(Circulation. 2000;102:2990.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Antioxidant Effect of Estrogen on Cytomegalovirus-Induced Gene Expression in Coronary Artery Smooth Muscle Cells
From the Cardiology Branch (E.S., R.O.C.) and Pathology Section (Z.-X.Y., K.T., V.J.F.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.
Correspondence to Edith Speir, National Institutes of Health, Building 10, Room 7B15, 10 Center Dr MSC-1650, Bethesda, MD 20892-1650. E-mail speire{at}nih.gov
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Abstract |
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Background—Pathogens infecting the arterial wall with resultant inflammation may contribute to atherogenesis. Human coronary artery smooth muscle cells (SMCs) infected with human cytomegalovirus (CMV) demonstrate a rapid increase in reactive oxygen species (ROSs), with activation of genes involved in viral replication and inflammation. Because estrogen appears to have antioxidant properties, we wished to determine whether this hormone attenuates SMC responses to CMV infection.
Methods and Results—Using confocal microscopy and an
intracellular fluorescent dye activated by ROSs, we
found that 17ß-estradiol (0.1 to 10 nmol/L) and its stereoisomer
17
-estradiol (which has low affinity for the estrogen receptor)
dose-dependently inhibited ROS generation in CMV-infected SMCs. These
effects were not blocked by the estrogen receptor inhibitor
ICI 182,780. 3-Methoxyestrone, which lacks the phenolic hydroxyl group,
did not interfere with ROS generation. We found that 17ß-estradiol
and 17-estradiol, but not 3-methoxyestrone, prevented binding of
nuclear factor (NF)-
B to DNA. Furthermore, in SMCs transfected with
the reporter constructs 3XB-CAT, MIEP-CAT, or ICAM-CAT,
cotransfection with a CMV-IE72 expression plasmid caused promoter and
CAT activation. Treatment with 17ß-estradiol and 17-estradiol, but
not 3-methoxyestrone, inhibited CAT activity and, in CMV-infected SMCs,
prevented IE72 and ICAM-1 protein expression and cytopathic
effects.
Conclusions—These findings indicate that estrogen molecules with
an A-ring hydroxyl group have estrogen receptor–independent anti-CMV
effects at physiological concentrations by
inhibiting ROS generation, NF-B activation, NF-B–dependent
transcription, and viral replication. To the extent that chronic
infection of the vascular wall with CMV contributes to atherogenesis,
these antioxidant actions of estrogen may be of therapeutic importance.
Key Words: atherosclerosis • viruses • hormones • antioxidants • adhesion molecules
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Introduction |
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An atheroprotective effect of estrogen therapy in healthy postmenopausal women has been suggested by several studies.1 2 Although most actions of estrogen are receptor-dependent, nongenomic effects may also be important. Thus, 17ß-estradiol (E2) has antioxidant effects in isolated microsomes and protects neurons from oxidative stress, and E2 or 17
-estradiol
(17-E2) can be effective in
physiological doses.3 4 5 Furthermore,
estradiol inhibits LDL peroxidation in postmenopausal
women.6 The antioxidant effect of estradiol in neurons and
lipid membranes appears to be dependent on the presence of the hydroxyl
group in the C3 position of the A-ring of the steroid
molecule.5
Human cytomegalovirus (CMV) may contribute to the development and
progression of atherosclerosis.7 We
previously detected accumulated amounts of the tumor suppressor protein
p53 in human coronary artery smooth muscle cells (SMCs) of
restenosis atherectomies, which correlated with the presence of
intracellular CMV.8 We further demonstrated that CMV
infects SMCs and initiates viral replication, with generation of
reactive oxygen species (ROSs).9 ROSs are important for
activation of NF-B,10 a transcription factor
that initiates expression of CMV proteins such as IE72 and IE84, and of
mediators of inflammation such as intercellular adhesion molecule-1
(ICAM-1).11 CMV infection causes release of
arachidonic acid, stimulation of xanthine/xanthine
oxidase,9 and calcium influx, which contribute to ROS
generation.
Because of the known effects of lipid and neuronal antioxidant
properties and of reported interaction with calcium and cAMP, we asked
whether estradiol might have effects with regard to ROS generation and
cAMP activation and thereby inhibit viral and cellular gene expression.
The partial estrogen antagonist tamoxifen has been shown to
have lipid membrane antioxidant effects by decreasing membrane
fluidity4 and by inhibiting protein kinase (PK)
C,12 an activator of NF-B. Because
interaction of CMV particles with the cell membrane
leads to activation of PKC,13 tamoxifen may interfere with
CMV-dependent activation of the host cell by several mechanisms.
Accordingly, we included tamoxifen, previously associated with reduced
cardiovascular risk, in our
experiments.14
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Methods |
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Cells, Viruses, Plasmids, Antibodies, and Drugs
Human coronary SMCs (38-year-old female donor) and optimal medium (SmGM) including 5% FBS were purchased from Clonetics. Cells and virus (CMV, Towne strain) were grown as described elsewhere.9 Virus and the plasmids pHD101CAT3 (MIEP-CAT), pRc/RSVIE72, and pRSVIE72 were gifts from E.S. Huang, University of North Carolina at Chapel Hill; 3X-
B-CAT and 3X-mutB-CAT
were gifts from A.S. Baldwin Jr, University of North Carolina at Chapel
Hill; the ICAM-1 constructs (-277/+1 and -182/+1; the former contains
an essential NF-B site, which is deleted in the latter) were gifts
from S.W. Caughman, Emory University, Atlanta, Ga.15 The
following supplies were purchased: E2,
17-E2, 3-methoxyestrone (3-ME), and tamoxifen
from Sigma; forskolin,
(R)-p-adenosine-3',5'-cyclic
phosphorothioate (Rpiso), and the calcium ionophore A32187 from
Calbiochem. The estrogen receptor (ER) inhibitor ICI
182,780 was kindly supplied by Zeneca Pharmaceuticals. Biotinylated
ICAM-1 antibody was obtained from R&D Systems, and
fluorescein-streptavidin and anti-streptavidin reagents
were obtained from Vector Laboratories. Fluo
3-AM was obtained from Molecular
Probes.
Assessment of H2O2-, CMV-, or
cAMP-Dependent Intracellular Redox State
Generation of ROSs after administration of
H2O2 (200 nmol/L) to SMCs
or during the first stages of CMV infection in SMCs was measured by use
of
2N,7N-dichlorodihydrofluorescein
diacetate (DCFH-DA; Molecular Probes). SMCs (passage 3) were grown in
4-well coverslips (Nunc) for 48 to 72 hours. HBSS containing
H2O2 freshly diluted from a
30% stock solution was added to SMCs for 10 minutes. Cells were washed
twice with HBSS, and DCFH-DA–containing HBSS was added. Preparations
for fluorescence monitoring were done in the darkened
microscopy room. Fluorescence was detectable after incubation
with DCFH-DA for 5 minutes. One 4-well slide was processed at a time.
Photomicrographs were collected within 10 minutes after addition of
DCFH-DA. Fluorescence was quantified by confocal laser scanning
microscopy (Leica TCS4D, Leica Lasertechnik). SMCs were grown in 4-well
chamber slides for 72 hours and treated in serum-free and phenol
red–free medium for 1 hour with one of the following:
E2, 17-E2, 3-ME (each
0.1 to 10 nmol/L), or tamoxifen (50 to 500 nmol/L), and
virus-containing medium was added for 1 hour. Free virus was then
washed out with HBSS, and cells were treated with 5 µmol/L
DCFH-DA in HBSS and monitored under the microscope. Fluorescent
signal was recorded and quantified by measurement of
fluorescence intensity of 3 fields per well with computer
software provided with the microscopy system. Results are tabulated as
relative fluorescence units. Five experiments were performed.
For intracellular cAMP assessment by enzyme immunoassay, SMCs were
grown in 6-well plates at 4x104 cells/well for
18 hours. SMCs in duplicate wells were treated with forskolin (5
µmol/L), E2, or 17-E2
(10 nmol/L), and then infected with CMV. Cells were washed, and 0.3 mL
of ethanol was added per well at 0.5 hour and 1 hour. Supernatants were
collected and evaporated in a Speedvac for 45 minutes, and residues
were taken up into the reagent buffer specified by the instructions
provided with the cAMP Bioassay kit (Amersham). Three experiments were
performed.
Nuclear Extracts and Electrophoretic Mobility Shift Assay
SMCs were grown to 90% confluence in
175-cm2 flasks. After removal of the medium, SMCs
were pretreated for 1 hour as follows: with serum-free medium and
ethanol vehicle or with E2 (1 and 10 nmol/L),
17-E2 (10 nmol/L), and 3-ME (10 nmol/L). After
1 hour, the medium was renewed, and cells were infected for 1 hour with
CMV at a multiplicity of infection (MOI) of 2. Cells were washed twice
and harvested with a cell scraper, and nuclear extracts were prepared.
For the duration of the experiment, cells were grown in 2%
charcoal–adsorbed FBS (Cocalico Biologicals) to limit the hormone
content of serum. Experiments were repeated twice with similar results.
Double-stranded NF-B oligonucleotide probes were
obtained commercially (Promega) and radiolabeled with
[
-32P]ATP and T4 polynucleotide
kinase.
Transfections and CAT Assays
SMCs were grown in optimal medium (5% FBS). For CAT assays,
cells were seeded in 100-mm plates (1x106) and
transfected 20 hours later in serum-free SmGM with 0.1 µg (per
3 mL total volume per dish) of MIEP-CAT. For cotransfections, we used
0.1 µg MIEP-CAT plus 1 µg pRc IE72; 1 µg of 3X-B-CAT or
3X-mutB-CAT plus 0.5 µg of the IE72; and 2 µg ICAM-1-CAT or
B-deleted ICAM-1-CAT plus increasing amounts of IE72. DOTAP
(N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium
methyl sulfate; Boehringer Mannheim) lipofection agent was used
as described before.9 The assay was repeated
twice.
Immunocytochemistry and Northern Blot Analysis for
ICAM-1
For immunofluorescence assays, SMCs were
infected as described above. Cells were fixed for 5 minutes in
-10°C methanol exposed to biotinylated primary antibodies for
2 hours, then to fluorescein streptavidin and to
anti-streptavidin antibodies for 1 hour. SMCs were then monitored under
the laser scanning microscope. For Northern blotting, cells were grown
to 80% confluence, and 1 flask each was pretreated with
E2 or 3-ME (1 nmol/L), with
N-acetylcysteine (NAC, 10 mmol/L, Sigma), or with
vehicle for 1 hour in serum-free medium. CMV infection was performed as
described above. SMCs were incubated with growth medium until harvested
at 3 hours after infection by scraping into 15 mL Trizol (phenol and
guanidine isothiocyanate, Gibco/BRL). The suspension was extracted with
chloroform according to directions supplied with the reagent. Total RNA
(15 to 20 µg/lane) was resolved on a 1% agarose/formaldehyde gel and
transferred overnight to nylon membranes. With ICAM-1 oligo-cocktail
(R&D Systems) end-labeled with 32P,
1x107 cpm of probe was used for overnight (12 to
16 hours) hybridization at 42°C. Three experiments were performed,
with similar results.
Immunoblotting
SMCs were pretreated for 1 hour with the estrogen compounds or
tamoxifen and then infected with 2 MOI of CMV for 16 hours. Cells were
then lysed, and equal aliquots of protein were subjected to SDS-PAGE as
described in detail elsewhere. Whole-cell extracts were prepared for
blotting with IE72 at 16 hours after CMV infection.
Viral Infectivity Assay and Cytopathic Effects
SMCs were infected with 5 MOI of CMV, as described before.
Estrogen compounds, tamoxifen, ethanol vehicle, or medium was added to
SMCs at 1 hour before infection. Free virus was then removed, cells
were washed, and growth medium was added. The medium was renewed the
next day and every 48 hours thereafter. Cytopathic effects and plaque
formation were assessed at 3 to 5 days after infection by counting the
number of foci (plaques) of infected cells exhibiting cytomegalic
changes.
Cell Viability
After each experiment described above, SMCs were returned to the
incubator for 3 to 7 days and monitored for cytopathic effects. Extra
wells and dishes were prepared for cell counts and trypan blue
exclusion. None of the treatments caused cell death.
Statistics
All experiments were performed in duplicate or triplicate with 3
to 6 separate SMC cultures, except for the immunoblots and
Northern blots, which were performed twice from 2 separate cultures.
Data are presented as mean±SD. InStat3 software was
used for calculation of probability values by 1-way ANOVA and for
Tukey-Kramer multiple comparison test.
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Results |
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Effects of Estrogen on ROS-Dependent Fluorescence
We examined the effect of estradiol on ROS generation by SMCs in response to exogenous H2O2 (substrate for DCFH-DA) or to CMV infection. E2 and its stereoisomer 17
-E2 dose-dependently
inhibited ROS-dependent fluorescence in SMCs (0.1, 0.5, and 1.0
nmol/L), whether induced by exogenous
H2O2
(Table
) or by CMV infection
(Figure 1, Table). Similar
inhibitory effects on ROS generation were seen with the
partial ER antagonist tamoxifen (50 to 500 nmol/L), but not
with 3-ME. Treatment with the ER inhibitor ICI 182,780
(1 µmol/L) had little effect on reduction of
fluorescence by E2 (Table). These
data indicate that in SMCs, estradiol has dose-dependent and
ER–independent inhibitory effects on ROSs derived from
H2O2, the substrate for
DCFH-DA. CMV infection causes cellular release of ROSs and
arachidonic acid and stimulates PKC.13 We
also assessed the effects of E2 on calcium by
loading SMCs with the calcium-specific fluorescence probe fluo
3-AM. Intracellular calcium, depicted as fluo 3-AM
fluorescence, was increased at 1 to 60 minutes after SMCs had
been exposed to (1) 50 nmol/L calcium chloride in the presence of the
ionophore A23187 and (2) CMV. E2 inhibited
calcium-dependent fluorescence in each case. ROSs can induce
calcium overload, loss of cAMP, and smooth muscle contraction, and
providing cAMP or antioxidants can restore cAMP-dependent PKA, which
can counteract ROS-induced effects.16 17
E2 has been shown to increase intracellular cAMP
levels,18 19 20 21 and we found that treatment of CMV-infected
cells with E2 or 17-E2
increased intracellular cAMP levels at 0.5 to 1 hour after infection.
We used forskolin (5 µmol/L), an adenylyl cyclase
activator that increases PKA, as a positive control (Figure 2). Increased cAMP has beneficial effects
on calcium homeostasis and cellular redox state, and this could be one
of the inhibitory mechanisms of E2 on
SMCs activated by CMV.
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Estrogen and CMV-Induced Binding of NF-B
CMV has been shown to cause ROS generation, with subsequent
activation and nuclear translocation of NF-B in a variety of
cells.9 10 Pretreatment of SMCs for 1 hour with
17-E2 or E2, but not
with 3-ME, caused a marked decrease in CMV-induced NF-B binding, as
shown by electrophoretic mobility shift assay (Figure 3).
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Estrogen and NF-B–Dependent Transcription
Because transcriptional mechanisms play an important role in
ROS/NF-B stimulation of viral and inflammatory gene expression, we
used transient transfections to examine E2
effects on transactivation by IE72 protein (Figure 4A through 4C) versus (1) reporters
containing 3 wild-type or 3 mutated NF-B–binding elements
(3XB-CAT, 3Xm-B-CAT); (2) the CMV promoter MIEP, which contains 4
NF-B binding sites and multiple other cellular transcription factor
binding sites; and (3) the effect of E2-induced
cAMP on NF-B–dependent transcription. In SMCs transfected with
3XB-CAT, cotransfection with IE72 caused a 5-fold increase in CAT
activity, and this activity was inhibited by forskolin (20
µmol/L) or E2 (10 nmol/L; bars 5 and 7,
respectively). Treatment of transfected cells with forskolin plus Rpiso
(10 µmol/L, PKA inhibitor) or
E2 plus Rpiso partially relieved the inhibition.
These findings indicate signaling between E2 and
cAMP that leads to anti-inflammatory effects in coronary SMCs.
cAMP has been shown to interfere with NF-B–dependent transcription
in human endothelial cells.22 We now
report that E2-induced cAMP reduces
NF-B–dependent transcription in SMCs.
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Estradiol Inhibits IE72 Protein Expression
Immunoblotting of lysates of CMV-infected cells with an
IE72-specific antibody demonstrated that treatment with E2
and 17-E2 but not 3-ME reduced steady-state levels of
IE72 protein at 16 hours after infection (Figure 5). These experiments
show that estradiol attenuates the expression of IE72. This blocking
effect is due, at least in part, to the antioxidant and
NFB-inhibitory effects of estradiol. Because IE72 is essential for
the expression of the early and late genes of CMV, inhibition of IE72
effectively decreases viral productivity.
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Estradiol Inhibits ICAM-1 Transcription and Protein
Expression
CMV-IE72 transactivates the ICAM-1 promoter via the intact
NF-B binding site, because deletion of this site results in lack of
response (Figure 6A). As shown by
Northern blotting, ICAM message was detectable at 3 hours after CMV
infection of SMCs, whereas baseline (0 hours) mRNA was undetectable
(Figure 6B). Pretreatment of SMCs with NAC (10 mmol/L) or
with E2 (10 nmol/L) reduced ICAM-1 mRNA, whereas
3-ME had little effect. Hybridization with the housekeeping gene
glucose-3-phosphate-dehydrogenase confirmed both viability of the cells
and equal sample loading. As seen by
immunofluorescence, pretreatment with
E2, but not with 3-ME, inhibits ICAM-1 protein
expression at 24 hours after CMV infection (Figure 6C). These
data indicate that E2 attenuates CMV-induced
ICAM-1 message by an antioxidant mechanism. This effect appears to be
independent of ERs, because similar effects were obtained with
17-E2.
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Viral Replication and Cytopathology
We next examined whether the inhibitory effect of
estradiol on viral transcription and on expression of IE72 would affect
viral replication. We infected SMCs with viral stock that had been
treated with ethanol vehicle or with increasing concentrations of
E2, 17-E2, 3-ME, or
tamoxifen. Infection in untreated and vehicle-treated wells was
similar. In contrast, E2 and
17-E2, but not 3-ME, each inhibited viral
infectivity in concentration-related fashion, and maximally (10 nmol/L)
by 65%, and 5 µmol/L tamoxifen reduced viral titer by 80%
(Figure 7). These data suggest
that estradiol and tamoxifen attenuate CMV infectivity and cytopathic
effects in human coronary SMCs.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have previously reported that infection of coronary artery SMCs with human CMV results in a rapid increase in ROSs.9 Here we show that E2, at physiological concentrations, inhibits ROSs induced by H2O2 or by CMV infection. E2 also inhibits intracellular calcium after (1) uptake of exogenous calcium chloride in the presence of ionophore A23187 and (2) accumulation during CMV infection (Speir et al, unpublished observations). Interestingly, in a recent report, sex-specific inhibition of calcium entry during vasoconstriction was E2-dependent.23 In a reperfusion model with nanomolar E2 plasma concentrations, E2 diminished hydroxyl radical production.24 In the present study, inhibition of ROSs by E2 and 17
-E2
is probably due to antioxidant properties, because 3-ME, a related
compound that lacks the phenolic group, does not reduce ROSs. These
properties of E2 and
17-E2 are independent of ligand/receptor
binding because (1) ERs in SMCs are not demonstrable by
immunocytochemistry or immunoblotting (data not shown);
(2) inhibition of H2O2- and
CMV-induced ROSs are similar for E2 and
17-E2, but the latter has weak ER affinity;
and (3) coincubation with the ER blocker ICI 182,780 does not diminish
the inhibitory actions of E2 on ROS
fluorescence. Estrogen also prevents SMC membrane peroxidation,
but micromolar E2 concentrations were
required.25 Although high concentrations of
E2 are generally required for ROS scavenging,
physiological doses of E2 can
be effective.3 24 This could depend on cell type and
experimental conditions. For example, in our experience, human
coronary SMCs do not tolerate
H2O2 concentrations
>1 µmol/L, whereas human aortic and rat SMCs tolerate 100 to
200 µmol/L H2O2.
Importantly, CMV-infected SMCs are in a highly activated state:
within minutes, CMV induces NF-B, phospholipases, calcium influx,
and release of arachidonic acid. Bioavailability of
E2 is another variable. Thus,
physiological concentrations of estradiol increased
nitric oxide bioavailability by inhibiting superoxide anion
production; however, an ER-dependent mechanism was
implied.26
Another finding is linked to E2 antioxidant
actions: E2 and 17-E2
stimulate intracellular cAMP (Figure 2
), which inhibits
NF-B–induced transcription (Figure 4C). The effects of
E2 on cAMP could account for important
ER-independent mechanisms of gene activation. Effects via hypothetical
membrane receptors have been reported,18 19 20 21 and our
findings suggest that redox effects, perhaps relieving oxidative
modification of cAMP-dependent PKA may play a role. In neurons,
protective effects by E2 have been linked to
increases in cAMP levels, and this was inhibited by
antagonists of cAMP/PKA, but not by the ER
antagonist ICI 182,780, involving
E2-binding sites that are distinct from both
ER and ERß.27 It is possible that SMCs express
binding sites similar to those in neurons. However, our data suggest
that E2 increased cAMP levels by scavenging ROSs
and subsequent protection of PKA from oxidation. Further experiments
are needed to support this hypothesis.
We demonstrate that estradiol inhibits important steps of viral gene
expression. Activation of the MIEP by mitogenic stimuli
leads to expression within 3 to 4 hours of IE72, which
transactivates the MIEP via NF-B binding sites. IE72
induction ensures expression of IE55 and IE84, and these 3 proteins
induce transcription of p65 and p50, the major subunits of NF-B,
which in turn activate the MIEP.28 We show by
transient transfection of SMCs that IE72 activates the MIEP,
and E2, 17-E2, and
tamoxifen, but not 3-ME, reduce this transcriptional activity partly by
interfering with the DNA binding of NF-B. Because the MIEP contains
sites for factors other than NF-B, we tested the effects of
E2 in SMCs transfected with IE72 and a reporter
containing 3 NF-B elements only. As was true for MIEP activation,
both stereoisomers of estradiol and tamoxifen, but not
3-methoxy-estrone, blocked IE72-dependent B activity. Thus,
estradiol inhibits NF-B–dependent activation by (1) reduced IE72
levels and subsequent reduced supply of the B subunit p65, (2)
inhibition of NF-B by scavenging ROSs, and (3) augmentation of
intracellular cAMP.
In latently infected vascular cells, frequent abortive reactivation
occurs, characterized by activation of the MIEP and expression of IE72,
which activates the MIEP and ICAM. Thus, suppression of NF-B
and IE72 prevents cell activation and progression of infection. As
evidence for this, both stereoisomers of estradiol and tamoxifen, but
not 3-methoxyestrone, reduced IE72 protein expression, viral titers,
and cytopathic effects in SMCs. Our results suggest that estrogen
treatment may inhibit viral and cellular inflammatory genes, which may
contribute to atherogenesis in susceptible women.
Received May 1, 2000; revision received June 6, 2000; accepted June 30, 2000.
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