Hydrogen peroxide stimulates tyrosine phosphorylation of focal
adhesion kinase in vascular endothelial cells
Suryanarayana
Vepa1,
William M.
Scribner2,
Narasimham L.
Parinandi1,
Denis
English3,
Joe G. N.
Garcia1, and
Viswanathan
Natarajan1
1 Division of Pulmonary and
Critical Care Medicine, Department of Medicine, Johns Hopkins
School of Medicine, Baltimore, Maryland 21224;
2 Department of Medicine,
Indiana University School of Medicine, and
3 Bone Marrow Transplantation
Laboratory, Methodist Hospital of Indiana, Indianapolis, Indiana
46202
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ABSTRACT |
Reactive oxygen species (ROS) are implicated in
the pathophysiology of several vascular disorders including
atherosclerosis. Although the mechanism(s) of ROS-induced vascular
damage remains unclear, there is increasing evidence for ROS-mediated
modulation of signal transduction pathways. Exposure of bovine
pulmonary artery endothelial cells to hydrogen peroxide
(H2O2)
enhanced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa
cellular proteins, which were determined by immunoprecipitation with
specific antibodies focal adhesion kinase
(p125FAK) and paxillin (p68).
Brief exposure of cells to a relatively high concentration of
H2O2
(1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation
of FAK, which reached maximum levels within 10 min (290% of basal
levels). Cytoskeletal reorganization as evidenced by the appearance of
actin stress fibers preceded H2O2-induced
tyrosine phosphorylation of FAK, and the microfilament disruptor
cytochalasin D also attenuated the tyrosine phosphorylation of FAK.
Treatment of BPAECs with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM attenuated
H2O2-induced
increases in intracellular Ca2+
but did not show any consistent effect on
H2O2-induced
tyrosine phosphorylation of FAK. Several tyrosine kinase inhibitors,
including genistein, herbimycin, and tyrphostin, had no detectable
effect on tyrosine phosphorylation of FAK but attenuated the
H2O2-induction of mitogen-activated protein kinase activity. We conclude that H2O2-induced
increases in FAK tyrosine phosphorylation may be important in
H2O2-mediated
endothelial cell activation.
signal transduction; tyrosine kinase; cytoskeleton; oxidants
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INTRODUCTION |
REACTIVE OXYGEN SPECIES (ROS) or oxidants
[superoxide anion, hydroxyl radical, hydrogen peroxide
(H2O2),
hypochlorous acid, and peroxynitrite] have been implicated in
cell and tissue damage initiated by a number of agents, including
inflammatory mediators, hyperbaric oxygen, and ischemic tissue
reperfusion (15). Several studies have provided clear evidence that
vascular disorders, including atherosclerosis, pulmonary hypertension,
and adult respiratory distress syndrome, result, at least in part, from
the activity of these highly reactive, short-lived oxidative agents
(5). When generated at relatively high levels by activated leukocytes, for example, ROS can cause cell injury through oxidative damage to
cellular macromolecules (15). Recent studies suggested that low levels
of ROS modulate signal transduction pathways in mammalian cells (41).
For example,
H2O2
stimulates protein phosphorylation (34), activates protein kinases
(PKs) (1, 3, 13, 19), inhibits tyrosine phosphatases (7), alters
intracellular Ca2+
(Ca2+i) (35), stimulates phospholipases
(24), and regulates transcription factors such as nuclear factor-
B and activator protein-1 (36).
H2O2
activated several members of the mitogen-activated protein kinase
(MAPK) family in vascular endothelial cells (ECs), fibroblasts, and
smooth muscle cells (1, 13, 19; Scribner, Vepa, and Natarajan,
unpublished data) through a tyrosine kinase-dependent mechanism.
H2O2
alone or in combination with vanadate, an inhibitor of protein tyrosine
phosphatases, mimics several of the metabolic and growth-promoting
effects of growth factors and insulin (18). The biochemical and
molecular pathways involved in the oxidant-mediated activation of PKs
and the contribution of these changes to oxidative stress are yet to be
clearly understood.
Focal adhesion kinase (FAK;
p125FAK) is a non-receptor
tyrosine kinase involved in the structure and function of focal
adhesions (12), which are critical for cellular integrity by
maintaining cell-cell and cell-matrix interactions. Tyrosine
phosphorylation of FAK and paxillin (p68) has been implicated as an
early event in signal transduction initiated by integrins (12), growth
factors (2), bioactive lipids such as lysophosphatidic acid (LPA), sphingosine and sphingosylphosphorylcholine (8, 32, 38), and
extracellular matrix proteins (4, 16). Tyrosine phosphorylation of
focal adhesion-associated proteins induced by these ligands is
accompanied by alterations in the actin cytoskeletal network and
relocation of focal adhesion-associated proteins (12, 27). Although ROS
are known to alter cytoskeletal organization (19), which is vital for
cell migration, differentiation, and cellular permeability changes, the
effects of these agents on the individual proteins involved in
cytoskeletal reorganization and focal adhesion have not been well
studied. A recent study (40) examined the role of FAK in
H2O2-induced
apoptosis in T98G cells and concluded that tyrosine phosphorylation of
FAK by
H2O2
acted as a suppressor of apoptosis. Gozin et al. (11) reported hydroxyl
radical-induced tyrosine phosphorylation of FAK and paxillin in human
umbilical vein endothelial cells. The present study was undertaken to
examine the ability of
H2O2
to stimulate tyrosine phosphorylation of focal adhesion proteins in
vascular ECs. Our data indicate that
H2O2 induces both FAK and paxillin tyrosine phosphorylation through tyrosine
phosphatase inhibition more than through activation of tyrosine kinases.
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METHODS |
Materials. Medium 199 (M199), fetal bovine serum, trypsin, Dulbecco's phosphate-buffered
saline (PBS),
H2O2,
12-O-tetradecanoylphorbol 13-acetate
(TPA), genistein, myelin basic protein (MBP) fragment, and cytochalasin
D were obtained from Sigma (St. Louis, MO). Herbimycin, tyrphostin 47, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and bisindolylmaleimide I were purchased from Calbiochem (La Jolla, CA). Monoclonal antibodies against FAK and paxillin were procured from Transduction Laboratories (Lexington, KY),
and anti-phosphotyrosine antibody 4G10 was purchased from Upstate
Biotechnology (Lake Placid, NY). MAPK antibodies [extracellular signal-regulated kinase (ERK) 1 and ERK2], polyclonal FAK
antibodies and protein A/G plus agarose were from Santa Cruz
Biotechnology (Santa Cruz, CA). Rhodamine-phalloidin and fura 2-AM were
obtained from Molecular Probes (Eugene, OR). Precast acrylamide gels
were purchased from NOVEX (San Diego, CA), and all electrophoretic reagents were from Bio-Rad (Hercules, CA). Bovine pulmonary artery ECs
(BPAECs; passage 16) were obtained
from American Type Culture Collection (Manassas, VA).
Cell culture. BPAECs were cultured as
previously described (24). Subcultured BPAECs (from
passages 18 to
21), used in all the experiments,
showed cobblestone morphology and stained positive for factor
VIII-related antigen. In our experiments, we have not observed any
differences in EC response to oxidants whether the cells are serum
starved or not. Thus, in all our experiments, we used cells grown in
serum-containing medium.
Immunoprecipitation and Western
blotting. BPAECs grown on
T-75cm2 flasks were washed once
with serum-free M199 and stimulated with H2O2
or other agents in serum-free M199 for specified time periods. The
cells were washed once in ice-cold PBS and again in ice-cold PBS
containing 1 mM sodium orthovanadate. The cells (5×
106) were scraped into 1 ml of
lysis buffer (20 mM Tris · HCl, pH 7.4, containing
0.5% deoxycholic acid, 0.5% SDS, 1% Triton X-100, 1% Nonidet P-40,
0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml of leupeptin, 5 µg/ml
of aprotinin, and 1 mM sodium orthovanadate). The samples were
centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatants
were used for immunoprecipitation with either anti-FAK, anti-paxillin,
or anti-MAPK antibody (2-4 µg) at 4°C for 4 h. Protein A/G
plus agarose (20 µl) was then added and incubated for an additional 3 h at 4°C. The antigen-antibody complex was pelleted, washed three
times with ice-cold lysis buffer, and either dissociated by boiling in
1× SDS sample buffer for 5 min or washed once with the
nondenaturing assay buffer used for kinase assays [50 mM PIPES,
pH 7.0, 10 mM MgCl2, 3 mM
MnCl2, and 0.1 mM dithiothreitol (DTT)]. The samples were then analyzed on 8 or 10% SDS-PAGE
gels. After SDS-PAGE, the proteins were transferred to Immobilon-P
membranes by electroblotting, blocked with blocking buffer (GIBCO BRL,
Life Technologies), and incubated for 18-24 h at 4°C with
either anti-FAK (1:1,000 dilution), anti-paxillin (1:5,000 dilution),
anti-phosphotyrosine (1 µg/ml), or anti-MAPK (1 µg/ml) antibody.The
membranes were washed four times with PBS containing 0.1% Tween 20 followed by incubation with goat anti-rabbit or anti-mouse IgG
conjugated to horseradish peroxidase (1:3,000 dilution) for 1 h at room
temperature, and the blots were developed with enhanced
chemiluminescence. Densitometric scanning of the blots was carried out
with a Bio-Rad model GS-700 densitometer and quantified with Molecular
Analyst software.
Rhodamine-phalloidin staining for visualization of
actin stress fibers. BPAECs grown on glass coverslips
were washed twice with M199 containing HEPES and incubated for the
indicated times at 37°C in medium with and without
H2O2.
Thereafter, for actin staining, the cells were washed twice with
ice-cold PBS and fixed in 3.0% formaldehyde in PBS for 10 min at room
temperature. The cells were rinsed three times with PBS and
permeabilized with PBS containing 0.1% Triton X-100 for 5 min at
22°C. The permeabilized cells were then washed three times in PBS
and subsequently incubated with rhodamine-phalloidin (1 U/coverslip)
for 30 min in the absence of light. Unbound rhodamine-phalloidin was
removed by washing with PBS three times. The coverslips were mounted
onto glass slides and sealed with nail polish. Fluorescent images of
actin fibers were obtained with confocal microscopy at a magnification
of ×100 (Bio-Rad model MRC1024 with a krypton/argon laser system)
as described elsewhere by Hart et al. (17) and Schaphorst et al.
(33).
In vitro kinase assays. For MAPK
assays, an MBP fragment was used as a substrate (13).
Immunoprecipitates obtained with anti-ERK1 and anti-ERK2 antibodies
were washed once with kinase assay buffer (50 mM PIPES, pH 7.0, 10 mM
MgCl2, 3 mM
MnCl2, and 0.1 mM DTT) and
incubated with a reaction mixture (50 µl) that contained 5 µg of
MBP, 10 µCi of
[
-32P]ATP
(10 µM), and kinase assay buffer. The reaction was
carried out at 37°C for 30 min and terminated by adding 10 µl of
6× Laemmli electrophoresis sample buffer and boiling for 5 min.
MBP phosphorylation was measured after SDS-PAGE (14% gels) and
autoradiography. In vitro kinase assays to determine the
autophosphorylation of FAK were performed according to the protocol
described by Rankin et al. (28). Briefly, immunoprecipitates of FAK
prepared from BPAEC lysates after various treatments were washed
extensively and suspended in kinase assay buffer (50 mM PIPES, pH 7.0, 10 mM MnCl2, and 1 mM DTT). The
reaction mixture was incubated for 10 min at 30°C in the presence
of 0.25 µCi
[
-32P]ATP/µl
reaction mixture. The reactions were stopped by the addition of
6× Laemmli electrophoresis sample buffer and boiling for 5 min.
In vitro phosphorylation of FAK was measured after SDS-PAGE (8% gels)
and autoradiography.
Measurement of
Ca2+i
concentration. BPAECs grown on glass coverslips were
loaded with fura 2-AM as described earlier (23). Fura 2-loaded BPAECs were challenged with different agents and studied in a
thermostat-regulated sample compartment of an SLM
spectrofluorometer controlled by a PC program to excite the fura
2-loaded cells alternately at 340 and 380 nm, and fluorescence was
monitored at 510 nm. The ratios of fluorescence intensities at 340 and
380 nm were calculated. In all experiments, measurements were corrected
for autofluorescence from cells not loaded with fura 2.
 |
RESULTS |
H2O2 increases
tyrosine phosphorylation in BPAECs.
To assess the effect of
H2O2
on protein tyrosine phosphorylation, BPAECs were incubated with varying
concentrations of
H2O2 for 10 min, and the cell lysates were subjected to SDS-PAGE and Western
blotting with anti-phosphotyrosine antibody. As shown in
Fig.1,
H2O2
in a time- and dose-dependent manner stimulated tyrosine
phosphorylation of several proteins between 50 and 200 kDa, including
heavily phosphorylated bands at 110-130 and 65-80 kDa. The
increase in tyrosine phosphorylation of 110- to 130- and 65- to 80-kDa
proteins reached a maximum (164 and 457% over basal levels,
respectively) at 10 min of treatment with 1.0 mM H2O2
(Fig. 1A) and was linear from 0.05 to 1 mM
H2O2
(Fig. 1B). However, significant
increases were observed from 0.1 mM
H2O2. The concentrations and time periods of
H2O2
employed in this present study have been found to be noncytotoxic (24),
and although the exact physiological relevance of these doses has not
been determined, it has been speculated that under certain conditions oxidants are released into a relatively sequestered microenvironment, thus creating a high-oxidant concentration (43).

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Fig. 1.
Time course and dose response of
H2O2-stimulated
tyrosine phosphorylation. Lysates (20 µg of protein) prepared from
bovine pulmonary artery endothelial cells (BPAECs) treated with either
1 mM
H2O2
for various time intervals (A) or
different concentrations of
H2O2
for 10 min (B) were subjected to
SDS-PAGE and membrane transfer and analyzed by Western blotting with
anti-phosphotyrosine antibody 4G10 followed by enhanced
chemiluminescence as described in
METHODS. Brackets, range of proteins
in which tyrosine phosphorylation was increased; arrows, protein bands
used in quantitation. Blots are representative of 3 independent
experiments. Nos. on left, molecular
mass.
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Stimulation of FAK and paxillin tyrosine phosphorylation by
H2O2.
Because ROS treatment of BPAECs produced a pattern of tyrosine
phosphorylation very similar to that induced by bombesin, sphingosine, and sphingophosphorylcholine in Swiss 3T3 cells (28, 38), we investigated whether FAK and paxillin were tyrosine phosphorylated in response to
H2O2
in BPAECs. The cell lysates from control and H2O2-treated
BPAECs were incubated with either anti-FAK or anti-paxillin monoclonal
antibodies, and the immunoprecipitates were probed with
anti-phosphotyrosine, anti-FAK, or anti-paxillin antibody. As shown in
Fig.2,
A and
C, at 1.0 mM
H2O2,
tyrosine phosphorylation of FAK was rapid, was detected as early as 30 s of exposure, was linear up to 10 min (290% of basal levels), and
declined thereafter, reaching near basal levels at 60 min.
H2O2-induced
FAK tyrosine phosphorylation was concentration dependent (330% of
control value with 1 mM
H2O2)
and was observed with concentrations of
H2O2
as low as 125 µM at 10 min (Fig. 2,
B and
D). Generation of ROS by xanthine
(100 µM)/xanthine oxidase (10 U/ml) also resulted in a time-dependent
increase in FAK phosphorylation (data not shown). In addition to FAK,
we also investigated the effect of
H2O2
on tyrosine phosphorylation of paxillin. Similar to FAK, treatment of
BPAECs with
H2O2
(1 mM) also enhanced tyrosine phosphorylation of paxillin as evidenced
by Western blotting analysis of anti-paxillin immunoprecipitates with
anti-phosphotyrosine (Fig.
3). Although H2O2-induced
tyrosine phosphorylation of paxillin also showed a time and
concentration dependence (Fig. 3), the time course of tyrosine
phosphorylation of paxillin was different compared with that of FAK.
These results indicate that
H2O2
induces tyrosine phosphorylation of FAK and paxillin in BPAECs, and on
the basis of these results, for all further experiments, ECs were
incubated with 1 mM
H2O2
for 10 min.


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Fig. 2.
H2O2-induced
tyrosine phosphorylation of focal adhesion kinase (FAK).
A: confluent BPAECs were treated with
1 mM
H2O2
for indicated time periods, and cell lysates (1 mg of protein) were
subjected to immunoprecipitation with anti-FAK monoclonal antibody
followed by immunoblotting (I.B.) of equal amount of immunoprecipitates
with anti-phosphotyrosine (A-PTyr) and anti-FAK (A-FAK) antibodies as
described in METHODS.
B: confluent BPAECs were exposed to
indicated concentrations of
H2O2
for 10 min. Cell lysates (1 mg of protein) were immunoprecipitated with
A-FAK, and equal aliquots of immunoprecipitates were further analyzed
by Western blotting with A-PTyr and A-FAK.
C and
D: densitometric analysis of blots in
A and
B, respectively, was performed as
described in METHODS, and tyrosine
phosphorylation was normalized for amount of FAK and is presented as
percent increase over control levels. Blots are representative of 3 independent experiments.
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Fig. 3.
H2O2-induced
tyrosine phosphorylation of paxillin.
A: confluent BPAECs were treated with
1 mM
H2O2
for indicated time periods, and cell lysates (1 mg of protein) were
subjected to immunoprecipitation with anti-paxillin monoclonal antibody
(A-Paxillin) followed by immunoblotting of equal amount of
immunoprecipitates with A-PTyr and A-Paxillin as described in
METHODS.
B: confluent BPAECs were exposed to
indicated concentrations of
H2O2
for 10 min. Cell lysates (1 mg of protein) were immunoprecipitated with
A-Paxillin, and equal aliquots of immunoprecipitates were further
analyzed by Western blotting with A-PTyr and A-Paxillin.
C and
D: densitometric analysis of blots in
A and
B, respectively, was performed as
described in METHODS, and tyrosine
phosphorylation was normalized for amount of paxillin and is presented
as percent increase over control levels. Blots are representative of 3 independent experiments.
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Effect of H2O2
on FAK autophosphorylation activity.
An increase in FAK phosphorylation by growth factors or sphingosine
derivatives has been associated with increased FAK activity (30, 31).
To examine whether the
H2O2-mediated
increase in FAK tyrosine phosphorylation was associated with a
concomitant increase in FAK activity, we tested the FAK
immunoprecipitates obtained from
H2O2-treated
EC lysates. As shown in Fig.
4, no increase in in vitro
autophosphorylation activity of FAK was observed in anti-FAK
immunoprecipitates from
H2O2-treated
ECs. Similar results were also obtained when we used a protocol
described by Rodriguez-Fernandez and Rozengurt (30) to carry out
autophosphorylation with a polyclonal FAK antibody (data not shown).
These data suggest that the sites that are being tyrosine
phosphorylated in response to
H2O2
probably are not involved in regulating the autophosphorylating activity of FAK.

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Fig. 4.
Effect of
H2O2
on autophosphorylating activity of FAK. Confluent BPAECs were treated
with
H2O2
for 10 min, and cell lysates (1 mg of protein) were subjected to
immunoprecipitation (I.P.) and I.B. with A-FAK. Equal amounts of
immunoprecipitate were subjected to autophosphorylation reaction with
[ -32P]ATP (10 µCi, 10 µM), separated by SDS-PAGE, and subjected to
autoradiography as described in
METHODS. This experiment is
representative of 3 independent determinations. Nos. on
right, molecular mass.
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H2O2
stimulates actin stress-fiber formation in BPAECs.
Tyrosine phosphorylation of FAK has been shown to be an early event in
cell adhesion that is crucial to the formation and assembly of focal
adhesion structures (12, 27) and is invariably preceded by actin
reorganization. We therefore examined whether ROS modulated actin
stress-fiber orientation and cytoskeletal organization in ECs. As shown
in Fig. 5, resting BPAECs contain very few
actin stress fibers, and treatment of BPAECs with
H2O2 (100 µM and 1 mM for 2 min) caused changes in the organization and
orientation of actin stress fibers. Within 2 min,
H2O2
caused reorganization of actin fibers throughout the cytoplasm (data not shown), and by 10 min, the cells were packed with numerous stress
fibers (Fig. 5). We examined whether disruption of the actin stress
fibers could affect the ROS-mediated increase in FAK tyrosine
phosphorylation. Preincubation of BPAECs for 30 min with cytochalasin D
(5-10 µM), a selective disruptor of the intracellular actin
cytoskeletal network, attenuated
H2O2-induced
tyrosine phosphorylation of FAK (Fig. 6),
suggesting a link between FAK activation and actin stress-fiber
formation.

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Fig. 5.
Effect of
H2O2
on actin stress-fiber formation. Confluent BPAECs grown on glass
coverslips were washed twice with medium 199 and incubated with 100 µM and 1 mM
H2O2
for 10 min. Cells were subsequently fixed in formaldehyde and stained
for F-actin with rhodamine-phalloidin. Changes in actin cytoskeleton
were visualized with a confocal microscope at a magnification of
×100 as described in METHODS.
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Fig. 6.
Effect of cytochalasin D on
H2O2-induced
tyrosine phosphorylation of FAK. Confluent BPAECs were treated for 45 min with cytochalasin D and then incubated with (+) and without
( )
H2O2
for 10 min. Cell lysates (1 mg of protein) were subjected to
immunoprecipitation with A-FAK, and immunoprecipitates were divided
into equal aliquots and analyzed by Western blotting with A-FAK or
A-PTyr. Blot is representative of 3 independent experiments.
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H2O2-mediated
increases in FAK tyrosine phosphorylation is independent of PKC.
We examined the role of PKC on tyrosine phosphorylation of FAK by
incubating cells with the PKC activator TPA. Treatment of BPAECs with
TPA (100 nM) for 10 min had no effect on the tyrosine phosphorylation
of FAK (Fig. 7), and the same concentration of TPA also
had no effect on
H2O2-induced
FAK tyrosine phosphorylation (Fig. 7). Preincubation of the cells for 1 h with the specific PKC inhibitor bisindolylmaleimide I followed by
treatment with H2O2
(1 mM, 10 min) did not have any effect on
H2O2-induced
FAK phosphorylation (data not shown).

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Fig. 7.
Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on
H2O2-induced
tyrosine phosphorylation of FAK. BPAECs were preincubated with either
medium 199 or medium 199 containing TPA for 30 min and challenged with
H2O2
for 10 min, and cell lysates were prepared as described in
METHODS. Cell lysates (1 mg of
protein) were subjected to immunoprecipitation with A-FAK, and equal
amounts of immunoprecipitates were analyzed by Western blotting with
A-PTyr or A-FAK. Blot is representative of 2 separate experiments.
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Effect of
Ca2+ on
H2O2-mediated
increases in FAK tyrosine phosphorylation.
Changes in Ca2+i have been shown to
modulate signaling pathways involving protein tyrosine phosphorylation (20). We therefore examined the role of
Ca2+i in
H2O2-induced
FAK phosphorylation.
H2O2
treatment of BPAECs grown on coverslips increased
Ca2+i (Fig.
8A) as
measured by the emitted fluorescence at 510 nm of fura 2-loaded cells
excited at 340 and 380 nm but was quenched by BAPTA-AM, a chelator of
free Ca2+i (24). As shown in Fig. 8B, treatment of BPAECs with BAPTA
showed no attenuation of
H2O2-induced FAK tyrosine phosphorylation. Although we have observed an increase of
tyrosine phosphorylation with high concentrations of BAPTA, this
increase was not reproducible. These data indicate that the H2O2-stimulated
tyrosine phosphorylation of FAK is independent of changes in
Ca2+i in ECs.

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Fig. 8.
Effect of Ca2+ on
H2O2-induced
tyrosine phosphorylation of FAK. A:
BPAECs grown on glass coverslips and loaded with fura 2 as described in
METHODS were treated with Hanks'
balanced salt solution (HBSS) or HBSS containing
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM for 30 min (30') and challenged with
H2O2.
Intracellular Ca2+ was measured
with a luminescence spectrometer as described in
METHODS.
B: confluent BPAECs were incubated
with medium 199 or medium 199 containing BAPTA-AM for 30 min followed
by a 10-min exposure to
H2O2.
Cell lysates (1 mg of protein) were subjected to immunoprecipitation
with A-FAK and analyzed by Western blotting for phosphotyrosine or FAK
with appropriate antibodies. Blot is representative of 3 independent
experiments. Densitometric analysis of blot was performed, and relative
intensities were calculated.
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H2O2-mediated
increases in FAK tyrosine phosphorylation is resistant to tyrosine
kinase inhibitors.
Because protein tyrosine phosphorylation is a balance between protein
tyrosine kinases and tyrosine phosphatases, we investigated the effect
of tyrosine kinase inhibitors on
H2O2-induced
FAK tyrosine phosphorylation. BPAECs pretreated with genistein (100 µM, 3 h), tyrphostin (100 µM, 3 h), or herbimycin (5 µM, 5 h)
before being challenged with
H2O2
showed no effect on
H2O2-induced
FAK phosphorylation (Fig. 9). However, under similar
experimental conditions, these tyrosine kinase inhibitors attenuated
H2O2-induced
MAPK activity as measured by MBP phosphorylation (Fig. 9). These
results suggest that
H2O2
differentially regulates MAPK activation and increases in tyrosine
phosphorylation of FAK.

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Fig. 9.
Effect of tyrosine kinase inhibition on mitogen-activated protein
kinase (MAPK) activity (A) and FAK
phosphorylation (B). BPAECs were
treated with either medium 199 or medium 199 containing genistein (3 h), herbimycin (5 h), or tyrphostin (3 h) followed by exposure to
H2O2
for 10 min. Cell lysates (1 mg of protein) were immunoprecipitated with
anti-extracellular signal-regulated kinases 1 and 2. Immunoprecipitates
were assayed for MAPK activity with myelin basic protein (MBP) and
[ -32P]ATP as
described in METHODS. Arrow, position
of [ -32P]MBP on
14% SDS-PAGE. Similarly, cell lysates (1 mg of protein) prepared as
described above were immunoprecipitated with A-FAK and subsequently
analyzed by Western blotting with A-PTyr. Data are representative of 3 experiments. Nos. on right, molecular
mass.
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DISCUSSION |
ROS-mediated alterations in cellular signaling pathways play an
important role in vascular dysfunction (5). Natarajan et al. (25)
previously reported a functional link between oxidant-induced phospholipase D activation and protein tyrosine phosphorylation. The
present work is an attempt to further identify individual proteins in
which tyrosine phosphorylation is altered on oxidant exposure and to
elucidate the contribution of these to the oxidant injury. The tyrosine
phosphorylation profile observed in the present study was very similar
to those described with LPA, bombesin, and sphingophosphorylcholine (8,
28, 31, 32, 38, 39). Because previous studies (2, 26, 28, 32, 38, 39) have shown that growth factors, hormones, and bioactive lipids stimulate tyrosine phosphorylation of FAK and paxillin in mammalian cells, we investigated the possible effect of
H2O2
in modulating the tyrosine phosphorylation of FAK and paxillin in ECs.
The results of the present study clearly show a dose- and
time-dependent increase in the tyrosine phosphorylation of FAK and
paxillin. However, our results suggest differences in the time course
of
H2O2-induced tyrosine phosphorylation of FAK and paxillin. Paxillin phosphorylation appears to be delayed compared with that of FAK. Recently,
H2O2-induced increases in tyrosine phosphorylation of FAK in the human glioblastoma cell line T98G were reported (40). In T98G cells, the
increase in tyrosine phosphorylation of FAK was apparent only after 30 min and continued up to 5 h, whereas in ECs with a relatively high
concentration of
H2O2
(1 mM), we observed increases as early as 30 s of oxidant exposure that
reached a plateau by 10-15 min. In addition, we observed a decline
in both tyrosine phosphorylation and cell viability after 2 h of
H2O2
exposure (data not shown). These results indicate clear differences
between cell types in response to
H2O2-induced
tyrosine phosphorylation of FAK. Gozin et al. (11) have shown increased
tyrosine phosphorylation of FAK and paxillin by ROS produced through
the xanthine/xanthine oxidase system in human umbilical vein ECs. This
study has identified the hydroxyl radical as the reactive species
responsible for the observed increases. However, the precise
biochemical mechanism by which ROS increase tyrosine phosphorylation of
these proteins is not clear. Because ROS are known to alter the redox
status of the cells by depleting thiols (37), it will be interesting to
examine the role of thiols in ROS-induced increases in tyrosine phosphorylation of focal adhesion proteins.
FAK is a non-receptor tyrosine kinase and possesses autophosphorylating
activity (6). An earlier study (31) with LPA, endothelin, bombesin, and
sphingophosphorylcholine showed a correlation between increases in FAK
tyrosine phosphorylation and autophosphorylating activity. However, in
our study, we were unable to demonstrate any increase in the
autophosphorylating activity of FAK in the immunoprecipitates obtained
after oxidant exposure of ECs. This may be due to differences in the
sites of FAK phosphorylation by oxidants compared with other agents.
Inhibition of the autophosphorylating activity of FAK by 1 mM
H2O2
is intriguing and may suggest that
H2O2-induced
phosphorylation of certain tyrosine residues is inhibitory to the
autophosphorylating activity. In addition, FAK immunoprecipitates
obtained from oxidant-treated cells have not revealed any increases in
tyrosine kinase activity compared with FAK immunoprecipitates obtained
from untreated cells (data not shown). These results suggest that
treatment of ECs with oxidants did not increase the association of PKs
with FAK. Earlier studies (12, 38, 39) have demonstrated a correlation between FAK activation and cytoskeletal rearrangement as measured by
the formation of actin stress fibers. Cytochalasin D, an inhibitor of
the microfilament network, attenuated tyrosine phosphorylation of FAK
and associated proteins. The results from the present study are
consistent with these earlier observations. Although the physiological relevance of actin stress fibers is not firmly established, the presence and formation of these fibers under in vivo situations were
demonstrated in ECs (10). The functional significance of ROS-induced actin stress-fiber formation requires further investigation.
H2O2
is a known activator of PKC in ECs (42), and the involvement of PKC in
tyrosine phosphorylation of FAK was demonstrated in certain cell types
(14, 21, 32, 39, 44). However, in ECs, we observed neither a direct
influence of PKC on tyrosine phosphorylation of FAK nor an indirect
influence of PKC on
H2O2-induced tyrosine phosphorylation of FAK. In contrast to the present findings in
ECs, in a previous study with platelets (14), bisindolylmaleimide, an
inhibitor of PKC, was shown to attenuate tyrosine phosphorylation of
FAK. Also, downregulation of PKC attenuated only phorbol
12,13-dibutyrate-induced, but not endothelin-mediated, FAK tyrosine
phosphorylation in fibroblasts (39). However, in Rat-1 fibroblasts,
Saville et al. (32) showed that endothelin- but not LPA-mediated FAK
tyrosine phosphorylation was attenuated by inhibiting PKC. These
results indicate that depending on the cell type, agonist-induced
tyrosine phosphorylation of FAK is either PKC dependent or independent.
Our results with tyrosine kinase inhibitors are in contrast to those
observed in Swiss 3T3 fibroblasts (8) and human neuroblastoma cells
(SK-N-SH clone SY54) (4) in which tyrosine phosphorylation of FAK was
found to be sensitive to one or more tyrosine kinase inhibitors
(erbstatin, tyrphostin, herbimycin, and/or genistein). Our data show
that under the current experimental conditions, the
H2O2-mediated
tyrosine phosphorylation of FAK in ECs is insensitive to tyrosine
kinase inhibitors and thus suggest alternate pathways of increased
tyrosine phosphorylation. However, the same inhibitors significantly
attenuated
H2O2-induced
overall tyrosine phosphorylation as measured by immunoblotting of total
cell lysates (data not shown) and also the tyrosine kinase-dependent
activation of MAPK as measured by MBP phosphorylation (Fig. 9). It is
possible that H2O2-induced
tyrosine phosphorylation of FAK in ECs may be primarily through the
inhibition of protein tyrosine phosphatases. Recent reports (9, 22)
indicated that inhibitors of tyrosine phosphatase, such as vanadate and
pervanadate, activate tyrosine phosphorylation of FAK and paxillin. In
fact, a role for protein tyrosine phosphatase activity in focal
adhesion and stress-fiber formation was demonstrated in Swiss 3T3 cells
(29). In addition, many studies (7, 18) have demonstrated
the inhibitory effect of
H2O2
on tyrosine phosphatases. Based on the published data, it is possible
that in ECs, ROS-mediated increases in FAK tyrosine phosphorylation
result, at least in part, from the inhibition of a protein tyrosine
phosphatase(s) specific to FAK. However, the present data do not
exclude the involvement of tyrosine kinases, which were not inhibited
under our experimental conditions, in the stimulation of FAK tyrosine phosphorylation. Because FAK is a known substrate for Src and ROS are
known to activate Src in various cell types including ECs (3), one has
to cautiously interpret the lack of inhibition of
H2O2-induced
FAK tyrosine phosphorylation by tyrosine kinase inhibitors.
In summary, the present results demonstrate that ROS stimulate tyrosine
phosphorylation of FAK and paxillin in vascular ECs. However, in
contrast to the effect of
H2O2
on MAPK activation, the increase in tyrosine phosphorylation of FAK
appears to be insensitive to tyrosine kinase inhibition. These findings
suggest a novel and differential regulation of FAK and MAPK by ROS.
Presently, the physiological significance of this differential
regulation by ROS remains unclear; however, recent reports (1, 13,
34) demonstrate the participation of ROS in signaling
cascades involving members of the MAPK and Syk tyrosine kinase
families. Consistent with this postulate, Huot et al. (19) recently
demonstrated a role for ROS-induced p38 kinase in the cytoskeletal
rearrangement in ECs. Future efforts need to focus on the role of
specific protein tyrosine phosphatases and protein tyrosine kinases in
the regulation of ROS-mediated tyrosine phosphorylation of focal
adhesion proteins and actin cytoskeleton assembly in vascular endothelium.
 |
ACKNOWLEDGEMENTS |
We thank Beverly Clark for excellent secretarial assistance.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-47671, HL-57260, and PO1-HL-58064 (to V. Natarajan) and by a
grant from the Methodist Showalter Foundation (to D. English).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Vepa, Dept.
of Medicine, Div. of Pulmonary and Critical Care Medicine, Johns
Hopkins School of Medicine, 4A-62, 5501 Hopkins Bayview Cir.,
Baltimore, MD 21224 (E-mail:svepa{at}welchlink.welch.jhu.edu).
Received 31 August 1998; accepted in final form 18 March 1999.
 |
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