From the Departments of Obstetrics and Gynecology and
Biochemistry, Osaka University Medical School, 2-2 Yamadaoka,
Suita, Osaka 565-0871 and the ¶ Department of Obstetrics and
Gynecology, Yamagata University School of Medicine, 2-2-2 Iidanishi,
Yamagata, Yamagata 990-9585, Japan
Received for publication, June 12, 2000, and in revised form, September 19, 2000
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ABSTRACT |
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Although estrogen is known to activate
endothelial nitric oxide synthase (eNOS) in the vascular endothelium,
the molecular mechanism responsible for this effect remains to be
elucidated. In studies of both human umbilical vein endothelial cells
(HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17 The inhibitory effect of estrogen on the development of
atherosclerosis has been suggested by abundant human epidemiological and animal experimental data (1-9). The incidence of atherosclerotic diseases is lower in premenopausal women than in men, steeply rises in
postmenopausal women, and is reduced to premenopausal levels in
postmenopausal women who receive estrogen therapy (10-12). Until
recently, the atheroprotective effects of estrogen were attributed
principally to the effects on serum lipid concentrations. However,
estrogen-induced alterations in serum lipids account for only
approximately one-third of the observed clinical benefits of estrogen
(12-14). Recent evidence suggests that the direct actions of estrogen
on blood vessels contribute to the cardioprotective effects of estrogen
(13, 15). There are many kinds of direct effects of estrogen on blood
vessels, such as estrogen-induced increases of vasodilatation and
inhibition of the response of blood vessels to injury and the
development of atherosclerosis. However, the molecular mechanism
underlying the estrogen-induced vasodilatation has not yet been
determined. Several studies suggest that a key mediator of this
vasodilator response could be the endothelium-derived relaxing factor
nitric oxide (NO), and that brief treatment with estrogen increases
basal NO release in endothelial cells without elevation of eNOS
mRNA or protein (16). Estrogen activates endothelial nitric oxide
synthase (eNOS) without altering expression of the eNOS gene in
vascular endothelium (17-20). However, the details of the mechanism of
the estrogen-induced eNOS activation are not yet well understood.
The serine/threonine kinase termed Akt or protein kinase B
(PKB)1 is an important
regulator of various cellular processes, including glucose metabolism
and cell survival (21, 22). Activation of receptor tyrosine kinases and
G-protein-coupled receptors, and stimulation of cells by mechanical
force, can lead to the phosphorylation and activation of Akt (23-25).
Akt was identified as a downstream component of survival signaling
through phosphatidylinositol 3-kinase (PI3K) (26-30). Akt may be
regulated by both phosphorylation and the direct binding of PI3K lipid
products to the Akt pleckstrin homology domain. Akt can then
phosphorylate substrates such as glycogen synthase kinase-3,
6-phosphofructo-2-kinase, and BAD. More recently, it was found
that eNOS is also an Akt substrate and is activated by
Akt-dependent phosphorylation to release NO in endothelial
cells (31-34).
The actions of estrogen can be mediated by the classical nuclear
receptors, ER Materials--
17 Cell Cultures--
TRLECs (41), kindly provided by Dr. K. Fukuo
and Dr. S. Morimoto (Osaka University Medical School, Japan), and
Chinese hamster ovary (CHO) cells, obtained from American Type Culture
Collection (Rockville, MD), were cultured at 37 °C in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum in a
water-saturated atmosphere of 95% O2 and 5%
CO2. HUVECs were isolated according to the method of Jaffe
et al. (42), plated in gelatin-coated tissue culture wells,
and grown in M199 medium containing 20% fetal bovine serum and 50 µg/ml endothelial cell growth supplement (Clonetics Corp., San Diego,
CA). HUVECs were used at passage 2 or 3.
Constructs--
The vectors encoding the various HA-tagged forms
of Akt, wild-type Akt (HA-Akt), kinase-inactive Akt (HA-AktK179M), and
constitutively active Akt (HA-m Assay of eNOS Activity--
Cells were serum-starved overnight
in phenol red-free medium before eNOS activity measurements. eNOS
activity was determined as the conversion of radiolabeled
L-arginine to L-citrulline by a method
described previously (50, 51) with a minor modification. Briefly, 10 µl of a sample was incubated for 10 min at 37 °C in a solution
consisting of 50 mM HEPES, 1 mM dithiothreitol,
1 mM CaCl2, 0.1 mM
tetrahydro-L-biopterin, 1 mM NADPH, 10 µg/ml
calmodulin, 10 µM FAD, and 1.55 µM
L-[guanidino-14C]arginine (pH
7.8), in a final volume of 100 µl. The reaction was terminated by the
addition of 200 µl of buffer A (100 mM HEPES, 10 mM EDTA, pH 5.2). The whole reaction mixture was then
applied to a 0.3-ml Dowex 50-WX column (Na+ form, 200-400
mesh) that had been equilibrated with buffer A. Citrulline was eluted
with 0.5 ml of buffer A, and then radioactivity was measured with a
liquid scintillation counter.
Assay of eNOS Activity Using a Transient Expression
System--
TRLECs cultured in 100-mm dishes were transfected with 1 µg of CMV-6, 1 µg of CMV-6 containing the gene for HA-AktK179M, 1 µg of pSG5, 1 µg of ER Assay of Akt Kinase Activity--
Cells were serum-starved
overnight in phenol red-free medium and then treated with various
materials. They were then washed twice with phosphate-buffered saline
and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM Assay of Akt Activity Using a Transient Expression
System--
CHO cells cultured in 100-mm dishes were transfected with
1 µg of pSG5, 1 µg of ER Preparation of Partially Purified eNOS--
Human eNOS was
overexpressed in Sf-21 cells, which had been infected with baculovirus
carrying human eNOS cDNA (56). Human eNOS was partially purified by
chromatography on 2',5'-ADP-Sepharose gel, and its specificity was
determined as described previously (57).
Assay of eNOS Phosphorylation--
TRLECs cultured in 100-mm
dishes were treated with 10 Statistics--
Statistical analysis was performed using
Student's t test, and p < 0.05 was
considered significant. Data are expressed as the mean ± S.E.
eNOS Activation by 17 Activation of Akt by 17
To determine whether this process involves rapid ER activation, the
effect of concomitant treatment with the pure ER antagonist ICI-182,780
was determined (Fig. 4B, lane 4). ICI-182,780
clearly caused a decrease in 17 Akt-dependent eNOS Phosphorylation and
Activation--
To determine whether 17 Role of Extracellular and Intracellular Ca2+ in
17 Effect of ER This study showed that Akt is activated in HUVECs and TRLECs by
17 Normal endothelium secretes nitric oxide, which relaxes vascular smooth
muscle and inhibits platelet activation (62). In cultured endothelial
cells, physiologic concentrations of estrogen cause a rapid release of
nitric oxide without altering gene expression (17, 18). The rapidity of
the activation of Akt (Fig. 3) and eNOS (Fig. 1) by 17 There are two estrogen receptors, estrogen receptor eNOS is a Ca2+/calmodulin-dependent enzyme, and
it has been reported that estrogen induces translocation of eNOS by a
Ca2+-dependent and receptor-mediated mechanism
(58). Because A23187 induces eNOS activation and produces
endothelium-dependent vascular relaxation, Ca2+
entry from the extracellular space into endothelial cells also plays a
key role (19, 59). Ca2+/calmodulin-dependent
protein kinase kinase can activate and phosphorylate Akt (74). In
addition, A23187 also activates Akt (75). More recently, it was also
reported that plasma-lemmal caveolae-associated ER Estrogen can cause the rapid activation of signaling pathways involving
c-src-related tyrosine kinases and MAP kinases in nonendothelial cells (77, 78). It has also been reported that tyrosine
kinase-MAP kinase signaling is involved in acute ER-estradiol (E2), but not 17
-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17
-E2
stimulated the activation of Akt, and the PI3K inhibitor
wortmannin blocked the 17
-E2-induced activation of Akt.
17
-E2-induced Akt activation was also inhibited by ICI-182,780, but
not by actinomycin D. Either treatment with wortmannin or exogenous
expression of a dominant negative Akt in TRLECs decreased the
17
-E2-induced eNOS activation. Moreover, 17
-E2-induced Akt
activation actually enhances the phosphorylation of eNOS.
17
-E2-induced Akt activation was dependent on both extracellular and
intracellular Ca2+. We further examined the
17
-E2-induced Akt activity in Chinese hamster ovary (CHO) cells
transiently transfected with cDNAs for estrogen receptor
(ER
) or estrogen receptor
(ER
). 17
-E2 stimulated the
activation of Akt in CHO cells expressing ER
but not in CHO cells
expressing ER
. Our findings suggest that 17
-E2 induced eNOS
activation through an Akt-dependent mechanism, which is
mediated by ER
via a nongenomic mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
(35, 36) or through other putative membrane
receptors. By definition, rapid effects of estrogen that involve
nongenomic mechanisms are independent of transcriptional activation by
the nuclear ERs. These rapid effects are believed to be mediated by
receptors located in or close to the plasma membrane (37, 38).
Estrogen-induced vasodilatation occurs 5-20 min after estrogen
administration (39, 40) and is not dependent on changes in gene
expression; this action of estrogen is sometimes referred to as
"nongenomic." Therefore, we sought to determine whether the
estrogen-induced eNOS activation is mediated by Akt activation and
which type of ER is involved in this effect using both human umbilical
vein endothelial cells (HUVECs) and simian virus 40-transformed rat
lung vascular endothelial cells (TRLECs) (41).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-E2, 17
-E2, E2-17-BSA (17
-estradiol
(17-hemisuccinate/BSA; 38 mol E2/mol BSA), actinomycin D, and
wortmannin were purchased from Sigma Chemical Co. (St. Louis, MO).
ICI-182,780 was obtained from Tocris (Ballwin, MO). ECL Western
blotting detection reagents were obtained from Amersham Pharmacia
Biotech (Arlington Heights, IL). Rabbit polyclonal anti-Akt antibody
and an Akt kinase assay kit, including GSK-3 fusion protein and a
phospho-specific GSK-3
/
antibody, were obtained from New England
BioLabs (Beverly, MA). Rabbit polyclonal anti-hemagglutinin (HA)
antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
4-129Akt) used in this study
have been described previously (26, 43, 45-47). The vectors encoding
the wild-type eNOS and mutant eNOS of serine 1179 to alanine (S1179A
eNOS) was a kind gift from Dr. W. C. Sessa (Yale University, New
Haven, CT) (31). The human estrogen receptor
(ER
) expression
vector, pSG5-HEGO, was a kind gift from Dr. P. Chambon (Institut de
Chimie Biologique, Strasbourg, France) (48). The plasmid pSG5-mER
encoding nucleotides 12-1469 of ER
(35) was kindly provided Dr.
E. R. Levin (University of California, Irvine, CA) via Dr. K. S. Korach (National Institutes of Health, Research Triangle Park, NC).
expression vector (pSG5-HEGO), or 1 µg of ER
expression vector (pSG5-mER
) using LipofectAMINE plus (Life
Technologies, Inc.) as described previously (52, 53). Seventy-two hours
after transfection, serum-deprived cells were incubated with
10
7 M 17
-E2 for 15 min, and the eNOS
activity was measured as described above.
-glycerolphosphate, 1 mM sodium
orthovanadate, 1 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride). The extracts were centrifuged to remove
cellular debris, and the protein content of the supernatants was
determined using the Bio-Rad protein assay reagent. 250 µg of protein
from the lysate samples was incubated with gentle rocking at 4 °C
overnight with immobilized anti-Akt antibody cross-linked to agarose
hydrazide beads. After Akt was selectively immunoprecipitated from the
cell lysates, the immunoprecipitated products were washed twice in lysis buffer and twice in kinase assay buffer (25 mM Tris,
pH 7.5, 10 mM MgCl2, 5 mM
-glycerolphosphate, 0.1 mM sodium orthovanadate, and 2 mM dithiothreitol), and the samples were resuspended in 40 µl of kinase assay buffer containing 200 µM ATP and 1 µg of GSK-3
fusion protein. The kinase reaction was allowed to
proceed at 30 °C for 30 min and stopped by the addition of Laemmli
SDS sample buffer (54). Reaction products were resolved by 15%
SDS-PAGE followed by Western blotting (55) with an
anti-phospho-GSK-3
/
antibody as described previously (47).
expression vector (pSG5-HEGO), or 1 µg of ER
expression vector (pSG5-mER
) using LipofectAMINE plus as described previously (52, 53). Seventy-two hours after transfection,
serum-deprived cells were incubated with 10
7
M 17
-E2 for 15 min, and the Akt activity was measured as
described above.
7 M 17
-E2 for
15 min. Cell lysates were subjected to immunoprecipitation with
anti-Akt antibody. For assay using a transient expression system,
TRLECs cultured in 100-mm dishes were transfected with 1 µg of
HA-Akt, 1 µg of HA-AktK179M, or 1 µg of HA-m
4-129Akt using
LipofectAMINE plus (Life Technologies, Inc.) as described previously
(52, 53). Seventy-two hours after transfection, serum-deprived cells
were incubated with 10
7 M 17
-E2 for 15 min, and lysates were immunoprecipitated with anti-HA antibody. The
immunoprecipitated products were washed once in lysis buffer and twice
in kinase assay buffer, and samples were resuspended in 30 µl of
kinase assay buffer containing 40 µM
[
-32P]ATP (1 µCi) and 5 µg of partially purified
eNOS, or 5 µg of recombinant wild-type or S1179A eNOS purified from
Escherichia coli. The kinase reaction was allowed to proceed
at room temperature for 5 min and stopped by the addition of Laemmli
SDS sample buffer (54). Reaction products were resolved by 8%
SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-E2--
To evaluate whether eNOS is
activated by 17
-E2 in TRLECs (Fig.
1A, upper panel)
and HUVECs (Fig. 1A, lower panel), cultured cells
were exposed to 17
-E2 for the indicated times. The increase in eNOS
activity induced by 10
7 M 17
-E2 reached a
plateau from 15 through 30 min and rapidly declined thereafter. The
dose dependence of 17
-E2-induced eNOS activation was also evaluated
in TRLECs (Fig. 1B). TRLECs were treated with various
concentrations of 17
-E2 for 15 min. In the range of
10
10 to 10
7 M, 17
-E2 induced
the activation of eNOS in a dose-dependent manner. A higher
concentration (10
6 M) of 17
-E2 did not
induce a stronger response (data not shown). The response was specific
for 17
-E2, because 17
-E2 had no effect (Fig.
2A). To determine whether this
response involves rapid ER activation, the effect of concomitant
treatment with the pure ER antagonist ICI-182,780 was determined (Fig.
2B). ICI-182,780 completely abolished the induction of eNOS
activation by 17
-E2. Moreover, the effects of E2-17-BSA, a
membrane-impermeable conjugate of E2, and actinomycin D, an inhibitor
of gene transcription, were tested to rule out the influence of genomic
events mediated by nuclear ERs (Fig. 2C). E2-17-BSA
stimulated an increase in eNOS activity similar to that induced by
17
-E2, and actinomycin D did not affect the induction of eNOS
activation by 17
-E2.
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Fig. 1.
Activation of eNOS in endothelial cells.
Cells were grown in 60-mm dishes. A, TRLECs (upper
panel) and HUVECs (lower panel) were treated with
10 7 M 17
-E2 for the indicated times.
B, TRLECs were treated with the indicated concentrations of
17
-E2 for 15 min. eNOS activity was measured by the conversion of
L-[guanidino-14C]arginine to
L-[guanidino-14C]citrulline, as
described under "Experimental Procedures." The basal activity of
eNOS was arbitrarily set at 1.0. Data are expressed as the mean -fold
activation ± S.E. of six separate experiments. *p < 0.05 and **p < 0.01 as compared with the control,
respectively.
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Fig. 2.
Specificity of the augmentation of eNOS
activation by 17 -E2. Cells were grown in
60-mm dishes. A, TRLECs were treated with 10
7
M 17
-E2 or 10
7 M 17
-E2 for
15 min. B, TRLECs were pretreated with 10
6
M ICI-182,780 for 15 min, followed by treatment with
10
7 M 17
-E2 for 15 min. C,
TRLECs were pretreated with or without 25 µg/ml actinomycin D
(Act-D) for 120 min, followed by treatment with
10
7 M 17
-E2 or 10
7
M E2-17-BSA for 15 min. eNOS activity was
measured by the conversion of
L-[guanidino-14C]arginine to
L-[guanidino-14C]citrulline, as
described under "Experimental Procedures." The basal activity of
eNOS was arbitrarily set at 1.0. Data are expressed as the mean -fold
activation ± S.E. of six separate experiments.
**p < 0.01 as compared with the control.
-E2--
To determine whether Akt is
activated by 17
-E2 in TRLECs and HUVECs, 17
-E2 was added to
cultured cells for the indicated times (Fig.
3A) and at the indicated
concentrations for 15 min (Fig. 3B). Cell lysates were
subjected to immunoprecipitation with immobilized anti-Akt antibody,
and then supplemented with GSK-3
fusion protein and analyzed by
Western blotting with anti-phospho-GSK-3
/
antibody. Activation of
Akt by 17
-E2 in both TRLECs and HUVECs reached a plateau at 15 min,
and declined thereafter (Fig. 3A). 17
-E2 induced the
activation of Akt in a dose-dependent manner in TRLECs
(Fig. 3B) and HUVECs (data not shown). The response was
specific for 17
-E2, because 10
10-10
7
M 17
- E2 had no effect (Fig.
4A). Because Akt is an
effector of survival signaling downstream of PI3K (26-30), we next
examined whether stimulation of TRLECs with 17
-E2 could increase the
activity of Akt through a PI3K-dependent mechanism. TRLECs
were stimulated with 17
-E2 in the presence or absence of wortmannin,
a PI3K inhibitor, and the kinase activity of Akt was assayed. The
induction of Akt activity by 17
-E2 was inhibited by wortmannin (Fig.
4B, lane 6). These results indicate that E2
activates Akt activity through a PI3K-dependent
mechanism.
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Fig. 3.
Activation of Akt by
17 -E2 in endothelial cells. Cells were
grown in 100-mm dishes. A, TRLECs (upper panel)
and HUVECs (lower panel) were treated with 10
7
M 17
-E2 for the indicated times. B, TRLECs
were treated with the indicated concentrations of 17
-E2 for 15 min.
Lysates were subsequently subjected to immunoprecipitation with
immobilized anti-Akt antibody, and the kinase reaction was carried out
in the presence of cold ATP and GSK-3
fusion protein, as described
under "Experimental Procedures." After the reactions were stopped
with Laemmli sample buffer, samples were resolved by 12% SDS-PAGE and
then analyzed by Western blotting with an anti-phospho-GSK-3
/
antibody. Experiments were repeated three times with essentially
identical results.
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Fig. 4.
Specificity of the augmentation of Akt
activation by 17 -E2. Cells were grown in
100-mm dishes. A, TRLECs were treated with 10
7
M 17
-E2 or the indicated concentrations of 17
-E2 for
15 min. B, TRLECs were pretreated with or without 2 × 10
7 M wortmannin for 15 min or
10
6 M ICI-182,780 for 15 min, followed by
treatment with 10
7 M 17
-E2 for 15 min.
C, TRLECs were pretreated with or without 25 µg/ml
actinomycin D (Act-D) for 120 min, followed by treatment
with 10
7 M 17
-E2 or 10
7
M E2-17-BSA for 15 min. Experiments were repeated three
times with essentially identical results.
-E2-induced Akt activation. Moreover, E2-17-BSA, a membrane-impermeable conjugate of E2, and actinomycin D,
an inhibitor of gene transcription, were used to rule out the influence
of genomic events mediated by nuclear ERs (Fig. 4C). E2-17-BSA also stimulated an increase in Akt activity, and actinomycin D did not affect the induction of Akt activity by 17
-E2.
-E2-induced Akt activation
is involved in the phosphorylation of eNOS, 10
7
M 17
-E2 was added to cultured cells for 15 min. Cell
lysates were subjected to immunoprecipitation with anti-Akt antibody, and then assayed in an immunocomplex kinase assay using purified eNOS
(57) as a substrate (Fig. 5A,
left panel). 17
-E2 directly increased the phosphorylation
of eNOS in anti-Akt immunoprecipitates. Moreover, we evaluated the
effect of exogenous expression of various forms of Akt on the in
vitro phosphorylation of purified eNOS. TRLEC transfected with
wild-type or mutant forms of hemagglutinin (HA)-tagged Akt were exposed
to 10
7 M 17
-E2 for 15 min, and extracts
from these cells were immunoprecipitated with anti-HA antibody and
assayed in an immunocomplex kinase assay for their ability to
phosphorylate purified eNOS. Akt constructs that were expressed in
TRLEC included HA-tagged wild-type Akt (HA-Akt), an Akt derivative
rendered kinase-inactive by point mutation within the Akt catalytic
domain (HA-AktK179M), and an Akt derivative rendered constitutively
active by targeting it to the plasma membrane with a myristoyl tag
(HA-m
4-129Akt) (26, 43, 45-47). 17
-E2 directly increased the
phosphorylation of eNOS in anti-HA immunoprecipitates prepared from
TRLEC transfected with wild-type Akt (Fig. 5A, lane
2). Anti-HA immunoprecipitates prepared from TRLEC transfected
with the kinase-inactive Akt failed to phosphorylate eNOS induced by
17
-E2 (Fig. 5A, lane 4). In addition, anti-HA
immunoprecipitates from TRLEC transfected with constitutively active
Akt were found to induce eNOS phosphorylation in immunocomplex kinase
assays (Fig. 5A, lane 5). Because it was reported
that Akt directly phosphorylated on serine 1179 (31), an immunocomplex
kinase assay with anti-Akt antibody was performed using recombinant
wild-type eNOS or mutant eNOS of serine 1179 to alanine (eNOS S1179A)
as a substrate (Fig. 5B). Mutation of serine 1179 to alanine
markedly reduced 17
-E2-induced phosphorylation of eNOS compared with
the wild-type protein. These results suggest that 17
-E2-induced Akt
activation actually increases the phosphorylation of eNOS. Next, we
sought to determine whether an Akt cascade is involved in the
regulation of the eNOS activation induced by 17
-E2 in the
endothelial cells. To examine whether the stimulation of the eNOS
activation by 17
-E2 is the result of activation of Akt, either
wortmannin (Fig. 5C) or an expression vector,
kinase-inactive HA-AktK179M, was used (Fig. 5D).
Pretreatment with 2 × 10
7 M wortmannin
for 15 min completely abolished the 17
-E2-induced eNOS activation
(Fig. 5C). In addition, transfection with HA-AktK179M clearly abolished the 17
-E2-induced eNOS activation, whereas transfection with control vector had no effect on the 17
-E2-induced eNOS activation (Fig. 5D). These results suggest that the
PI3K-Akt cascade is involved in the 17
-E2-induced eNOS
activation.
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Fig. 5.
Akt-dependent eNOS
phosphorylation and activation. A, the effect of
expressed Akt on the phosphorylation of purified eNOS induced by
10 7 M 17
-E2 for 15 min was examined.
Immunocomplex kinase assays were performed using anti-Akt
immunoprecipitates from TRLECs (left panel) or using anti-HA
immunoprecipitates from TRLECs expressing HA-tagged Akt constructs
encoding HA-Akt (Wild-type Akt), kinase-inactive HA-AktK179M
(Inactive Akt), or constitutively active HA-m
4-129Akt
(Active Akt) expressed in TRLECs (right panel).
B, the effect of 17
-E2 on the phosphorylation of
recombinant wild-type eNOS or eNOS S1179A was examined. Immunocomplex
kinase assays were performed using anti-Akt immunoprecipitates from
TRLECs treated with or without 10
7 M 17
-E2
for 15 min. C and D, the effect of wortmannin and
kinase-deficient Akt on 17
-E2-induced eNOS activation was examined.
TRLECs were grown in 60-mm dishes. Cells were pretreated with or
without 2 × 10
7 M wortmannin for 15 min, followed by treatment with 10
7 M E2 for
15 min (C) or cells were transfected with control vector
(CMV-6) or kinase-inactive HA-AktK179M (Inactive
Akt) and, after 72 h, were stimulated with 10
7
M E2 for 15 min (D). eNOS activity was measured
as described in the legend for Fig. 1. The basal activity of eNOS of
parent cells (C) or cells transfected with CMV-6
(D) was arbitrarily set at 1.0. Data are expressed as the
mean -fold activation ± S.E. of six separate experiments.
**p < 0.01 as compared with the control.
-E2-induced Akt and eNOS Activation--
eNOS is a
Ca2+/calmodulin-dependent enzyme, and it has
been reported that estrogen induces translocation of eNOS in a
Ca2+-dependent and receptor-mediated manner
(58). A23187 induces eNOS activation and produces
endothelium-dependent vascular relaxation (19, 59). Thus,
eNOS activity is largely regulated by Ca2+ mobilization. We
therefore evaluated the role of extracellular and intracellular
Ca2+ in 17
-E2-induced Akt and eNOS activation in TRLECs
(Fig. 6). Elimination of extracellular
Ca2+ by treatment with 3 mM EGTA for 1 min
clearly blocked the A23187-induced Akt (Fig. 6A, upper
panel) and eNOS (Fig. 6A, lower panel)
activation, and similarly, treatment with 3 mM EGTA for 1 min clearly inhibited the 17
-E2-induced Akt (Fig. 6A,
upper panel) and eNOS (Fig. 6A, lower
panel) activation, indicating that Ca2+ influx is
required for 17
-E2-induced Akt and eNOS activation. Next, the effect
of intracellular Ca2+ on 17
-E2-induced Akt and eNOS
activation was examined (Fig. 6B). Treatment with 50 µM
1,2-bis(o-amino-phenoxy)ethane-N,N,N'-tetraacetic-acetoxymethyl ester (BAPTA-AM) for 20 min to eliminate intracellular Ca2+
(52, 53) completely blocked the 17
-E2-induced Akt (Fig. 6B, upper panel) and eNOS (Fig. 6B,
lower panel) activation. Moreover, elimination of both
extracellular and intracellular Ca2+ by treatment with 3 mM EGTA for 15 min (52, 53, 60) abolished the
17
-E2-induced Akt (Fig. 6B, upper panel) and
eNOS (Fig. 6B, lower panel) activation,
indicating that intracellular Ca2+ is also required for
17
-E2-induced Akt and eNOS activation. Thus, these results suggest
that Ca2+ mobilization mediated by both extracellular and
intracellular Ca2+ is required for the 17
-E2-induced Akt
and eNOS activation.
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Fig. 6.
Role of extracellular and intracellular
Ca2+ in 17 -E2-induced Akt and eNOS
activation. A, cells were pretreated with 3 mM EGTA for 1 min, and then treated with 10
6
M A23187 or 10
7 M 17
-E2 for 15 min. B, cells were pretreated with 50 µM
BAPTA-AM for 20 min or 3 mM EGTA for 15 min, and then
treated with 10
7 M 17
-E2 for 15 min. Akt
activity (upper panel) and eNOS activity (lower
panel) were measured as described in the legends for Figs. 3 and
1, respectively. The basal activity of eNOS was arbitrarily set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six
separate experiments. **p < 0.01 as compared with the
control.
or ER
Expression on 17
-E2-induced Akt and
eNOS Activation--
The potential role of ER
or ER
in
17
-E2-induced Akt activation was evaluated. Transfection of ER
into TRLECs had no effect on 17
-E2-induced Akt activation compared
with transfection of control vector (Fig.
7A, upper panel).
On the other hand, transfection of ER
into TRLECs caused an increase
in both basal and 17
-E2-induced Akt activation compared with
transfection of control vector (Fig. 7A, upper
panel). Moreover, transfection of ER
into TRLECs caused an
increase in 17
-E2-induced eNOS activation compared with transfection of control vector or of ER
(Fig. 7A, lower
panel). We confirmed that both ER
and ER
were expressed in
TRLECs (data not shown). Therefore, CHO cells, which do not express
ER
or ER
(61), were used to examine which of these receptors is
involved in 17
-E2-induced Akt activation. In CHO cells transfected
with control vector or ER
, 17
-E2 had no effect on Akt activity
(Fig. 7B). However, in cells transfected with ER
, there
was an apparent increase in Akt activity upon brief stimulation with
17
-E2 (Fig. 7B). These results indicate that 17
-E2
induces Akt activity through ER
.
View larger version (13K):
[in a new window]
Fig. 7.
Effect of ER or
ER
expression on
17
-E2-induced Akt and eNOS activation.
Cells were grown in 60-mm dishes. A, TRLECs were transfected
with control vector (pSG5), ER
expression vector
(pSG5-HEGO), or ER
expression vector
(pSG5-mER
) and, after 72 h, were stimulated with
10
7 M 17
-E2 for 15 min. Akt activity
(upper panel) and eNOS activity (lower panel)
were measured as described in the legend for Figs. 3 and 1,
respectively. The basal activity of eNOS of transfected cells was
arbitrarily set at 1.0. Data are expressed as the mean -fold
activation ± S.E. of six separate experiments.
**p < 0.01 as compared with the control. B,
CHO cells were transfected with empty vector (pSG5), ER
expression vector (pSG5-HEGO), or ER
expression vector
(pSG5-mER
) and, after 72 h, were stimulated with
10
7 M 17
-E2 for 15 min. Akt activity was
measured as described in the legend for Fig. 3. Experiments were
repeated three times with essentially identical results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-E2 and that Akt activation is required for the activation of eNOS
following brief treatment with 17
-E2: Treatment with wortmannin, a
PI3K inhibitor, attenuated the 17
-E2-induced Akt and eNOS
activation, and TRLECs expressing inactive Akt showed less induction by
17
-E2 of eNOS activity. A pure ER antagonist, ICI-182,780 completely
inhibited the 17
-E2-induced Akt and eNOS activation. Moreover, in
CHO cells transfected with ER
, there was an apparent increase in Akt
activity upon brief stimulation with 17
-E2. These results suggest
that a 17
-E2-induced PI3K-Akt cascade stimulates the activation of
eNOS through ER
.
-E2 along with
the fact that the activation was not altered by the inhibition of gene
transcription by actinomycin D indicate that the process may not
require the classical nuclear effects of the estrogen. However, the
acute response of Akt (Fig. 4B) and eNOS (Fig.
2B) to 17
-E2 was fully inhibited by concomitant treatment
with the pure ER antagonist ICI-182,780, suggesting that the response
requires a rapid ER activation. Are these rapid effects of estrogen
mediated by an unidentified estrogen receptor or by the known estrogen
receptors acting in a novel way? The existence of rapidly acting
membrane receptors for steroid hormones in both nonvascular and
vascular cells has been suggested for over two decades (63, 64), but no
such receptors have been isolated or cloned. Alternatively, the rapid
effects of estrogen on vascular cells could be mediated by a known
estrogen receptor, perhaps located in the plasma membrane (64), which
is able to activate nitric oxide synthase in a nongenomic manner (19,
20). In addition, estrogen increases the expression of genes for
important vasodilatory enzymes such as prostacyclin synthase and nitric oxide synthase (65, 66). Some of the effects of estrogen may therefore
be due to longer-term increases in the expression of the genes for
these enzymes in vascular tissues.
(ER
) and
estrogen receptor
(ER
), both of which are members of the superfamily of steroid hormone receptors (35, 67). Genetic disruption
of ER
in mice leads to lower levels of vascular nitric oxide (69).
In addition, ER
can directly activate endothelial nitric oxide
synthase (19, 20). We found that there was an apparent increase in Akt
activity upon brief stimulation with 17
-E2 in CHO cells transfected
with ER
, whereas 17
-E2 had no effect on Akt activity in cells
transfected with control vector or ER
(Fig. 7B). These
findings suggest that ER
is capable of mediating the acute response
and rapid vasodilatation caused by estrogen. What is the role of ER
in endothelial cells? In addition to forming homodimers, ER
and
ER
can form heterodimers with each other (70), adding a further
degree of complexity in the regulation of gene expression by estrogen
in cells expressing both receptors. Estrogen continues to provide
protection against vascular injury in mice in which ER
has been
disrupted (71), and the expression of ER
, but not ER
, is elevated
after vascular injury in male rats (72). Estrogen also provides
protection against vascular injury in mice in which ER
has been
disrupted (73), suggesting the possibility that either of the two known estrogen receptors is sufficient to protect against vascular injury or
that some other unknown signaling pathway is involved.
mediates the
acute estrogen-stimulated NO production which occurs via a novel
Ca2+-dependent signaling pathway (20). We
confirmed that 17
-E2-induced eNOS activation was dependent on both
extracellular and intracellular Ca2+ (Fig. 6, lower
panel). Interestingly, although it was reported that
phosphorylation of eNOS by Akt represents a novel
Ca2+-independent regulatory mechanism for activation of
eNOS (31-34, 76), the current findings indicate that 17
-E2-induced
Akt activation was dependent on both extracellular and intracellular
Ca2+ (Fig. 6, upper panel).
-mediated eNOS
activation in endothelial cells (19, 79). Although no direct evidence
that eNOS is one of the substrates of MAP kinase has been reported, it
has been reported very recently that eNOS is a substrate of Akt in
endothelial cells (31-34), and that PI3K and Akt contribute to the
production of NO stimulated by insulin in endothelial cells (80). In
addition, estrogen stimulates the activation of Akt, and Akt is a
downstream effector of estrogen-dependent proliferation and
survival in hormone-responsive MCF-7 breast carcinoma cells (81).
However, until recently there had not been any studies addressing the
role of the PI3K-Akt cascade in estrogen-induced eNOS activation. This
is the first report showing that estrogen stimulates the activation of
the PI3K-Akt cascade in endothelial cells and that this cascade might
be involved in the eNOS activation induced by estrogen. Is there any
cross-talk between the MAP kinase and PI3K-Akt signaling cascades?
Although we2 and other groups
(44, 49, 68) reported that MAP kinase and PI3K-Akt signaling cascades
converge at BAD to suppress the apoptotic effect of BAD, further
investigations are necessary to examine whether MAP kinase and PI3K-Akt
signaling cascades converge at eNOS to stimulate the release of NO. We
are currently investigating this possibility.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Michael E. Greenberg and
Sandeep Robert Datta for the gift of the vectors encoding the various
HA-tagged forms of Akt, Dr. P. Chambon for the gift of the human ER
expression vector (pSG5-HEGO), Drs. E. R. Levin and K. S. Korach for the gift of the mouse ER
expression vector (pSG5-mER
),
and Dr. W. C. Sessa for wild-type and S1179A eNOS.
![]() |
FOOTNOTES |
---|
* 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. Tel.: 81-6-879-3354; Fax: 81-6-879-3359; E-mail: masa@gyne.med.osaka-u.ac.jp.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M005036200
2 Hayakawa, J., Ohmichi, M., Kurachi, H., Kanda, Y., Hisamoto, K., Nishio, Y., Adachi, K., Tasaka, K., Kanzaki, T., and Murata, Y. (2000) Cancer Res. 60, 5988-5994.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Akt/PKB, protein
kinase B;
E2, estradiol;
NO, nitric oxide;
eNOS, endothelial nitric
oxide synthase;
ER, estrogen receptor;
ER, estrogen receptor
;
ER
, estrogen receptor
;
TRLECs, simian virus 40-transformed rat
lung vascular endothelial cells;
HUVECs, human umbilical vein
endothelial cells;
CHO, Chinese hamster ovary;
MAP kinase, mitogen-activated protein kinase;
PI3K, phosphatidylinositol 3-kinase;
PAGE, polyacrylamide gel electrophoresis;
BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-acetoxymethyl ester;
CMV, cytomegalovirus;
HA, hemagglutinin;
BSA, bovine serum albumin;
BAD, Bcl-2-associated death
promoter.
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