Molecular Basis of Cell Membrane Estrogen Receptor
Interaction With Phosphatidylinositol 3-Kinase in
Endothelial Cells
Tommaso Simoncini, Elena Rabkin, James K. Liao
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Objective—Nontranscriptional signaling mechanisms mediate some of the biological effects of estrogen, such as the rapid
actions on the blood vessels. By interacting with phosphatidylinositol 3-kinase (PI3K), estrogen receptor (ER) ␣ leads
to activation of protein kinase Akt and to subsequent increase in endothelial nitric oxide synthase activity. Because PI3K
is mainly a cytoplasmic complex, we studied the cellular site of interaction between this enzyme and ER␣, and we
dissected the molecular mechanisms that mediate this interaction.
Methods and Results—By using cultured human saphenous vain endothelial cells, we found that cell membrane– bound ER␣
colocalizes with PI3K and may be responsible for PI3K activation. Furthermore, we characterized the subsequent steps in the
activation of the PI3K/Akt signaling cascade, comparing the molecular events that follow insulin or estradiol activation of
PI3K.
Conclusions—We provide novel evidence for an important role of nonnuclear estrogen receptor in rapid, nontranscriptional
responses of human endothelial cells to estrogen. (Arterioscler Thromb Vasc Biol. 2003;23:198-203.)
Key Words: estrogen 䡲 estrogen receptor 䡲 nontranscriptional signaling 䡲 phosphatidylinositol 3-kinase 䡲 endothelium
E
strogen signaling has traditionally been identified with
the transcriptional control of target genes via the binding
of nuclear estrogen receptors to genomic consensus sequences.1 Nonetheless, in the past few years, several biological
actions of estrogen have been identified that are too rapid to
be compatible with transcriptional mechanisms.2
Estrogen has cardiovascular protective effects that largely
depend on nontranscriptional regulation of the vessel wall.3,4
The most prominent nongenomic action of estrogen at this level
is the induction of rapid vasorelaxation, which partially depends
on the modulation of cell membrane ion channels in endothelial
and smooth muscle cells. In vascular smooth muscle cells
(VSMCs), estradiol treatment inhibits voltage-dependent L-type
Ca2⫹ channels.5 17␤-estradiol also controls potassium efflux in
VSMCs by opening Ca2⫹- and voltage-activated K⫹ channels via
cGMP-dependent phosphorylation.6 However, a major role is
played by acute activation of NO synthesis in endothelial cells,7,8
which mediates estrogen effects also in humans.9
Rapid induction of NO synthesis by estrogen largely depends
on activation of the endothelial isoform of NO synthase
(eNOS).10 Estrogen receptor (ER) ␣ is involved in this phenomenon, which is in part attributable to mitogen-activated protein
(MAP) kinases or tyrosine kinase activation.7
In addition, we recently described the interaction of ER␣ with
phosphatidylinositol 3-kinase (PI3K), showing that this mecha-
nism accounts for the major part of eNOS activation in human
endothelial cells.8 Moreover, in a mice model, estrogen decreases vascular leukocyte accumulation after ischemia/reperfusion injury in an eNOS-, PI3K-, and ER-dependent manner,8 and
nongenomic recruitment of PI3K has marked anti-ischemic
effects in a myocardial infarction model,11 confirming the
pathophysiological importance of this nongenomic pathway.
PI3K is a lipid kinase mediating the cellular effects of cell
membrane– bound receptor-dependent molecules.12 PI3K is predominantly a heterodimer formed by an 85-kDa (p85␣) adapter/
regulatory subunit and by a 110-kDa (p110) catalytic subunit.13
By phosphorylating the D-3 position of the phosphatidylinositol
ring, PI3K synthesizes phosphatidylinositol 3-phosphates
(PtdIns-3-P, PtdIns-3,4-P2, PtdIns-3,4,5-P3),12 which regulate the
activity of kinases containing pleckstrin homology domains such
as phosphatidylinositol-dependent kinases and protein kinase
Akt.14,15 The serine/threonine kinase Akt represents the principal
downstream effector of PI3K, triggering several of its cellular
effects, including activation of eNOS.16,17
The site of interaction between PI3K and ER␣ is unclear.
Because PI3K is mainly cytoplasmic, it may be possible that
cytoplasmic or cell membrane– bound ERs are responsible for
the recruitment of PI3K. Indeed, extranuclear ERs have been
described long since,18 and, very recently, membrane-bound
Received September 9, 2002; revision accepted October 24, 2002.
From the Cardiovascular Division, Department of Medicine (T.S., J.K.L.) and Department of Pathology (E.R.), Brigham & Women’s Hospital and Harvard
Medical School, Cambridge, Mass, and Molecular and Cellular Gynecological Endocrinology Laboratory (T.S.), Department of Reproductive Medicine and
Child Development, University of Pisa, Italy.
Correspondence to Tommaso Simoncini, MD, PhD, Molecular and Cellular Gynecological Endocrinology Laboratory (MCGEL), Department of Reproductive
Medicine and Child Development, University of Pisa, Via Roma, 67, 56100, Pisa, Italy. E-mail [email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000053846.71621.93
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PI3K Interacts With Cell Membrane ER
199
Figure 1. Acute activation of eNOS by
estrogen: relative role of PI3K, Tyr
kinases, and MAP kinases. A, Kinetics of
eNOS activation in HSVECs by E2 (10
nmol/L) or insulin (100 nmol/L). B, Effects
of Tyr kinases inhibitor genistein (GS) (50
␮mol/L) or of MAP kinases inhibitor PD
98059 (PD) (5 ␮mol/L) versus wortmannin (WM) on E2-induced eNOS activation
in HSVECs. *P⬍0.05 compared with
time-corresponding E2 only–treated cells.
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ERs have been identified in endothelial cells and implicated in
the regulation of NO production.19
Moreover, there is lack of information on the specific molecular mechanisms that link PI3K activation to interaction with
ER␣ and on the signaling apparatus that is recruited by estrogen.
The aim of this study was therefore to characterize the
subcellular site of interaction of ER␣ and PI3K as well as the
specific mechanisms through which ER couples to and transactivates PI3K.
Methods
Cell Cultures
Human saphenous and bovine aortic endothelial cells (BAECs) were
harvested with type IA collagenase. MCF-7 cells were from ATCC,
and p85␣⫹/⫹ mice fibroblasts were a gift of Dr L. Cantley. All cells
were cultured and stimulated under serum-starved conditions consisting of phenol red-free medium 199 or DMEM (Gibco BRL, Life
Technologies) with 0.4% charcoal-stripped FCS.
Nitrite and eNOS Activity Assays
NO accumulation was determined by a modified nitrite assay using
2,3-diaminonaphthalene, as described.8 Endothelial cells were harvested in PBS containing 1 mmol/L EDTA, and cell lysates were
assayed for eNOS activity, as described.8
Immunoprecipitations and PI3K Assays
Endothelial cell protein extracts were immunoprecipitated with Abs
versus progesterone receptor (Santa Cruz, clone C-20), IRS-1 (Santa
Cruz, clone E-12) or IRS-2 (Santa Cruz, clone M-19), ER␣ (NeoMarkers, clone TE111), eNOS (Transduction Laboratories, clone 3)
or p85␣ (Pharmingen, clone U15), as described.8 The immunoprecipitates were either used for immunoblotting or for PI3K assays, as
described.8 The labeled phospholipids were extracted with chloroform/methanol, and the organic phase, containing the PI3K products,
was separated by borate TLC according to Walsh.20
Immunoblotting
Endothelial cell lysates were separated by SDS-PAGE, and immunoblottings were performed with standard technique. The Abs used
were progesterone receptor (Santa Cruz, clone C-20), p85␣ (Pharmingen, clone U15), P-Tyr (Santa Cruz, clone PY99), G␣q (Santa
Cruz, clone E-17), Sp1 (Santa Cruz, clone PEP 2), ER␣ (NeoMarkers, clone TE111), wild-type Akt (catalogue No. 06-558) and
Thr308-P-Akt (catalogue No. 06-678) or Ser473-P-Akt (catalogue No.
06-801) (all from Upstate Biotechnology), eNOS (Transduction
Laboratories, clone 3), inducible NOS (iNOS) (Transduction Laboratories, clone 6), wild-type ERK 1/2 (catalogue No. 442704), or
Tyr204-P-ERK 1/2 (catalogue No. 442705) (Calbiochem).
Cell Immunofluorescent Analysis
Endothelial cells were fixed with 3% paraformaldehyde. Cells were
incubated overnight with anti-p85␣ antibody (Pharmingen, clone
U15, 1:50 in 1% BSA) and mouse anti-ER␣ (NeoMarkers, clone
TE111, 1:50 in 1% BSA) at 4°C. Immunofluorescent staining was
then performed as described.21
Transfection Assays
The Akt constructs have been described previously.22 BAECs and
murine wild-type fibroblasts were transfected using the Lipofectamine reagent (Gibco BRL). To control for transfection efficiency, pCMV.␤-Gal plasmid containing the ␤-galactosidase gene
was cotransfected in all experiments. ␤-galactosidase staining indicated that transfection efficiency was 30% to 35%. Cells (60% to
70% confluent) were assayed for eNOS and ␤-galactosidase activities as described.8
Statistical Analysis
All values are expressed as mean⫾SD. Statistical differences between mean values were determined by ANOVA, followed by the
Fisher’s protected least-significance difference test for comparison
of mean values. Two-group comparisons were performed by the
unpaired Student’s t test.
A detailed Methods section is available online at
http://atvb.ahajournals.org.
Results
In human endothelial cells, physiological concentrations of
17␤-estradiol (E2) acutely increase NO release via an ER and
PI3K-dependent mechanism. 8 Different from insulindependent activation, type III NOS is recruited in a biphasic
manner (EC50 value of ⬇0.1 nmol/L), showing an early
induction within 2 minutes, followed by a more substantial
increase after 15 to 20 minutes (Figure 1A). The initial
increase is mediated by tyrosine kinases and MAP kinases,7
because it is partially prevented by genistein or by PD 98059
(Figure 1B), whereas the later increase can be blocked by
wortmannin (Figure 1B) as well as by ICI 182,780. MAP
kinase involvement in the early estrogen activation of eNOS is
suggested also by the time-consistent ERK-1/2 phosphorylation
(Figure IA, available online at http://atvb.ahajournals.org).
Because many effects of estrogen depend on transcriptional regulation, we investigated the effect of RNA
synthase inhibitors, actinomycin D and 5,6dichlorobenzimidazole riboside (DRB), and protein synthesis inhibitor, cycloheximide, on estrogen-induced eNOS
activity. Actinomycin D, DRB, or cycloheximide have no
effect on basal or E2-stimulated eNOS activity (Figure IB,
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Arterioscler Thromb Vasc Biol.
February 2003
Figure 2. Mechanism of estrogenstimulated eNOS and PI3K activation. A,
HSVEC lysates were treated for 30 minutes with E2 (10 nmol/L) or with insulin
(100 nmol/L), immunoprecipitated with
either anti-ER␣ or anti-IRS-1 Abs
(respectively), and then immunoblotted
for the detection of associated p85␣. B,
HSVECs were treated for different times
with E2 (10 nmol/L) or with insulin (100
nmol/L), immunoprecipitated with either
anti-IRS-1 or anti-IRS-2 Abs, and then
immunoblotted for the detection of IRS1/2 tyrosine phosphorylation with a specific Ab. C, Effect of E2 (10 nmol/L, 30
minutes) or insulin (100 nmol/L, 30 minutes) on IRS-1/2 associated PI3K activity
in HSVECs. D, Effect of insulin (100
nmol/L, 30 minutes) on basal or on
estrogen-activated ER␣-associated PI3K
in HSVECs.
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available online at http://atvb.ahajournals.org). Furthermore, the
inhibitors have no effect on eNOS activation by insulin (Figure
IB) or acetylcholine (data not shown), pathways that do not
involve transcriptional or translational mechanisms.23,24 Using a
promoter construct containing a tandem estrogen-response element linked to a luciferase gene, we found that although
actinomycin D does not block estrogen-stimulated eNOS activity, it blocks estrogen-induced gene transcription (Figure IC,
available online at http://atvb.ahajournals.org), suggesting that
E2 activates eNOS through nontranscriptional mechanisms.
E2 increases PI3K activity, with the greatest increase occurring after 20 minutes.8 The increase in ER␣-associated PI3K is
linked to increased association between ER␣ and the adapter/
regulatory subunit p85␣ (Figure IIA). This interaction is specific
for ER␣, because no p85␣ coimmunoprecipitation can be
detected with progesterone receptor (Figure IIB). Estrogen
induces ER␣-p85␣ interaction similarly to insulin-triggered
association between the phosphotyrosine adapter insulin receptor substrate-1 (IRS-1) and p85␣ (Figure 2A). However, ER␣p85␣ interaction is not mediated by IRS-1 or IRS-2, because
estrogen treatment is not associated with IRS-1/2 tyrosine
phosphorylation (Figure 2B).
Estrogen-dependent ER␣-p85␣ interaction is associated with
activation of ER␣-bound PI3K.8 However, only a minority of
PI3K associates with ER␣. In fact, we did not detect changes in
PI3K activity in the total p85␣ immunoprecipitates, as opposed
to ER␣ coimmunoprecipitates (Figure IIC). In agreement with
the data on eNOS, PI3K activation by E2 is independent of gene
transcription or protein synthesis, as shown with DRB and
cycloheximide (Figure IID). Confirming the immunoprecipitation studies, E2 does not recruit PI3K via IRS-1/2, because no
activation of IRS-1/2–associated PI3K can be seen on estrogen
challenge (Figure 2C). Furthermore, insulin is unable to trigger
the activation of ER␣-associated PI3K, nor to increase estrogeninduced PI3K activation (Figure 2D), suggesting that the 2
hormones recruit PI3K through distinct mechanisms.
Because ERs are prevalently localized in the nucleus, whereas
PI3K is mainly cytoplasmic, the site of interaction is unclear.
ER␣ staining in untreated cells is mostly nuclear, but after E2
treatment, ER␣ staining in the cytoplasmic/cell membrane compartment increases (Figures 3A and 3B) time-consistently with
eNOS and PI3K activation.
Accumulation of ER␣ in the cytoplasm/cell membrane is
prevented by tamoxifen (Figures 3A and 3B) and seems to be a
feature of endothelial cells, because it is not detectable in MCF-7
cells in similar conditions (Figure 3B). PI3K staining is not
affected by E2, but E2 favors the colocalization of ER␣ and p85␣
in the cytoplasmic/cell membrane compartment (Figure 3A),
although a nuclear interaction cannot be completely excluded by
these experiments. To confirm these observations, we obtained
purified nuclear and cytoplasmic/cell membrane fractions from
endothelial cells and studied the distribution of ER␣ and p85␣.
The purity of the fractions was confirmed by immunoblotting for
the cell membrane–associated G␣q and for the nuclear transcription factor Sp1. ER␣ is mainly nuclear, but there is a slight
enhancement of the cytoplasmic/cell membrane distribution
after E2 treatment (Figure 3C). p85␣, instead, is mainly cytoplasmic, and its distribution does not change with exposure to
estrogen (Figure 3C).
Because cell membrane ERs have been described in
endothelial cells, and they have been involved in eNOS
regulation,19 we studied whether the membrane-impermeable
E2-BSA complex was able to activate eNOS. Compared with
E2, E2-BSA is still able to activate eNOS, but the kinetics are
slightly different (Figure 3D). Indeed, the early eNOS activation, which is sensitive to MAP kinase inhibitors, is
comparable, but the later, wortmannin-sensitive increase is
reduced (Figure 3D). From these experiments, there is evidence that cell membrane– bound estrogen receptors are
important for binding and activation of PI3K.
One of the prominent downstream targets of PI3K is phosphorylation and activation of protein kinase Akt by
phosphatidyl-dependent kinase (PDK)-1 and -2.14,15 Estrogen
increases Akt kinase activity,8 but the mechanism is unclear. Akt
is activated by 2 independent phosphorylations on serine47315 and
threonine308.14 Under basal conditions, there is little threonine or
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Figure 3. Cell membrane/cytoplasmic
ER␣ is responsible for interaction with
p85␣. A and B, HSVECs (A and B) and
MCF-7 (B) cells were stimulated with E2
(10 nmol/L) with and without tamoxifen
(TM, 1 ␮mol/L), and immunofluorescent
staining using antibodies to ER␣ (FITC,
green) or p85␣ (rhodamine, red), alone or
in combination, was performed. Note
that between 10 and 20 minutes after E2
stimulation, there is increased ER␣ staining in the cytoplasm/cell membrane of
endothelial cells, where it colocalizes
with p85␣ (arrows), whereas this does
not happen in MCF-7 cells (B). C, Purified cytoplasmic/cell membrane or
nuclear protein extracts from HSVECs
treated with E2 (10 nmol/L) or with vehicle (ethanol) were immunoblotted for the
membrane-associated G protein G␣q, for
the nuclear-associated Sp1, for ER␣ or
for p85␣. D, Effect of cell membrane
impermeable BSA-conjugated E2 (E2BSA) (10 nmol/L) versus E2 (10 nmol/L)
on eNOS activation in HSVECs in the
presence or absence of wortmannin
(WM, 30 nmol/L) or ICI 182,780 (ICI, 10
␮mol/L). *P⬍0.05 compared with timecorresponding E2-BSA only–treated cells.
serine phosphorylation of Akt (Figure 4A). E2 causes Akt
threonine and serine phosphorylation in a time-delayed manner
similar to E2-stimulated PI3K and eNOS activation. Phosphorylation of Akt by E2 is inhibited by wortmannin and ICI 182,780
(Figures 4A). These results demonstrate that activation of PI3K
by ER␣ is associated with phosphorylation of Akt, which
represents the mechanism for Akt activation by estrogen. To
understand if Akt phosphorylation may happen in the microenvironment of a complex involving ER␣ and PI3K, we performed
immunoprecipitations for these 2 latter molecules and examined
Figure 4. Activation of Akt through Ser/
Thr phosphorylation mediates estrogeninduced eNOS activation. A, Effect of
17␤-estradiol (E2, 10 nmol/L), 17␣estradiol (␣E2, 10 nmol/L), or insulin (Ins,
100 nmol/L) on the serine-threonine
phosphorylation of Akt in the presence
or absence of ICI 182,780 (ICI, 10
␮mol/L) or wortmannin (WM, 30 nmol/L)
in HSVECs. The experiment was performed 3 times with similar results. B,
Effect of transfection of empty vector,
wild-type Akt, or constitutively active or
negative dominant Akt constructs in
BAECs (shown in the middle box) on
ER␣, p85␣, eNOS, or iNOS protein
expression. Two separate experiments
yielded similar results.
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February 2003
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whether Akt coprecipitates with them. Our results show that this
is not the case (Figure IIIA), suggesting that Akt phosphorylation and eNOS activation take place as separate processes
respect to ER␣/PI3K interaction. This hypothesis is additionally
supported by the evidence that ER␣ and p85␣ do not associate
with eNOS in basal conditions nor after E2 treatment (Figure
IIIB).
Akt mediates eNOS activation by estrogen,8 as confirmed by
the transient transfection of BAECs and murine fibroblasts with
Akt mutant constructs. BAECs express both ER␣ and eNOS,
and the p85␣⫹/⫹ fibroblasts were cotransfected with ER␣ and
eNOS cDNAs. The transfection of the wild-type form of Akt or
of the two myristylated, constitutively active Akt mutants
(myr-Akt and ⌬PH-myr-Akt [deletion of the pleckstrin homology domain]) markedly increased eNOS activity (Figure IIIC).
Transfection of a kinase-inactive dominant-negative Akt mutant
with a point mutation in the ATP binding domain, Akt
(K179M),22 does not affect basal eNOS activity but decreases
E2-stimulated eNOS activity by 50% (Figure IIIC), compatible
with transfection efficiency. Because transfection experiments
require longer periods of incubations in the presence of the
constructs, we checked whether transfection itself may be
inducing eNOS or iNOS expression or alter ER␣ or p85␣ levels.
BAECs overexpressing the different Akt constructs have unchanged amounts of ER␣, p85␣, and eNOS, and no expression
is present for iNOS in any condition (Figure 4B). These
experiments confirm that acute regulation of eNOS by Ser/Thrphosphorylated Akt accounts for the rapid production of NO
after estrogen treatment in endothelial cells.
Discussion
The role of cell membrane– bound estrogen receptors has
been discussed for several years, since the first identification
of membrane binding sites for 17␤-estradiol.18 Since then,
estrogen receptors localized on the cell membrane have been
proposed to be involved with the transduction of the nongenomic effects of estrogen, that is with that variety of
actions that estrogen exerts in different tissues, which are too
rapid to be compatible with gene transcription and protein
synthesis. Nonetheless, there is still uncertainty about the real
nature of these receptors, as well as about the signaling
mechanisms that they activate on binding with estrogen.25
Several intracellular signaling cascades have been associated
with rapid estrogen-dependent effects: the adenylate cyclase
pathway,26 the phospholipase C pathway,27 the G-protein–
coupled receptor-activated pathways,28 as well as the MAP
kinase pathway.29
At the vascular level, cell-surface ERs have been proposed to
mediate estrogen-dependent rapid activation of NO synthesis,19,30 and this seems to be partially dependent on MAP kinase
activation.30 We have shown that rapid eNOS regulation by
estrogen in endothelial cells is played through modulation of the
PI3K/Akt pathway via a direct interaction of ER␣ with the
regulatory subunit of PI3K, p85␣.8
Our present data add to these findings, defining a role for
cell-membrane estrogen receptors as the potential subpopulation
of ERs that interacts with PI3K. These findings significantly
broaden the role of cell membrane ERs, potentially involving
these receptors with a variety of intracellular activities triggered
by this lipid kinase.12,31,32
The debate is open on the origin of cell membrane ERs.
Recently, transfection studies have shown that nuclear and cell
membrane ERs derive from the same transcripts,28 but still no
data are available on possible conformational differences between these subpopulations. We have shown that ER␣ interacts
with p85␣ after a conformational change dependent on estradiol
binding and that this phenomenon does not require adapter
molecules.8 Our present data show that BSA-conjugated E2
activates eNOS through PI3K/Akt. However, we find differences in the profile of eNOS activation when comparing the
effects of E2-BSA versus natural E2. A possible explanation may
be that the steric hindrance imposed by the BSA may partially
prevent the association between the engaged ER␣ and p85␣,
supporting the concept that, as for ER-dependent nuclear effects,
a correct conformational change on ligand binding is necessary
for the nongenomic signaling of cell membrane– bound ERs.
Alternatively, it may be that part of the ER␣/PI3K interaction
takes place in the cytoplasm, and therefore E2-BSA may be less
potent than E2 because of lack of recruitment of non–membranebound cytoplasmic ER␣.
An intriguing finding is the apparent increase in the ER␣
amount in the cytoplasm after estradiol exposure in endothelial
but not MCF-7 cells. Although the mechanisms that regulate
steroid receptors cytoplasmic/nuclear shuttling are not completely clear, there are hints that interaction with specific
coactivators or the phosphorylation status of RNA polymerase II
C-terminal domain may induce cyclic assembly and disassembly
of ER transcription complexes in the nucleus,33 therefore inducing ER cycling on and off the nucleus, which may possibly serve
to create a frequent sampling of the extracellular hormonal
milieu.33 If this is the case, cell-specific coactivators may induce
preferential ER cytoplasmic or nuclear localization and may
provide the basis for the different relevance of nongenomic
versus genomic signaling of ER␣ in distinct tissues.
We provide evidence that ER␣ recruits PI3K independently
by adapter molecules mediating insulin signaling, such as
IRS-1/2 or by tyrosine kinases pathways, therefore additionally
characterizing the molecular events that link ER␣ to PI3K. We
also show that interaction with PI3K is specific for ER␣ and
does not extend to progesterone receptor. ER␤ does not interact
with PI3K,8 as well, and this reinforces the possibility that the
two isoforms may have partially distinct roles in some tissues,
such as the vascular wall. This has been previously shown using
vascular injury models, where estrogen protective effects are
entirely mediated by ER␣,34 although ER␤ (and not ER␣)
expression increases steeply after the injury,35 suggesting distinct roles for the 2 receptors. The differential interaction with
PI3K may therefore represent the first example of the different
role of ER␣ and ER␤ on nongenomic signaling pathways.
When looking at the amount of PI3K activated by ERs, it
turns out that the recruitment of a small fraction of total PI3K is
sufficient to give rise to the visible regulatory effects that ensue
on estrogen treatment of endothelial cells (ie, eNOS activation
and NO release). This is strongly suggested by the fact that
modulation of ER␣-mediated PI3K activity is not discernible in
p85␣-immunoprecipitates, because of the high basal activity
contributed by total cellular PI3K. Thus, because most of PI3K
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is potentially active and resides in the cytoplasm under basal
conditions, ER␣-associated PI3K must have some specificity to
activate the signal for eNOS activation. A possible explanation
for this may be represented by the localization to the cell
membrane of the ER subpopulation, which may favor signal
transduction to eNOS.
Localization to the cell membrane may also facilitate rapid
activation of Akt, which is responsible for estrogen-dependent
eNOS activation. Indeed, PtdIns-3,4,5-P3 production by PI3K
depends on the local availability of lipid substrates, which is
maximal at the cell membrane and is necessary for the activation
of PDK-1 and -2. These 2 kinases mediate the serine and
threonine phosphorylation of Akt.14,15 We show that estrogendependent activation of Akt through PI3K is mediated through
phosphorylation on these residues, suggesting that the localization of ERs to cell membrane and their recruitment of PI3K may
create a microenvironment at this site that facilitates eNOS
activation (which is prevalently localized to the cell membrane,
as well) by activated Akt.
In conclusion, our experiments provide evidence for an
important role of cell membrane– bound estrogen receptors for
binding and activation of PI3K, which leads to eNOS activation.
These findings suggest an important function for the extranuclear fraction of estrogen receptors in mediating rapid, nontranscriptional signaling of estrogen.
Acknowledgments
This work was supported by grants from the National Institutes of
Health (HL48743, HL70274, and HL52233), the Ministero per
l’Università e la Ricerca Scientifica e Tecnologica (MURST), and
the University of Pisa. Dr Liao is an Established Investigator of the
American Heart Association.
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Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Molecular Basis of Cell Membrane Estrogen Receptor Interaction With
Phosphatidylinositol 3-Kinase in Endothelial Cells
Tommaso Simoncini, Elena Rabkin and James K. Liao
Arterioscler Thromb Vasc Biol. 2003;23:198-203; originally published online December 26,
2002;
doi: 10.1161/01.ATV.0000053846.71621.93
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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T. Simoncini et al., Fig. IA (online)
IA
49
P -ERK
36
49
ERK
36
E2
PD
Time (min)
+
0
+
+
2
+
+
+
5
+
+
+
10
Acute activation of eNOS by estrogen: relative role of PI3K, Tyr kinases and MAP kinases.
ERK-1/2 phosphorylation after E2 (10 nM) treatment of HSVEC and effect of PD 98059 (PD) (5 µM).
T. Simoncini et al., Fig. IB (online)
IB
6
No stimulation
eNOS activity
*
5
*
*
*
E2
*
*
4
Insulin
*
*
3
2
1
0
Control
ACT
DRB
CHX
Acute activation of eNOS by estrogen: relative role of PI3K, Tyr kinases and MAP kinases.
Effect of actinomycin D (ACT, 5 µM), 5,6-dichlorobenzimidazole riboside (DRB, 50 µM), and
cycloheximide (CHX, 10 µM) on E2 (10 nM)- or insulin (100 nM)-stimulated eNOS activity.
HSVEC were pre-treated with ACT, DRB, or CHX for 2 hours prior to stimulation. The eNOS
activity was measured 30 minutes after stimulation and standardized to control (no stimulation).
*indicates p<0.05 compared to control.
T. Simoncini et al., Fig. IC (online)
IC
*
Promoter activity
12
10
8
6
4
2
0
Vector
pERE
pERE
+ E2
pERE +
E2 + ACT
Acute activation of eNOS by estrogen: relative role of PI3K, Tyr kinases and MAP kinases.
Bovine aortic endothelial cells were transfected with vector or a luciferase reporter gene construct
containing a tandem palindromic estrogen-response element (pERE) and luciferase activity was
measured after E2 (10 nM) stimulation for 30 minutes in the presence or absence of actinomycin D
(ACT, 5 µM). Cells were co-transfected with pCMV.β-Gal and pre-treated with actinomycin D for
2 hours prior to E2 stimulation. The pERE luciferase activity was standardized to the corresponding
β-galactosidase activity and the values were normalized to that of vector transfection (fold
induction). *indicates p<0.01 compared to transfection with pERE.
T. Simoncini et al., Fig. IIA (online)
IIA
107
IP p85a
74
ERα
49
Time (min)
0
5
10
E2
20
20
E2 +
TM
Mechanism of Estrogen-stimulated eNOS and PI3K activation.
HSVEC were treated for different times with E2 (10 nM) in the presence or absence of 100 nM
tamoxifen (TM) and cell lysates were immunoprecipitated with a monoclonal Ab vs. p85α ad then
used for immunoblotting with ERα Ab.
T. Simoncini et al., Fig. IIB (online)
IIB
IP PR
107
PR
74
IgGH
49
IP PR
107
p85α
74
IgGH
49
Cont Prog
Mechanism of Estrogen-stimulated eNOS and PI3K activation. HSVEC lysates were
immunoprecipitated with a specific progesterone receptor (PR) Ab and then Western analysis was
performed with either an anti-PR or an anti-p85α Ab.
T. Simoncini et al., Fig. IIC (online)
IIC
IP ERα
PIP3
IP p85α
PIP3
Control E2
E2 + αE2
ICI
Mechanism of Estrogen-stimulated eNOS and PI3K activation.
Relative ratio between ERα-recruited PI3K and total cellular PI3K and their responsiveness to
estrogen treatment in HSVEC.
T. Simoncini et al., Fig. IID (online)
IID
IP ERα
PIP3
Cont
E2
E2 + E2 + E2 +
ICI DRB CHX
Mechanism of Estrogen-stimulated eNOS and PI3K activation.
Effect of ICI 182,780 (ICI, 10 µM), 5,6-dichlorobenzimidazole riboside (DRB, 50 µM) and
cycloheximide (CHX, 10 µM) on ERα-associated PI3K activity in HSVEC. Inhibitors were added 2 h
before E2 stimulation.
T. Simoncini et al., Fig. IIIA (online)
IIIA
IP ERα
Akt
74
IgGH
49
107
IP p85α
Akt
74
IgGH
49
Con
E2
Activation of Akt through Ser/Thr phosphorylation mediates estrogen-induced eNOS activation.
Lack of co-immunoprecipitation of Akt, ERα and p85α in HSVEC.
T. Simoncini et al., Fig. IIIB (online)
IIIB
107
IP eNOS
ERα
74
IgGH
49
IP eNOS
p85α
107
IgGH
49
74
Con
E2
Activation of Akt through Ser/Thr phosphorylation mediates estrogen-induced eNOS activation.
Absence of co-immunoprecipitation of eNOS, ERα and p85α in HSVEC.
T. Simoncini et al., Fig. IIIC (online)
eNOS activity/β-Gal activity
IIIC
8
*
7
*
*
*
BAEC
6
FB p85a +/+
5
*
4
*
* *
3
** **
2
1
0
Control
WT
myr ∆PH-myr K179M Vector K179M
+ E2
+ E2
Akt accounts for eNOS activation by estrogen in endothelial cells.
Effect of E2 (10 nM, 30 min) on eNOS activity in bovine aortic endothelial cells (BAEC) and murine
fibroblasts (FB p85α +/+) transfected with empty vector (control), wild-type Akt (WT),
constitutively-active Akt mutants (myr and ∆PH-myr), or a dominant-negative Akt mutant (K179M).
The fibroblasts were also co-transfected with ERα and eNOS and all cells were co-transfected with
pCMV.β-Gal. The eNOS activity was standardized to the corresponding β-galactosidase activity and
the values were normalized to that of control. *indicates p<0.05 compared to control and **indicates
p<0.05 compared to that of E2 stimulation. Results shown are from three separate experiments
performed in duplicates.
ATVB MS# S02-3601 R1
T. Simoncini et al.
Detailed Methods (Online)
Cell cultures
Human saphenous and bovine aortic endothelial cells were harvested with Type IA
collagenase (1 mg/mL) as described 1. Endothelial cells were maintained in phenol red-free
Medium 199 (Gibco BRL, Life Technologies, Gaithersburb, MD), containing HEPES (25
mmol/L), heparin (50 U/mL), endothelial cell growth factor (ECGF) (50 µg/mL), Lglutamine (2 mmol/L), antibiotics, and 5% estrogen-deprived fetal bovine serum. Fetal
bovine serum was deprived of estrogen by activated charcoal-stripping. Once grown to
confluence the cells were replated on 1.5% gelatin-coated flasks at 20000 cells/cm2.
HSVEC or PAEC isolated by this technique form a confluent monolayer of polygonal cells
and express von Willebrand factor as determined by their content of immunoreactive
protein. HSVEC were used for all experiments excluding transfections, where BAEC were
used. MCF-7 cells were from American Tissue Type Collection, and p85α positive mice
fibroblasts were a gift of Dr. L. Cantley (Boston, USA). MCF-7 and p85α positive mice
fibroblasts were grown in DMEM (Gibco BRL) with standard nutrients. Cell number was
assessed by direct cell counting of adherent cells, after trypsine detachment, in a Neubauer
hemocytometer (VWR Scientifics). The percentage of cells excluding Trypan Blue after
staining with the compound was taken as a measure of cell viability. All cells were
stimulated under serum-starved conditions consisting of phenol red-free Medium 199 or
DMEM with 0.4% charcoal-stripped fetal calf serum.
1
ATVB MS# S02-3601 R1
T. Simoncini et al.
Nitrite assay
NO accumulation from serum-starved endothelial cells was determined by a modified
nitrite assay using 2,3-diaminonaphthalene, as described 2. Briefly, fluorescence of 1-(H)naphtotriazole was measured with excitation and emission wavelenghts of 365 and 450 nm,
respectively. Standard curves were constructed with known amounts of sodium nitrite.
Nonspecific fluorescence was determined in the presence of LNMA (3 mM).
eNOS activity assay
Endothelial cells were harvested in PBS containing 1 mM EDTA, and cell lysates were
assayed for eNOS activity as described 2. HSVEC were grown to confluence in 55 cm2
culture dishes, treated accordingly with the experimental protocol, and harvested in icecold PBS containing 1 mM EDTA. Cell lysates were pelleted in a microfuge (2 minutes,
13000 rpm, 4°C) and subsequently homogenized in a buffer containing 25 mM Tris-HCl,
pH 7.4, 1 mM EDTA, 1 mM EGTA. Endothelial nitric oxide synthase activity was detected
by measuring the conversion of [3H]L-arginine to [3H]L-citrulline with the nitric oxide
synthase assay kit (Calbiochem, La Jolla, CA), according to the manufacturer’s
instructions. Rat cerebellum extracts, containing elevated amounts of iNOS, were used as
positive controls, while endothelial cell extracts incubated in the presence of the
competitive NOS inhibitor L-NAME (1 mM) were used to subtract the nonspecific activity.
2
ATVB MS# S02-3601 R1
T. Simoncini et al.
Immunoprecipitations
Endothelial cell lysates were prepared by harvesting cell monolayers with 20 mM Tris, pH
7.4, 10 mM EDTA, 100 mM NaCl, 0.5 % NP-40 and protease inhibitors. Equal amount of
lysates were incubated with 1 µg of precipitating antibody for 1 hour at 4°C under gentle
agitation. 25 µL of a 1:1 protein A-agarose slurry were added to the tubes, and the samples
were rolled at 4°C for another hour. The samples were then pelleted and washed 5 times
with 20 mM Tris, pH 7.4, 10 mM EDTA, 150 mM NaCl, 0.5 % NP-40 containing protease
inhibitors. After the washes, the pellets were resuspended in 50 µL of 2X Laemmli buffer
and boiled for 5 min. The supernatants were separated on a SDS-PAGE gel, transferred to a
nylon membrane and immunoblotted.
PI3K assays
PI3 kinase activity in endothelial cell lysates was assayed using the borate thin layer
chromatography method, as described 3. Following a 6-hour starvation in medium 199
containing 0.4% estrogen deprived-FBS, cells were treated according to the experimental
protocol, washed and harvested in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris-HCl,
pH 7.4, 1 mM CaCl2, 1 mM Mg Cl2, 0.1 mM Na3VO4, 1% NP-40). After pelleting the cell
debris, the supernatant was incubated for 1 hour at 4°C with 1 µg of a specific precipitating
antibody. Protein/Ab complexes were subsequently immunoprecipitated with the addition
of 25 µL of a 1:1 slurry of protein A-agarose for 1 hour at 4°C and subsequent
centrifugation. The immunoprecipitates were washed three times with lysis buffer, three
times with 0.1 M Tris-HCl, pH 7.4, 5 mM LiCl, 0.1 mM Na3VO4, followed by two washes
3
ATVB MS# S02-3601 R1
T. Simoncini et al.
with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1 mM Na3VO4. The
immunoprecipitates were then mixed with 50 µL Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM
EDTA, 20 µg of PtdIns-4,5-P2 (Sigma, Saint Louis, MO), 10 µL 100 mM MgCl2 and 5µL
of a 0.88 mM ATP, 20 mM Mg Cl2 solution, containing 30 µCi of [γ-32P]ATP (3000
Ci/mmol; NEN Life Science Products, Boston, MA). The reaction was incubated at 37°C
for 10 minutes, and subsequently blocked by the addition of 20 µL of 6N HCl. The
phospholipids were extracted with 160 µL of chloroform/methanol (1:1, v/v). 50 µL of the
organic phase, containing the labeled PI3 kinase products were separated by borate thin
layer chromatography on glass-backed Silica Gel 60 plates (EM Separations, Gibbstown,
NJ) pretreated with a solution containing 25 mM trans-1,2-diaminocyclohexane-N,N,N’N’tetraacetic acid (CDTA, Sigma, Saint Louis, MO), 66% (v/v) ethanol and 0.06 N NaOH,
dried for 1 hour and then baked at 100°C for 15 minutes. The chromatography was
developed with a solution containing 37.5% (v/v) methanol, 30% (v/v) chloroform, 22.5%
(v/v) pyridine (Sigma, Saint Louis, MO), 1.33% (v/v) formic acid, 1 M boric acid and 8.5
mM butylated hydroxitoluene (Sigma, Saint Louis, MO), briefly dried and exposed to
autoradiography.
Immunoblotting
After the different treatments, endothelial cells were rinsed once with ice-cold PBS before
addition of the lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 1 mM
sodium orthovanadate, 1 mM NaF, and 1 mM phenylmethylsulfonylfluoride) to the dishes
on an ice tray. The cell lysates were scraped, boiled, and centrifuged for 2 min at 13000
4
ATVB MS# S02-3601 R1
T. Simoncini et al.
rpm. Total cell lysates (40 µg of protein) and low range molecular weight markers (BIORAD, Hercules, CA) were separated by SDS-polyacrylamide gel electrophoresis (12%
running, 4% stacking) and electrophoretically transferred to polyvinylidene fluoride
membranes (Immobilon P, 0.45 µm pore size) and incubated overnight at 4°C with
blocking solution (5% skim milk in TBS - 0.1% Tween 20). Affinity-purified rabbit
antibodies (0.4 µg of IgG/ml) were incubated with the blots overnight at 4 °C in TBS 0.1% Tween 20 containing 5% BSA. The blots were washed three times with TBS - 0.1%
Tween 20 and then treated with specific secondary antibodies (1:2000 dilution) coupled to
horseradish peroxidase. Immunodetection was accomplished using the enhanced
chemoluminescence kit (ECL Kit, Amersham Corp.).
Cell immunofluorescent analysis
Endothelial cells were grown on gelatin-coated coverslips in 6-well plates. After
treatments, endothelial cells were fixed and permeabilized with 3% paraformaldehyde.
Blocking was performed with 3% normal goat serum for 20 min. Cells were incubated
overnight with anti-p85α antibody (1:50 in 1% BSA) and mouse anti-ERα (1:50 in 1%
BSA) at 4 °C. Immunofluorescent staining was then performed as described 4. Briefly, a
biotinylated secondary antibody was used. After 45 min of incubation with the secondary
antibody, streptavidin-fluorescein isothiocyanate was added for 45 min.
Immunofluorescence was visualized using an Olympus BX 60F microscope. Photographic
images were taken from different random fields.
5
ATVB MS# S02-3601 R1
T. Simoncini et al.
Transfection assays
The wild type as well as the mutant Akt constructs have been described previously 5.
BAEC and murine wild type fibroblasts were transfected with each plasmid (4 µg) using
the Lipofectamine reagent system (Gibco BRL). As an internal control for transfection
efficiency, pCMV.β-Gal plasmid (2 µg), containing the β-galactosidase gene was cotransfected in all experiments. β-galactosidase staining indicated that cellular transfection
efficiency was approximately 30-35%. Cells (60-70% confluent) were harvested and cell
extracts were prepared using lysis buffer (100 µg/ml leupeptin, 50 µg/ml aprotinin, 0.1 mM
phenylmethylsulfonyl fluoride, 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 5 mM TrisHCl, pH 7.4), as described 2. Briefly, cell extracts were centrifuged at 12,000 x g for 10
min, and the supernatant was used for eNOS activity and β-galactosidase assays. The
relative eNOS activity was calculated as the ratio of eNOS to β-galactosidase activity.
β-galactosidase assays
β-Galactosidase activity was assayed spectrophometrically (absorption at 410 nm) and
compared to a standard curve using known amounts of purified β-galactosidase (Sigma) as
described previously 6.
Statistical analysis
All values are expressed as the means ± SD. Statistical differences between mean values
were determined by ANOVA, followed by the Fisher’s protected least significance
6
ATVB MS# S02-3601 R1
T. Simoncini et al.
difference (PLSD) test for comparison of mean values. Two-group comparisons were
performed by the unpaired Student’s t-test.
7
ATVB MS# S02-3601 R1
T. Simoncini et al.
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8