Download Human Vascular Smooth Muscle Cells Contain Functional Estrogen

1943
Human Vascular Smooth Muscle Cells Contain
Functional Estrogen Receptor
Richard H. Karas, MD, PhD; Bruce L. Patterson, MD; Michael E. Mendelsohn, MD
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Background The decreased incidence of coronary artery
disease observed in postmenopausal women given estrogen
(E2) replacement demonstrates an atheroprotective effect of
E2 that is generally believed to be mediated by indirect,
E2-induced changes in cardiovascular risk factor profiles. We
hypothesized that the atheroprotective effect of E2 may be in
part mediated by a direct effect of E2 on vascular smooth
muscle cells (VSMCs). Therefore, a series of experiments was
performed to determine whether human VSMCs contain a
competent E2 receptor, a ligand-activated transcription factor
known to mediate E2-induced effects in nonvascular cells.
Methods and Results Ribonuclease protection assays, with a
probe derived from the human E2 receptor, were used to
demonstrate E2-receptor mRNA in human saphenous vein
VSMCs. To show that VSMCs contain E2-receptor protein as
well as message, immunoblotting and immunofluorescence
studies with a monoclonal anti-E2-receptor antibody were
performed, and E2-receptor protein was detected by both
methods. Transient transfection assays using a specific E2responsive reporter system were used next to determine
whether the VSMC E2 receptor is capable of E2-induced
transcriptional transactivation. Initial studies using mammary
artery-derived VSMCs resulted in a 2.4-fold increase in
reporter activity in response to 10-7 mol/L E2. Subsequent
studies using saphenous vein VSMCs demonstrated increasing
levels of reporter activation as the concentration of E2 was
increased from 10` mol/L (1.3-fold increase; SEM, 0.07;
P=.05, n=3) to 10~7 mol/L (1.6-fold increase; SEM, 0.04;
P=.002, n=6). The specificity of the E2-induced transactivation of the reporter gene was shown by dose-dependent
inhibition of transactivation by the pure E2 antagonist ICI
164,384 and by enhancement of the transactivation by simultaneous overexpression of the E2 receptor.
Conclusions We have demonstrated for the first time that
human VSMCs express E2-receptor mRNA and protein and
that the E2 receptor in VSMCs is capable of estrogendependent gene activation. These data suggest a mechanism
by which estrogen may directly alter VSMC function.
(Circulation. 1994;89:1943-1950.)
Key Words * hormones * smooth muscle * cells .
estrogen * genetics
C ardiovascular disease is the leading cause of
mortality in US women. However, the biology
of atherosclerotic disease and ischemic cardiovascular events in women is poorly understood. The low
incidence of vascular events in premenopausal women
and the marked increase in myocardial infarction following menopause were first recognized more than 50
years ago' and suggested a role for steroid sex hormones
in the development of atherosclerosis. Since then, a
number of studies have shown that estrogen (E2) administration inhibits the development of experimentally
induced atherosclerosis in animal models.2,3 Recent
observational studies have demonstrated that oophorectomized women have an increased mortality from
cardiovascular disease if they are not given E2 replacement.4 Furthermore, postmenopausal E2 use is associated with a decreased incidence of angiographically
documented coronary artery disease,5 myocardial infarction,6 and death from cardiovascular disease.6-9 A
recent meta-analysis of the effects of hormonal replacement therapy on mortality in women concluded that
postmenopausal E2 replacement reduces the risk of
death from cardiac causes by 35%,10 prompting recom-
mendations for the widespread use of E2 replacement
Received January 19, 1994; revision accepted February 8, 1994.
From the Molecular Cardiology Research Center, New England
Medical Center, Tufts University School of Medicine, Boston,
Mass.
Correspondence to Michael E. Mendelsohn, MD, Molecular
Cardiology Research Center, New England Medical Center, 750
Washington St, Box 80, Boston, MA 02111.
therapy.1,112
The atheroprotective effects of E2 are generally believed to be due to E2-induced changes in classic cardiovascular risk factors. For example, E2 use has been
associated with increased high-density lipoprotein levels,5'13,14 decreased low-density lipoprotein levels,5 13 and
improved glucose metabolism with decreased serum insulin levels.13 Acute E2 administration has recently been
shown to prevent impairment of endothelium-dependent
dilation of atherosclerotic arteries in a primate model,
suggesting a direct effect of E2 on vascular reactivity.15
However, the mechanism mediating this effect is not yet
clear. A number of animal studies have demonstrated
specific binding of E2 to vascular cells,16-20 supporting the
hypothesis that vascular tissue is E2 sensitive. These
studies have relied primarily on detection of intravenously injected radiolabeled hormone in vascular tissues.
However, the nature of the E2-binding proteins present,
the cells responsible for hormone binding, and direct
biological effects of E2 on specific vascular cells have not
yet been characterized.
Most of the biological effects of steroid hormones are
mediated through intracellular hormone receptors that
act as ligand-activated transcription factors (reviewed in
References 21 through 23). In the presence of ligand, the
estrogen receptor (E2 receptor) binds in a highly specific
fashion to DNA containing a cis-acting transcriptional
enhancer (the E2-response element [ERE]24), resulting
in an altered pattern of gene expression (reviewed in
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References 25 through 27). The E2-receptor gene has
been cloned and sequenced28 and codes for a 66-kd zinc
finger protein containing hormone binding, DNA binding, and transcriptional activation domains (reviewed in
Reference 22). This receptor has been shown to upregulate or downregulate the expression of several important genes, including c-myc,29 cyclin,30 neis31 progesterone receptor,32 and HSP2733 in an E2-dependent
fashion.
Vascular smooth muscle cells (VSMCs) are one of
several cell types within vascular tissue that play a
critical role in the pathogenesis of atherosclerosis.
VSMC proliferation, induced and sustained by a complex array of growth factors, is the hallmark of myointimal hyperplasia and lesion progression (reviewed in
Reference 34). Because of the importance of VSMC
proliferation in the development of atherosclerosis and
the known growth-regulatory effects of E2 on several
cell types,35-39 we hypothesized that the atheroprotective effect of E2 may be in part mediated by a direct
effect of E2 on VSMCs. To begin to explore this
hypothesis, we undertook a series of experiments to
determine whether human VSMCs contain transcriptionally competent E2 receptor. The results we present
establish the presence of both E2-receptor mRNA and
protein in human VSMCs and demonstrate further,
using transient transfection assays, the functional competence of the E2 receptor in VSMCs.
Methods
Cell Culture Techniques
VSMCs were cultured from surgical specimens of human
saphenous vein and mammary artery essentially as described.40 For all experiments, VSMCs were grown in media
that did not contain phenol red because of the recognized
estrogenic effects of this compound.37 Cells were maintained in
DMEM containing 10% fetal bovine serum in which the E2
content was determined to be less than 2.6x10-11 mol/L
(Hyclone). VSMCs were allowed to migrate from the primary
explants and were subsequently passaged at confluence. Cultured cells were a uniform population of smooth muscle cells
identified both by their morphology and by immunostaining
for smooth muscle-specific a-actin (Sigma Chemical Co; data
not shown). VSMCs were harvested at passage 2 for all experiments described below. MCF-7 human breast tumor cells
(kindly provided by M. Brown), used as a positive control in
several experiments because they are known to express high
levels of E2 receptor,41,42 were maintained in DMEM and 10%
fetal bovine serum.
RNase Protection Assay
Total cellular RNA was harvested by a commercially available guanidinium thiocyanate method according to the manufacturer's instructions (Biotecx Laboratories). A radiolabeled
RNA probe, complementary to 123 base pairs (bp) of the E
region (hormone-binding domain) of the human E2-receptor
sequence, was constructed by in vitro transcription (MaxiScript, Ambion) and gel purified on 5% SDS-polyacrylamide
gels. RNase protection assays were performed with a commercially available kit (RPAII, Ambion) according to the manufacturer's instructions. The products of the RNase digestion
were resolved on 5% SDS-polyacrylamide gels, and relative
intensities of protected E2-receptor probe fragments were
quantified using the volume integration mode on a PhosphorImager (Molecular Dynamics).
Western Blotting
VSMCs were suspended by gentle scraping and lysed in the
following buffer: 20 mmol/L Tris (pH 7.4), 50 mmol/L NaCl,
50 mmol/L NaF, 50 mmol/L EDTA, 20 mmol/L Na pyrophosphate, 1 mmol/L Na orthovanadate, 1% Triton X-100, 1
mmol/L PMSF, 0.6 mg/mL leupeptin, and 10 ,g/mL aprotinin.
The lysis buffer was supplemented with both EDTA and
multiple protease inhibitors to prevent the partial proteolysis
of the E2-receptor protein observed in preliminary studies. To
fractionate subcellular components, total lysates were centri-
fuged at 16 OOOg for 10 minutes, and aliquots of the supernatant and cell pellet were solubilized in a high salt buffer (10%
SDS, 500 mmol/L NaCI21) and subjected to SDS-PAGE on
10% gels. Immunoblotting was performed with a well-characterized monoclonal anti-E2-receptor antibody (D547), specific
for mammalian E2 receptor43 (kindly provided by G. Greene),
that was developed by Enhanced Chemiluminescence (ECL,
Amersham).
Immunofluorescent Staining
Immunofluorescent staining of human saphenous VSMCs
(HSVSMCs) grown from explants onto glass coverslips was
performed using minor modifications of an established protocol.44 Briefly, cells were fixed in 3.7% formaldehyde, incubated
for 1 hour with D547 anti-E2-receptor antibody43 (1: 5 to 1:10
dilutions), washed, exposed to FITC-labeled goat anti-rat IgG
(Boehringer-Mannheim) (1:20 to 1:100 dilutions), and examined by fluorescence microscopy. Control studies in which
primary antibody was omitted were included in all immunofluorescent studies and were consistently negative (data not
shown).
Transfection Assays
Transfection conditions for VSMCs were optimized during
preliminary experiments by counting 3-galactosidase-positive
cells after transfection with a constitutively expressed (-galactosidase gene (data not shown). The reporter plasmids ERE-Luc
(containing three copies of the Xenopus vitellogenin ERE24
proximal to the thymidine kinase promoter driving expression of
firefly luciferase cDNA)45 or TK-Luc (identical to ERE-Luc but
lacking the ERE; both plasmids were the kind gift of C. Glass)
were electroporated (BioRad, Gene Pulser; 220 V, 960 1,F) into
cells obtained from one subconfluent T-150 flask. In a subset of
experiments, co-transfection of either the expression plasmid
pHEGO,46 which contains the wild-type human E2-receptor
cDNA driven by an SV-40 enhancer element (kind gift of M.
Brown), or an equal quantity of carrier DNA (salmon sperm
DNA), was used to determine the effect of overexpression of E2
receptor on the activity of the reporter plasmids. To maintain
identical levels of transfection for each experimental condition
within an assay, cells pooled from a single electrophoration
were aliquoted evenly into a 12-well plate in the presence of 5
mmol/L butyric acid. Thirty-six hours after transfection, the
cells were treated with media in the presence or absence of
either 17p-estradiol (Sigma Chemical Co) or the pure E2
antagonist ICI 164,38447 (Zeneca Pharmaceuticals; kind gift of
A. Wakeling), each dissolved in ethanol. Control cells were
treated with ethanol alone to a concentration identical to that of
treated cells (final concentration of ethanol, <0.05%). After 24
hours of hormone exposure, luciferase activity was determined
in triplicate on a Monolight 2010 luminometer (Analytic Luminescence Laboratories) as described."8
Statistical Analysis
Values are reported as mean+SEM. All comparisons were
made with the Student's t test. A value of Ps.05 was considered significant.
Karas et al Human Vascular SMC Estrogen Receptor
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FIG 1. Human saphenous vein smooth muscle cells (HSVSMCs)
contain E2-receptor mRNA. Shown is a Phosphorimage from an
RNase protection assay demonstrating the presence of E2-receptor mRNA in total RNA from HSVSMCs and MCF-7 breast cancer
cells. In each case, a radiolabeled RNA probe from the E region of
the human E2-receptor gene is protected (arrowhead). The source
and quantity of RNA loaded in each lane are shown above the lane.
One of eight similar RNase protection assays is shown.
Results
HSVSMCs Express the E2-Receptor Gene
To demonstrate the presence of E2-receptor message
in VSMCs, an RNase protection assay was performed
with total RNA prepared from HSVSMCs and a riboprobe from the hormone-binding domain of the E2
receptor. RNase protection assays were used rather
than Northern blots because of the anticipated low
abundance of the E2-receptor message in nonreproductive tissue.49 In Fig 1, the appearance of a 123-bp
protected fragment (arrowhead) demonstrates the presence of E2-receptor mRNA in HSVSMCs. MCF-7
breast cancer cell RNA was included in the assay as a
positive control and also protected the riboprobe (Fig
1). Yeast RNA (free of human E2-receptor mRNA)
served as a negative control and did not protect the
riboprobe (data not shown). Quantification of the intensity of the signal for E2-receptor mRNA demonstrated 42- to 53-fold less E2-receptor message in
HSVSMCs than in MCF-7 cells, consistent with the
known supraphysiological expression of E2 receptor in
these tumor cells.42
HSVSMCs Contain E2-Receptor Protein
To demonstrate that VSMCs contain E2-receptor protein as well as message, we analyzed lysates of HSVSMCs
by Western blotting with monoclonal anti-E2-receptor antibody. The presence of E2-receptor protein in
HSVSMCs is demonstrated in Fig 2. E2-receptor protein was detected consistently by immunoblotting in
HSVSMCs derived from both male and female donors
and in one study with lysates from VSMCs derived from
mammary artery (data not shown). To determine the
subcellular distribution of E2-receptor protein in VSMCs,
cell lysates were solubilized with high salt conditions used
classically to define nuclear and nonnuclear compartmen-
FG 2. Human saphenous vein smooth muscle cells (HSVSMCs)
contain E2-receptor protein. Total cellular lysate or cytoplasmic
(supematant) and nuclear (pellet) fractions from a high salt lysis21 of
cultured HSVSMCs were subjected to SDS-PAGE, immunoblotted
with anti-E2-receptor antibody, and developed by chemiluminescence techniques. In all such studies, MCF-7 cell protein lysates
were used as a positive control in adjacent lanes and produced a
band identical in size to that seen in the VSMCs (not shown). The
majority of the E2-receptor protein was recovered consistentiy in
the cell pellet. One of four similar studies is shown.
talization of steroid hormone receptors.21 The E2-receptor protein was recovered predominantly from the nuclear
pellet (Fig 2). In all such studies, MCF-7 cell protein
lysates were used as a positive control in adjacent lanes
and produced a band identical in size to that seen in the
VSMCs (data not shown). A larger, 72-kd band of unknown identity was seen in some experiments (Fig 2,
nuclear pellet lane), as reported in other cells.50 E2receptor protein was also found by immunoblotting with a
different anti-E2-receptor monoclonal antibody (D7543;
kindly provided by G. Greene; data not shown).
To confirm the subcellular distribution of the E2 receptor observed by immunoblotting, immunofluorescent
staining for E2 receptor was also undertaken. Fig 3 shows
both the phase-contrast appearance (panel A) and immunofluorescent staining for E2 receptor (panel B) in freshly
cultured HSVSMCs. The predominantly nuclear localization of E2 receptor in HSVSMCs is consistent with the
pattern observed in similar immunohistochemical studies
of E2 receptor in other nonvascular cells.43.5'-53 Control
studies in which nonimmune serum was substituted for
primary antibody demonstrated only faint and diffuse
background staining (data not shown).
E2 Receptor in Human VSMCs Is
Transcriptionally Competent
The data presented thus far demonstrate that VSMCs
contain both E2-receptor mRNA and protein but do
not establish the functional integrity of the receptor. To
determine whether the human VSMC E2 receptor is
capable of specific, E2-induced transcriptional transactivation, the E2 responsive reporter plasmid ERE-Luc
or the control plasmid TK-Luc was introduced into
human VSMCs. Exposure of VSMCs derived from
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Circulation Vol 89, No 5 May 1994
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FIG 3. Photomicrograph of immunofluorescent identification of E2 receptor in human saphenous vein smooth muscle cells
(HSVSMCs). The intracellular distribution of E2 receptor is demonstrated in freshly cultured HSVSMCs. A, Phase-contrast appearance;
B, immunofluorescent staining with anti-E2-receptor antibody. Note the predominantly nuclear distribution of the E2 receptor. (x40;
bar=2 gum.)
mammary arteries to 10` mol/L E2 had no effect on the
activity of the TK-Luc plasmid, which lacks the ERE
(Fig 4). However, exposure of VSMCs derived from
mammary arteries to 10-7 mol/L E2 resulted in a
2.4-fold increase in the activity of the ERE-Luc plasmid
(Fig 4). E2 also induced increases in ERE-driven luciferase activity in experiments using saphenous vein
VSMCs derived from several different donors. The
induction of reporter activity in HSVSMCs increased in
a dose-dependent fashion, from 1.3-fold (SEM, 0.07;
P=.05, n=3) at 10` mol/L E2 to 1.6-fold (SEM, 0.04;
P=.002, n=6) at 10-7 mol/L E2 (Fig 4). These data
provide direct evidence for the functional integrity of
endogenous E2 receptor in VSMCs.
To define further the specificity of the E2-induced
activation of the ERE in VSMCs, a separate series of
competition experiments was conducted with E2 and
the pure antiestrogen ICI 164,384. In mammary arteryderived VSMCs transfected with the ERE-Luc plasmid,
10-7 mol/L E2 activated the ERE 1.5-fold in the presence of low-dose (10-7 mol/L) ICI 164,384 (Fig 5,
P=.01). Treatment with increasing doses of ICI 164,384
led to a dose-dependent inhibition of the E2-induced
transactivation, which was complete at 10-5 mol/L ICI
164,384 (Fig 5). These data are consistent with the
relative potencies of E2 and ICI 164,384 previously
reported54 and directly support the specificity of the
E2-mediated transactivation of the ERE reporter (see
'Discussion"). Treatment with ICI 164,384 alone had
no effect on the activity of the plasmid TK-Luc, which
lacks the ERE (data not shown).
Taken together, these data demonstrate that human
VSMCs contain E2 receptor with transcriptional activation that is specifically regulated by E2, although the
magnitude of the transactivation of the reporter system
noted in Figs 4 and 5 is relatively modest. To determine
whether this result is due to the low abundance of E2
receptor in VSMCs, we examined the effect of increased
expression of the E2 receptor on the magnitude of the
E2-induced transactivation of the ERE in HSVSMCs.
In these studies, increased expression of the E2 receptor was accomplished by co-transfection of an expression plasmid for human wild-type E2 receptor
(pHEGO) with either ERE-Ltuc or TK-Luc. In the
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FIG 4. Human vascular smooth muscle cells (VSMCs) contain
transcriptionally competent E2 receptor. VSMCs derived from
either mammary artery or saphenous vein (HSVSMCs) were
transiently transfected with a reporter plasmid containing an
E2-response element driving expression of the luciferase gene
(ERE-Luc) or a control plasmid that lacks the E2-response
element (TK-Luc) and subsequently grown for 24 hours in media
alone (control state; C), or with either 10-9 mol/L 17f-estradiol
(E2 10-9 mol/L) or 10-7 mol/L 17f-estradiol (E2 10-7 mol/L). Bars
represent the mean relative luciferase activity with the SEM
where appropriate (n=5 mammary artery/TK-Luc; n=2 mammary artery/ERE-Luc; n=3 to 6 HSVSMCs/ERE-Luc). Luciferase
activity is shown relative to control cells that were not exposed to
hormones. *P=.05, **P=.002 vs HSVSMCs/ERE-Luc control.
Karas et al Human Vascular SMC Estrogen Receptor
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FIG 5. Activation of the E2-response element reporter in human
vascular smooth muscle cells (VSMCs) is competitively inhibited
by the antiestrogen ICI 164,384. Mammary artery VSMCs transfected with the ERE-Luc plasmid were grown in the absence or
presence of 1 0- mol/L 17p-estradiol (E2) and exposed to
various concentrations of the pure antiestrogen ICI 164,384 (IC1).
Bars represent the mean luciferase activity relative to control
cells that were not exposed to E2 (+SEM; n=3). #P=.01 vs bar
A; *P=.05 vs bar B; **P=.001 vs bar B and P=NS vs bar A.
absence of overexpression of the E2 receptor, treatment of HSVSMCs with iO-` mol/L E2 again resulted
in a 1.5-fold increase in ERE activity (Fig 6; cf Fig 4).
However, in HSVSMCs co-transfected with the E2receptor expression plasmid, E2 exposure resulted in a
7.5-fold increase in ERE activity (Fig 6). Of note,
co-transfection of the E2-receptor plasmid had no
effect on the activity of the TK-Luc (control) plasmid
(Fig 6).
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FiG 6. Overexpression of the E2 receptor in human saphenous
vein smooth muscle cells (HSVSMCs) enhances E2-induced transactivation of the E2-response element. HSVSMCs were treated and
transiently transfected with a reporter plasmid containing an E2response element driving the expression of the luciferase gene
(ERE-Luc) or the control plasmid that lacks the E2-response
element (TK-Luc) as in Fig 5. Cells were co-transfected either with
an E2-receptor expression plasmid (ER plasmid) or carrier DNA
and grown for 24 hours in the absence (hatched bars) or presence
(solid bars) of 10-7 mo/L 173-estradiol and assayed for luciferase
activity. Bars represent the mean luciferase activity relative to
control cells that were not exposed to E2 (+SEM). Note that
overexpression of the E2 receptor enhances E2-induced activation
of the E2-response element reporter (ERE-Luc) fivefold but has no
effect on the control plasmid (TK-Luc) lacking this element.
1947
Discussion
The data presented here demonstrate for the first
time that human VSMCs contain the biochemical machinery known to mediate E2 effects on the transcription of E2-responsive genes in E2-responsive tissues.55
The results demonstrate E2-receptor mRNA by RNase
protection assay and E2-receptor protein by both Western blotting and immunofluorescent staining in
HSVSMCs. Furthermore, the majority of VSMC E2
receptor is shown to be localized to the nucleus by both
immunoblotting (Fig 2) and immunofluorescence (Fig
3), although some cytosolic receptor was also detected.
These findings are consistent with other immunologically based detection methods demonstrating E2 receptor primarily in the nucleus of nonvascular cells.43,51-54
Although the data presented above demonstrate that
human VSMCs contain E2 receptor, they also suggest
that the receptor is present at a low level. The RNase
protection assay demonstrates that E2-receptor mRNA
is approximately 50-fold less abundant in HSVSMCs
than in MCF-7 breast cancer cells. In addition, several
lines of evidence suggest that E2-receptor protein levels
were substantially less in HSVSMCs than in MCF-7
cells; preliminary experiments demonstrated that E2receptor protein was difficult to detect by non-ECLbased immunoblotting techniques, and compared with
MCF-7 cells, lysates containing 25- to 50-fold more
VSMCs were required for detection of E2 receptor by
immunoblotting (R.H. Karas, M.E. Mendelsohn, unpublished observations). Finally, the E2-receptor overexpression studies, demonstrating that co-transfection
of an E2-receptor expression plasmid increases the
magnitude of E2-induced activation of the ERE in
VSMCs (Fig 6), further support the conclusion that
VSMCs contain a relatively low abundance of functional
E2 receptor.
Demonstration of E2-receptor mRNA and protein in a
cell is not sufficient to establish the functional integrity of
the receptor in that cell because of the complex regulatory
system controlling transcriptional activation of steroid
hormone receptors. A number of intracellular events are
known to modulate steroid hormone receptor function.
For example, several proteins have been found to associate with estrogen and other steroid receptors.23-56-61 Some
such interactions appear to be critical for efficient transactivation of steroid-responsive genes,62 whereas others
may inhibit transcriptional activation.57-59 Thus, cells may
contain E2 receptor in a transcriptionally inefficient or
inactive state due to interactions with and/or absence of
associated proteins. The transient transfection assays presented in Figs 4 through 6 demonstrate conclusively that
the E2 receptor in human VSMCs is transcriptionally
competent. The specificity of the E2-induced transactivation in VSMCs is demonstrated by (1) control studies with
the control plasmid TK-Luc, which establish that the ERE
is necessary for transactivation of the reporter; (2) the
dose-dependent increase in transactivation of ERE-Luc by
E2; and (3) dose-dependent inhibition of the E2-induced
increase in ERE-driven reporter activity by the specific E2
antagonist ICI 164,384.
VSMC proliferation is a central event in atherosclerosis and may have a different biology in women than in
men. Demonstration that VSMCs contain functional E2
receptor raises the possibility that E2 may alter patterns
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Circulation Vol 89, No 5 May 1994
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of gene expression important to VSMC growth. E2 can
promote or inhibit proliferation, depending on the type
of cell studied. Both neuroblastoma cells and 3T3
fibroblasts transfected with E2-receptor expression vectors are growth suppressed by exposure to E2,35.63 and
E2 has been reported to inhibit the growth of VSMCs
derived from rabbit aorta,6" rat pulmonary artery,65 and
porcine coronary artery.68 These studies and the known
atheroprotective effect of estrogen in postmenopausal
women are consistent with the hypothesis that in some
settings E2 may directly inhibit the proliferation of
VSMCs. Although our data demonstrate E2-induced
activation of an exogenous ERE in VSMCs, it will be
important next to demonstrate that E2 directly activates
the expression of endogenous VSMC genes. In nonvascular cells, only a few E2 receptor-activated genes have
thus far been identified, including HSP27, the progesterone receptor, c-fos, and c-myc.23'32'67'68 If E2 directly
modulates the growth response of VSMCs, the protooncogenes are candidate genes whose expression may
be modulated by estrogen.
How might the VSMC E2 receptor mediate a growthinhibitory effect of E2? One logical target for E2mediated growth regulation is proto-oncogene expression. E2-dependent regulation of the expression of
proto-oncogenes is well described in breast cancer
cells.29'31'69 Some of the proto-oncogenes known to be
regulated by E2 in breast cancer cells (eg, c-myc29,67) are
also important regulators of VSMC proliferation.70,71
Another mechanism by which steroid receptors might
participate in growth control is suggested by recent
reports demonstrating cross-talk between steroid hormone receptor and cell surface receptor-mediated signaling pathways.39'59'60'72-75 Steroid and cell surface receptor-mediated signaling may interact in several ways.
For example, steroid receptors may be activated directly
by mitogens that act through membrane-spanning receptors, as in the recent report of epidermal growth
factor-induced transactivation of an ERE-reporter system in endometrial adenocarcinoma cells.76 In addition,
steroid receptors may have complex interactions with
other transcription factors known to regulate proliferation, such as those between the glucocorticoid receptor
and the c-fos protein.58-60'68 The E2 receptor has also
been shown to interact specifically with AP-1.77,78 Such
studies raise the intriguing possibility that E2 receptormediated effects on gene expression in VSMCs may
converge in several ways with growth factor-mediated
pathways known to regulate the proliferation of
VSMCs, a critical component in the pathogenesis of
atherosclerosis.4
In summary, we have demonstrated for the first time
that human VSMCs express E2-receptor mRNA and
protein and have shown further that E2 receptor in
VSMCs is capable of transcriptional transactivation.
Human VSMCs therefore possess the requisite biochemical machinery to respond directly to E2 exposure.
Our model provides a novel system with which to study
the role of estrogen and the estrogen receptor in VSMC
biology. Given the expanding investigations into the
role of antiestrogens in the treatment of breast cancer,
the controversy regarding the cardiovascular effects of
oral contraceptives, and the growing recognition of the
atheroprotective effects of E2, definition of the biolog-
ical effects of E2 on VSMCs
clinical implications.
may
also have important
Acknowledgments
We thank Myles Brown for helpful discussions and critical
review of the manuscript. We also thank R. Sanders Williams
and Deeb Salem for their support and guidance. We are
grateful to Peter Libby for help in establishing human VSMC
cultures in our laboratory. We are also grateful to Sarah
O'Neill, Wendy Baur, and Laura Tassi for excellent technical
support and to Patricia Nayak for expert preparation of the
manuscript. We thank Marshall A. Wolf for generous support
provided to Bruce Patterson. Richard Karas is the recipient of
a Pfizer Postdoctoral Fellowship in Cardiology. This work is
dedicated to the memory of Sheldon M. Wolff.
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Human vascular smooth muscle cells contain functional estrogen receptor.
R H Karas, B L Patterson and M E Mendelsohn
Circulation. 1994;89:1943-1950
doi: 10.1161/01.CIR.89.5.1943
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