Src Kinase Mediates Phosphatidylinositol
3-Kinase/Akt-dependent Rapid Endothelial Nitric-oxide
Synthase Activation by Estrogen*
M. Page
Haynes
§,
Lei
Li
§,
Diviya
Sinha
§,
Kerry S.
Russell
§,
Koji
Hisamoto
§,
Roland
Baron¶,
Mark
Collinge
§,
William C.
Sessa
§, and
Jeffrey R.
Bender
§**
From the
Sections of Cardiovascular Medicine and
Immunobiology, Departments of
Pharmacology, ¶ Cell Biology
and Orthopedics, and the § Vascular Biology and
Transplantation Program, Boyer Center for Molecular Medicine, Yale
University School of Medicine, New
Haven, Connecticut 06536
Received for publication, October 23, 2002
 |
ABSTRACT |
17
-Estradiol activates endothelial nitric
oxide synthase (eNOS), enhancing nitric oxide (NO) release from
endothelial cells via the phosphatidylinositol 3-kinase
(PI3-kinase)/Akt pathway. The upstream regulators of this pathway are
unknown. We now demonstrate that 17
-estradiol rapidly activates eNOS
through Src kinase in human endothelial cells. The Src family kinase
specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17
-estradiol- but not
ionomycin-stimulated NO release. Consistent with these results, PP2
blocked 17
-estradiol-induced Akt phosphorylation but did not
inhibit NO release from cells transduced with a constitutively active
Akt. PP2 abrogated 17
-estradiol-induced activation of PI3-kinase,
indicating that the PP2-inhibitable kinase is upstream of PI3-kinase
and Akt. A 17
-estradiol-induced estrogen receptor/c-Src
association correlated with rapid c-Src phosphorylation. Moreover,
transfection of kinase-dead c-Src inhibited 17
-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to
induce Akt phosphorylation, whereas cells maintaining c-Src expression
demonstrated estrogen-induced Akt activation. Estrogen rapidly
activated c-Src inducing an estrogen receptor, c-Src, and P85
(regulatory subunit of PI3-kinase) complex formation. This complex
formation results in the successive activation of PI3-kinase, Akt, and
eNOS with consequent enhanced NO release, implicating c-Src as a
critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.
 |
INTRODUCTION |
The cardioprotective effects of estrogen are diverse, including
both rapid non-genomic and delayed genomic effects on the blood vessel
wall (reviewed in Ref. 1). Specific, rapid vascular effects, such as
moderation of vasomotor tone, have been linked to an
estrogen-stimulated increase in bioavailable nitric oxide (NO)1 (2-4). 17
-estradiol
(E2) treatment of human endothelial cells (EC) induces rapid release of
NO by estrogen receptor (ER)-dependent activation of
endothelial nitric oxide synthase (eNOS) (5). Many factors regulate
eNOS enzyme activity, including fatty acid modification, subcellular
localization, and binding to numerous proteins and cofactors, including
calmodulin, caveolin-1, the 90-kDa heat shock protein (HSP90), and
tetrahydrobiopterin (see Ref. 6 for review). eNOS is a
Ca2+/calmodulin-dependent enzyme, the activity
of which is also regulated by phosphorylation. Specific phosphorylation
of eNOS by the serine/threonine kinase Akt renders the enzyme more
active at much lower Ca2+ concentrations (7, 8). We
demonstrated previously that the ER-dependent activation of
eNOS occurs at resting Ca2+ concentrations and requires
activation of the phosphatidylinositol-3-OH kinase (PI3-kinase)/Akt
pathway (9). The regulatory subunit of PI3-kinase, P85, acts to
stabilize and inhibit the catalytic activity of PI3-kinase. Recently,
ER was shown to specifically bind to P85 in vitro (10). The
E2-induced association correlated with increases in PI3-kinase activity
in EC. However, the specific mechanism for E2 activation of PI3-kinase
is not known.
Evidence is emerging that membrane forms of steroid hormone receptors
exist and participate in signaling pathways (11-14). The activity of
E2 at the cell membrane has been shown in EC, neurons, and breast
cancer cell lines. We previously determined that rapid E2 activation of
eNOS and MAP kinase occurs through a membrane-associated ER (9, 12).
The EC line EAhy.926 used in these experiments exhibits rapid
E2-induced signaling but is unable to stimulate
ER-dependent gene transactivation. Additionally, EAhy.926
cells do not express the traditional 66-kDa ER
or ER
but express
a 46-kDa protein immunoreactive with C-terminal ER antibodies.
Recently, a protein of similar size reactive with E2 and anti-ER
antibodies was found to be associated with the plasma membrane in MCF-7
cells (13, 14). Additionally, a 46-kDa putative ER, reactive with
anti-ER antibodies, was found in wild-type and in the initial ER
knockout mice. This form of the receptor was thought to be responsible
for E2 enhancement of basal NO production in the initial ER
knockout
mice, because this E2 effect was lost in the complete ER
knockout
mouse (15). In human ECs expressing both the 66- and the 46-kDa
receptor, both rapid signaling to MAP kinase and gene transactivation
of estrogen-responsive element-luciferase reporter was
stimulated with E2 treatment (12). As previously indicated, the
specific mechanism of membrane-associated ER coupling to P85 is
unknown. E2-mediated actions are sensitive to serine/threonine and
tyrosine kinase inhibition. Previously, the activation of the tyrosine
kinase c-Src was associated with rapid E2 effects in breast cancer
cells (16, 17). Src activation induces MAP kinase through a
Shc/Grb2/Ras signaling cascade. In addition to MAP kinase, Ras-GTP has
been shown to bind and activate PI3-kinase. Because E2 rapidly
activates both EC MAP kinase and PI3-kinase, we investigated the
ability of E2 to activate Src kinase in human EC and whether the
consequences of this activation include activation of PI3-kinase, Akt,
eNOS, and MAP kinase. Here, we present evidence that the non-receptor
tyrosine kinase, c-Src, is rapidly activated in EC upon stimulation by
E2. This activation leads to formation of a functional signaling
complex composed of ER, c-Src, and P85.
 |
EXPERIMENTAL PROCEDURES |
Materials--
E2 and ionomycin were purchased from Sigma. Stock
solutions were prepared in ethanol with final ethanol concentrations
less than 0.1%. Stock solutions of LY294002 (Calbiochem) and ICI
182,780 (Zeneca Pharmaceuticals) were prepared in Me2SO,
with final Me2SO concentrations less than 0.1%.
Anti-phosphorylated Akt, anti-Akt, anti-phosphorylated p60, and
anti-phosphorylated eNOS were purchased from Cell Signaling. Anti-P85
and anti-c-Src were purchased from Santa Cruz. Anti-eNOS antibody was
purchased from BD Transduction Laboratories. All other reagents were
purchased from Sigma unless otherwise noted.
Cell Culture--
The EC line EAhy.926, described previously (9,
18), was maintained in DMEM and 10% fetal bovine serum, supplemented
with 5 mM hypoxanthine, 0.8 mM thymidine, and
20 µM aminopterin. Human umbilical vein EC (HUVEC) were
isolated and maintained as described previously (5). Murine embryonic
fibroblasts derived from embryos deficient in c-Src, yes, and fyn
(SYF
/
) or fibroblasts derived from control animals
lacking both yes and fyn but maintaining normal levels of c-Src
(YF
/
S+/+), described previously (19), were
maintained in DMEM and 10% fetal bovine serum. Before E2 stimulation,
cells were cultured in E2-free medium consisting of phenol red-free
DMEM and 10% gelding horse serum and were subsequently serum-starved
in phenol red-free DMEM containing 0.1% fatty acid-free bovine serum albumin.
NO Release--
EC monolayer NO release was quantified by
NO-specific chemiluminescence using potassium iodide and acetic acid
reflux, as described previously (8, 9). Cells were stimulated with E2
and ionomycin for 30 min at 37 °C, and supernatants were collected for NO analysis. The Src family kinase inhibitor, PP2, or vehicle was
added 30 min before agonist stimulation and NO collection.
Immunoprecipitation and Western Blotting--
Cell monolayers
were stimulated as described in the figure legends. Cells were either
lysed directly in SDS-PAGE sample buffer or in 20 mM
Tris-HCl, pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% Nonidet P-40, 1 mM Na3VO4, 1 mM NaPiPO4, supplemented with a
protease inhibitor mixture (Roche Molecular Biochemicals). Lysates were either directly subjected to SDS-PAGE or first incubated with the
appropriate primary antibody, immunoprecipitated with protein A/G
agarose (Santa Cruz) subjected to SDS-PAGE and immunoblotting. Immunoblots were probed with horseradish peroxidase-coupled
species-specific secondary antibodies and visualized by enhanced chemiluminescence.
Phosphoinositide 3-Kinase Assay--
The PI3-kinase assay was
performed essentially according to the manufacturer's instructions
(Upstate Biotechnology). Briefly, monolayers of EAhy.926 cells were
E2-deprived for 48 h and serum-starved in 0.25% bovine serum
albumin overnight before E2-stimulation. Some plates were additionally
preincubated with inhibitors or vehicle control before stimulation.
Cells were washed and lysed in the presence of a protease inhibitor
mixture (Roche Molecular Biochemicals), 1 mM
Na3VO4, and 1 mM NaF. The
supernatant was collected and precleared by irrelevant mouse IgG.
Approximately 500 µg of soluble proteins were subjected to
immunoprecipitation with anti-P85 or anti-ER
(Santa Cruz
Biotechnology, Santa Cruz, CA) antibodies for 2 h at 4 °C. The
immunocomplexes were harvested by protein A/G agarose, washed, and
incubated with 15 µg of phosphatidylinositol (Avanti Polar Lipids,
Inc) for 10 min at room temperature. PI3-kinase activity as monitored
at 37 °C for 15 min after addition of 20 µCi of
[
-32P]ATP (3000 Ci/nmol; 0.88 mM ATP) and
20 mM MgCl2 to the reaction. The reaction was
terminated by 6N HCl, after which the lipids were extracted by
chloroform/methanol (1:1) and fractionated by thin layer chromatography
in chloroform/methanol/water/ammonium hydroxide (129:114:21:5). The
thin layer chromatography plate was then air-dried and subjected to autoradiography.
Adenoviral Infection--
Recombinant adenoviruses expressing
-galactosidase (
-gal), or the membrane-targeted, myristoylated
Akt (myr-Akt), described previously (8), were obtained from K. Walsh (St. Elizabeth's Medical Center, Boston, MA). Monolayers were
incubated with the recombinant adenoviruses at a multiplicity of
infection of 100 for
-gal and myr-Akt. After infection, E2-free
medium was added for the cell recovery period followed by serum
starvation in phenol red-free DMEM plus 0.1% bovine serum albumin.
Adenovirally infected cells were stimulated as described in the figure legends.
Transient Transfection--
Cell monolayers were incubated with
empty vector (pcDNA3), kinase dead Src kinase (Src K295M), or
constitutively active Src kinase (Src Y527F) and Fugene (Roche
Molecular Biochemicals) at a 1:6 DNA-to-lipid ratio according to the
manufacturer's directions in E2-free medium. After transfection, cells
were serum-starved in phenol red-free DMEM and 0.1% bovine serum
albumin before stimulation with E2.
 |
RESULTS |
Effect of Src Family Kinase Inhibition on NO Release--
We
demonstrated previously that induced NO release occurs through
activation of E2-stimulated PI3-kinase and Akt activation, resulting in
phosphorylation and enhanced activation of eNOS (9). Once Akt is
targeted to the membrane via a PI3-kinase-dependent mechanism, it can be phosphorylated on at least two residues, Ser473 and Thr308. Phosphorylation of
Thr308 is thought to be largely constitutive, whereas
Ser473 phosphorylation is highly inducible. Thus
"activation" of Akt is almost exclusively measured by
Ser473 phosphorylation (20). Recently, in addition to
phosphorylation of Ser473 and Thr308, Akt has
been shown to be phosphorylated on Tyr315 and
Tyr326 by Src kinase (21). This tyrosine phosphorylation is
thought to be important for full activation of Akt but is independent of serine/threonine phosphorylation. These authors demonstrated that
the activity of a constitutively active Akt (myr-Akt) was further
augmented by transfection of a constitutively active Src kinase
(Src527F). Therefore, we attempted to determine whether Src kinase was
a primary upstream mediator of the signal transduction pathway leading
to E2-mediated NO release. EC were pretreated with PP2, a
pharmacological inhibitor specific for Src family tyrosine kinases (22,
23), or vehicle for 30 min before agonist stimulation and NO
collection. As in our prior work, the EAhy.926 EC line was used,
largely because the cells are phenotypically homogeneous, contain good
levels of eNOS, and display rapid signaling responses to estrogen (9,
12). PP2 completely abrogated E2-induced NO release but had no effect
on ionomycin-stimulated NO release (Fig.
1). This demonstrates that a member of
the Src kinase family is involved in E2- but not calcium
ionophore-enhanced eNOS activation.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of Src kinase inhibition on stimulated
NO release. EC monolayers that were E2-deprived for 48 h were
pretreated with PP2 (10 µM) or vehicle for 30 min before
agonist stimulation in supplemented Hanks' balanced salt
solution. The agonists, E2 (10 ng/ml), or ionomycin (2 µM) were added for 30 min, after which the medium was
collected and NO was measured by NO-specific chemiluminescence. *,
p 0.01 difference compared with control cells
(C).
|
|
Effect of PP2 on Constitutively Active Akt-enhanced NO
Release--
To begin dissecting the level at which Src kinase
transduces the aforementioned E2-stimulated responses, EAhy.926 cells
were infected with recombinant adenovirus encoding either
-gal as a
control or a membrane targeted and thus constitutively active myr-Akt.
After infection, the cells were pretreated with PP2, followed by E2 or
vehicle stimulation for 30 min, and the NO release was quantified. PP2
has no effect on NO release induced by constitutively active Akt (Fig.
2), indicating that Akt itself is not a
critical substrate for Src kinase. That is, Src kinase-mediated Akt
tyrosine phosphorylation is not a required step in
Akt-dependent eNOS activation. This demonstrates that a Src
kinase is playing a role upstream of Akt-mediated eNOS phosphorylation.
Because PP2 was unable to prevent NO release induced by constitutively
active Akt, it was important to determine where in the pathway Src
kinase was involved. PP2 abrogates E2-induced phosphorylation of Akt on
Ser473 in immortalized EAhy.926 EC (Fig.
3A) and in HUVEC (Fig.
3B), indicating Src kinase involvement in the primary
activation of Akt (Fig. 3). Phosphorylation of a downstream target of
E2-activated Akt, eNOS Ser1177, was inhibited by
pretreatment of HUVEC with PP2 (Fig. 3C), correlating with
PP2 inhibition of E2-stimulated NO release (Fig. 1). These data also
indicate that the E2-induced signaling responses seen in the
immortalized EAhy.926 cells are functionally identical to that seen in
primary HUVEC.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of PP2 on constitutive Akt
activity. EC were virally transduced with either control -gal
or myr-Akt. After E2 deprivation and serum starvation, the indicated EC
were preincubated with PP2 (10 µM) and stimulated with E2
(10 ng/ml) for 30 min, after which the medium was collected and NO was
measured by NO-specific chemiluminescence. *, p 0.01 difference compared with -gal control cells (C,
-gal).
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of PP2 on induced Akt and eNOS
phosphorylation. EC (EAhy.926 (A) and HUVEC
(B)) were pretreated with PP2 (10 µM) or
vehicle for 20 min, followed by E2 (10 ng/ml) activation for 15 min. EC
were washed, lysed, subjected to SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with phosphorylation-specific Akt
antibody (pAKT) and reprobed with total Akt antibody
(AKT). C, HUVEC lysates were
additionally subjected to SDS-PAGE, transferred to nitrocellulose,
immunoblotted with phosphorylation-specific eNOS antibody
(peNOS) and reprobed with total eNOS antibody
(eNOS).
|
|
Effect of E2 Stimulation on EC c-Src--
Although the PP2 data do
not define the precise Src kinase involved, c-Src is rapidly
phosphorylated in response to E2 in mammary tumor cell lines and
osteoblasts (17, 24-26). In these cells, Src kinase activation results
in the induction of the Shc/Ras/Erk signal transduction pathway. We
have previously demonstrated rapid EC Erk1/2 activation in
response to E2 (12). Human EC, including EAhy.926 cells, contain easily
detectable levels of c-Src. Therefore, E2-induced c-Src activation was
evaluated. c-Src activation kinase requires carboxy-terminal
Tyr530 dephosphorylation and subsequent kinase domain
Tyr416 autophosphorylation (27). Fig.
4A demonstrates induced c-Src Tyr416 phosphorylation within 2 min of E2 stimulation. As
with all other E2-stimulated rapid signaling responses we have observed
in human EC (5, 9, 12, 28), this activation is completely inhibited by
the conventional ER antagonist ICI 182,780, indicating that this is an
ER-mediated event (see below). PP2 also abrogates E2-induced c-Src
phosphorylation, consistent with the requirement for
autophosphorylation. However, E2-induced c-Src phosphorylation was not
inhibited by the specific PI3-kinase inhibitor LY294002, indicating E2
activation of Src kinase occurs before activation of the PI3-kinase/Akt
pathway.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of E2 on c-Src phosphorylation and
c-Src/ER association. EC monolayers were pretreated with ICI
182,780 (ICI, 10 µM), LY294002
(LY, 10 µM), or PP2 (10 µM) for 30 min before activation. E2 was added to the
monolayer in serum-free medium for the indicated times. A,
cells were washed, lysed and immunoblotted with
phosphorylation-specific c-Src antibody (pSrc).
Membranes were reprobed with total c-Src antibody (c-Src).
B, E2 (10 ng/ml) was added to the monolayer in serum-free
medium for the indicated times. Cells were washed, lysed, and
immunoprecipitated with anti-c-Src antibody-agarose conjugate.
Immunoprecipitates (IP) were immunoblotted with anti-ER
antibody and reprobed with anti-c-Src antibody. The ratio of ER that
was co-immunoprecipitated with c-Src was determined by densitometry
based upon total c-Src detected in the immunoblots. WB,
Western blot.
|
|
Although c-Src phosphorylation/activation is clearly E2-induced in EC
(Fig. 4A) and in osteoclasts (16, 25, 29), the precise
mechanism has not been defined. In osteoclasts, an interaction between
ligand-activated steroid receptors and c-Src seems required for kinase
activity (16, 25, 29). Western blots performed on coimmunoprecipitates
from E2-stimulated EAhy.926 cells demonstrated a rapidly induced
c-Src/ER association (Fig. 4B). The anti-ER antibody used is
a monoclonal antibody directed at the carboxyl terminus of ER
. In
human EC, including EAhy.926, the antibody blots/immunoprecipitates a
46-kDa protein that we believe to be the predominate
membrane-associated ER in these cells (Ref. 12, and below). Thus, E2
stimulation promotes the formation of a putative signaling complex
between the 46-kDa, signal-transducing ER and c-Src. As might be
expected from our previous results, PP2 also inhibited E2- but not
ionomycin-stimulated Erk1/2 activation (data not shown).
These data support the idea that E2-induced c-Src activation results in
parallel activation of the MAP kinase (Erk1/2) and Akt
pathways, the latter of which results in eNOS phosphorylation and
augmented NO release.
The Role of Src Kinase in E2-induced PI3-kinase Activation--
We
have previously demonstrated that E2-activated NO release can be
completely inhibited by the PI3-kinase inhibitor LY294002 (9),
indicating an absolute requirement for PI3-kinase. It was recently
demonstrated that ER
can associate with the regulatory subunit of
PI3-kinase, P85, and that this association correlates with increased
production of phosphatidylinositol 3,4,5-phosphates (10). The mechanism
by which ligand-induced ER/P85 association activates PI3-kinase remains
to be determined. In EAhy.926 cells, which do not express the
full-length ER, we thus evaluated whether this alternative form of ER
could associate with P85. E2-treated cell lysates were
immunoprecipitated with anti-ER
antibodies and Western blotted for
the presence of P85. E2 rapidly stimulated ER/P85 association in
EAhy.926 cells (Fig. 5A). This
association was blocked by the ER antagonist, ICI 182,780 and PP2,
indicating a role for Src kinase in this complex formation (Fig.
5B). Furthermore, c-Src was inducibly associated with P85 in
response to E2 (Fig. 5C), an effect that was also inhibited
by ICI 182,780 and PP2. Because PP2 treatment inhibited the apparent
upstream ER/P85 association, as well as downstream Akt and eNOS
activation, it was important to determine the effect of Src kinase
inhibition on E2-induced PI3-kinase activity. EAhy.926 cells were
pretreated with ICI 182,780, LY294002, PP2, or vehicle control for 30 min before E2 stimulation, after which PI3-kinase was
immunoprecipitated from stimulated cells with anti-P85 (Fig.
6) or anti-ER
antibodies. The
production of PI3-kinase generated D3-phosphoinositides (PIP) was
determined by an in vitro kinase assay. Fig. 6 demonstrates
that in P85 immunoprecipitates, E2 stimulation rapidly increased the
production of PIP, with maximum levels achieved in 10 min. These
increases in PIP were completely abrogated by ER antagonist ICI
182,780, LY294002, and PP2. Identical results were obtained when
E2-induced PI3-kinase activity was immunoprecipitated with anti-ER
antibodies. E2-stimulated increased production of PIP in ER
immunoprecipitates was also inhibited by pretreatment with ICI 182,780, LY294002 and PP2 (data not shown). This indicates that Src kinase is
required for the E2-mediated increase in PI3-kinase activity and that
Src is a component of the activated ER/PI3-kinase signaling complex.
Notably, PI3-kinase inhibition with LY294002 did not inhibit E2
activation of Src kinase (Fig. 4A), suggesting a sequential
activation cascade in which ER/c-Src association induces Src kinase
activity and the activated ER/c-Src complex consequently associates
with P85, effecting increased PI3-kinase activity.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of E2 on ER/P85/c-Src
interaction. A, E2-deprived and serum-starved EC
monolayers were stimulated with E2 (10 ng/ml) for 15 min. Cells were
washed, lysed, and immunoprecipitated with anti-P85 antibody.
Immunoprecipitates (IP) were immunoblotted with anti-ER
antibody and reprobed with anti-P85 antibody. WB, Western
blot. B and C, E2-deprived and serum-starved EC
monolayers were pretreated with ICI 182,780 (ICI, 10 µM), PP2 (10 µM), or vehicle for 30 min and
then stimulated with E2 for 10 min. Cells were washed, lysed, and
immunoprecipitated with anti-ER or anti-c-Src antibodies.
Immunoprecipitates were immunoblotted with anti-P85 antibody. Membranes
were reprobed with anti-ER antibody or anti-c-Src antibody.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of PP2 on E2-stimulated PI3-kinase
activity. EC monolayers were preincubated with ICI 182,870 (ICI, 10 µM), LY294002 (LY, 10 µM), PP2 (10 µM), or vehicle for 30 min
before E2 (10 ng/ml) stimulation for the indicated times. Cells were
then lysed and PI3-kinase was immunoprecipitated with anti-P85
antibody. PI3-kinase activity was determined by an in vitro
kinase assay. Thin layer chromatography of phosphorylated PIP is
shown.
|
|
The Role of c-Src in E2-stimulated Akt Activation--
E2-induced
phosphorylation of Akt was absent in fibroblasts devoid of Src family
kinases c-Src, fyn, and yes (SYF
/
). However, in cells
lacking fyn and yes, but maintaining normal expression of c-Src
(YF
/
S+/+), E2 markedly stimulated Akt
phosphorylation (Fig. 7A)
further implicating c-Src involvement in E2-induced Akt activation. To further document a critical role for c-Src in E2 activation of the
PI3-kinase/Akt pathway, EC were transiently transfected with either
control vector (Fig. 7B, lanes 1-3), a
kinase-dead c-Src (SrcK295M) (Fig. 7B, lanes 4 and 5), or a constitutively active c-Src (Src527F) (Fig.
7B, lanes 6 and 7). Cells were then
stimulated with E2, and the activation of Akt was determined by
presence of Ser473 phosphorylation. E2 treatment of
endothelial cells transfected with control vector resulted in increased
phosphorylation of Akt (Fig. 7B, compare lane 1 with lanes 2 and 3). In cells expressing the
kinase-dead Src, E2 was unable to stimulate Akt phosphorylation (Fig.
7B, compare lanes 4 and 5). Moreover,
cells transfected with the constitutively active c-Src in the absence
of additional stimulation demonstrated increased basal Akt
phosphorylation (Fig. 7B, compare lane 1 with
lanes 6 and 7). These data demonstrate that c-Src
can mediate the E2-induced Akt/eNOS activation response and that
activated c-Src, by itself, can effect Akt activation.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of c-Src mutant expression on
E2-induced Akt activation. A, either
YF / S+/+or SYF / murine
embryonic fibroblast monolayers were E2-deprived, serum-starved, and
stimulated with E2 for 15 min. Monolayers were washed, lysed, subjected
to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with
phosphorylation-specific Akt antibody (pAKT) and reprobed
with anti-Akt antibody (AKT). B, EC monolayers
were transiently transfected with either control vector (pcDNA3)
(lanes 1-3), kinase-dead c-Src (Src-K295M) (lanes
4 and 5) or constitutively active c-Src (Src 527F)
(lanes 6 and 7). Cells were E2-deprived,
serum-starved, and stimulated with E2 for 15 min. Cells were then
washed, lysed, and immunoblotted with phosphorylation-specific Akt
antibody (pAKT) and reprobed with anti-Akt antibody
(AKT). Lanes 2 and 3 represent
duplicate E2 stimulation of control cells.
|
|
 |
DISCUSSION |
There are now numerous reports of estrogen-induced endothelial NO
release in vitro and vasodilation in vivo (5, 9,
12, 28, 30-32). Although we have learned a great deal about the
downstream effectors in this important signaling pathway, the paradox
that a steroid hormone receptor can, upon engagement, be responsible for triggering rapid "transmembrane" signal transduction remains. In particular, the most proximal molecular components of this pathway
remain obscure. We previously demonstrated that estrogen, similar to
shear stress and insulin, can stimulate the enhancement of eNOS
activity in an ER-dependent fashion that does not require an intracellular Ca2+ flux (5). Since that initial
observation, we and others have demonstrated that E2 treatment of EC
results in rapid phosphorylation and activation of Akt with consequent
phosphorylation of eNOS on Ser1177. This phosphorylation
enhances electron flux through the eNOS reductase domain with a reduced
rate of calmodulin dissociation at low (resting) Ca2+
levels (7, 9). This provided the first mechanistic explanation for
E2-stimulated NO release in the absence of a Ca2+ flux.
However, the mechanism of E2-induced Akt phosphorylation remains unknown.
Our data, and those of others, have defined PI3-kinase as a critical
upstream activator in the E2-stimulated Akt/eNOS activation pathway (9,
10). In fact, a direct interaction between ER and P85, the regulatory
subunit of PI3-kinase, has been demonstrated, correlating with
increased PI3-kinase activity (10). However, the E2-stimulated
molecular switches responsible for the ER/PI3-kinase association are
not defined. There are several reasons to suspect that Src family
kinases could be the link between ER and PI3-kinase. First, we and
others have demonstrated, in parallel to Akt activation, that E2
stimulation of EC results in rapid ERK1/2 activation (12, 30). This response resembles that mediated by receptor tyrosine kinases, which, in some cases, recruit Src family kinases as a part of
a MAP kinase cascade. Second, P85 has been shown to be a Src kinase
(lck and abl) substrate (33-35), and fyn, lyn, and lck can, through
their SH3 domains, interact with P85 (36-41). Third, estrogen-induced
c-Src phosphorylation has been demonstrated in osteoclasts and breast
cancer cell lines (24, 29).
Here, we provide the first demonstration of ER-dependent
c-Src activation in EC, and that this activation provides a functional link between ER engagement and the PI3-kinase/Akt/eNOS pathway. A
pharmacological inhibitor of the Src family tyrosine kinases inhibited
not only Akt activation and NO release but also PI3-kinase dependent
generation of phosphatidylinositol phosphates, indicating that Src
activation is upstream of PI3-kinase. The c-Src specificity was
documented by inhibiting E2-induced Akt phosphorylation with a
kinase-dead c-Src. We now demonstrate an estrogen-stimulated molecular
complex formation, between ER, P85, and c-Src, that includes activated
c-Src, phosphorylated within 2 min of E2 treatment. The basis and
direct consequence of a P85/c-Src interaction remain to be determined,
although several possibilities exist. As noted above, P85 was shown to
be specifically phosphorylated on Tyr688 by the Src kinases
lck and abl (33-35), and PI3-kinase has been shown to be a
preferential substrate for c-Src (42, 43). It is also possible that
estrogen-activated c-Src could tyrosine phosphorylate docking proteins
containing binding sites for the SH2 domain of P85, thus alleviating,
upon interaction, the inhibitory constraint on the PI3-kinase P110
catalytic subunit (44, 45). Alternatively, the SH3 domains of several
Src kinases have been shown to bind directly to P85 and regulate its
activity (36-41). This includes c-Src that, in osteoclasts, interacts
directly through its SH3 domain with P85, in response to colony
stimulating factor-1 (46).
Although we believe that these rapid signaling responses to estrogen
have important implications in vascular tissue, other ligand-activated
steroid hormone receptors may have similar effects. Engagement of the
androgen receptor, but not the progesterone receptor, can result in
phosphatidylinositol 3,4,5-phosphate generation (10). As might be
expected, ER and androgen receptor have been shown to directly couple
with c-Src, whereas the progesterone receptor has not (25, 29),
consistent with the notion that steroid hormone receptor-induced
PI3-kinase activation is c-Src-dependent. In contrast, if
steroid hormone receptors heteromultimerize, responses can be
diversified. For example, PR and ER can associate in the absence of
ligand; in this setting, either progestins or estrogens can rapidly
trigger c-Src activation (25). Also, a ternary androgen receptor/ER/c-Src complex has been demonstrated, through an
ER-pTyr537/c-Src-SH2 and androgen receptor/c-Src-SH3
interaction (29). Whether ER-pTyr537 is constitutively or
inducibly (by estrogen) phosphorylated remains unclear.
The expectation is that those ER-dependent sequential
c-Src/PI3-kinase/Akt activation events are rapidly catalyzed at the plasma membrane. This brings the focus back to that of a
non-conventional, membrane-localized steroid hormone receptor-signaling
pathway. There is an impressive and growing list of membrane steroid
hormone-mediated responses in a variety of cells (9, 11-14, 47-55).
We have recently taken advantage of the EAhy.926 EC line, which, under
the described culture conditions, does not express the 66-kDa,
estrogen-responsive element-enhancing ER but does express a 46-kDa ER
that is capable of transducing the signals we have described previously
(9, 12). We are currently identifying the requirements for membrane localization and preferentially expressed forms of ER in vascular tissue, which are responsible for ligand-induced c-Src activation and
consequent NO release. As we come closer to identifying the most
proximal components of this signal transduction cascade, the
feasibility of targeting reagents to positively modulate cardiovascular responses expands.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Lynn O'Donnell for
technical assistance and all those providing valuable reagents,
including K. Walsh for the recombinant adenoviruses used.
 |
FOOTNOTES |
*
This work was supported in part by the National Institute of
Health Grant HL61782 (to J. R. B.).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.
**
To whom correspondence should be addressed: 295 Congress Ave, New
Haven, CT 06536. Tel.: 203-737-2223; Fax: 203-737-2293; E-mail:
jeffrey.bender@yale.edu.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M210828200
 |
ABBREVIATIONS |
The abbreviations used are:
NO, nitric oxide;
E2, 17
-estradiol;
EC, endothelial cells;
ER, estrogen receptor;
eNOS, endothelial nitric-oxide synthase;
PI3-kinase, phosphatidylinositol 3-kinase;
MAP, mitogen-activated protein;
DMEM, Dulbecco's modified Eagle's medium;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
-gal,
-galactosidase;
myr-Akt, membrane-targeted, myristoylated
Akt;
HUVEC, human umbilical vein endothelial cell;
PIP, phosphatidylinositol phosphate.
 |
REFERENCES |
1.
|
Mendelsohn, M. E.,
and Karas, R. H.
(1999)
N. Engl. J. Med.
340,
1801-1811[Free Full Text]
|
2.
|
Rubanyi, G. M.,
Freay, A. D.,
Kauser, K.,
Sukovich, D.,
Burton, G.,
Lubahn, D. B.,
Couse, J. F.,
Curtis, S. W.,
and Korach, K. S.
(1997)
J. Clin. Invest.
99,
2429-2437[Abstract/Free Full Text]
|
3.
|
Guetta, V.,
Quyyumi, A. A.,
Prasad, A.,
Panza, J. A.,
Waclawiw, M.,
and Cannon, R. O., 3rd
(1997)
Circulation
96,
2795-2801[Abstract/Free Full Text]
|
4.
|
Best, P. J.,
Berger, P. B.,
Miller, V. M.,
and Lerman, A.
(1998)
Ann. Intern. Med.
128,
285-288[Abstract/Free Full Text]
|
5.
|
Caulin-Glaser, T.,
Garcia-Cardena, G.,
Sarrel, P.,
Sessa, W. C.,
and Bender, J. R.
(1997)
Circ. Res.
81,
885-892[Abstract/Free Full Text]
|
6.
|
Fulton, D.,
Gratton, J. P.,
and Sessa, W. C.
(2001)
J. Pharmacol. Exp. Ther.
299,
818-824[Abstract/Free Full Text]
|
7.
|
McCabe, T. J.,
Fulton, D.,
Roman, L. J.,
and Sessa, W. C.
(2000)
J. Biol. Chem.
275,
6123-6128[Abstract/Free Full Text]
|
8.
|
Fulton, D.,
Gratton, J. P.,
McCabe, T. J.,
Fontana, J.,
Fujio, Y.,
Walsh, K.,
Franke, T. F.,
Papapetropoulos, A.,
and Sessa, W. C.
(1999)
Nature
399,
597-601[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Haynes, M. P.,
Sinha, D.,
Russell, K. S.,
Collinge, M.,
Fulton, D.,
Morales-Ruiz, M.,
Sessa, W. C.,
and Bender, J. R.
(2000)
Circ. Res.
87,
677-682[Abstract/Free Full Text]
|
10.
|
Simoncini, T.,
Hafezi-Moghadam, A.,
Brazil, D. P.,
Ley, K.,
Chin, W. W.,
and Liao, J. K.
(2000)
Nature
407,
538-541[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Pappas, T. C.,
Gametchu, B.,
and Watson, C. S.
(1995)
FASEB J
9,
404-410[Abstract/Free Full Text]
|
12.
|
Russell, K.,
Haynes, M.,
Sinha, D.,
Clerisme, E.,
and Bender, J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5930-5935[Abstract/Free Full Text]
|
13.
|
Marquez, D. C.,
and Pietras, R. J.
(2001)
Oncogene
20,
5420-5430[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Chambliss, K. L.,
Yuhanna, I. S.,
Mineo, C.,
Liu, P.,
German, Z.,
Sherman, T. S.,
Mendelsohn, M. E.,
Anderson, R. G.,
and Shaul, P. W.
(2000)
Circ. Res.
87,
E44-52[Medline]
[Order article via Infotrieve]
|
15.
|
Pendaries, C.,
Darblade, B.,
Rochaix, P.,
Krust, A.,
Chambon, P.,
Korach, K. S.,
Bayard, F.,
and Arnal, J. F.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
2205-2210[Abstract/Free Full Text]
|
16.
|
Migliaccio, A., Di,
Domenico, M.,
Castoria, G.,
de Falco, A.,
Bontempo, P.,
Nola, E.,
and Auricchio, F.
(1996)
EMBO J.
15,
1292-1300[Abstract]
|
17.
|
Migliaccio, A.,
Pagano, M.,
and Auricchio, F.
(1993)
Oncogene
8,
2183-2191[Medline]
[Order article via Infotrieve]
|
18.
|
Edgell, C. J.,
McDonald, C. C.,
and Graham, J. B.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
3734-3737[Abstract]
|
19.
|
Klinghoffer, R. A.,
Sachsenmaier, C.,
Cooper, J. A.,
and Soriano, P.
(1999)
EMBO J.
18,
2459-2471[Abstract/Free Full Text]
|
20.
|
Persad, S.,
Attwell, S.,
Gray, V.,
Mawji, N.,
Deng, J. T.,
Leung, D.,
Yan, J.,
Sanghera, J.,
Walsh, M. P.,
and Dedhar, S.
(2001)
J. Biol. Chem.
276,
27462-27469[Abstract/Free Full Text]
|
21.
|
Chen, R.,
Kim, O.,
Yang, J.,
Sato, K.,
Eisenmann, K. M.,
McCarthy, J.,
Chen, H.,
and Qiu, Y.
(2001)
J. Biol. Chem.
276,
31858-31862[Abstract/Free Full Text]
|
22.
|
Hanke, J. H.,
Gardner, J. P.,
Dow, R. L.,
Changelian, P. S.,
Brissette, W. H.,
Weringer, E. J.,
Pollok, B. A.,
and Connelly, P. A.
(1996)
J. Biol. Chem.
271,
695-701[Abstract/Free Full Text]
|
23.
|
Salazar, E. P.,
and Rozengurt, E.
(1999)
J. Biol. Chem.
274,
28371-28378[Abstract/Free Full Text]
|
24.
|
Kousteni, S.,
Bellido, T.,
Plotkin, L. I.,
O'Brien, C. A.,
Bodenner, D. L.,
Han, L.,
Han, K.,
DiGregorio, G. B.,
Katzenellenbogen, J. A.,
Katzenellenbogen, B. S.,
Roberson, P. K.,
Weinstein, R. S.,
Jilka, R. L.,
and Manolagas, S. C.
(2001)
Cell
104,
719-730[Medline]
[Order article via Infotrieve]
|
25.
|
Migliaccio, A.,
Piccolo, D.,
Castoria, G., Di,
Domenico, M.,
Bilancio, A.,
Lombardi, M.,
Gong, W.,
Beato, M.,
and Auricchio, F.
(1998)
EMBO J.
17,
2008-2018[Free Full Text]
|
26.
|
Brubaker, K. D.,
and Gay, C. V.
(1999)
J. Cell. Biochem.
76,
206-216[Medline]
[Order article via Infotrieve]
|
27.
|
Bjorge, J. D.,
Jakymiw, A.,
and Fujita, D. J.
(2000)
Oncogene
19,
5620-5635[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Russell, K. S.,
Haynes, M. P.,
Caulin-Glaser, T.,
Rosneck, J.,
Sessa, W. C.,
and Bender, J.
(2000)
J. Biol. Chem.
275,
5026-5030[Abstract/Free Full Text]
|
29.
|
Migliaccio, A.,
Castoria, G., Di,
Domenico, M.,
de Falco, A.,
Bilancio, A.,
Lombardi, M.,
Barone, M. V.,
Ametrano, D.,
Zannini, M. S.,
Abbondanza, C.,
and Auricchio, F.
(2000)
EMBO J.
19,
5406-5417[Abstract/Free Full Text]
|
30.
|
Chen, Z.,
Yuhanna, I. S.,
Galcheva-Gargova, Z.,
Karas, R. H.,
Mendelsohn, M. E.,
and Shaul, P. W.
(1999)
J. Clin. Invest.
103,
401-406[Abstract/Free Full Text]
|
31.
|
Lantin-Hermoso, R. L.,
Rosenfeld, C. R.,
Yuhanna, I. S.,
German, Z.,
Chen, Z.,
and Shaul, P. W.
(1997)
Am. J. Physiol.
273,
L119-L126[Abstract/Free Full Text]
|
32.
|
Kirsch, E. A.,
Yuhanna, I. S.,
Chen, Z.,
German, Z.,
Sherman, T. S.,
and Shaul, P. W.
(1999)
Am. J. Respir. Cell Mol. Biol.
20,
658-666[Abstract/Free Full Text]
|
33.
|
von Willebrand, M.,
Williams, S.,
Saxena, M.,
Gilman, J.,
Tailor, P.,
Jascur, T.,
Amarante-Mendes, G. P.,
Green, D. R.,
and Mustelin, T.
(1998)
J. Biol. Chem.
273,
3994-4000[Abstract/Free Full Text]
|
34.
|
Cuevas, B., Lu, Y.,
Watt, S.,
Kumar, R.,
Zhang, J.,
Siminovitch, K. A.,
and Mills, G. B.
(1999)
J. Biol. Chem.
274,
27583-27589[Abstract/Free Full Text]
|
35.
|
Cuevas, B. D., Lu, Y.,
Mao, M.,
Zhang, J.,
LaPushin, R.,
Siminovitch, K.,
and Mills, G. B.
(2001)
J. Biol. Chem.
276,
27455-27461[Abstract/Free Full Text]
|
36.
|
Herrera-Velit, P.,
and Reiner, N. E.
(1996)
J. Immunol.
156,
1157-1165[Abstract]
|
37.
|
Kapeller, R.,
Prasad, K. V.,
Janssen, O.,
Hou, W.,
Schaffhausen, B. S.,
Rudd, C. E.,
and Cantley, L. C.
(1994)
J. Biol. Chem.
269,
1927-1933[Abstract/Free Full Text]
|
38.
|
Mak, P., He, Z.,
and Kurosaki, T.
(1996)
FEBS Lett.
397 (2-3),
183-5[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Prasad, K. V.,
Janssen, O.,
Kapeller, R.,
Raab, M.,
Cantley, L. C.,
and Rudd, C. E.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7366-7370[Abstract]
|
40.
|
Prasad, K. V.,
Kapeller, R.,
Janssen, O.,
Duke-Cohan, J. S.,
Repke, H.,
Cantley, L. C.,
and Rudd, C. E.
(1993)
Philos. Trans. R. Soc. Lond-Biol. Sci.
342,
35-42[Medline]
[Order article via Infotrieve]
|
41.
|
Susa, M.,
Rohner, D.,
and Bichsel, S.
(1996)
Biochem. Biophys. Res. Commun.
220,
729-734[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Cantley, L. C.,
Auger, K. R.,
Carpenter, C.,
Duckworth, B.,
Graziani, A.,
Kapeller, R.,
and Soltoff, S.
(1991)
Cell
64,
281-302[Medline]
[Order article via Infotrieve]
|
43.
|
Carpenter, C. L.,
Duckworth, B. C.,
Auger, K. R.,
Cohen, B.,
Schaffhausen, B. S.,
and Cantley, L. C.
(1990)
J. Biol. Chem.
265,
19704-19711[Abstract/Free Full Text]
|
44.
|
Lee, A. W.,
and States, D. J.
(2000)
Mol. Cell. Biol.
20,
6779-6798[Abstract/Free Full Text]
|
45.
|
Shinohara, M.,
Kodama, A.,
Matozaki, T.,
Fukuhara, A.,
Tachibana, K.,
Nakanishi, H.,
and Takai, Y.
(2001)
J. Biol. Chem.
276,
18941-18946[Abstract/Free Full Text]
|
46.
|
Grey, A.,
Chen, Y.,
Paliwal, I.,
Carlberg, K.,
and Insogna, K.
(2000)
Endocrinology
141,
2129-2138[Abstract/Free Full Text]
|
47.
|
Brubaker, K. D.,
and Gay, C. V.
(1999)
Calcif. Tissue Int.
64,
459-462[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Falkenstein, E.,
Heck, M.,
Gerdes, D.,
Grube, D.,
Christ, M.,
Weigel, M.,
Buddhikot, M.,
Meizel, S.,
and Wehling, M.
(1999)
Endocrinology
140,
5999-6002[Abstract/Free Full Text]
|
49.
|
Germain, P. S.,
Metezeau, P.,
Tiefenauer, L. X.,
Kiefer, H.,
Ratinaud, M. H.,
and Habrioux, G.
(1993)
Anticancer Res.
13,
2347-2353[Medline]
[Order article via Infotrieve]
|
50.
|
Kelly, M. J.,
and Levin, E. R.
(2001)
Trends Endocrinol. Metab.
12,
152-156[CrossRef][Medline]
[Order article via Infotrieve]
|
51.
|
Pietras, R. J.,
and Szego, C. M.
(1977)
Nature
265,
69-72[Medline]
[Order article via Infotrieve]
|
52.
|
Razandi, M.,
Pedram, A.,
Greene, G. L.,
and Levin, E. R.
(1999)
Mol. Endocrinol.
13,
307-319[Abstract/Free Full Text]
|
53.
|
Razandi, M.,
Pedram, A.,
and Levin, E. R.
(2000)
J. Biol. Chem.
275,
38540-38546[Abstract/Free Full Text]
|
54.
|
Watson, C. S.,
and Gametchu, B.
(1999)
Proc. Soc. Exp. Biol. Med.
220,
9-19[Abstract]
|
55.
|
Watters, J. J.,
Campbell, J. S.,
Cunningham, M. J.,
Krebs, E. G.,
and Dorsa, D. M.
(1997)
Endocrinology
138,
4030-4033[Abstract/Free Full Text]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.