From the Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118 and the § Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642
Received for publication, December 28, 2000, and in revised form, February 12, 2001
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ABSTRACT |
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The phenotypic properties of the endothelium are
subject to modulation by oxidative stress, and the c-Jun N-terminal
kinase (JNK) pathway is important in mediating cellular responses to stress, although activation of this pathway in endothelial cells has
not been fully characterized. Therefore, we exposed endothelial cells to hydrogen peroxide (H2O2)
and observed rapid activation of JNK within 15 min that involved
phosphorylation of JNK and c-Jun and induction of AP-1 DNA binding
activity. Inhibition of protein kinase C and phosphoinositide 3-kinase
did not effect JNK activation. In contrast, the tyrosine kinase
inhibitors, genistein, herbimycin A, and
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) significantly attenuated H2O2-induced JNK
activation as did endothelial cell adenoviral transfection with a
dominant-negative form of Src, implicating Src as an upstream activator
of JNK. Activation of JNK by H2O2 was also
inhibited by AG1478 and antisense oligonucleotides directed against the
epidermal growth factor receptor (EGFR), implicating the EGFR in this
process. Consistent with this observation, H2O2
stimulated EGFR tyrosine phosphorylation and complex formation with
Shc-Grb2 that was abolished by PP2, implicating Src in
H2O2-induced EGFR activation. Tyrosine
phosphorylation of the EGFR by H2O2 did not
involve receptor autophosphorylation at Tyr1173 as assessed
by an autophosphorylation-specific antibody. These data indicate that
H2O2-induced JNK activation in endothelial cells involves the EGFR through an Src-dependent pathway
that is distinct from EGFR ligand activation. These data represent one
potential pathway for mediating oxidative stress-induced phenotypic changes in the endothelium.
Abundant evidence indicates that cardiovascular disease is
characterized by a state of excess oxidative stress and increased production of reactive oxygen species
(ROS)1 within the arterial
wall. Traditionally, ROS such as superoxide, hydrogen peroxide, and
hydroxyl radical have been viewed within the context of inflammation.
More recently, however, it has become clear that ROS may mediate
specific cellular functions such as the response to growth factors (1),
hypertrophy (2), and apoptosis (3). Since blood vessels from
atherosclerotic animals exhibit an increased flux of ROS (4),
understanding the cellular signals elicited by ROS should provide
insight into the pathogenesis of cardiovascular disease.
Among the cellular signals subject to regulation by ROS in the vascular
wall, members of the mitogen-activated protein (MAP) kinases are
perhaps the best characterized. Three major subfamilies of MAP kinases
have been described, the extracellular signal-regulated kinases
(ERK1/2), c-Jun NH2-terminal kinases (JNKs), and p38 kinase (5). The ERKs are typically involved in the response to growth factors
(i.e. proliferation, hypertrophy, and differentiation) and
are activated in response to ROS such as superoxide (6) and hydrogen
peroxide (7). The JNKs and p38 kinases are primarily involved in the
cellular stress responses (5) and are activated by hydrogen peroxide in
smooth muscle cells (8).
The MAP kinase pathway has been implicated in a number of phenomena
associated with cardiovascular disease. For example, dual activation of
ERK1/2 and p38 kinase is required for the hypertrophic response to
angiotensin II in smooth muscle cells (9). Cytokine stimulation of
fibroblasts results in activation of both p38 kinase and ERK1/2, either
of which is sufficient for up-regulation of matrix metalloproteinase-1
activity (10). Dual localization of p53 and activated JNK in
smooth muscle cells within apoptotic regions of atherosclerotic plaque
suggests that JNK activation may contribute to plaque rupture (11).
Thus, MAP kinase activation in smooth muscle cells and fibroblasts is
associated with pathologic changes in the arterial wall.
In contrast to fibroblasts and smooth muscle cells, ROS-mediated
activation of MAP kinases in endothelial cells is less well characterized. Treatment of endothelial cells with hydrogen peroxide is
associated with activation of ERK1/2 (12) and p38 kinase (13); however,
the effect on JNK is unclear. Since JNK activation has been observed in
other cells in response to ROS (7), we sought to define the effect of
ROS on endothelial cell JNK activity and to identify the signaling
pathways involved.
Materials--
Cell culture reagents including medium
M-199 and Dulbecco's modified Eagle's medium were obtained from Life
Technologies, Inc. Protein A/G-agarose was from Pierce. The EGF
receptor-specific inhibitor tyrphostin AG1478, the platelet-derived
growth factor receptor-specific inhibitor tyrphostin AG1295,
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2), genistein, herbimycin A, LY294002, and calphostin C were obtained from Calbiochem. Anti-phosphotyrosine antibody (clone PY20),
anti-Shc, and anti-Grb2 antibodies were from Transduction Laboratories
(Lexington, KY). The anti-EGF receptor antibody was from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies directed
against phospho-JNK, phospho-c-Jun, phospho-ERK1/2, phospho-p38, and
secondary peroxidase-labeled antibodies were from New England Biolabs
(Beverly, MA). The anti-phospho-EGF receptor (Tyr1173)
antibody and anti-Src antibody (GD11) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). [ Cell Culture--
Porcine aortic endothelial cells (PAECs) were
harvested and grown up to passage 6 in M-199 supplemented with 15%
fetal bovine serum, 10 µg/ml heparin sulfate, 100 units/ml
penicillin, and 100 µg/ml streptomycin as described (15). Human
umbilical vein endothelial cells (HUVECs) were obtained from Clonetics,
Inc., and grown on 0.2% gelatin-coated tissue culture plates in
endothelial cell growth medium (Clonetics) and used between passages 2 and 4. COS-7 cells were utilized in some experiments due to their greater EGF receptor density and were purchased from the American Type
Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Prior to experiments, subconfluent cells were cultured in their respective medium containing 1% serum for 16 h and equilibrated for 30 min in HEPES-buffered physiologic salt solution containing 22 mM HEPES (pH 7.4), 124 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 0.16 mM HPO4,
0.4 mM H2PO4, 5 mM
NaHCO3, and 5.6 mM glucose in order to minimize
extracellular oxidation.
Immunoprecipitation and Western Blotting--
After treatments,
cells were washed with ice-cold phosphate-buffered saline twice and
incubated in lysis buffer containing 50 mM Tris-HCl (pH
8.0), 120 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 100 µg/ml
phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, and 1 mM EDTA for 30 min on
ice followed by brief sonication for 10 s. Cell lysates were then
centrifuged at 13,600 × g for 10 min, and the
supernatants were incubated with primary antibody for 16 h at
4 °C followed by a 2-h incubation with protein A/G-agarose. Following a brief centrifugation, pellets were washed twice with lysis
buffer and twice with phosphate-buffered saline and were resuspended in
loading buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS,
200 mM dithiothreitol, 20% glycerol, and 0.2% bromphenol blue. Western blot analysis was performed as described previously (16).
Proteins were detected using an enhanced chemiluminescence detection
kit (New England Biolabs, Beverly, MA). Densitometric analysis of
Western blots was carried out using the PDI Imageware System
(Huntington Station, NY).
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared from PAECs using the method of Dignam and colleagues (17).
Double-stranded oligonucleotides corresponding to the AP-1 consensus
sequence (5'-CGCTTGATGAGTCAGCCGGAA-3') were labeled with
[ Kinase Assay--
After various treatments, cells were lysed as
described above, and JNK activity was determined using a commercially
available assay kit (New England Biolabs). Briefly, the cell lysate was precipitated with GST-c-Jun-(1-89) fusion protein bound to
glutathione-Sepharose beads overnight at 4 °C. Washed
immunoprecipitates were then incubated for 30 min at 30 °C with 100 µM ATP in 30 µl of assay buffer. Reactions were stopped
by the addition of 3× gel loading buffer (150 mM Tris-HCl
(pH 6.8), 6% SDS, 600 mM dithiothreitol, 30% glycerol,
and 0.6% bromphenol blue), and samples were boiled for 3 min prior to
SDS-polyacrylamide gel electrophoresis. Phosphorylation of c-Jun was
determined by Western blotting (as described above) with antibody
specific for the phosphorylated form of c-Jun.
Transfections--
The protocol for HUVEC transfection with
KI-Src adenovirus (Ad.KI-Src) and a control adenovirus (Ad.LacZ)
encoding Reverse Transcription and Polymerase Chain
Reaction--
The procedures for semiquantitative reverse
transcription-polymerase chain reaction were essentially as
described (18) using forward (5'-CAGCCTCCAGAGGATGTTCA-3') and reverse
(5'-GGTGGCACCAAAGCTGTATT-3') primers corresponding to human EGF
receptor mRNA. Constitutively expressed Statistical Analysis--
All blots are representative of three
or four experiments. Comparisons among treatment groups were performed
with one-way analysis of variance and a post hoc Dunnett's
or Student's Newman-Keuls comparison as appropriate. Statistical
significance was accepted if the null hypothesis was rejected with
p < 0.05.
H2O2-induced JNK Activation in
PAECs--
We observed time-dependent JNK and c-Jun
phosphorylation in response to H2O2 (Fig.
1, A and B). We
also observed concentration-dependent c-Jun phosphorylation
beginning with only 50 µM H2O2
(Fig. 1C). We found that H2O2
concentrations of H2O2-induced JNK Activation Involves Src
Tyrosine Kinase Activity--
Although JNK activation has been linked
to the activity of protein kinase C (19) or PI 3-kinase (20) in some
systems, we did not observe any effect of protein kinase C or PI
3-kinase inhibition with calphostin C and LY294002, respectively, on
H2O2-induced JNK activation (data not shown).
Similarly, the inhibitor of growth factor receptor ligand binding
suramin had no effect (Figs. 3, A and B). In contrast, general tyrosine kinase
inhibition with genistein or herbimycin A significantly attenuated
H2O2-induced c-Jun phosphorylation as did the
specific Src family kinase inhibitor PP2 (Fig. 3, A and
B). We also found that PP2 inhibited the entire time course
of JNK activation (up to 2 h, data not shown), indicating that Src
activation is an early event in this process. To examine the role of
Src specifically, we overexpressed kinase-inactive c-Src in endothelial
cells and found an inverse relation between KI-Src overexpression and
H2O2-induced JNK activation (Fig.
3C), consistent with this being a Src-dependent
process.
The EGF Receptor Is Involved in
H2O2-induced JNK Activation--
There is
increasing evidence that cellular stresses such as UV irradiation and
osmotic shock can transactivate growth factor receptors as a component
of their cellular signaling (21). Accordingly, we investigated receptor
involvement in H2O2-induced endothelial cell
JNK activation using the receptor tyrosine kinase inhibitors AG1478
(for the EGF receptor) and AG1295 (for the platelet-derived growth
factor receptor). As shown in Fig. 4,
AG1478 inhibited H2O2-induced c-Jun
phosphorylation in a dose-dependent manner, whereas AG1295
did not, implicating the EGF receptor in JNK activation induced by
H2O2. We found that AG1478 inhibited the entire
time course of JNK activation (up to 2 h, data not shown),
indicating that the EGF receptor is an early event in
H2O2-induced JNK activation. To confirm a role
for the EGF receptor in this process, we inhibited EGF receptor
mRNA expression in HUVECs with antisense oligonucleotides (Fig.
5A), and this also attenuated
H2O2-induced JNK activation (Fig.
5B). We were able to demonstrate a reduction in EGF receptor expression with antisense oligonucleotide treatment in COS-7 cells (due
to higher EGF receptor density) that also inhibited JNK activation by
H2O2 (Fig. 5C). These data indicate
that the EGF receptor is involved in
H2O2-induced JNK activation.
H2O2 Stimulates ERK1/2, but Not p38, via an
Src- and EGF Receptor-dependent Mechanism--
To
determine the involvement of Src and the EGF receptor in
H2O2-induced activation of other MAP kinase
pathways, we investigated H2O2-induced
stimulation of ERK1/2 and p38 in PAECs. We found that
H2O2 stimulated activation of both ERK1/2 (Fig.
6, A and B,
p < 0.05) and p38 (Figs. 6, C and
D, p < 0.05), although only the former was
inhibited by PP2 and AG1478. Substituting COS-7 cells for PAECs yielded
similar results (data not shown). Thus, H2O2-induced activation of ERK1/2 but not p38
involves both Src and the EGF receptor.
H2O2-stimulated JNK Activation Involves
Src-mediated EGF Receptor Transactivation--
To investigate
ligand-independent EGF receptor activation, we treated HUVECs
(available EGFR antibodies are unreactive in PAECs) with
H2O2 and observed rapid tyrosine
phosphorylation (2 min) of the EGF receptor (Fig.
7A) that was accompanied by
phosphorylation of the adapter protein Shc (Fig. 7B). Using
COS-7 cells, we found that H2O2-induced EGF
receptor tyrosine phosphorylation produced receptor activation
manifested as complex formation with the adapter proteins Shc and Grb2,
similar to that observed with authentic EGF (Fig. 7C). This
EGF receptor activation by H2O2 was inhibited by AG1478 only at high concentrations (50 µM) but readily
inhibited by PP2 (Fig. 7C), consistent with Src-mediated EGF
receptor activation. In contrast, EGF-induced EGFR-Shc-Grb2 complex
formation was inhibited by AG1478 at a conventional concentration of 1 µM.
EGF Receptor Autophosphorylation Is Not Involved in
H2O2-mediated JNK Activation--
Activation
of the EGF receptor typically involves receptor tyrosine kinase
activity and autophosphorylation of the EGF receptor at tyrosine
residue 1173 (22). To examine this process in
H2O2-mediated EGF receptor activation, we
employed an antibody specific for the autophosphorylation site
(Tyr1173) of the activated EGF receptor. We did not observe
EGF receptor autophosphorylation in response to
H2O2 despite significant tyrosine phosphorylation of the receptor (Fig. 8).
In contrast, both antibodies detected a strong signal from
EGF-stimulated cells (Fig. 8). Therefore, these data confirm that
H2O2-mediated JNK activation involves an
autophosphorylation-independent mechanism that is distinct from EGF
receptor signaling itself.
The principal finding of this study is that
H2O2 activates JNK in endothelial cells through
Src-dependent EGF receptor transactivation. We found that
nontoxic concentrations of H2O2 rapidly
activate the JNK pathway in endothelial cells, leading to c-Jun
phosphorylation and activation of the transcription factor, AP-1.
Endothelial cell JNK activation by H2O2
involved tyrosine kinase activity but not protein kinase C or
phosphoinositide 3-kinase. The specific tyrosine kinase(s) involved in
H2O2-mediated JNK activation were Src and the
EGF receptor. Moreover, we found that
H2O2-induced EGF receptor activation in
endothelial cells was dependent upon Src and could be inhibited by
AG1478 at concentrations of The activation of JNK by H2O2 has been observed
in a number of cell types and transformed cell lines (7, 23-25).
However, little is known about oxidative stress-induced JNK activation in endothelial cells. Leptin binding to HUVECs is associated with ROS
production and activation of the JNK pathway (26). Laminar shear stress
on endothelial cells is associated with
peroxynitrite-dependent activation of JNK (20). However,
neither of these studies provided insight into the mechanisms of JNK
activation. The data presented here extend these previous studies to
H2O2-induced JNK activation in endothelial
cells. The precise role of the JNK pathway in modulating the
endothelial cell phenotype in vascular disease is not completely clear.
Overexpression of c-Jun in cultured endothelial cells results in
apoptosis (27), and human atherosclerotic plaques contain apoptotic
endothelial cells (28). Although the functional consequences of JNK
activation have thus far focused on cell death and apoptosis (29), it
is reasonable to speculate that JNK may play some role in nonlethal
phenotypic changes in the endothelial cell.
Activation of the JNK group of MAP kinases by cytokines has been
examined in some detail. Tumor necrosis factor binding to the tumor
necrosis factor receptor-1 induces receptor aggregation and recruitment
of TRADD (tumor necrosis factor receptor-1 death domain protein) and
TRAF2 (tumor necrosis factor receptor-associated factor 2) (30).
Activation of JNK by TRAF2 may be mediated by either MAP kinase kinase
4 or 7 through pathways that may involve MAP kinase/ERK kinase 1 or
apoptosis signal-regulating kinase (reviewed in Ref. 31). In contrast
to cytokines, the upstream signals linking environmental stress to JNK
activation are less well characterized. In the present study, we found
that H2O2-induced JNK activation was dependent
upon Src. This finding is in keeping with observations that Src
tyrosine kinases are activated by oxidative events (32-34) and that
Src is involved in JNK activation (19, 35). Yoshizumi et al.
(36) have recently demonstrated that H2O2-mediated JNK activation in fibroblasts is
dependent upon Src and Cas. The data presented here extend these
findings to involve the EGF receptor in Src-dependent
signaling. The precise role of Cas in this scheme vis-à-vis the
EGF receptor will require further investigation.
Previous studies in vascular smooth muscle cells and fibroblasts
indicated that H2O2-induced JNK activation
involved Src, whereas activation of ERK1/2 and p38 did not (36). In
contrast, mucoepidermoid carcinoma cells treated with
H2O2 exhibit EGF receptor activation that is
manifest as stimulation of ERK1/2 (37). The data presented here suggest
that endothelial cells respond to H2O2 in a
manner similar to the mucoepidermoid cells in that
H2O2-induced ERK1/2 stimulation was
significantly inhibited by both PP2 and AG1478, implicating both Src
and EGF in this process. The mechanism(s) responsible for this distinct
pattern of responses between endothelial and smooth muscle cells is not
clear but certainly warrants further investigation.
A novel aspect of this work is the involvement of the EGF receptor in
H2O2-induced JNK activation. We found that
H2O2-mediated c-Jun phosphorylation was
significantly inhibited by the EGF receptor tyrosine kinase inhibitor
AG1478 and down-regulation of the EGF receptor by antisense
oligonucleotides. Although EGF has been shown previously to activate
the JNK pathway (35), our results indicate that
H2O2 activates the EGF receptor via a mechanism that is distinct from EGF receptor autophosphorylation (Fig. 8) and
requires Src-mediated tyrosine phosphorylation (Fig. 7). Such ligand-independent "transactivation" of the EGF receptor has been described with respect to a number of diverse stimuli including G-protein-coupled receptors, cytokines, and cellular stress (38). The
data presented here are in agreement with one other report of
H2O2-induced EGF receptor transactivation (37).
In that study, EGF receptor transactivation by peroxide was found to
mediate ERK1/2-dependent mucin synthesis in NCI-H292 cells, a
mucoepidermoid carcinoma cell line. However, there was no attempt in
that study to investigate the effect of H2O2 on
nonreceptor tyrosine kinases or the JNK family of MAP kinases.
The precise mechanism of H2O2-induced EGF
receptor transactivation is not clear, although receptor modification
by H2O2 has been observed. Gamou and Shimizu
(39) found that H2O2 induced EGF receptor
tyrosine phosphorylation in both intact cells and isolated membranes
with the major site of phosphorylation being Tyr1173. In
contrast, EGF induced both tyrosine and serine phosphorylation in its
receptor, resulting in quantitatively twice the phosphorylation as that
observed with H2O2 (39). Goldkorn et
al. (40) also observed that H2O2
preferentially induced EGF receptor tyrosine phosphorylation, and this
effect was inhibited by genistein, implicating a nonreceptor
protein-tyrosine kinase. Our data extend these findings and indicate
that H2O2-induced EGF tyrosine phosphorylation
is mediated by Src (Fig. 8C) but does not appear to involve
Tyr1173 under our experimental conditions. This contention
is not without precedent, since Src has been shown to mediate
phosphorylation of the EGF receptor at tyrosine residues 845 and 1101, the former residue being critical for EGF-mediated stimulation of DNA
synthesis (41). These two Src phosphorylation sites are not among the autophosphorylation sites in the carboxyl terminus of the protein (41),
consistent with the results presented here (Fig. 8). With this in mind,
if H2O2-induced EGF receptor tyrosine
phosphorylation were exclusively mediated by Src, one would not expect
AG1478 to inhibit H2O2-induced receptor
tyrosine phosphorylation as reported here (Fig. 7C). It is
worth noting that AG1478 was only effective against
H2O2-induced responses at a concentration
50-fold higher than required to inhibit EGF-mediated receptor
activation (Fig. 8C). As yet undescribed actions of AG 1478 against Src kinase activity or EGFR-Src complex formation merit
consideration as potential explanations for these observations.
The nature of Src stimulation by oxidative stress is not
known. In this study, we used H2O2, a
two-electron oxidant, as our source of oxidative stress. Once formed,
H2O2 participates in two major oxidation
reactions relevant to biologic systems. In the first of these
reactions, H2O2 may oxidize sulfur atoms to the
corresponding sulfoxide via SN2 reaction of the
sulfur with the O-O bond of H2O2 (42). If the
sulfur atom is reduced, H2O2 can oxidize the
thiols to the corresponding disulfide or sulfenic acid (42). This
latter activity may be particularly germane to the activation of
protein-tyrosine kinases such as Src. There is a growing appreciation
that protein-tyrosine kinases are under a tonic inhibition by protein
tyrosine phosphatases that typically contain thiol groups critical to
their activity. Moreover, H2O2 has been shown
to reversibly inactivate protein tyrosine phosphatases both in
vitro (43, 44) and in cells (45) by virtue of thiol oxidation.
Based upon these reports, it is attractive to speculate that
H2O2 signaling is mediated through the
inactivation of protein-tyrosine phosphatases, and such speculation
could easily apply to Src family kinases. However, since all protein
phosphatases share a similar active site motif consisting of a cysteine
and an arginine separated by five residues (CXXXXXR),
it is difficult to understand why H2O2
demonstrates relative signal specificity in many systems. It is also
not clear if other single-electron (e.g. superoxide, lipid
peroxyl radicals, hydroxyl) or two-electron (e.g.
peroxynitrite, hypochlorous acid) oxidants will produce similar or
diverse patterns of signal transduction.
In summary, the data presented here indicate that
H2O2 readily induces JNK activation in
endothelial cells. This H2O2-mediated JNK
activation involves both Src family kinases and transactivation of the
EGF receptor. Given the emerging importance of EGF receptor transactivation in a number of heterologous signaling systems, this may
represent a new pathway for JNK activation in the setting of oxidative stress.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was
from PerkinElmer Life Sciences. The adenovirus containing kinase-inactive chicken c-Src (Ad.KI-Src; Lys295 to Met
mutation) was prepared as described (14). All other reagents were
obtained from Sigma or as described.
-32P]ATP using a T4 polynucleotide kinase kit
(Promega). The binding reactions and electrophoresis of DNA-protein
complexes were performed as described previously (16).
-galactosidase was described previously (14).
Antisense and control oligonucleotides directed against human EGF
receptor (catalog no. S12116) were obtained from Sequitur (Natick, MA).
Cells were seeded (2 × 105/well) in six-well plates
and allowed to reach 70-80% confluence (~16 h). Cells were washed
with fresh Dulbecco's modified Eagle's medium, and oligonucleotides
were introduced by incubation in 2 ml of Dulbecco's modified Eagle's
medium containing 6.6 µl of oligofectin I (Sequitur, Natick, MA) and
100 nM oligonucleotides for 4 h. Cells were then
washed and cultured in endothelial cell growth medium (Clonetics) for
48 h before experiments. Under these conditions, transfection
efficiency was ~70% as judged by parallel controls with fluorescent oligonucleotides.
-actin mRNA was
amplified with forward (5'-TCACCCTGAAGTACCCCATC-3') and reverse
(5'-CACACGCAGCTCATTGTAGA-3') primers in a similar fashion.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
500 µM were toxic and thus employed
only nontoxic H2O2 concentrations in subsequent
studies. As expected, c-Jun phosphorylation (Fig. 1B)
correlated closely with H2O2-induced JNK
activity by immune complex kinase assay (Figs.
2, A and B) and
AP-1 DNA binding activity (Fig. 2C). These data confirm
intact activation of the JNK signaling cascade in response to
H2O2 in endothelial cells.
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Fig. 1.
H2O2 stimulates JNK
and c-Jun phosphorylation. PAECs were treated with 200 µM H2O2 for the indicated periods
of time, and total cell lysates were subjected to immunoblotting with
phospho-JNK (A) and phospho-c-Jun antibodies (B),
respectively. C, PAECs were treated with the indicated
concentration of H2O2 for 30 min, and cell
lysates were analyzed by immunoblotting with phospho-c-Jun antibody as
in B. Data are representative of three independent
experiments.
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Fig. 2.
H2O2 activates c-Jun
kinase activity and enhances AP-1 DNA binding. PAECs were
treated with H2O2 (200 µM) for
the indicated periods of time. A, cell lysates were
immunoprecipitated with GST-c-Jun fusion protein bound to Sepharose
beads. The immunoprecipitates were then subjected to kinase activity
assay, followed by Western blot analysis using anti-phospho-c-Jun
antibody. B, composite JNK activity determined by
densitometric analysis of c-Jun phosphorylation normalized to 0 µM H2O2. Results represent
mean ± S.D. from three independent experiments. *,
p < 0.05 compared with 0 µM
H2O2 by analysis of variance with post
hoc Dunnett's test. C, PAECs were treated as in
A, and electrophoretic mobility shift assay was performed as
described under "Experimental Procedures." Data are representative
of three independent experiments.
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Fig. 3.
H2O2-induced c-Jun
phosphorylation involves Src tyrosine kinase activity. PAECs
were pretreated with suramin (0.3 mM), genistein (50 µM), herbimycin A (10 µM), PP2 (25 µM), or catalase (300 units/ml) for 30 min prior to
stimulation with H2O2 (200 µM)
for 30 min (A). Immunoblot (IB) was performed on
cell lysates using anti-phospho-c-Jun antibody. B, composite
densitometric analysis of phospho-c-Jun from three independent
experiments. Data are presented as mean ± S.E.; *,
p < 0.05 versus 0 µM
H2O2 by analysis of variance and post
hoc Dunnett's comparison. C, HUVECs were transfected
with adenovirus encoding -galactosidase (Ad.Ctl) or the avian
kinase-inactive c-Src (Ad.KI-Src) for 48 h and then stimulated
with H2O2 (200 µM) for 30 min.
Cell lysates were resolved with SDS-polyacrylamide gel electrophoresis,
transferred to membranes, and probed with an antibody against
phospho-c-Jun and c-Src.
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Fig. 4.
Effect of EGF receptor and platelet-derived
growth factor receptor tyrosine kinase inhibitors on
H2O2-induced c-Jun phosphorylation.
PAECs were pretreated (30 min) with the EGF receptor tyrosine kinase
inhibitor AG1478 (A) or the platelet-derived growth factor
receptor tyrosine kinase inhibitor AG1295 (B) at the
indicated concentrations prior to stimulation with
H2O2 (200 µM) for 30 min. Cell
lysates were subjected to immunoblot analysis (IB) with
anti-phospho-c-Jun antibody. Composite densitometric analysis is
presented in (C) as mean ± S.D. from three independent
experiments. *, p < 0.05 versus 200 µM H2O2 alone-treated group by
analysis of variance with a post hoc Dunnett's test.
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Fig. 5.
EGF receptor down-regulation inhibits
H2O2-stimulated c-Jun
phosphorylation. A, HUVECs were treated with
vehicle alone or with antisense or control oligonucleotides directed
against the EGF receptor as described under "Experimental
Procedures." After 24 h, total RNA was extracted, and reverse
transcription-polymerase chain reaction was performed to estimate EGF
receptor and -actin mRNA expression. B, HUVECs were
treated as in A, but after 48 h cells were exposed to
the indicated concentration of H2O2 and lysed,
and resolved proteins were probed with antibodies directed at either
phospho-c-Jun or
-actin as a loading control. C, COS-7 cells were
transfected with antisense or control oligonucleotides as above and
treated with H2O2 (200 µM) for 30 min. Cell lysates were resolved with SDS-polyacrylamide gel
electrophoresis and subjected to immunoblot analysis with antibodies
specific for the EGF receptor, phospho-c-Jun, and
-actin antibodies
in the same blots. Data are representative of three independent
experiments.
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Fig. 6.
H2O2-stimulated
ERK1/2 activation involves Src and the EGF receptor. PAECs were
pretreated with vehicle alone (control), PP2 (25 µM),
AG1478 (1 or 50 µM) or catalase (300 units/ml) for 30 min
prior to stimulation with H2O2 (200 µM) for 15 (A and B) or 30 min
(C and D). After stimulation, cells were lysed,
proteins were resolved by SDS-polyacrylamide gel electrophoresis, and
activation of ERK1/2 (A) or p38 (C) was assessed
by immunoblot (IB) with phosphospecific antibodies as
described under "Experimental Procedures." Immunoblots for ERK1/2
(A) and p38 (C) served as loading controls.
Composite densitometric analysis of phospho-ERK1/2 (B) and
phospho-p38 (D) normalized to control are also shown and
represent mean ± S.E. from three independent experiments (*,
p < 0.05 versus 200 µM
H2O2 by analysis of variance and post
hoc Dunnett's comparison).
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[in a new window]
Fig. 7.
H2O2-stimulated EGF
receptor tyrosine phosphorylation and Shc-Grb2 complex
formation. A, HUVECs were treated with
H2O2 (200 µM) for the indicated
periods of time. Cell lysates were then immunoprecipitated
(IP) with anti-EGF receptor antibody, and immunoblots
(IB) were performed with anti-phosphotyrosine antibody
(PY20). B, cell lysates from ECs treated as in A
were immunoprecipitated (IP) with PY20 and immunoblotted
(IB) with anti-Shc antibody. C, COS-7 cells were
stimulated with H2O2 (200 µM) or
EGF (50 ng/ml) for 5 min with or without pretreatment (30 min) with
AG1478 (1 or 50 µM) or PP2 (25 µM). Cell
lysates were subjected to immunoprecipitation with anti-EGF receptor
antibody and immunoblotting with phosphotyrosine, Shc, and Grb2
antibodies, respectively. Data are representative of three independent
experiments.
View larger version (39K):
[in a new window]
Fig. 8.
H2O2-mediated
tyrosine phosphorylation of the EGF receptor is distinct from
ligand-induced autophosphorylation. COS-7 cells were stimulated
with H2O2 (200 µM) or EGF (50 ng/ml) for 5 min. Cell lysates were subjected to immunoprecipitation
with anti-EGF receptor antibody. The immunoprecipitates were
immunoblotted with phospho-EGF receptor (Tyr1173) antibody
and reprobed with anti-phosphotyrosine and EGF receptor antibodies.
Data are indicative of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 µM. Taken together,
these data are consistent with Src as an upstream signal for EGF
receptor activation in response to H2O2.
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FOOTNOTES |
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* This work was supported by National Institute of Health Grants HL53398 and HL52936 (to J. A. V.); HL49192 and HL18645 (to B. C. B.); and HL59346, HL55854, and DK55656 (to J. F. K).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.
Established Investigator of the American Heart Association.
¶ To whom correspondence should be addressed: Boston University School of Medicine, Whitaker Cardiovascular Institute, 715 Albany St., Rm. W507, Boston, MA 02118. Tel.: 617-638-4894; Fax: 617-638-5437; E-mail: jkeaney@bu.edu.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M011766200
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ABBREVIATIONS |
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The abbreviations used are: ROS, reactive oxygen species; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; PAEC, porcine aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; PP2, 4-amino-5-(4-chlorophenyl)-7- (t-butyl)pyrazolo[3,4-D]pyrimidine.
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