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INTRODUCTION |
Vascular endothelial growth factor
(VEGF)1 is a secreted
glycoprotein specific for endothelial cells (1-3). VEGF is angiogenic in vivo (4, 5) and in vitro (6); its importance
in vasculogenesis and angiogenesis has been established through gene
deletion studies (7, 8). Flk-1/KDR and Flt-1 are the two receptor
tyrosine kinases that regulate the actions of VEGF and are expressed in endothelial cells (9-13), while the related receptor, Flt-4, is found
on lymphatic endothelium. The expression pattern of Flt-4 suggests it
may play a role during lymphangiogenesis (14).
All of the three VEGF receptors belong to the PDGFR-
family of
receptor tyrosine kinases (15). Upon activation, these receptors dimerize and/or oligomerize, following which autophosphorylation and
transphosphorylation of their tyrosine residues in the intracellular domain occurs. There are four putative tyrosine phosphorylation sites
(Tyr-951, Tyr-996, Tyr-1054, and Tyr-1059) in the KDR intracellular domain (16). These phosphorylated tyrosine molecules act as docking
sites for adaptor signaling molecules and non-receptor tyrosine
kinases, thereby generating a signal cascade that culminates in a
cellular response. The signal transduction pathways involved in
mediating the various biological functions of VEGF on endothelial cells
such as migration, proliferation, differentiation, or survival remain
to be completely defined.
The Ras-MAP (mitogen-activated protein) kinase pathway is a key
component in the transduction of signals leading to growth and
transformation. It consists of a linear cascade of protein kinases,
Raf, MAP kinase kinase, and MAP kinase, which are also called
extracellular-regulated kinases (Erks). Erk-1 and Erk-2 are acutely
activated upon growth factor stimulation (17).
Phosphatidylinositol (PI) 3-kinase, a heterodimer of an 85-kDa (p85)
adaptor subunit and a 100-kDa (p110) catalytic subunit (18-21), is
activated by most growth factors and has been implicated as a critical
factor in the control of cell proliferation and cell survival. PI
3-kinase phosphorylates the D-3 position of the inositol ring of
phosphoinositides, which in turn act as second messengers. The p85
subunit contains two Src homology 2 (SH2) domains, which bind to
tyrosine-phosphorylated receptors after stimulation of cells with
growth factors and in this manner recruit p110 into the complex at the
cell membrane. The region between the two SH2 domains, the iSH2 region,
mediates the association with p110, and this interaction is required
for the enzymatic activity of p110 (22). Phosphorylation of the p85
subunit of PI 3-kinase upon VEGF stimulation (23) is suggestive of a
potential role for PI 3-kinase in VEGF-mediated signaling. Given this
observation, we hypothesized that PI 3-kinase might play a critical
role in VEGF signaling, including the Ras-MAP kinase pathway. In this report, we demonstrate for the first time the functional significance of PI 3-kinase in VEGF signaling from Flk-1/KDR leading to MAP kinase
activation, followed by transcriptional activation of the c-Fos serum
response element that eventually culminates in endothelial cell proliferation.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human umbilical vein endothelial cells
(HUVECs) were purchased from Clonetics (San Diego, CA) and were
passaged in medium M199 (Life Technologies, Inc.) containing 20% fetal
bovine serum, 4 mM L-glutamine, penicillin,
streptomycin, and 2 ng/ml basic fibroblast growth factor on 80-mm
diameter dishes coated with 0.2% gelatin (Sigma). HUVECs were not
used after the sixth passage. For experimental purposes, HUVECs were
plated on 0.2% gelatin-coated dishes and allowed to form a monolayer.
Cultures were serum-starved with unsupplemented medium M199 containing
0.5% fetal bovine serum for 16-18 h. Serum-starved cultures were left
untreated or treated with VEGF 165 at the indicated concentrations and
time in 5.0 ml of medium M199 at 37 °C and harvested in appropriate
lysis buffer at 4 °C.
Materials--
Anti-Flk-1, Flt-1, and Erk-2 polyclonal
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA), anti-phospho-Erk-1/2 polyclonal antibodies were from New England
Biolabs, and anti-p85 and anti-phosphotyrosine antibodies (4G10) were
from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-hemagglutinin
(HA) antibody (12C5A),
-galactosidase and luciferase assay kits, and
cell proliferation assay kits containing BrdUrd and anti-BrdUrd
antibody were purchased from Boehringer Mannheim. Wortmannin was
purchased from Sigma, and PD98059 was bought from Calbiochem. PI
3-kinase substrate, phosphatidylinositol (sodium salt), was bought from
Avanti Polar Lipids, Inc. (Alabaster, AL). The ECL reagent was obtained
from Amersham Pharmacia Biotech. VEGF 165 was procured from R&D
(Minneapolis, MN). Wild type Flk-1 cDNA construct in CMV
promoter-based plasmid was from Dr. Axel Ullrich (Max-Planck-Institut
für Biochemie, München, Germany). Hemagglutinin (HA)-tagged
Erk-2 (pCMV-HA-Erk2) in pCMV, SRE-Fos-luciferase (SRE-Fos-Luc), and
pCMV-
-galactosidase plasmids were from Dr. Joseph Schlessinger (New
York University Medical Center, New York, NY). Dominant-negative (DN)
p85 mutant plasmid pSG5 p85
iSH2-C was from Dr. Julian Downward
(Imperial Cancer Research Fund, London, UK), and DN Ras (Ras N17) was
from Dr. E. Skolnik (New York University Medical Center, New York, NY).
Treatment with Wortmannin or PD98059--
Stock solutions of
wortmannin and PD98059 in Me2SO were kept at
20 °C.
Stocks were diluted in serum-free medium prior to use. Wortmannin,
PD98059, or the Me2SO carrier control was added to the
cells for 15-20 min before the addition of growth factor.
Protein Estimation--
Protein was estimated using the BCA
protein assay reagent (Pierce) as described by the manufacturer.
Transfections--
HUVECs were transiently transfected with 5 µg of SRE-Fos-Luc and 1.0 µg of pCMV-
-galactosidase plasmids
using Superfect according to the manufacturer's instruction (Qiagen).
24 or 48 h later, cultures were serum-starved as indicated earlier
and treated as mentioned in the legends. Cells were harvested in
appropriate lysis buffer.
For dominant negative studies, HUVECs were transiently transfected with
2.0 µg of HA-Erk-2, 1.0 µg of CMV-
-galactosidase, and increasing
concentrations of DN p85 plasmid. The concentration of DNA per
transfection was kept constant by supplementing with the vector alone.
For co-immunoprecipitation of p85 with Flk-1, HUVECs were transiently
transfected with 10 µg of Flk-1 plasmid.
For studies of cell cycle progression with DN p85, cells were
transiently transfected with 2 µg of CMV-
-gal and increasing concentrations of either DN p85 or DN Ras plasmids.
Immunoprecipitations and Immunoblotting--
HUVECs were lysed
in cell extraction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 1% Triton X-100, 1%
Nonidet P-40 and 0.25% sodium deoxycholate, and protease inhibitors,
namely 1 mM phenylmethylsulfonyl fluoride, 0.15 units/ml
aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A), clarified
by centrifugation (16,000 × g, 10 min, 4 °C), and
equal amount of lysate was immunoprecipitated with anti-p85 or
anti-Flk-1 antibodies and 25 µl of protein A-agarose for 4 h at
4 °C. Immunoprecipitates were washed three times with cold lysis
buffer, and beads were boiled and resolved in 8% SDS-PAGE. Proteins
were transferred to a nitrocellulose membrane and immunoblotted with
either anti-Flk-1 or anti-p85 antibodies. Blots were developed using
the ECL reagent.
In Vitro Kinase (Tyrosine Phosphorylation) Assay--
HUVECs
were lysed in modified RIPA (cold) buffer (50 mM HEPES, pH
7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 2 mM Na3VO4, 10 mM NaF, 5 mM sodium pyrophosphate) containing the above mentioned
protease inhibitors. Lysates were precleared and equal amounts of
protein were immunoprecipitated for 3 h with either anti-Flk-1 or
anti-p85 polyclonal antibodies. Immunoprecipitates were washed three
times with cold modified RIPA buffer, followed by two washes in kinase
assay buffer (50 mM Tris, pH 7.4, 100 mM NaCl,
1.5 MgCl2) at room temperature. In vitro kinase
assay was initiated by adding 30 µl of kinase assay buffer containing 50 µmol of cold ATP and 10 µCi of [
-32P]ATP
(>4000 µCi/mmol; Amersham Pharmacia Biotech) and shaken in a vortex
for 15 min at room temperature. Kinase complexes were resolved in an
8% SDS-PAGE. Gels were treated with KOH for 2 h at 60 °C to
remove serine and threonine phosphorylation. Gels were fixed, dried,
and autoradiographed.
In Vitro PI 3-Kinase Assay--
PI 3-kinase activity was assayed
essentially as described by Krook et al. (24). Serum-starved
HUVECs were treated with VEGF for 5 min. Anti-phosphotyrosine antibody
4G10 was used for immunoprecipitation at a concentration of 4 µg/mg
of lysate. PI 3-kinase assay was carried out in kinase assay buffer (30 mM HEPES, pH 7.4, 30 mM MgCl2, 50 µM ATP, and 400 µM adenosine) containing
0.2 mg/ml phosphatidylinositol and 10 µCi of
[
-32P]ATP for 15 min at 25 °C. Reactions were
terminated by the addition of 100 µl of 1 M HCl. The
reaction products were extracted with 200 µl of
CHCl3/methanol (1:1). The chloroform phase was
collected, separated by thin layer chromatography, autoradiographed,
and quantified using a phosphorimager.
MAP (Erk1/2) Kinase Assay--
Cells were washed twice with
ice-cold phosphate-buffered saline and extracted in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 25 mM NaF, 5 mM
sodium pyrophosphate, 1 mM Na3VO4,
1% Nonidet P-40, and protease inhibitors). Equal amount of lysate was
resolved in 10% SDS-PAGE. Following transfer to nitrocellulose
membrane, the blots were immunoblotted with anti-phosphospecific
Erk-1/2 antibodies that recognize phosphorylation of Thr202
and Tyr204. For inhibition studies, serum-starved cells
were preincubated with the indicated concentrations of wortmannin or
PD98059 for 15 min prior to VEGF treatment.
For assays of transfected HA-Erk-2, affinity-purified anti-HA
monoclonal antibody was used for immunoprecipitations (IP). The amount
of cell lysate used in the IP was normalized for
-galactosidase activity levels to account for transfection efficiencies. Erk-2 immune
complex assays were initiated by adding 30 µl of kinase assay buffer
(50 mM Tris, pH 7.5, 10 mM MgCl2)
containing 5 µCi of [
-32P]ATP (>4000 µCi/mmol)
and 2.5 µg of bovine brain-derived myelin basic protein (MBP). The
kinase complex was resolved in 14% SDS-PAGE and analyzed by
autoradiography. MBP runs as an 18-kDa protein.
SRE-Fos-Luc Analysis--
HUVECs transiently transfected with
SRE-Fos-Luc and CMV-
-galactosidase plasmids were pretreated with
wortmannin at the concentration mentioned in the figure legend and
stimulated with VEGF. Cultures were harvested 6 h later in
reporter lysis buffer and analyzed for luciferase and
-galactosidase activities.
Cell Proliferation Assay--
HUVECs were plated on
gelatin-coated coverslips in 12-well plates at a density of 5 × 104 cells, allowed to adhere for 12 h, and
serum-starved for 24 h. After 12 h of incubation in
serum-free medium M199 supplemented with 10 ng/ml VEGF containing 10 µM 5'-bromodeoxyuridine (BrdUrd), cells were fixed and
stained with anti-BrdUrd monoclonal antibody and alkaline
phosphatase-conjugated anti-mouse immunoglobulins.
For DN studies, cells transiently transfected with CMV-
-gal and DN
p85 or DN Ras were serum-starved for 36 h and then incubated in
medium M199 containing 10 µM BrdUrd and 10 ng/ml VEGF for
10 h. Cells were fixed and stained with X-gal, followed by
anti-BrdUrd as described above. The percentage of X-gal-positive cells
that had incorporated BrdUrd was evaluated using a light microscope.
Statistical Analysis--
Statistical analysis was carried out
by Student's t test. Results are expressed as mean ± standard deviation of the mean.
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RESULTS |
p85 Constitutively Associates with Flk-1/KDR and Undergoes Tyrosine
Phosphorylation in VEGF-treated Endothelial Cells--
In order to
investigate the mitogenic response to VEGF in a physiologically
relevant model, we chose to study the response in HUVECs rather than
cell line. Unfortunately, available antibodies did not
immunoprecipitate KDR from HUVECs, so we transfected these cells with a
cDNA encoding Flk-1, the murine homologue of KDR. Transfection
efficiencies in HUVECs varied from 20% to 40%, and transfection of
HUVECs did not change their gross morphology. We demonstrate the
co-immunoprecipitation of Flk-1/KDR with p85 from VEGF unstimulated and
stimulated human endothelial cells following immunoprecipitation with
anti-p85 antibodies (Fig. 1A). In contrast to these findings, the anti-Flk-1 antibodies did not immunoprecipitate p85 (Fig. 1B). We conclude from these
results that p85 can directly and constitutively associate with
Flk-1/KDR. p85 has been shown to associate with Flt-1 in a yeast
two-hybrid system (25). We therefore looked for Flt-1 association with p85 and Flk-1/KDR in the above immunoprecipitates by immunoblotting with Flt-1 specific antibodies. Under our experimental conditions, we
could not detect Flt-1 in either p85 or Flk-1 immunoprecipitates (data
not shown).

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Fig. 1.
Association of p85 with Flk-1/KDR in
HUVECs. Serum-starved HUVECs were either left untreated ( ) or
treated (+) with 10 ng/ml VEGF for 5 min; extracts were
immunoprecipitated with either anti-p85 (A) or anti-Flk-1
(B) polyclonal antibodies, followed by immunoblotting with
antibodies to Flk-1 and p85. Immunoprecipitation of Flk-1 with p85 is
demonstrated.
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We next confirmed previous observations that KDR, which has intrinsic
kinase activity, was tyrosine-phosphorylated as a result of VEGF
treatment (Fig. 2A). We found
that, in addition to KDR, two other proteins migrating with approximate
molecular masses of 46 and 70 kDa were also tyrosine-phosphorylated.
Concomitant phosphorylation of the p85 subunit of PI 3-kinase in
response to VEGF stimulation was demonstrated by in vitro
kinase assay following immunopecipitation with a specific anti-p85
antibody (Fig. 2B).

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Fig. 2.
Tyrosine phosphorylation of Flk-1/KDR and p85
in HUVECs in response to VEGF stimulation. Serum-starved HUVECs
were either left unstimulated ( ) or stimulated (+) with VEGF (10 ng/ml for 5 min) and lysed in modified RIPA buffer. ~800 µg of
total protein was immunoprecipitated with Flk-1 polyclonal antibodies
(A) or with p85 polyclonal antibodies (B).
Immunocomplex kinase assays were performed using
[ -32P]ATP, and complexes were resolved in an 8%
SDS-PAGE. Tyrosine phosphorylation of KDR and p85 is indicated.
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VEGF Promotes PI 3-Kinase Activation--
We sought to examine
whether p85 phosphorylation in response to VEGF treatment was able to
regulate the catalytic counterpart of the PI 3-kinase enzyme, p110. We
found VEGF treatment resulted in about 80 ± 25% increase in PI
3-kinase activity when examined by an in vitro kinase assay
using phosphatidylinositol as a substrate (Fig.
3, A and B). This
increase in PI 3-kinase activity could be blocked when endothelial
cells were preincubated with wortmannin (10 and 100 nM), a
fungal metabolite that is a potent inhibitor of PI 3-kinase
(p < 0.01 versus VEGF-stimulated).
Wortmannin also significantly inhibited the unstimulated background PI
3-kinase at 100 nM concentration (p < 0.001 versus control).

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Fig. 3.
VEGF promotes PI 3-kinase activation in
HUVECs. A, serum-starved HUVECs were left untreated or
treated with PI 3-kinase inhibitor wortmannin at indicated
concentrations for 20 min and were either unstimulated ( ) or
stimulated (+) with VEGF (10 ng/ml) for 5 min. Cells were harvested,
equal amount of lysates were immunoprecipitated with
anti-phosphotyrosine monoclonal antibodies (4G10), and immunocomplexes
were assayed for their ability to phosphorylate PI to
phosphatidylinositol phosphate (PIP) using
[ -32P]ATP. Data are mean ± standard deviation of
the percentage of intensity of control from three separate experiments.
@, p < 0.001 versus control; *,
p < 0.01 versus VEGF-stimulated.
B, a representative autoradiogram of the PI 3-kinase assay.
The position of PI (origin) and phosphatidylinositol phosphate
(PIP) are indicated.
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Wortmannin and DN PI 3-Kinase Inhibit VEGF-stimulated MAP Kinase
Activation--
MAP kinase plays a central role in controlling signals
for growth from most growth factor receptor tyrosine kinases. We looked at the kinetics of MAP kinase activation in VEGF-stimulated endothelial cells and found a time-dependent response, which was
maximum at 10 min (data not shown).
To assess the role of PI 3-kinase in MAP kinase activation by VEGF,
cells were preincubated with different concentrations of wortmannin
prior to VEGF stimulation. Interestingly, wortmannin in a
dose-dependent manner inhibited VEGF-induced Erk1/2
activation (Fig. 4A),
indicating that PI 3-kinase is involved in MAP kinase activation by
VEGF. About 70% inhibition of MAP kinase activation was observed with
100 nM wortmannin (concentration relevant to PI 3-kinase
inhibition). The MAP kinase kinase inhibitor PD98059, was used a
positive control (Fig. 4A). The blot was stripped and reprobed with Erk-2 antibodies to confirm equal loading of protein (Fig. 4B).

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Fig. 4.
Wortmannin and PD98059 inhibit VEGF
stimulated Erk activation in HUVECs. Serum-starved HUVECs were
either left untreated ( ) or pretreated with wortmannin or PD98059 at
indicated concentrations for 20 min and stimulated with VEGF (10 ng/ml)
for 10 min. Cells were harvested, and equal amounts of protein were
resolved in SDS-PAGE and immunoblotted with phosphospecific Erk
antibodies (A). Equal amount of protein loading was
confirmed by stripping the blot and reprobing with Erk-2 antibodies
(B).
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To confirm that the PI 3-kinase pathway was responsible for activating
MAP kinase in response to VEGF, cells were co-transfected with HA
epitope-tagged Erk-2 and increasing concentrations of a DN form of p85
(p85
iSH2-C). This DN p85 lacks the amino acids 475-523 in the iSH2
domain and therefore does not bind p110, the catalytic subunit of PI
3-kinase (26). Co-expression of p85 with HA-tagged Erk-2 was necessary
to facilitate the evaluation of dominant negative effects in only the
transfected cells. As expected, stimulation with VEGF caused an
activation of the tagged Erk-2. Importantly, overexpression of a DN p85
mutant inhibited VEGF-induced HA-Erk-2 activation in a
dose-dependent manner. Expression of HA-Erk-2 and DN p85 at
a 1:5 ratio suppressed Erk-2 activation almost 70% (Fig.
5).

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Fig. 5.
Dominant negative p85
(p85 iSH2-C) inhibits Erk-2 activation in
response to VEGF stimulation in HUVECs. HUVECs were transiently
co-transfected with HA-Erk-2 (2 µg), pCMV- -galactosidase (1 µg)
plasmids and either a control plasmid or 2, 6, or 9 µg of DN
p85 iSH2-C mutant plasmid. 48 h after transfection, cells were
serum-starved for 16 h and treated with VEGF (10 ng/ml) for 10 min, equal amount of -galactosidase extracts were immunoprecipitated
with HA (12CA5) monoclonal antibody, and immunoprecipitate kinase assay
was performed using MBP as a substrate in presence of
[ -32P]ATP. Kinase complex was resolved in SDS-PAGE.
Phosphorylation of MBP, which runs at 18 kDa, is indicated.
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PI 3-Kinase Activity Is Required for VEGF-induced Transcription
from the c-Fos Serum Response Element and Entry into
S-phase--
Erk-1/2 can regulate transcription from the c-Fos serum
response element (SRE) by phosphorylating the ternary complex factors Elk-1 and SAP-1, which control induction of immediate early genes (27).
To ascertain whether PI 3-kinase was essential for the nuclear events
initiated following VEGF stimulation, HUVECs were transiently
transfected with a vector containing the Fos-SRE promoter element
linked to the luciferase gene. As expected, treatment with VEGF
resulted in a 7-fold increase in transcription from the SRE promoter.
Wortmannin at a concentration of 20 nM inhibited the SRE
transcriptional activity by 47 ± 3.5%. Higher wortmannin concentration (100 nM) did not further inhibit the
transcriptional activity (Fig. 6). These
results suggest that PI 3-kinase activation following VEGF stimulation
can induce the transcription of c-Fos from SRE and presumably other
immediate early genes.

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Fig. 6.
Inhibition of VEGF-induced c-Fos
SRE-dependent transcription by wortmannin. HUVECs were
transiently transfected with 5 µg of SRE-c-Fos-Luc plasmid and 1 µg
of pCMV- -gal. 24 h after transfection, cells were serum-starved
for 16 h, pretreated with indicated concentrations of wortmannin,
and stimulated with VEGF for 6 h. Cells extracts were assayed for
luciferase and -galactosidase activity. The data presented are the
amount of SRE-Luc activity divided by the -galactosidase activity
present in the cell extracts. Data shown are the mean ± standard
deviation of three samples. This is a representative experiment
performed independently three times. *, standard deviation was less
than 1.
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To examine the biological effects of VEGF-activated PI 3-kinase
signaling events further downstream, experiments were carried out to
determine BrdUrd incorporation as a measure of VEGF-induced growth.
Following VEGF treatment, 65 ± 4.6% of the serum starved HUVECs
entered into the S-phase as detected by staining with anti-BrdUrd antibody. Wortmannin blocked VEGF-induced BrdUrd incorporation by
approximately 40% at 10 nM and about 50% at 100 nM concentration (Fig.
7A). Nonspecific effects of
wortmannin were eliminated by studies of cell cycle progression with DN
p85. Interestingly, DN p85 inhibited in a dose-dependent
manner entry of the cells into S-phase (Fig. 7B). DN Ras was
used as positive control. Both DN p85 and DN Ras failed to block cells
entering into the S-phase of the cell cycle in media containing 20%
serum (data not shown).

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Fig. 7.
Effects of wortmannin and dominant-negative
p85 on VEGF-induced cell cycle progression. A, HUVECs
were plated onto wells coated with 0.2% gelatin, allowed to adhere for
12 h, and then serum-starved overnight. Cells were either left
untreated ( ) or treated with wortmannin at the indicated
concentration for 20 min, and then stimulated with 10 ng/ml VEGF in the
presence of 10 µM BrdUrd in serum-free medium M199. Cells
were fixed 12 h later and immunostained with anti-BrdUrd
monoclonal antibodies and alkaline phosphatase-conjugated secondary
antibodies. Cells were counterstained with eosin. Data represent
mean ± standard deviation of four samples. This is a
representative experiment performed independently two times in
quadruplicate. B, HUVECs were transiently transfected with 2 µg of CMV- -gal plasmid; 5 and 10 µg of DN p85 plasmid
(p85 iSH2-C) or 3 and 6 µg of DN Ras (N17
Ras). 12 h after transfection, cells were serum-starved
for 36 h and stimulated with 10 ng/ml VEGF in M199 containing 10 µM BrdUrd. Cells were fixed 10 h later and stained
for X-gal followed by anti-BrdUrd. Values represent mean ± standard error of four samples. *, p < 0.02 versus VEGF-stimulated; @, p < 0.003 versus VEGF-stimulated.
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 |
DISCUSSION |
To further our knowledge of the signal transduction pathways from
VEGF receptor, we have analyzed the functional importance of PI
3-kinase in VEGF signaling from Flk-1/KDR. It has been shown that p85,
the adaptor subunit of PI 3-kinase, is capable of associating with the
VEGF receptor (23, 25). The functional significance of PI 3-kinase
activation in VEGF signaling has, however, not been defined clearly. In
our study, we show that phosphorylation of p85 in response to VEGF
regulates its catalytic counterpart, i.e. p110, and
furthermore demonstrate that PI 3-kinase activation contributes to MAP
kinase activation, transcription of c-Fos SRE, and cell cycle
progression in human endothelial cells.
Several reports have shown that activated Flk-1/KDR receptors will
associate with signaling intermediates such as phospholipase C-
,
GTPase-activating protein, Nck-Grb2, and Shc-Grb2 (23, 28-30). These
interactions result in tyrosine phosphorylation of some of the
potential substrates. The Ras-MAP kinase pathway has been reported to
signal for VEGF-induced growth, as has the PKC-MAP kinase pathway (30,
31). Our results show for the first time that PI 3-kinase not only gets
phosphorylated upon VEGF stimulation but also contributes to cell cycle
progression following MAP kinase activation.
One of the early events observed upon interaction of VEGF with its
receptor Flk-1/KDR is activation of the receptor's intrinsic tyrosine
kinase activity. In agreement with previous studies (28, 30), Flk-1/KDR
was strongly phosphorylated in response to VEGF in our study. Two other
proteins phosphorylated and recruited to the activated receptor had
approximate molecular masses of 46/47 and 67/70 kDa. The 46/47-kDa
protein (p46/47) phosphorylated in our study could either be p46Shc or
p47Nck, both of which are known to bind to Flk-1/KDR (30) as well as to
other receptor tyrosine kinases like PDGFR and epidermal growth factor
receptor (32-34). Although we did not characterize the identity of
these proteins, we subscribed to the idea that these were SH2
domain-containing tyrosine-phosphorylated proteins, which potentially
couple to the activation of Ras-MAP kinase pathway (35).
We also show that Flk-1/KDR constitutively associates with p85 as this
association was independent of VEGF stimulation. Flk-1/KDR has a
candidate motif YXXM in its intracellular region which is a
potential site for p85 to bind. It should be noted here that p85
contains two SH2 domain and a SH3 domain, the SH2 domain of p85 is
responsible for interaction with phosphorylated tyrosine in the context
of above motif. However, mutational analysis of tyrosine residues in
Flk-1/KDR would be required to verify this notion. Flt-1, the other
receptor for VEGF, has also been shown to associate with p85 in a
two-hybrid system, the binding site for which has been identified as a
YVNA motif (25). However, Flt-1 does not apparently signal for
proliferation, which has been underscored by knockout studies wherein
only the tyrosine kinase domain of Flt-1 has been deleted (36). Flt-1
tyrosine kinase homozygous mice (Flt-1TK
/
) develop
normal blood vessels and survive, which suggests that Flt-1 tyrosine
kinase domain does not signal for migration, proliferation, and
differentiation, essential features of endothelial cells during vasculogenesis. Although activation of the kinase counterpart of PI
3-kinase may or may not require p85 to be phosphorylated (37), we found
that VEGF treatment promoted p85 phosphorylation that in turn increased
PI 3-kinase activity.
In accordance with published literature (30, 38, 39), we have also
observed MAP kinase activation in endothelial cells upon VEGF
stimulation. We demonstrate for the first time in our study a link
between PI 3-kinase and the downstream activation of MAP kinase in VEGF
stimulated cells by 1) significantly inhibiting MAP kinase activation
with wortmannin, a potent PI 3-kinase inhibitor; and 2) blocking MAP
kinase activation with a dominant negative p85 mutant. These
observations suggest that the lipid products generated by the PI (3)
kinase following VEGF stimulation may bind to a signaling protein which
could activate MAP kinase. Candidate proteins could be pleckstrin
homology domain-containing proteins that require PI 3-kinase products
to be fully activated (35, 40).
Finally, we demonstrate for the first time the critical role of PI
3-kinase activation in generating a maximal mitogenic response to
VEGF. These observations are consistent with previous
evidence supporting a role for PI 3-kinase in PDGF-induced and
granulocyte-macrophage colony-stimulating factor-induced mitogenic
signaling in smooth muscle cells and macrophages, respectively (41,
42). PI 3-kinase activation has been linked to a number of biologically
diverse processes such as cell survival, membrane trafficking, and
insulin-stimulated glucose transport (43-45). Very recently, the
constitutively active forms of PI 3-kinase have been ingeniously used
to identify and study responses specifically induced by PI 3-kinase, an
approach that has shown that PI 3-kinase activation alone is sufficient to promote entry into S phase of the cell cycle (46). The fact that our
inhibition studies with either DN p85 or wortmannin did not abolish
VEGF-induced MAP kinase activation and mitogenesis suggests the
presence of additional PI 3-kinase-independent pathways in VEGF-induced
growth promoting effects, such as the PKC-MAP kinase (31) and the
Ras-MAP kinase pathway (30). In summary, results of our study identify
PI 3-kinase as an important mediator of VEGF-induced MAP kinase
activation and subsequent endothelial cell proliferation.