibitors of Ras,
suggesting that the ERK pathway might be a downstream effector of Ras
in mediating store-mediated Ca2+ entry in human platelets.
In addition, we have found that store depletion stimulated ERK
activation does not require PKC activity. This study demonstrates for
the first time a novel mechanism for regulation of store-mediated
Ca2+ entry in human platelets involving the ERK cascade.
In platelets and other nonexcitable cells, stimulation by various
agonists results in an increase in the intracellular free Ca2+ concentration
([Ca2+]i),1
which consist of two components: Ca2+ release from internal
stores and Ca2+ entry across the plasma membrane (1). The
main mechanism for Ca2+ influx is store-mediated
Ca2+ entry (SMCE), where the filling state of the internal
Ca2+ stores regulates the entry of Ca2+ (2).
Although the mechanisms involved in the activation of this pathway are
still not well understood, recent studies suggest that a secretion-like
coupling model is compatible with the mechanisms underlying SMCE in
several cell types (3, 4), including human platelets (5). The
secretion-like coupling model proposes a reversible trafficking and
coupling of the endoplasmic reticulum with the plasma membrane (3-5).
In human platelets we have recently provided the first direct evidence
of coupling stimulated by depletion of the intracellular
Ca2+ stores, which involves type II linositol
trisphosphate receptors in the endoplasmic reticulum and hTRP1
channels in the plasma membrane (6).
As with secretion, the actin cytoskeleton plays a role in the
activation (3, 5, 7, 8) and the maintenance of SMCE in platelets (5).
However, little is known about the signaling mechanisms involved in the
activation of this process. A role for protein tyrosine phosphorylation
in the activation of SMCE has been reported in several cell types,
including platelets (9-11), where tyrosine phosphorylation is required
for actin polymerization (12). In addition, small GTP-binding proteins
have been proposed as candidates for the activation of SMCE (13-15).
In human platelets, depletion of the Ca2+ stores stimulates
translocation and association of Ras with the plasma membrane (16),
which is essential for Ras activation (17). In these cells the role of
Ras proteins in SMCE is partially mediated by the reorganization of the
actin cytoskeleton (16). Activated Ras interacts with several signaling
proteins, which include Raf-1, phosphoinositide kinases, diacylglycerol
kinase, and MEK (18). Phosphoinositide kinases have recently been shown to modulate actin filament polymerization and SMCE (19). The serine/threonine kinase Raf-1 activates MEK and subsequently the ERK
cascade of MAP kinases (18). Since the characterization of ERK1 and
ERK2 it has become clear that these proteins are among the protein
kinases most commonly activated in signal transduction pathways from
cell proliferation to many other events including the production of
insulin in pancreatic We report here that depletion of the intracellular Ca2+
stores evokes Ca2+-independent activation of ERK kinases
and that the ERK kinase pathway is involved in the activation of SMCE
in human platelets, probably as a downstream effector of Ras proteins.
Materials--
Fura-2 acetoxymethyl ester (fura-2/AM) was from
Texas Fluorescence (Austin, TX). Apyrase (grade VII), aspirin, bovine
serum albumin, paraformaldehyde, Nonidet P-40, sodium vanadate,
fluorescein isothiocyanate-labeled phalloidin, thrombin,
methyl-2,5-dihydroxycinnamate, and thapsigargin (TG) were
from Sigma (Poole, Dorset, United Kingdom). 1,4-Diamino-2,3-dicyano-1,4-bis-(phenylthio)butadiene
(U0126), 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide (PD 184352), LY294002, phenylarsine oxide, Ro-31-8220, and cytochalasin D (Cyt D) were from Calbiochem (Nottingham, UK). Farnesylthioacetic acid (FTA) and PP1 were from Alexis Corp. (Nottingham, UK).
Phospho-p44/42 ERK monoclonal antibody (E10) was from New England
Biolabs (Beverly, MA). Horseradish peroxidase-conjugated ovine
anti-mouse IgG antibody (NA931) was from Amersham Pharmacia Biotech
(Little Chalfont, Bucks., UK).
Dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester was from Molecular Probes (Leiden, The
Netherlands). All other reagents were of analytical grade.
Platelet Preparation--
Fura-2-loaded platelets were prepared
as described previously (5). Briefly, blood was obtained from healthy
volunteers and mixed with one-sixth volume of acid/citrate dextrose
anticoagulant containing (in mM): 85 sodium citrate, 78 citric acid, and 111 D-glucose. Platelet-rich plasma was
then prepared by centrifugation for 5 min at 700 × g
and aspirin (100 µM) and apyrase (40 µg/ml) added.
Platelet-rich plasma was incubated at 37 °C with 2 µM
fura-2/AM for 45 min. For loading with dimethyl-BAPTA, cells were
incubated for 30 min at 37 °C with 10 µM
dimethyl-BAPTA AM. Cells were then collected by centrifugation at
350 × g for 20 min and resuspended in HEPES-buffered
saline containing (in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO4, pH 7.45, and
supplemented with 0.1% (w/v) bovine serum albumin and 40 µg/ml apyrase.
Measurement of Intracellular Free Calcium Concentration
([Ca2+]i)--
Fluorescence was recorded
from 1.5-ml aliquots of magnetically stirred platelet suspension
(108 cells/ml) at 37 °C using a Cairn Research
Spectrophotometer (Cairn Research Ltd., Sittingbourne, Kent, UK) with
excitation wavelengths of 340 and 380 nm and emission at 500 nm.
Changes in [Ca2+]i were monitored using the
fura-2 340/380 fluorescence ratio and calibrated according to the
method of Grynkiewicz et al. (21).
Determination of Ca2+
Entry--
Ca2+ influx in platelets that had been store
depleted using TG was estimated using the integral of the rise in
[Ca2+]i for 21/2 min after addition of
CaCl2 (5). When platelets were preincubated with
inhibitors, Ca2+ entry was corrected by subtraction of the
rise in [Ca2+]i due to leakage of the indicator.
TG-induced Ca2+ release was estimated using the integral of
the rise in [Ca2+]i for 3 min after its addition.
Thrombin-evoked Ca2+ influx was measured as the integral of
the rise in [Ca2+]i above basal for 11/2
min after addition of thrombin in the presence of external
Ca2+, corrected by subtraction of the integral over the
same period for stimulation in the absence of external Ca2+
(with 100 µM EGTA).
Measurement of F-actin Content--
The F-actin content of
resting and activated platelets was determined according to a
previously published procedure (16). Briefly, washed platelets (2 × 108 cells/ml) were activated in HEPES-buffered saline.
Samples of platelet suspension (200 µl) were transferred to 200 µl
of ice-cold 3% (w/v) formaldehyde in phosphate-buffered saline for 10 min. Fixed platelets were permeabilized by incubation for 10 min with 0.025% (v/v) Nonidet P-40 detergent dissolved in phosphate-buffered saline. Platelets were then incubated for 30 min with fluorescein isothiocyanate-labeled phalloidin (1 µM) in
phosphate-buffered saline supplemented with 0.5% (w/v) bovine serum
albumin. After incubation the platelets were collected by
centrifugation in an MSE Micro-Centaur Centrifuge (MSE Scientific
Instruments, Crawley, Sussex, UK) for 60 s at 3000 × g and resuspended in phosphate-buffered saline. Staining of
2 × 107 cells/ml was measured using a PerkinElmer
Fluorescence Spectrophotometer (PerkinElmer Life Sciences, Norwalk,
CT). Samples were excited at 496 nm and emission was at 516 nm.
Western Blotting--
Platelets stimulation was terminated by
mixing with an equal volume of 2 × Laemmli's buffer (22) with
10% dithiothreitol followed by heating for 5 min at 95 °C.
One-dimensional SDS electrophoresis was performed with 10%
polyacrylamide minigels and separated proteins were electrophoretically
transferred, for 2 h at 0.8 mA/cm2, in a semi-dry
blotter (Hoefer Scientific, Newcastle, Staffs., UK) onto nitrocellulose
for subsequent probing. Blots were incubated overnight with 10% (w/v)
bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBST)
to block residual protein-binding sites. Blocked membranes were then
incubated with the phospho-p44/42 MAP kinase monoclonal antibody (E10)
diluted 1:1500 in TBST for 1 h. The primary antibody was removed
and blots washed six times for 5 min each with TBST. To detect the
primary antibody, blots were incubated with horseradish
peroxidase-conjugated ovine anti-mouse IgG antibody diluted 1:10000 in
TBST, washed six times in TBST, and exposed to enhanced
chemiluminescence reagents for 1 min. Blots were then exposed to
preflashed photographic film. Densitometric measurements were made
using a Quantimet 500 densitometer (Leica, Milton Keynes, UK).
Statistical Analysis--
Analysis of statistical significance
was performed using Student's t test. For multiple
comparisons, one-way analysis of variance combined with the Dunnett
test was used.
Kinetics and Concentration Dependence of the Ability of
Thapsigargin to Activate ERK in Dimethyl-BAPTA-loaded Human
Platelets--
The activation of ERK was analyzed by Western blotting
using a mouse monoclonal phosphospecific anti-ERK antibody, which only detects the diphosphorylated and so activated form of ERK (23-25). Human platelets were loaded with the Ca2+ chelator
dimethyl-BAPTA so as to eliminate Ca2+- but not store
depletion-dependent responses. We checked that the rise in
[Ca2+]i evoked by TG, a specific inhibitor of the
endomembrane Ca2+-ATPase (sarco/endoplasmic reticulum
Ca2+-ATPase) (26), was abolished by BAPTA loading
(data not shown, but see Ref. 12). Treatment of dimethyl-BAPTA-loaded
platelets with 1 µM TG caused rapid activation of both
isoforms of ERK (p44 ERK1 and p42 ERK2). As shown in Fig.
1A, ERK activation was
detectable 1 min after treatment with TG and reached a maximum within 3 min with an increase of 318.4 ± 13.0% of control. At later times
ERK activation decreased such that it was near basal 30 min after TG
addition (Fig. 1A, n = 4). The effect of TG
on ERK activation was concentration-dependent (Fig.
1B). After treatment of platelets for 3 min with TG a
detectable increase in ERK activity was observed at 30 nM,
the effect was half-maximal at 151.9 ± 14.3 nM, and maximal at 1 µM (Fig. 1B, n = 4).
U0126 Inhibits TG-induced ERK Activation--
U0126 is a cell
permeant, potent and specific inhibitor of MEK1 and MEK2, the kinases
upstream of ERK (27). The effect of U0126 on ERK activation is shown in
Fig. 1C. Treatment of dimethyl-BAPTA-loaded human platelets
for 30 min at 37 °C with U0126 inhibited the TG-induced ERK
activation in a concentration-dependent manner, with an
IC50 of 55.4 ± 1.7 nM and complete
inhibition at 10 µM.
Effect of U0126 on TG-evoked SMCE--
In a Ca2+-free
medium TG evoked a prolonged elevation of [Ca2+]i
in platelets due to release of Ca2+ from the intracellular
stores. Subsequent addition of Ca2+ (final concentration
300 µM) to the external medium resulted in a sustained
increase in [Ca2+]i indicative of SMCE (Fig.
1D). Pretreatment of human platelets for 30 min at 37 °C
with different concentrations of U0126 decreased TG-evoked
Ca2+ entry in a concentration-dependent manner
(Fig. 1D). U0126-treated cells retained their ability to
respond to TG, which indicates that this treatment did not affect the
ability of platelets to store Ca2+ in intracellular
compartments. However, complete inhibition of ERK activation by 10 or
100 µM U0126 reduced Ca2+ entry by only 50%
(Fig. 1D). These results suggest that SMCE is only partially
dependent on ERK activation in human platelets.
Effect of PD 184352 on TG-evoked ERK Activation and SMCE--
To
further assess the involvement of the ERK cascade in SMCE in human
platelets we examined the effect of PD 184352, a selective inhibitor of
MEK1 which is structurally unrelated to U0126 (28, 29). Pretreatment of
human platelets for 30 min at 37 °C with different concentrations of
PD 184352 (100 nM and 3 µM) decreased TG-evoked ERK activation in a concentration-dependent
manner (data not shown). Treatment with PD184352 inhibits TG-evoked ERK
activation by 67% at a concentration of 100 nM while
reaching a complete inhibition of ERK activation at 3 µM
(n = 4). Consistent with the above, treatment of
platelets with PD 184352 reduced TG-induced Ca2+ entry in a
concentration-dependent manner. Treatment of platelets for
30 min with 100 nM PD 184352 reduced SMCE by 34.1 ± 5.5%. However, as shown for U0126, complete inhibition of TG-evoked ERK activation by 3 µM PD184352 reduced Ca2+
entry by only 57% (p < 0.01; n = 6).
As with U0126, PD 184352 did not modify the ability of platelets to
respond to Ca2+ mobilizing agents, such as TG (data not shown).
U0126 Reduces Thrombin-evoked ERK Activation and
[Ca2+]i Elevation--
Thrombin is a
physiological agonist that stimulates a large number of processes in
platelets (1). Previous studies have demonstrated that thrombin induces
activation of ERK (30). Here we report that thrombin evokes activation
of ERK in dimethyl-BAPTA-loaded platelets, indicating that this
response in independent of rises in [Ca2+]i. As
shown in Fig. 2A, treatment of
dimethyl-BAPTA-loaded platelet for 3 min with 1 unit/ml thrombin
induced an increase on ERK diphosphorylation of 445.5 ± 27.5% of
control (Fig. 2A; n = 3). As shown for TG,
preincubation with 10 µM U0126 for 30 min abolished
thrombin-induced ERK activation (Fig. 2A; p < 0.001; n = 3).
Treatment of human platelets for 30 min at 37 °C with 10 µM U0126 reduced the rise in
[Ca2+]i evoked by thrombin (1 unit/ml) in a
medium containing 1 mM Ca2+ (Fig.
2B). The initial peak [Ca2+]i
elevation above basal after agonist stimulation was significantly
reduced from 368.4 ± 45.1 to 207.1 ± 21.1 nM
(p < 0.001; n = 6). If we consider the
entry of Ca2+ stimulated by thrombin (see "Experimental
Procedures"), treatment of platelets with 10 µM U0126
significantly reduced thrombin-evoked Ca2+ entry by
50.2 ± 3.8 (p < 0.001; n = 6).
However, in the absence of external Ca2+ (100 µM EGTA added), 10 µM U0126 was without
effect on the thrombin-induced rise in [Ca2+]i.
The initial peak elevation in [Ca2+]i above basal
after agonist stimulation was 160.8 ± 16.6 nM in
control cells and 165.0 ± 22.6 nM in U0126-treated cells (Fig. 2C; n = 6).
Effect of U0126 on the Maintenance of SMCE--
To investigate the
role of the ERK pathway in the maintenance of SMCE we examined the
effect of U0126 on Ca2+ entry in platelets after SMCE had
been previously stimulated using TG.
Fig. 3 shows the effect of the addition
of U0126 to store-depleted human platelets. 10 µM U0126
or the vehicle were added 3 min after TG and cells were then incubated
for a further 30 min before the addition of Ca2+ to the
medium (final concentration 300 µM) to initiate
Ca2+ entry. As shown in Fig. 3, at the time when U0126 was
added Ca2+ entry was already stimulated (Control
t = 3 min). Addition of U0126 after activation of SMCE did
not significantly alter Ca2+ entry (Fig. 6;
p = 0.77; n = 6). These observations
suggest a role for the ERK cascade in the activation but not in the
maintenance of SMCE. In addition, these findings demonstrate that U0126
does not act either as a Ca2+ channel blocker or a
Ca2+ chelator.
Role of ERK Pathway in TG-induced Actin Polymerization in Human
Platelets--
A role for the actin cytoskeleton in SMCE has been
suggested in several cell types (7, 31). We have previously shown that
TG induces actin polymerization in dimethyl-BAPTA-loaded human
platelets (16). Stimulation of dimethyl-BAPTA-loaded platelets with 1 µM TG in a Ca2+-free medium raised F-actin
content by 34.1 ± 2.9% compared with control unstimulated cells.
Treatment of human platelets with U0126 at concentrations of 100 nM or 10 µM did not significantly alter the
F-actin content of resting or TG-treated platelets (Table I; p = 0.80;
n = 6) suggesting that the role of ERK in SMCE is not
mediated via actin polymerization. Similar results were obtained when
PD 184352 was used. Treatment of platelets with 100 nM or 3 µM PD 184352 did not modify the F-actin content either in
unstimulated or in TG-treated cells (data not shown). Consistent
with the results reported above, treatment of human platelets for 30 min with 100 nM or 10 µM U0126 after SMCE had
been activated by the addition of TG did not significantly modify
TG-evoked increase in the F-actin content (Table I; p = 0.82; n = 6).
Since depolarization of the membrane potential has been reported to
reduce the driving force for agonist- and store-depletion-evoked Ca2+ entry (32), we have investigated whether the effect of
U0126 could be attributed to changes in membrane potential. We studied the effects of this inhibitor on SMCE in the presence of the
K+ ionophore valinomycin, which stabilizes the platelet
membrane potential close to the K+ equilibrium potential
(33). Treatment of platelets with 10 µM U0126 inhibited
SMCE to the same extent in the presence or absence of valinomycin (3 µM; data not shown). This finding indicates that the
effect of U0126 is not due to a reduction in the membrane potential and
in agreement with previous studies (27, 29) indicates that the
inhibition of SMCE by U0126 is likely to be explained by blockade of
the ERK cascade in human platelets.
Involvement of the ERK Cascade in the Secretion-like Coupling
Model--
As reported previously, secretion-like coupling is the
model that best describes the mechanism of activation of SMCE in
several cell types (3, 4), including platelets (5, 6). A number of
studies have suggested a role for Ras proteins in the activation of
SMCE (13-16). We have recently reported that depletion of the intracellular stores using TG in dimethyl-BAPTA-loaded platelets induced translocation and association of Ras with the plasma membrane, which has been shown to be essential for its activation (17). This
process was prevented by treatment with the farnesylcysteine analog,
FTA (16). In these cells, Ras activity is required for both the
activation of SMCE, by a mechanism partially dependent on the actin
cytoskeleton, and its maintenance (16). To investigate whether the ERK
cascade is a downstream effector of Ras in the activation of SMCE, we
examined the effect of FTA on TG-induced ERK activation. As shown in
Fig. 4A, treatment of
dimethyl-BAPTA-loaded platelets with 40 µM FTA for 10 min
had no effect on basal ERK activity but significantly reduced
TG-induced ERK activation. FTA treatment reduced TG-induced ERK
activation to 7.1 ± 4.3% of control (p < 0.001;
n = 4). In contrast, treatment of human platelets for
40 min with 10 µM Cyt D, conditions that prevent actin
polymerization in these cells (5, 16, 34), did not modify either basal
or TG-stimulated ERK activation. These findings suggest that Ras is an
upstream regulator of the ERK cascade, an effect which might belong to
the actin cytoskeleton-independent branch of the
Ras-dependent activation of SMCE in these cells.
We further investigated if other components of the secretion-like
coupling model, such as the phosphatidylinositol 3- and 4-kinases (PI3K
and PI4K; Ref. 19) or protein tyrosine kinases (12), modulate the
activity of ERK. As shown in Fig. 4B, incubation of human
platelets at 37 °C in the presence of either 1 µg/ml methyl-2,5-dihydroxycinnamate for 30 min, a treatment that
we have recently show to abolish store depletion-induced tyrosine phosphorylation (12), or 10 µM PP1 for 10 min, to inhibit
the tyrosine kinases of the Src family, had no effect on either basal or store depletion-induced ERK activation (n = 3).
Furthermore, we have found that inhibition of protein-tyrosine
phosphatases by treatment of platelets for 30 min with 10 µM phenylarsine oxide or 100 µM sodium
vanadate had no effect on U0126-induced inhibition of SMCE (Fig.
4C; n = 4), providing further evidence for
the independence of the ERK cascade from the protein tyrosine
phosphorylation/dephosphorylation process. Preincubation of platelets
with 100 µM LY294002 for 30 min has been shown to inhibit
both PI3K and PI4K in human platelets (19). Treatment of platelets with
100 µM LY294002 did not alter basal or store
depletion-stimulated ERK activation (Fig. 4B,
n = 3). Since both tyrosine kinases and
phosphatidylinositol kinases have been shown to be involved in the
cytoskeleton-dependent branch of Ras-dependent
activation of SMCE (12, 19), these findings further suggest that the
ERK cascade might be a component of the actin cytoskeleton-independent
pathway of Ras-mediated SMCE in platelets.
Protein kinase C (PKC), has been shown to be important in mediating
Gq-dependent activation of the ERK cascade
(18). To investigate whether PKC activity is required for store
depletion-induced ERK activation, we examined the effect of the PKC
inhibitor Ro-31-8220. Treatment of human platelets for 5 min with
Ro-31-8220 (3 µM) was without effect on either basal or
store depletion-induced ERK activation (Fig. 4B;
n = 3), indicating that store depletion-stimulated ERK
activation does not require PKC activity.
Complementary Effects of ERK and the Actin Cytoskeleton in
Ras-dependent SMCE in Human Platelets--
To further
investigate whether the ERK cascade and actin polymerization are two
independent pathways involved in Ras-dependent activation
of SMCE, we have investigated the combined effect of both mechanisms on
the activation of SMCE. Human platelets were incubated at 37 °C in
the absence or presence of 10 µM U0126 for 30 min, 10 µM Cyt D for 40 min, or both agents in combination or 40 µM FTA for 10 min. Platelets were then stimulated with TG (200 nM) in a Ca2+-free medium and 3 min later
CaCl2 (final concentration 300 µM) was added
to initiate Ca2+ entry. As reported previously (16),
treatment of platelets with Cyt D reduced SMCE by 50%. Similar results
were obtained with U0126 (Fig. 5).
Interestingly, the combined treatment with both Cyt D and U0126 reduced
SMCE to a similar extent to that observed with FTA (Fig. 5). The
greater inhibition observed when Cyt D and U0126 were used in
combination compared with that seen with FTA, could be explained by the
fact that treatment of platelets with FTA was unable to completely
inhibit actin polymerization (16).
ERKs are thought to act as a point of convergence of multiple
cellular signaling pathways since they are activated by a broad range
of biochemical signals. The ERK cascade contains at least three protein
kinases that work in series. The first of these three kinases is a Raf
isoform, commonly Raf-1, which is usually activated by Ras. Once
activated Raf-1 phosphorylates MEKs, a family of dual-specificity
protein kinases that phosphorylate two residues, a threonine and a
tyrosine, to activate their ERK targets (18, 35).
Blood platelets, which are anucleate nondifferentiating cells with no
growth potential, are a useful model for studying the involvement of
ERK in cellular signaling, independently of nuclear DNA-dependent pathways. Two forms of ERK have been
identified in human platelets, p44 ERK1 and p42 ERK2, stimulated by
thrombin, collagen, or phorbol esters (36, 37). However, the ERK signal pathway in platelets remains largely uncharacterized.
The results presented here demonstrate that depletion of the
intracellular Ca2+ stores using TG induced activation of
ERK1 and ERK2, a process which, to our knowledge, is for the first time
here shown to be independent of rises in [Ca2+]i.
In this respect, our data differ from those reported by Chao et
al. (38) in human foreskin fibroblast (HSWP) cells and A431 cells.
In that study, TG-induced ERK activation was Ca2+
dependent; however, epidermal growth factor increased ERK
activity was found to be independent of [Ca2+]i
elevation. This may reflect the fact that the ERK cascade is regulated
by different mechanisms in different cell types, although the
discrepancy may also be due to differences in the experimental
protocol. Chao et al. (38) studied the activation of ERK
after a 15-min treatment with EGTA, which was shown to fully deplete
the intracellular Ca2+ stores, while we have found that in
platelets ERK activation declines significantly 3 min after depletion
of the Ca2+ stores has commenced.
The development in recent years of inhibitors of the ERK cascade, such
as U0126 and PD 184352, two potent and highly specific inhibitors of
MEK (27-29), have been useful for identifying the cellular functions
of the ERK pathway. Indeed, both U0126 and PD 184352 inhibited ERK
activation stimulated by depletion of the Ca2+ stores using
TG. To evaluate the possible involvement of the ERK pathway in SMCE we
examined the effect of these inhibitors on TG-evoked SMCE. Our results
demonstrate that both agents reduced SMCE in a
concentration-dependent manner. However, we found that complete inhibition of ERK activation reduced Ca2+ entry
only by 50%, suggesting that the ERK cascade is only partially required for the activation of SMCE. The finding that U0126 and PD
184352 inhibit Ca2+ entry was confirmed with the use of
thrombin, a physiological agonist that stimulates a large number of
processes in human platelets, including calcium mobilization and ERK
activation (1, 36, 37). In addition, the present study shows that
thrombin-induced ERK activation does not require rises in
[Ca2+]i. Our results clearly show that U0126
inhibited thrombin-induced Ca2+ elevation in the presence
of 1 mM external Ca2+ without having any effect
on the release of Ca2+ from the intracellular stores. In
agreement with the observations reported above, these findings suggest
that the role of ERK on thrombin-induced Ca2+ mobilization
occurs entirely through the modulation of Ca2+ entry.
It remains to be elucidated how the ERK pathway acts to mediate
SMCE. The actin cytoskeleton has been proposed to play a key role in
the activation of SMCE in several cell types (3, 7, 8), including human
platelets (31). Hence, we have investigated the effect of the ERK
cascade inhibitors, U0126 and PD 184352, on store depletion-induced
actin polymerization. Our results clearly show that neither U0126 nor
PD 184352 modify TG-induced actin polymerization in human platelets.
These observations indicate that ERK-mediated activation of SMCE is
unlikely to occur as a result of modulation of actin reorganization.
We have previously reported that different mechanisms are
required for the activation and maintenance of SMCE in platelets (12,
31). Since the ERK cascade participates in the activation of SMCE we
also investigated its possible role in the maintenance of the process.
Addition of the MEK inhibitor U0126 after the activation of SMCE did
not reverse either actin polymerization or Ca2+ entry
activated by TG. This suggests that the ERK cascade is not required for
the maintenance of SMCE. The lack of effect of ERK on the maintenance
of SMCE and actin reorganization is consistent with previous studies
reporting that the actin cytoskeleton is essential for the maintenance
of the secretion-like coupling process underlying SMCE (5).
The results presented above indicate that the effect of U0126 on
Ca2+ entry is not likely to be mediated by nonspecific
effects as a Ca2+ chelator or Ca2+ channel
blocker, since it did not have any effect on Ca2+ entry
when added after depletion of the stores. In addition, our results
indicate that these inhibitors are effective at selectively inhibiting
Ca2+ entry, as demonstrated by the lack of effect on
Ca2+ storage or agonist-evoked Ca2+ release.
Depolarization of the membrane potential has been shown to decrease the
driving force for Ca2+ entry (32). To address this issue we
have conducted experiments in the presence of valinomycin, a potassium
ionophore that clamps the platelet membrane potential close to the
potassium equilibrium potential (33). Our results indicate that U0126
inhibited Ca2+ entry to the same extent in the absence or
presence of valinomycin. Furthermore, U0126 was effective at reducing
the activation but not the maintenance of SMCE, hence, the effect of
this inhibitor cannot be attributable to a modification of membrane
potential. Therefore, these results together with others presented in
this paper indicate that the effect of U0126 on SMCE is more likely to
be explained by inhibition of the ERK pathway. These observations are
in agreement with previous studies reporting the high specificity of
U0126 and PD 184352 as inhibitors of MEK over many other protein kinases (29).
A role for the Ras family of small GTPases in SMCE has been suggested
in several cell types, including platelets (13-16). In human platelets
we have recently reported that the involvement of Ras in SMCE is
mediated via two different, actin cytoskeleton-dependent and cytoskeleton-independent pathways (16). Ras is commonly required
for the activation of Raf-1, the first kinase of the ERK cascade (39).
Hence, the possibility that Ras could mediate TG-induced ERK activation
was investigated. We conducted a series of experiments wherein
platelets were incubated with the farnesylcysteine analog FTA at a
concentration that we have previously reported to inhibit membrane
association and thus activation of Ras (16). Treatment of platelets
with FTA caused no changes in basal ERK activity but abolished
TG-induced ERK activation. In contrast, no effect was observed after
treatment of platelets for 40 min with 10 µM Cyt D, which
completely inhibits TG-induced actin polymerization (16). Thus,
TG-induced ERK activation is likely to be mediated by Ras but is
independent of the actin cytoskeleton. Therefore, the ERK cascade is a
possible downstream effector of Ras that might be involved in the
cytoskeleton-independent branch of the Ras-mediated SMCE mechanism.
Consistent with the above, we have found that the activity of
phosphatidylinositol 3- and 4-kinases or tyrosine kinases, which
modulate SMCE through the reorganization of the actin cytoskeleton (12,
19), is not required for store depletion-induced ERK activation in
platelets. Furthermore, inhibition of protein-tyrosine phosphatases did
not impair U0126-induced inhibition of SMCE. These findings provide
further evidence for the involvement of the ERK cascade on the actin
cytoskeleton-independent branch of the Ras-dependent
activation of SMCE in human platelets.
In agreement with the above, we have previously reported (16) that
treatment with FTA resulted in a massive reduction in SMCE while Cyt D
or U0126 were about half as effective as FTA. Furthermore, the effects
of Cyt D and U0126 were found to be additive. This indicates that the
actin cytoskeleton and the ERK cascade mediate SMCE by independent
pathways. Our results allow us to propose that the ERK pathway might be
a candidate for the cytoskeleton-independent branch of Ras-mediated
SMCE in human platelets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 1.
Thapsigargin-induced ERK activation is
important for the activation of store-mediated Ca2+ entry
in human platelets. Dimethyl-BAPTA-loaded human platelets were
treated with either 1 µM TG for various periods of time
(A) or for 3 min with various concentrations of TG (10 nM-1 µM) as indicated (B) or were
preincubated for 30 min at 37 °C with various concentrations of
U0126 (10 nM to 100 µM) as indicated and then
treated with 1 µM TG for 3 min (C) and then
lysed. Samples were subjected to SDS-PAGE and Western blotting with the
specific phospho-p44/42 ERK monoclonal antibody (E10) as described
under "Experimental Procedures." Bands were revealed using
chemiluminescence, and were quantified using scanning densitometry. The
panels show results from one experiment representative of
three others. D, fura-2-loaded human platelets were
incubated for 30 min in the presence of different concentrations of
U0126 (10 nM to 100 µM) at 37 °C and then
stimulated with TG (200 nM) in a Ca2+-free
medium. Three min later CaCl2 (final concentration 300 µM) was added to the medium to initiate Ca2+
entry. Elevations in [Ca2+]i were monitored using
the 340/380 nm ratio and traces were calibrated in terms of
[Ca2+]i. Traces shown are representative of six
separate experiments.
View larger version (23K):
[in a new window]
Fig. 2.
Effect of U0126 on thrombin-evoked ERK
activation and [Ca2+]i elevations in the presence
and absence of external Ca2+. A,
dimethyl-BAPTA-loaded human platelets were preincubated for 30 min at
37 °C in the absence or presence of U0126 (10 µM) as
indicated and then treated with 1 unit/ml thrombin for 3 min and lysed.
Samples were subjected to SDS-PAGE and Western blotting with the
specific phospho-p44/42 ERK monoclonal antibody (E10) as described
under "Experimental Procedures." Bands were revealed using
chemiluminescence, and were quantified using scanning densitometry. The
panel shown is representative of three independent experiments.
B and C, fura-2-loaded human platelets were
incubated for 30 min at 37 °C in the presence of either U0126 (10 µM) or dimethyl sulfoxide (Control). At the
time of experiment either 1 mM CaCl2
(B) or 100 µM EGTA (C) were added.
Cells were then stimulated with thrombin (1 unit/ml) at the time
indicated. Elevations in [Ca2+]i were monitored
using the 340/380 nm ratio and traces were calibrated in terms of
[Ca2+]i. Traces shown are representative of six
separate experiments.
View larger version (22K):
[in a new window]
Fig. 3.
Effect of U0126 on the maintenance of
store-mediated Ca2+ entry. Fura-2-loaded human
platelets were suspended in a Ca2+-free medium (100 µM EGTA added) as described under "Experimental
Procedures." Cells were then stimulated with TG (200 nM)
and 3 min later 10 µM U0126 or the vehicle (dimethyl
sulfoxide; control t = 33 min) were added as indicated
by the thick arrow. CaCl2 (final concentration
300 µM) was added to the medium at the same time, as a
control (control t = 3 min) or 30 min after the
addition of U0126 or the vehicle to initiate Ca2+ entry.
Traces are representative of six independent experiments.
Lack of effect of U0126 on the F-actin content of washed and
preactivated platelets
View larger version (34K):
[in a new window]
Fig. 4.
Involvement of the ERK cascade in the
secretion-like coupling model. A, dimethyl-BAPTA-loaded
human platelets were preincubated at 37 °C in the absence or
presence of either 40 µM FTA for 10 min or 10 µM Cyt D for 40 min as indicated and then treated with 1 µM TG for 3 min and lysed. B,
dimethyl-BAPTA-loaded human platelets were preincubated at 37 °C in
the absence or presence of 1 µg/ml
methyl-2,5-dihydroxycinnamate (M-2,5-DHC) for 30 min, 10 µM PP1 for 10 min, 100 µM LY294002
for 30 min, or 3 µM Ro-31-8220 for 5 min as indicated and
then stimulated with 1 µM TG for 3 min and lysed. Samples
were subjected to SDS-PAGE and analyzed by Western blotting with the
specific phospho-p44/42 ERK monoclonal antibody (E10) as described
under "Experimental Procedures." Bands were revealed using
chemiluminescence. The panels show results from one
experiment representative of three others. C, fura-2-loaded
human platelets were incubated at 37 °C for 30 min in the presence
of 10 µM phenylarsine oxide (PAO), 100 µM sodium vanadate or the vehicle and then with U0126 (10 µM) or the vehicle (Control) for a further 30 min. Cells were then stimulated with TG (200 nM) in a
Ca2+-free medium. Three min later CaCl2 (final
concentration 300 µM) was added to the medium to initiate
Ca2+ entry. Elevations in [Ca2+]i
were monitored using the 340/380 nm ratio and traces were calibrated in
terms of [Ca2+]i. Traces shown are representative
of four separate experiments.
View larger version (38K):
[in a new window]
Fig. 5.
Role of ERK and the actin cytoskeleton in
store-mediated Ca2+ entry in human platelets.
Fura-2-loaded human platelets were incubated at 37 °C either in the
presence of 10 µM U0126 for 30 min, 10 µM
Cyt D for 40 min, both agents for the times indicated, 40 µM FTA for 10 min, or the vehicles as control. At the
time of experiment 100 µM EGTA was added. Cells were then
stimulated with 200 nM TG and 3 min later CaCl2
(final concentration 300 µM) was added to the medium to
initiate Ca2+ entry. Elevations in
[Ca2+]i were monitored using the 340/380 nm ratio
and traces were calibrated in terms of [Ca2+]i.
Histograms represent the percentage of Ca2+ entry, measured
as described under "Experimental Procedures," relative to control
for each of the treatments. Values shown are mean ± S.E. of six
separate experiments. *, p < 0.05 compared with the
combined treatment with U0126 and Cyt D.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Grant of Junta de Extremadura-Consejería de
Educación y Juventud and Fondo Social Europeo, Spain.
§ To whom correspondence should be addressed: Dept. of Physiology, Downing Street, University of Cambridge, Cambridge CB2 3EG, United Kingdom. Tel.: 44-1223-333870; Fax: 44-1223-333840; E-mail: sos10@cam.ac.uk.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M009218200
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ABBREVIATIONS |
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The abbreviations used are: [Ca2+]i, intracellular free calcium concentration; SMCE, store-mediated calcium entry; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; TG, thapsigargin; Cyt D, cytochalasin D; FTA, farnesylthioacetic acid; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; BAPTA, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid.
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