From the Departments of § Obstetrics and Gynecology,
Molecular and Medical Pharmacology, and
** Medicine, University of California,
Los Angeles, California 90095-1740
Received for publication, August 1, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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17- The mechanism by which estrogens attenuate the development of
atherosclerosis is not known, although various actions of estrogens have been suggested to mediate this effect (1). We have previously demonstrated that 17- Some effects of estrogens are mediated through non-genomic mechanisms,
whereas others require transcriptional activation of genes (3). These
latter actions usually require that estrogens combine with specific
receptors. Two types of estrogen receptors (ER) have been described.
They are the "classical" ER, ER The present work was therefore undertaken to test whether certain
estrogenic compounds, including an estrogen metabolite, with different
agonistic potencies for the two types of estrogen receptors were more
potent than 17- Materials--
The culture medium M199, HEPES buffer, and
collagenase-type 1 from porcine skin were obtained from
Invitrogen. Gelatin, endothelial-derived growth factor,
genistein, TNF Cell Culture--
For isolation of human umbilical vein
endothelial cells (HUVEC), umbilical cords from female fetus were
selected as we observed increased expression of ER Reverse Transcription-PCR for ER Northern Blot Analysis for VCAM-1 and Endothelial Nitric-oxide
Synthase (eNOS) mRNA--
10 µg of total RNA was loaded on 1%
agarose-formaldehyde gel, electrophoresed, and transferred to Hybond
membrane (Amersham Biosciences) overnight by capillary action. The RNA
was UV-cross-linked using GS Gene Linker (BioRad). VCAM-1 (Research
Genetics) and eNOS (a gift from Dr. Thomas Michel, Brigham and
Women's Hospital, Boston, MA) cDNAs were labeled with
[ Western Blot Analysis for eNOS and ELISA for VCAM-1
Protein--
Following the treatments, cells were lysed in a buffer
containing 50 mM HEPES (pH 7.5), 1 mM DTT, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 10%
glycerol, 10 mM Electrophoretic Mobility Shift Assay for
NF Data Analysis--
Values are expressed as mean ± S.D.
obtained from three separate experiments in each group. Differences
between groups were assessed by one-way analysis of variance and
Newman-Keuls multiple comparison test where appropriate. p
values < 0.05 were considered as significant.
Effect of TNF
The effects of preincubation of HUVEC with different concentrations of
17- Effect of 17-Epiestriol on TNF Intermediate Role of Nitric Oxide in the Action of
17-Epiestriol--
To determine whether NO played an intermediate role
in attenuating TNF Effect of 17-Epiestriol on TNF The primary objective of this study was to assess whether some
selected estrogenic compounds, including a phytoestrogen, a synthetic
estrogen, and an estrogen metabolite with different agonistic potencies
for ER Our results indicate that 17- In this regard, in both the present study and in those previously
reported (8, 15, 16), the concentrations of 17- In contrast, EE, which is a more potent and selective agonist for ER No estrogen-responsive element in the promoter region of the VCAM-1
gene has yet been identified (20), and other indirect mechanisms need
to be considered. The VCAM-1 promoter contains consensus binding sites
for the nuclear transcription factor NF On this basis we wanted to assess the possibility that estrogens may
have increased the synthesis of NO, which in turn may have led to the
suppression of DNA binding of transcription factors, thereby leading to
inhibition of VCAM-1 expression. There is a lot of similarity between
the actions of estrogens and NO on various transcription factors that
modulate VCAM-1 expression. NO donors inhibited cytokine-induced
expression of VCAM-1 and repressed VCAM-1 gene transcription in part by
inhibiting nuclear binding protein NF We previously demonstrated that estrogens can increase NO production
(12), and subsequently others have demonstrated that it can occur
through both genomic (26) and non-genomic (27, 28) mechanisms. We
therefore decided to assess whether 17-epiestriol at concentrations
that attenuated VCAM-1 mRNA expression also increased eNOS protein
and whether this was at least in part due to increased eNOS mRNA.
Our results indicated that 17-epiestriol also had a biphasic effect on
eNOS protein expression. 17-Epiestriol at a concentration that
maximally suppressed TNF In conclusion, our results indicate that estrogens most likely
inhibited VCAM-1 mRNA and protein expression by an action mediated by ER These results may have important clinical implications. An important
area of research with relation to estrogen replacement therapy is
identifying the role of ER estradiol (17-
E2)
attenuates the expression of vascular cell adhesion molecule 1 (VCAM-1)
in vivo at physiological levels (pg/ml), whereas
supraphysiological concentrations of 17-
E2 (ng/ml) are
required in vitro. We assessed whether a metabolite of
estrogen, which could only be generated in vivo, might be a more potent inhibitor of VCAM-1 expression and thereby explain this
discrepancy. We report here that 17-epiestriol, an estrogen metabolite
and a selective estrogen receptor (ER)
agonist, is ~400×
more potent than 17-
E2 in suppressing tumor necrosis
factor (TNF)
-induced VCAM-1 mRNA as well as protein expression
in human umbilical vein endothelial cells. Genistein, an ER
agonist, at low concentrations (1 and 10 nM) also
suppressed TNF
-induced VCAM-1 mRNA expression. These actions of
17-epiestriol and genistein were significantly attenuated in the
presence of the estrogen receptor antagonist ICI-182780. Other
estrogenic compounds such as ethinyl estradiol and estrone did not have
any effect on TNF
-induced VCAM-1 expression at the concentrations
tested. We further show that, 1) 17-epiestriol induces the expression
of endothelial nitric-oxide synthase mRNA and protein, 2)
17-epiestriol prevents TNF
-induced migration of NF
B into the
nucleus, 3) NG-nitro-L-arginine methyl ester,
an inhibitor of NO synthesis, abolishes 17-epiestriol-mediated
inhibition of TNF
-induced VCAM-1 expression and migration of NF
B
from the cytoplasm to the nucleus. Our results indicate that
17-epiestriol is more potent than 17-
E2 in suppressing
TNF
-induced VCAM-1 expression and that this action is modulated at
least in part through NO.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
estradiol (17-
E2) in
vivo inhibits the adhesion of monocytes to endothelial cells of
the rabbit aorta (2). We also demonstrated that following a
cholesterol-enriched diet to ovariectomized rabbits, expression
of VCAM-11 protein was
induced in the aorta, and this was attenuated by administration of
17-
E2 (2). Treatment of cultured rabbit aortic
endothelial cells with 17-
E2 also attenuated the
lysophosphatidylcholine-induced expression of VCAM-1 protein. However,
the concentrations of 17-
E2 required to suppress VCAM-1
expression in vitro were in the supraphysiological range
when compared with the physiological levels required for in
vivo studies. We therefore postulated that one mechanism by which
only relatively low concentrations of 17-
E2 were
required for in vivo studies when compared with in
vitro studies was that 17-
E2 might be converted to
a more potent metabolite in vivo (2).
and the "novel" more
recently described ER
(4). Both ER
and ER
have been detected
in endothelial cells and in vascular smooth muscle cells (5, 6). The
affinity of various estrogenic compounds for the two ER subtypes
markedly differ. 17-
E2 binds to ER
and ER
with
similar affinity. 17-
ethinyl estradiol (EE), a synthetic
estrogen widely used in oral contraceptive formulations, has an
ER
-selective agonist potency, whereas 17-epiestriol, an estrogen
metabolite, and genistein, a phytoestrogen, at nM
concentrations have ER
-selective agonist potency (7).
E2 in suppressing VCAM-1 mRNA
expression and the potential mechanism(s) involved. Information obtained from these studies could lead to the development of compounds that attenuate atherogenesis with fewer side effects than those observed with 17-
E2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, NG-nitro-L-arginine methyl
ester (L-NAME), diethyl pyrocarbonate, salmon testes DNA,
Denhardt's solution, and SDS were obtained from Sigma; the steroids
17-
E2, EE, and 17-epiestriol were also obtained from
Sigma with ~99% purity as assessed by thin layer chromatography.
Fetal bovine serum (FBS) charcoal/dextran-treated was obtained from
HyClone Laboratories. FBS was obtained from Atlas, Fort Collins, CO,
and ICI-182780 was obtained from Tocris Cookson Ltd., Ballwin, MO. The
human NF
B p65 Nushift kit (2006760) was obtained from Geneka
Biotechnology (Montreal, Canada).
and ER
mRNA when compared with those obtained from male fetus. HUVEC were
isolated from freshly collected umbilical cords (female fetuses) as
previously described (8) and cultured on 0.1% gelatin-coated 75-mm
flasks in M199 medium supplemented with 20% FBS, 5 µg/ml
endothelial-derived growth factor, 100 µg/ml heparin, 100 units of
penicillin G sodium, 100 µg/ml streptomycin sulfate, 0.25 µg/ml
amphotericin-B and 10 mM HEPES buffer, pH
7.5. Prior to the experiments, cells were shifted to phenol red-free M199
medium supplemented with 2% charcoal/dextran-treated FBS and the
antibiotics mentioned above. Only second or third passage cells were
utilized in all the current studies.
and ER
mRNA--
RNA
extraction and purification were done using RNeasy mini kit as
described by the manufacturer's protocol (Qiagen, Chatsworth, CA).
Total RNA (3 µg per sample) was subjected to reverse transcription reaction with 50 units of Moloney murine leukemia virus reverse transcriptase at 42 °C for 25 min as previously described (9). The
resulting cDNA samples were PCR-amplified using Gene Amp RNA PCR
kit (PerkinElmer Life Sciences) according to the manufacturer's protocol. Oligonucleotide primers were designed for simultaneous PCR
amplification (9) of specific DNA fragments contained in both ER
and
ER
using the following set of primers: forward primer sequence,
5'-AAG AGC TGC CAG GCC TGC CG-3'; reverse primer sequence, 5'-GCC CAG CTG ATC ATG TGA ACC A-3'.
-32P]dCTP (ICN Biochemicals) using the random priming
method. Membranes were prehybridized at 42 °C overnight followed by
hybridization with respective labeled probes for another 24 h at
42 °C. The membranes were washed twice at room temperature with 2×
sodium chloride and sodium citrate (SSC) buffer and 0.5% SDS followed by washing at 65 °C for 30 min twice with 0.5× SSC and 0.1% SDS, and respective bands were quantitated using a Phosphoimager (Molecular Dynamics). The VCAM-1 band was normalized with the GAPDH band as an
internal control.
-glycerophosphate, 1 mM NaF, 0.1 mM orthovanadate, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride (10).
10 µg of total protein were subjected to SDS-PAGE and transferred to
polyvinylidene difluoride membrane using a semi-dry transfer apparatus.
The membranes were incubated at room temperature with eNOS monoclonal
antibody (1:1000) or GAPDH (1:5000) antibody for 60 min. The membranes were then incubated in diluted (1:1000) horseradish peroxidase-linked secondary antibody (1:1000) for 60 min at room temperature. The proteins were detected using ECL Western blotting kit following exposure of the membrane to autoradiography film (Hyperfilm-ECL, Amersham Biosciences). The relative intensities were quantified by
densitometric analysis (Personal Densitometer, SI, Molecular Dynamics).
Membrane-bound VCAM-1 protein was measured by ELISA as described
previously (11).
B--
After appropriate treatments, 108 cells were
washed twice with ice-cold phosphate-buffered saline, resuspended in 1 ml of ice-cold harvest buffer (10 mM HEPES, pH 7.9, 5 mM MgCl2, 10 mM NaCl, 0.3 M sucrose, 0.1 mM EGTA, O.5 mM DTT,
0.5 mM PMSF, 1 µg/ml each of antipain, aprotinin, and
leupeptin), homogenized in a Dounce homogenizer (20-30 strokes), and
centrifuged for 30 s at 21,000 × g. The pellet
was resuspended in 400 µl of nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.3 mM KCl, 0.2 mM EGTA, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF), rocked gently at 4 °C for 30 min, and centrifuged again for 30 min at 21,000 × g at 4 °C.
Nuclear proteins in the supernatant were dialyzed (20 mM
HEPES, pH 7.9, 0.1 M KCl, 0.2 mM EDTA, 20%
glycerol, 0.5 mM DTT, 0.5 mM PMSF) overnight at
4 °C. The debris was pelleted by centrifugation, and nuclear proteins in the supernatant were quantitated by Bradford reagent (BioRad). Using 10 µg of nuclear extract from each condition, NF
B
electrophoretic mobility shift assay was performed as described in the
manufacturer's protocol utilizing NF
B p65 Nushift kit (Geneka Biotechnology).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
on VCAM-1 mRNA Expression and the Modulating
Role of ER Agonists--
Second and third passage HUVEC, which
were utilized for all our studies, expressed both ER
and ER
mRNA (data not shown). No basal VCAM-1 mRNA expression was
detected in the unstimulated cells or those treated with the different
estrogenic compounds alone. The structures of the different estrogens
studied are shown in Fig. 1. Preliminary
experiments indicated that HUVEC when exposed to 10 ng/ml of TNF
for
4 h led to maximal VCAM-1 mRNA expression. This concentration
and time period were utilized for all the experiments.
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Fig. 1.
Structure of various estrogen receptor
ligands.
E2, EE (100 and 300 pM, 1, 3, 10, 30, 100, 300 nM), 17-epiestriol (10, 30, 100, 300 pM and 1, 3, 10 nM), and genistein (1 and 10 nM) on TNF
(10 ng/ml)-induced VCAM-1 mRNA expression
are shown in Figs. 2-5. 17-
E2 at concentrations ranging from 1 to 300 nM
attenuated the TNF
-induced VCAM-1 expression, whereas lower
concentrations did not have any significant effect when compared with
that observed with TNF
alone (Figs. 2 and 3). EE did not have any significant
effect on VCAM-1 mRNA expression (Figs. 2 and 3). 17-epiestriol
showed a response that was different from that of 17-
E2
and EE (Fig. 4). At lower concentrations (30-300 pM), there was significant attenuation of VCAM-1
mRNA expression when compared with that observed with TNF
alone.
The maximal attenuation was observed with 100 and 300 pM.
The calculated IC50 (mean ± S.D.) for 17-
E2 and 17-epiestriol were 16.3 ± 6.8 nM
and 36 ± 5.3 pM, respectively. Interestingly,
at higher concentrations (1 and 10 nM), the
effect of 17-epiestriol was gradually lost, and the VCAM-1
expression started returning to baseline values. However, the magnitude
of VCAM-1 expression at 3 nM of 17-epiestriol was still
significantly less than that observed with TNF
alone. Estriol at
similar concentrations to that of 17-epiestriol had no effect (data not
shown). Genistein (1 and 10 nM) attenuated VCAM-1 mRNA
expression (Fig. 5). The calculated
IC50 (mean ± S.D.) for genistein was 7.16 ± 3 nM. The effects of all these estrogen receptor agonists
were significantly attenuated in the presence of the ER antagonist
ICI-182780 (1 µM), and the results for 17-epiestriol are shown in Fig. 6.
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Fig. 2.
Representative pictures of Northern blot
analysis showing the effects of different concentrations of
17- estradiol (17-
E2) (panel A) and
17
ethinyl 17-
estradiol (EE) (panel B) on
TNF
-induced VCAM-1 mRNA expression.
Human umbilical vein endothelial cells were preincubated with either
17-
E2 or EE at various concentrations for 48 h,
and TNF
(10 ng/ml) was added to the culture media for the last
4 h. Following this, the cells were collected and total RNA was
extracted. RNA was loaded on 1% agarose-formaldehyde gel,
electrophoresed, and visualized under UV light after ethidium bromide
staining. GAPDH was used as internal control. The treatment regimens
and the concentration of the steroids are also shown and are described
in detail under "Experimental Procedures."
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Fig. 3.
Quantitation of VCAM-1 mRNA expression
using the Phosphoimager of the various treatments shown in Fig. 2.
Quantitation is expressed as mean ± S.D. arbitrary densitometric
units. Panels A and B show the effects of 17-
E2 and EE respectively on TNF
-induced VCAM-1 mRNA
expression. * signifies significant difference (p < 0.05) from TNF
only (lane 2) -treated cells.
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Fig. 4.
Representative picture of a Northern blot
analysis showing the effects of different concentrations of
17-epiestriol on TNF -induced VCAM-1 mRNA
expression (panel A) and quantitation by Phosphoimager
(panel B). Quantitation is expressed as mean ± S.D. of arbitrary densitometric units obtained from three separate
experiments. GAPDH was used as internal control. * signifies
significant difference (p < 0.05) from TNF
only
(lane 2) -treated cells.
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Fig. 5.
Representative picture of a Northern blot
analysis showing the effects of different concentrations of genistein
on TNF -induced VCAM-1 mRNA expression
(Panel A) and quantitation by Phosphoimager
(Panel B). Quantitation is expressed as mean ± S.D. of arbitrary densitometric units obtained from three separate
experiments. GAPDH was used as internal control. * signifies
significant difference (p < 0.05) from TNF
(lane 2) -treated cells.
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Fig. 6.
Representative picture of Northern blot
analysis showing the effect of 300 pM 17-epiestriol on
TNF -induced (10 ng/ml) VCAM-1 and GAPDH
mRNA expression in the presence and absence of an ER antagonist
ICI-182780 (1 µM). The ER
antagonist was added to the culture medium containing HUVEC 60 min
before the addition of 17-epiestriol.
-induced VCAM-1 Protein
Expression--
We next examined the effect of 17-epiestriol on
TNF
-induced VCAM-1 protein expression. HUVEC were preincubated with
different concentrations of 17-epiestriol for 48 h. Six hours
after the addition of TNF
, the cells were fixed and assayed for
cell-bound VCAM-1 protein by ELISA as described previously (11). A
significant reduction in VCAM-1 protein was observed at a
300-pM concentration of 17-epiestriol (Fig.
7). Consistent with the observation
related to VCAM-1 mRNA, TNF
-induced VCAM-1 protein expression
also showed a biphasic response to 17-epiestriol.
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Fig. 7.
Effect of different concentrations (30-300
pM and 10 nM) of 17-epiestriol on
TNF -stimulated (10 ng/ml) VCAM-1 protein
expression as analyzed by ELISA. Values represent absorbance
(OD) at 490 nM. Quantitation is expressed as
mean ± S.E. obtained from three separate experiments. *
significant difference (p < 0.05) from that observed
with TNF
alone.
-induced VCAM-1 expression, we first analyzed the
effect of 17-epiestriol on eNOS expression. Treatment of HUVEC with
17-epiestriol showed a biphasic response on eNOS protein expression.
The maximal increase (68 ± 8%) was observed at 300 pM for both eNOS protein (Fig.
8) and mRNA expression (35%, data
not shown). We next assessed the effect of three different
concentrations of 17-epiestriol on TNF
-induced VCAM-1 mRNA
expression in the absence and presence of L-NAME (3 × 10
4 M), an inhibitor of NO synthesis. This
concentration of L-NAME has been previously demonstrated by
us to significantly attenuate basal- and agonist-stimulated NO release
by endothelial cells (12). L-NAME either alone or along
with TNF
did not affect VCAM-1 expression. 17-epiestriol (100 and
300 pM and 1 nM) attenuated VCAM-1 mRNA
expression, and this effect was not observed in the presence of an
inhibitor of NO synthesis (Fig. 9).
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Fig. 8.
Representative picture of a Western blot
analysis performed eNOS (top) and GAPDH
(bottom) in the absence and presence of varying
concentrations (30-300 pM and 1 and 10 nM) of
17-epiestriol. HUVEC were incubated with 17-epiestriol for 48 h (at concentrations indicated). Ten micrograms of protein were loaded
onto each lane. The proteins were separated on 7.5% polyacrylamide
gel. eNOS and GAPDH were analyzed using monoclonal eNOS- and
GAPDH-specific antibodies. The treatment regimen of the various groups
of cells in each of the lanes is also indicated.
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Fig. 9.
Representative picture of a Northern blot
analysis demonstrating the effects of different concentrations of
17-epiestriol (100 and 300 pM and 1 nM) on
TNF -induced VCAM-1 mRNA expression in the
absence and presence of an inhibitor of nitric oxide synthase inhibitor
L-NAME. The various treatment regimens in each of the
lanes are also shown. GAPDH was used as the internal control.
-induced NF
B
Activation--
To determine whether 17-epiestriol regulated
TNF
-induced VCAM-1 expression by inhibiting NF
B migration to the
nucleus, we performed gel-shift assays using oligonucleotides
corresponding to the tandem-
B sites on the VCAM-1 promoter. We also
assessed whether NO played an intermediate role in this action by
17-epiestriol. 17-epiestriol at concentrations of 100 pM
(data not shown) and 300 pM (Fig.
10, lane 5) significantly
decreased the intensity of the shifted band produced by nuclear
extracts obtained from HUVEC treated with TNF
. However, 10 nM of 17-epiestriol was less effective in decreasing the
intensity of the shifted band when compared with 300 pM
(Fig. 10, lane 6). Specificity of the complexes was
determined by competition with an excess of unlabelled oligonucleotide and with a labeled mutant oligonucleotide when no band was observed (Fig. 10, lane 4). The specificity was further confirmed by
supershift analysis (Fig. 10, lane 3) with an
affinity-purified polyclonal antiserum to p65. Nuclear translocation of
NF
B following treatment with 17-epiestriol in the presence of
L-NAME was similar to that observed in the absence of
17-epiestriol and in the presence of L-NAME alone (Fig. 10,
lanes 7 and 8). This suggests that the inhibitory effect of
17-epiestriol on the translocation of NF
B to the nucleus following
stimulation of HUVEC with TNF
was mainly dependent on NO
synthesis.
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Fig. 10.
Representative picture of an electrophoretic
mobility shift assay showing the effect of
TNF -induced NF
B
activation in HUVEC and the modulating role of 17-epiestriol (300 pM and 10 nM) in the absence and presence of
L-NAME (3 × 10
4
M), an inhibitor of nitric oxide synthase. Cells
were incubated with 17-epiestriol (300 pM and 10 nM) or vehicle for 48 h. TNF
(10 ng/ml) was added
to the HUVEC for the last 4 h of incubation with either vehicle or
17-epiestriol. L-NAME was added 30 min prior to the
addition of TNF
. Experiments were also performed in the presence of
either wild type oligonucleotide (Wt Oligo) or mutant
oligonucleotide (Mut Oligo). This picture is representative
of four such experiments. The specificity of the assay was evaluated by
incubating the extracts with p65 antibody (lane
3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
were more potent than 17-
E2 in
attenuating TNF
-induced VCAM-1 expression of HUVEC in
vitro. 17-
E2 is the major circulating unconjugated
estrogen in premenopausal women. It binds to human ER
and ER
with
approximately similar affinity (7) and was therefore used
as the prototype with which the comparisons to the other estrogens were
made. EE, a synthetic estrogen, was selected since it is widely used as
the estrogenic component in oral contraceptives and has ER
-selective
agonistic potency (7). 17
epiestriol was selected because it is an
estrogen metabolite (13), which has an ER
-selective agonist potency (7). Estriol was selected as its concentration markedly increases during pregnancy and is structurally very similar to 17
epiestriol except that the 17-OH group in estriol is in the 17-
position, whereas that of 17
epiestriol is in the
position (Fig. 1). This
difference makes it a very weak agonist for both ER
and ER
(7).
The isoflavone phytoestrogen genistein binds to ER
almost as well as
17-
E2, whereas its affinity to ER
is considerably lower than that of 17-
E2 (14).
E2 did not affect VCAM-1
mRNA expression at lower concentrations (100 and 300 pM), whereas at higher concentrations (1-300
nM) it decreased VCAM-1 mRNA in a
concentration-dependent manner (Figs. 2 and 3). This action of 17-
E2 in vitro has been studied by other
investigators; however, the results are controversial. In studies using
HUVEC, 17-
E2 suppressed the induction of VCAM-1
mRNA expression induced by either lipopolysaccharide (2, 15) or by
interleukin-1
(8, 16). In contrast, Cid et al. (18)
observed that 17-
E2 did not affect basal VCAM-1
expression but caused a 30-50% increase in the presence of TNF
and
had no effect when HUVEC were stimulated with interleukin-1. It has
also been reported that 17-
E2 had no effect on the
TNF
-induced expression of VCAM-1 in cultured endothelial cells (19).
These contrasting observations may be due to differences in the extent
to which the endothelial cells used in the different studies expressed
the estrogen receptors, differences in the concentration of 17-
E2 utilized, or to differences in the agents used to induce
VCAM-1 expression.
E2
required for suppression of VCAM-1 expression in vitro were in the supraphysiological range (1 nM-10 µM).
The physiological concentration of 17-
E2 is in the
pM range, whereas that of 17-epiestriol has not been
reported in humans.
when compared with 17-
E2, did not attenuate
TNF
-induced VCAM-1 mRNA expression at any of the concentrations
studied (Figs. 2 and 3). One potential explanation for the absence of
any effect of EE may be that ER
mediates the estrogen-induced
decrease in VCAM-1 mRNA expression, whereas ER
has a negligible
role. This may be the case as 17-epiestriol, which has an
ER
-selective agonistic potency, decreased VCAM-1 mRNA expression
and the IC50 was ~400× lower than that observed with
17-
E2. At present, we cannot explain why higher
concentrations of 17-epiestriol were gradually less effective. A
similar biphasic effect of 17-epiestriol on VCAM-1 protein expression
(Fig. 7) and a shift of NF
B to the nucleus (Fig. 10) was also
observed. Estriol, which is a very weak agonist for both ER
and
ER
(7), did not affect VCAM-1 mRNA expression. This indicates
that the alignment of the 17-OH group in the
position is an
important requirement for 17-epiestriol for its selective ER
agonistic activity. The ability of the various estrogens to attenuate
VCAM-1 mRNA expression was not observed in the presence of the
anti-estrogen 1C1-182780. This confirmed that the action of the
estrogens were receptor-mediated. Further and more specific assessment
of the precise ER type involved in mediating the VCAM-1 mRNA
expression by estrogens will have to await the discovery of specific
ER
and ER
receptor antagonists. Genistein at nanomolar concentrations has binding affinity to ER
, which is similar to that
of 17-
E2, whereas its affinity to ER
is considerably
less (14). Our observation that genistein at nM
concentrations (1 and 10 nM) decreased VCAM-1 mRNA
expression supports the concept that ER
is most likely involved in
the attenuation of VCAM-1 expression in endothelial cells and that
ER
probably has very little role in this phenomenon. At these
concentrations, genistein has very little tyrosine kinase inhibitory
activity (14) and, hence, the effects are likely due to its action on
the ER
.
B, the GATA family of
transcription factors, and an AP-1 site (15). NF
B is a
proinflammatory transcription factor up-regulating several genes
involved in endothelial activation (21). It has recently been
demonstrated that a very high concentration of 17-
E2
(10 µM) suppressed VCAM-1 expression indirectly by
inhibiting the nuclear translocation and DNA binding of NF
B, and
this was also associated with a reduction of AP-1 and GATA
transcription factors binding to the VCAM-1 promoter (15). These
investigators also demonstrated that in human endothelial cells, NF
B
inhibition by E2 was associated with decreased I
B-
degradation.
B (22, 23). NO also stabilized
the NF
B inhibitor, I
B-
(24), and cellular treatment with NO
donor compounds also inhibited AP-1 binding to DNA (25).
-induced VCAM-1 mRNA expression also
increased eNOS protein (Fig. 8) and mRNA, suggesting that NO may
play an intermediary role in this action of 17-epiestriol. This was
confirmed by observing that the suppression of VCAM-1 mRNA
expression by 17-epiestriol was not observed following inhibition of NO
synthesis (Fig. 9). Our results also suggest that 17-epiestriol-induced
NO production played an intermediate role in inhibition of the nuclear
translocation and DNA binding of NF
B in HUVEC as this inhibitory
effect of 17-epiestriol was significantly attenuated in the presence of
an inhibitor of NO synthase. Higher concentrations (1 and 10 nM) of 17-epiestriol attenuated the eNOS protein expression
when compared with 300 pM, and this may explain the
biphasic response of 17-epiestriol on VCAM-1 mRNA and protein
expression. Our study did not address the precise molecular
mechanism(s) by which NO production stimulated by 17-epiestriol
inhibited the translocation of NF
B to the nucleus. It is also
possible that the differences in actions observed between estrogenic
compounds with different affinities for ER
and ER
receptors could
be mediated by interaction with the AP-1 site. The interactions of
ER
and ER
with the AP-1 site could lead to signaling in opposite
ways and thus may play opposite roles in gene regulation (29). It would
also be interesting to assess whether the reduction of AP-1 and GATA
binding to the VCAM-1 promoter by estrogens is also mediated by an
increase in NO production, thereby indicating an important intermediary
role of NO in the regulation of some transcription factors by estrogens.
and not by ER
and that NO played an intermediate role in
this process. Our results further suggest that 17-epiestriol, an
estrogen metabolite, is more potent than 17-
E2. This
may be one potential explanation as to why much lower concentrations of
17-
E2 are required to suppress VCAM-1 expression
in vivo (where various metabolites of estrogens are formed)
when compared with those required in vitro.
and ER
in mediating the antiatherosclerotic action of estrogens. It has been demonstrated that
17-
E2 can inhibit responses to vascular injury in an
acute carotid artery endothelium-denuded model in ovariectomized
ER
/
(30) and ER
/
(31) animals to
the same extent as in wild type animals. On this basis it has been
suggested that ER
and ER
can complement each other in
vivo to mediate the vasoprotective action of estrogens following
vascular injury. Recently, it has been demonstrated that genistein,
which has selective binding affinity to ER
, provided vasoprotective
actions that were equally efficacious to that observed with 17-
E2 in an acute carotid artery endothelium-denuded rat model
and yet were devoid of effects on the uterus, which mainly have ER
receptors (32). In the acute carotid artery endothelium-denuded model,
in contrast to the atherosclerosis model, the endothelial cells are
denuded, and it would therefore be difficult to assess the actions of
estrogens exerted specifically via the endothelial cells. It would
therefore be interesting to assess whether 17-epiestriol is as
effective or more potent than 17-
E2 in attenuating
atherogenesis and yet is devoid of effects on the reproductive system
when compared with 17-
E2. Further studies are in
progress to assess this in vivo in an animal model. Our
results demonstrating the biphasic action of 17-epiestriol in
modulating VCAM-1 gene and protein expression indicate that estrogens
may have paradoxical effects in inflammatory processes. This may be one
potential explanation for the controversy in relation to the effects of
estrogen replacement therapy on cardiovascular morbidity (17).
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ACKNOWLEDGEMENTS |
---|
We thank Srirupa Mukhopadhyay, Julie Gage, Janis Cuevas, and Svetlana Arutyunova for technical assistance.
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FOOTNOTES |
---|
* This work was supported, in part, by NIA, National Institutes of Health Grant AG-15857.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of the Pfizer/Society for Women's Health Research Scholars Grant for Faculty Development in Women's Health.
Recipient of a graduate scholarship from The American Heart
Association Southern California Chapter.
To whom correspondence should be addressed: Depts. of
Obstetrics & Gynecology and Molecular & Medical Pharmacology, David Geffen School of Medicine at UCLA, Center for Health Sciences, 10833 Le
Conte Ave., Los Angeles, CA 90095-1740. Tel.: 310-206-6575; Fax:
310-206-2057; E-mail: gchaudhuri@mednet.ucla.edu.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M207800200
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ABBREVIATIONS |
---|
The abbreviations used are: VCAM, vascular cell adhesion molecule; ER, estrogen receptors; EE, ethinyl estradiol; L-NAME, NG-nitro-L-arginine methyl ester; TNF, tumor necrosis factor; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cells; eNOS, endothelial nitric-oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Nathan, L., and Chaudhuri, G. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 477-515[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Nathan, L.,
Pervin, S.,
Singh, R.,
Rosenfeld, M.,
and Chaudhuri, G.
(1999)
Circ. Res.
85,
377-385 |
3. | Haynes, M. P., Russell, K. S., and Bender, J. R. (2000) J. Nucl. Cardiol. 7, 500-508[Medline] [Order article via Infotrieve] |
4. | Kuiper, G. G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 90, 5925-5930[CrossRef] |
5. | Register, T. C., and Adams, M. R. (1998) J. Steroid Biochem. Mol. Biol. 64, 187-191[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Lindner, V.,
Kim, S. K.,
Karas, R. H.,
Kuiper, G. G.,
Gustafsson, J. A.,
and Mendelsohn, M. E.
(1998)
Circ. Res.
83,
224-229 |
7. |
Barkhem, T.,
Carlsson, B.,
Nilsson, Y.,
Enmark, E.,
Gustafsson, J.,
and Nilsson, S.
(1998)
Mol. Pharmacol.
54,
105-112 |
8. |
Caulin-Glaser, T.,
Watson, C. A.,
Pardi, R.,
and Bender, J. R.
(1996)
J. Clin. Invest.
98,
36-42 |
9. |
Hodges, Y. K.,
Tung, L.,
Yan, X. D.,
Graham, J. D.,
Horwitz, K. B.,
and Horwitz, L. D.
(2000)
Circulation
101,
1792-1798 |
10. |
Singh, R.,
Pervin, S.,
Karimi, A.,
Cederbaum, S.,
and Chaudhuri, G.
(2000)
Cancer Res.
60,
3305-3312 |
11. |
Mukherjee, T. K.,
Dinh, H.,
Chaudhuri, G.,
and Nathan, L.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
4055-4060 |
12. | Hayashi, T., Fukuto, J. M., Ignarro, L. J., and Chaudhuri, G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11259-11263[Abstract] |
13. | Yang, N. N., Venugopalan, M., Hardikar, S., and Glasebrook, A. (1996) Science 273, 1222-1225[Abstract] |
14. |
Kuiper, G. G.,
Lemmen, J. G.,
Carlsson, B.,
Corton, J. C.,
Safe, S. H.,
van der Saag, P. T.,
van der Burg, B.,
and Gustafsson, J. A.
(1998)
Endocrinology
139,
4252-4263 |
15. |
Simoncini, T.,
Maffei, S.,
Basta, G.,
Barsacchi, G.,
Genazzani, A. R.,
Liao, J. K.,
and De Caterina, R.
(2000)
Circ. Res.
87,
19-25 |
16. | Nakai, K., Itoh, C., Hotta, K., Itoh, T., Yoshizumi, M., and Hiramori, K. (1994) Life Sci 54, PL221-PL227[Medline] [Order article via Infotrieve] |
17. |
Writing Group for the Women's Health Initiative Investigators.
(2002)
JAMA
288,
321-333 |
18. | Cid, M. C., Kleinman, H. K., Grant, D. S., Schnaper, H. W., Fauci, A. S., and Hoffman, G. S. (1994) J. Clin. Invest. 93, 17-25[Medline] [Order article via Infotrieve] |
19. | Aziz, K. E., and Wakefield, D. (1996) Cell. Immunol 167, 79-85 |
20. |
Iademarco, M. F.,
McQuillan, J. J.,
Rosen, G. D.,
and Dean, D. C.
(1992)
J. Biol. Chem.
267,
16323-16329 |
21. | De Caterina, R., and Gimbrone, M. A., Jr. (1995) in n-3 Fatty acids-Prevention and Treatment in Vascular Disease (Kristensen, S. D. , Schmidt, E. B. , De Caterina, R. , and Endres, S., eds) , pp. 9-24, Springer Verlag, London |
22. | De Caterina, R., Libby, P., Peng, H. B., Thannickal, V. J., Rajavashisth, T. B., Gimbrone, M. A., Jr., Shin, W. S., and Liao, J. K. (1995) J. Clin. Invest. 96, 60-68[Medline] [Order article via Infotrieve] |
23. | Spiecker, M., Darius, H., Kaboth, K., Hubner, F., and Liao, J. K. (1998) J. Leukoc. Biol. 63, 732-739[Abstract] |
24. |
Katsuyama, K.,
Shichiri, M.,
Marumo, F.,
and Hirata, Y.
(1998)
Arterioscler. Thromb. Vasc. Bio.
18,
1796-1802 |
25. | Tabuchi, A., Sano, K., Oh, E., Tsuchiya, T., and Tsuda, M. (1994) FEBS Lett. 351, 123-127[CrossRef][Medline] [Order article via Infotrieve] |
26. | Weiner, C. P., Lizasoain, I., Baylis, S. A., Knowles, R. G., Charles, I. G., and Moncada, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5212-5216[Abstract] |
27. |
Caulin-Glaser, T.,
Garcia-Cardena, G.,
Sarrel, P.,
Sessa, W. C.,
and Bender, J. R.
(1997)
Circ. Res.
81,
885-892 |
28. |
Chen, Z.,
Yuhanna, I. S.,
Galcheva-Gargova, Z.,
Karas, R. H.,
Mendelsohn, M. E.,
and Shaul, P. W.
(1999)
J. Clin. Invest.
103,
401-406 |
29. |
Paech, K.,
Webb, P.,
Kuiper, G. G. J. M.,
Nilsson, S.,
Gustafsson, J.,
Kushner, P. J.,
and Scanlan, T. S.
(1997)
Science
277,
1508-1510 |
30. | Iafrati, M. D., Karas, R. H., Aronovitz, M., Kim, S., Sullivan, T. R., Jr., Lubahn, D. B., O'Donnell, T. F., Jr., Korach, K. S., and Mendelsohn, M. E. (1997) Nat. Med. 3, 545-548[Medline] [Order article via Infotrieve] |
31. |
Karas, R. H.,
Hodgin, J. B.,
Kwoun, M.,
Krege, J. H.,
Aronovitz, M.,
Mackey, W.,
Gustafsson, J. A.,
Korach, K. S.,
Smithies, O.,
and Mendelsohn, M. E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
15133-15136 |
32. |
Makela, S.,
Savolainen, H.,
Aavik, E.,
Myllarniemi, M.,
Strauss, L.,
Taskinen, E.,
Gustafsson, J. A.,
and Hayry, P.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7077-7082 |