© 2001 American Heart Association, Inc.
Vascular Biology |
Progesterone, but Not Medroxyprogesterone, Inhibits Vascular Cell Adhesion Molecule-1 Expression in Human Vascular Endothelial Cells
From the Department of Molecular Medicine, Osaka University Graduate School Medicine (M.O., H.S., X.X., S.S., H.K., S.K.), and Osaka University (T.K.), Suita-City, Osaka, Japan.
Correspondence to Soji Kasayama, MD, PhD, Department of Molecular Medicine, Osaka University Graduate School of Medicine (C-4), 2-2 Yamada-oka, Suita-City, Osaka 565-0871, Japan. E-mail kasayama{at}imed3.med.osaka-u.ac.jp
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Abstract |
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Abstract—It has been shown that ovarian steroid hormones can reduce the incidence of cardiovascular disease in postmenopausal women. As hormone replacement therapy for postmenopausal women, progestins are added to estrogens to eliminate the increased risk of endometrial cancer. However, the effects of progestins on the atherogenic process have not been well understood. In the present study, we examined the effects of progestins on the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Immunocytochemical analysis revealed the presence of progesterone receptors in HUVECs. Progesterone clearly inhibited tumor necrosis factor-
–activated expression of VCAM-1 protein and its mRNA
in HUVECs. Synthetic progesterone receptor agonist R5020 also inhibited
the tumor necrosis factor-–activated VCAM-1 expression,
whereas medroxyprogesterone acetate (MPA) failed to
do so. Electrophoretic mobility shift assays demonstrated that
progesterone, but not MPA, inhibited DNA binding of the transcription
nuclear factor-
B, which is critical for the inducible expression of
VCAM-1. Because the expression of VCAM-1 is one of the earliest events
that occurs in the atherogenic process, this adhesion molecule might be
a target molecule for progesterone on vascular walls. The contrasting
effects of progesterone and MPA seem clinically important, inasmuch as
MPA is a widely used progestin in the regimen of hormone replacement
therapy.
Key Words: progesterone • medroxyprogesterone • vascular cell adhesion molecule-1 • endothelial cells • progesterone receptors
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Introduction |
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Compared with men of similar age and postmenopausal women, premenopausal women are known to be protected against cardiovascular diseases.1 2 3 Ovarian steroid hormones have been reported to play an important role in the protection against cardiovascular diseases. There are many reports indicating that estrogens have beneficial effects on atherosclerotic vascular diseases in postmenopausal women.4 5 6 7 8 9 10 In contrast, the effects of progestins, which are used to eliminate the increased risk of endometrial cancer by estrogen treatment,11 on vascular walls have not been well understood. Recently, Grodstein et al12 have shown that a combination therapy with estrogens and progestins lowered the incidence of cardiovascular disease in postmenopausal women compared with therapy with estrogens alone. In addition, it has been shown that in ovariectomized baboons, the administration of 17ß-estradiol and progesterone together resulted in fewer vascular lesions than the administration of 17ß-estradiol alone.13 These results suggest that progestins independently exert antiatherogenic effects.
It has been shown that progesterone induces endothelium-independent relaxation in rabbit coronary arteries14 and also inhibits the induction of platelet calcium responses.15 A recent study by Lee et al16 has clearly shown that progesterone inhibits arterial smooth muscle cell proliferation via downregulating the expression of cyclins A and E. Cheng et al17 have demonstrated the effects of progesterone on macrophages: inhibition of cholesterol ester synthesis and block of glucocorticoid-induced increases in cholesterol ester synthesis. In addition, Miyagawa et al18 have shown that progesterone plus 17ß-estradiol protects against coronary vasospasm in ovariectomized rhesus monkeys. Thus, the studies concerning the effects of progestins on atherogenesis have mainly focused on vascular smooth muscles as well as lipid metabolism. However, to date, there is no evidence of the direct effects of progestins on vascular endothelial cells.
Atherosclerosis is characterized by endothelial cell injury, which in turn leads to the adhesion of mononuclear leukocytes to the endothelium, the initial migration and proliferation of smooth muscle cells, and extracellular matrix deposition.19 Various adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, E-selectin, and platelet endothelial cell adhesion molecule, have been demonstrated to be expressed in atherosclerotic lesions, which might be involved in mononuclear leukocyte adhesion to the vascular endothelium.20 21 22 23 24 Thus, to repress the expression of these adhesion molecules on vascular endothelium may make it a molecular target of antiatherogenic agents. Indeed, it has been recently shown that some activators of the nuclear receptors, such as estrogen receptors25 26 and peroxisome proliferator–activated receptors,27 28 target the adhesion molecules in vascular endothelial cells. In the present investigation, we studied whether progesterone and/or medroxyprogesterone acetate (MPA) influences VCAM-1 expression in cultured vascular endothelial cells.
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Methods |
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Materials and Cell Culture
Promegestone (R5020) was obtained from NEN Life Products. 17
-Hydroxy-11-(4-dimethyl-aminophenyl)-17-propenyl-estra-4.5-diene-3-one
(RU486) was from Biomol. Onapristone (ZK98299) was provided by
Schering. All other steroid compounds were purchased from Sigma
Chemical Co. Human umbilical vein endothelial cells (HUVECs, Cascade Biologics) were maintained in MCDB131 medium with 10% FCS (JRH Biosciences) and 2 ng/mL basic fibroblast growth factor (Kaken Pharmaceutical) (growth medium). The cells have the characteristics of vascular endothelial cells in terms of cobblestone appearance at confluence and von Willebrand factor expression.
Whole-Cell ELISA
HUVECs were plated onto 96-well collagen-coated
dishes in the growth medium. After 24 hours, the culture medium was
changed to DMEM–15% gelding horse serum (Sigma) without basic
fibroblast growth factor. The cells were pretreated with test compounds
or vehicle (0.1% ethanol) for 24 hours and thereafter were stimulated
with 20 ng/mL tumor necrosis factor (TNF)- (Dainippon
Pharmaceutical) for 4 hours. ELISA for cell surface VCAM-1 protein was
performed as described below. After treatment with 2%
paraformaldehyde, the cells were washed twice with 1%
BSA in PBS and then incubated for 1 hour with a primary antibody
specific to VCAM-1 (Genzyme) diluted 1/1000 in 3% BSA in PBS. Then the
cells were washed 4 times and incubated for 1 hour with
peroxidase-conjugated sheep anti-mouse IgG (Amersham) diluted 1:1000 in
3% BSA in PBS. After the cells were washed, the second antibody
binding was detected by reaction of tetramethylbenzidine with
H2O2 (TMB peroxidase EIA
substrate kit, Bio-Rad). The reaction was stopped by the addition of 25
µL of 8N sulfuric acid, and plates were read on an ELISA reader at an
optical density of 450 nm. In parallel, HUVECs were treated as
described above. Cell extracts were prepared from these cells in lysis
buffer and assayed for protein content by a BCA protein assay reagent
kit (Pierce).
Northern Blot Analysis
HUVECs were grown in the growth medium on 10-cm
collagen-coated dishes until they were subconfluent. After the culture
medium was changed to DMEM–15% gelding horse serum (without basic
FGF), the cells were treated with test compounds and/or TNF-. Total
RNA extracted by the acid guanidinium thiocyanate–phenol-chloroform
method was electrophoresed in 1% agarose–2% formaldehyde gels. The
gels were transferred to nylon membranes (Hybond
N+, Amersham), and the membranes were
UV-irradiated. A 32P-labeled cDNA probe for
human VCAM-1 was prepared by a random priming method (Amersham).
Hybridization was carried out by using Quick hybridization buffer
(Stratagene).
Nuclear Extraction and EMSA
HUVECs were treated with test compounds and/or
TNF-, as described above. The nuclear extracts were prepared by the
method of Schreiber et al.29
An electrophoretic mobility shift assay (EMSA) for nuclear factor
(NF)-B was performed, as
described.30 The sequence of
double-stranded oligonucleotides used to determine the
DNA binding abilities of NF-B was that derived from the human VCAM-1
gene promoter. The NF-B consensus sequence is shown in bold face:
5'-CTGCCC-TGGGTTTCCCCTTGAAGGGATTTCCCTCCGCC-3'.31
Immunocytochemistry
After HUVECs were treated with 2%
paraformaldehyde, the cells were incubated with 0.1
mol/L glycine in PBS and then with 0.1% Triton X-100 in PBS. After
they were washed, the cells were incubated with 5% goat serum in PBS
for 1 hour and thereafter with antibody against the progesterone
receptor (PgR, Santa Cruz) diluted 1:200, followed by incubation with
biotinylated anti-rabbit IgG antibody. The next steps were performed
with the use of Vectastain ABC kit (Vector Laboratories).
For immunostaining of NF-B p65 protein,
formaldehyde-fixed HUVECs were incubated with antibody against p65
(Santa Cruz) diluted 1:200, followed by incubation with FITC-conjugated
second antibody (Jackson Immunoresearch). Probes were excited with the
492-nm line of an argon-ion laser. Emitted light was detected at
520 nm.
Statistical Analysis
All values represent mean±SD. When the
significant difference was discussed, an unpaired Student
t test was
used.
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Results |
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PgRs in HUVECs
We examined whether PgRs are expressed in HUVECs. Immunocytochemical analysis using PgR-specific antibody (Figure 1
) revealed perinuclear localization of PgRs in
HUVECs. When HUVECs were treated with progesterone, the staining was
translocated into nuclei. The staining was blocked when the cells were
treated with anti-PgR antibody preabsorbed with excess PgR antigen.
Thus, the staining was indicated specific for
PgR.
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Effects of Progestins on VCAM-1 Expression
in HUVECs
When HUVECs were exposed to TNF-, cell surface
VCAM-1 levels were
upregulated.32 The
TNF-–induced VCAM-1 levels were correlated with the cell numbers
plated between 1x103 and
2x104 cells per well (please see Figure I,
published online and available at http://atvb.ahajournals.org).
Therefore, in the following experiments, we determined the VCAM-1
levels when the cells were plated at a density of
2x104 cells per well. Treatment with
progesterone repressed the TNF-–induced increase of VCAM-1 protein
in a dose-dependent manner
(Figure 2). Significant repression was observed at
concentrations
10-7 mol/L.
At 10-5 mol/L,
progesterone inhibited TNF-–induced stimulation of VCAM-1 levels by
83%. However, progesterone did not show any significant change of
unstimulated VCAM-1 levels
(Figure 2). The synthetic PgR agonist R5020
(10-5 mol/L)
inhibited the TNF-–induced increase of VCAM-1 by 44%
(Figure 3). By contrast, MPA showed no effect on the
TNF-–induced stimulation of VCAM-1 levels. Neither
17-hydroxyprogesterone, pregnenolone, 17-hydroxypregnenolone,
nor 5ß-pregnane-3,20-dione demonstrated inhibition of the
TNF-–induced VCAM-1 expression
(Figure 3). Progesterone-mediated inhibition of VCAM-1
expression was not blocked by PgR antagonists RU486 and
ZK98299 (please see Figure II, published online and available at
http://atvb.ahajournals.org). As shown in Figure III (published online
and available at http://atvb.ahajournals.org), 17ß-estradiol also
reduced the TNF-–induced VCAM-1 levels, but it failed to augment
progesterone-mediated inhibition of the VCAM-1 expression.
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Next, to examine the effects of progestins on the levels of
VCAM-1 mRNA in HUVECs, Northern blot analyses were performed.
As shown in
Figure 4, TNF- increased VCAM-1 mRNA accumulation.
Treatment of the cells with progesterone significantly reduced the
TNF-–induced accumulation of VCAM-1 mRNA. By contrast, MPA failed
to reduce the VCAM-1 mRNA accumulation.
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Effects of Progestins on Activation of
NF-B in HUVECs
TNF-–induced activation of VCAM-1 transcription in
vascular endothelial cells has been shown to depend, at
least in part, on the activation of the transcription factor
NF-B.31 32 Thus,
we examined the effect of progestins on TNF-–induced activation of
NF-B in HUVECs. EMSA with the nuclear extracts from HUVECs and the
oligonucleotide probe containing NF-B binding sites
in the human VCAM-1 gene promoter showed that TNF- enhanced 2 of the
gel-retarded bands
(Figure 5, lanes 1 and 2). The gel-retarded bands were
specific for NF-B, inasmuch as it disappeared in the presence of
excess unlabeled oligonucleotides but not unrelated
oligonucleotides
(Figure 5, lanes 3 and 4). The addition of NF-B p65
antibody supershifted both bands
(Figure 5, lane 5), whereas the addition of NF-B p50
antibody supershifted mainly the lower band
(Figure 5, lane 6). The addition of both antibodies almost
completely supershifted the gel-retarded bands
(Figure 5, lane 7). These results indicate that the upper
band consists of p65-p65 homodimers and that the lower band consists
mainly of p65-p50 heterodimers. Treatment of HUVECs with progesterone
or R5020 repressed the TNF-–induced increase in the intensity of
the gel-retarded bands
(Figure 5, lanes 8 through 10). By contrast, MPA had no
effect on the intensity of the gel-retarded bands
(Figure 5, lane 11).
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Effects of Progesterone on Nuclear
Translocation of NF-B in HUVECs
Translocation of NF-B proteins from the cytoplasm to
nuclei is a known prerequisite for the activation of
NF-B.33 To examine whether
progesterone prevents the nuclear translocation of the NF-B
proteins, subcellular localization of NF-B p65 was investigated. As
demonstrated in
Figure 6, TNF- facilitated the translocation of the p65
protein from the cytoplasm into nuclei. Progesterone failed to prevent
the TNF-–induced translocation of the p65 protein. Treatment with
progesterone alone did not show any effect on the localization of the
p65 protein.
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Discussion |
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In the present investigation, we for the first time demonstrated that progesterone significantly repressed levels of cell surface VCAM-1 protein enhanced by TNF-
in human vascular
endothelial cells. The synthetic PgR agonist R5020 also
inhibited the TNF-–induced VCAM-1 expression. By contrast,
17-hydroxyprogesterone, pregnenolone, 17-hydroxypregnenolone, and
5ß-pregnane-3,20-dione, which can activate the pregnane X
receptor (PXR) as does
progesterone,34 did not show
any effect on the TNF-–induced VCAM-1 expression.
Immunocytochemical study revealed the expression of PgRs in HUVECs.
Thus, the inhibitory effect of progesterone on VCAM-1
expression is suggested to be mediated via PgR but not the pregnane X
receptor, although the involvement of other receptors cannot be
completely ruled out. In this relation, PgR has been shown to be
present in canine vascular
tissue,35 baboon and human
aorta,36 37 and
human arterial smooth muscle
cells.16
Progesterone significantly exerts its inhibitory
effect at a concentration of
10-7 mol/L,
although higher concentrations are necessary to induce more profound
inhibition of VCAM-1 expression. The effective concentration of
progesterone
(10-7 mol/L)
seems higher than its physiological concentration
in human sera. Therefore, a pharmacological concentration of
progesterone may be needed to exert the inhibitory effect
on adhesion molecule expression. Alternatively, it is also possible
that a physiological concentration of progesterone
is enough to demonstrate such an effect in vivo, because of intact
vascular microenvironment or the prolonged exposure to progesterone. In
similar instances, other studies on VCAM-1 regulation by estrogens and
glucocorticoids25 26
have also noted that their inhibitory effects were more
evident at concentrations that were higher than their
physiological concentrations.
VCAM-1 is known to play an important role in mediating mononuclear leukocyte-selective adhesion to vascular endothelium.20 38 This adhesion molecule is expressed in the vascular lesions in early atherosclerosis,21 and its soluble form was elevated in sera from patients with early atherosclerosis,39 40 indicating that VCAM-1 is one of the key molecules involved in the atherogenic process. Therefore, the progesterone-induced repression of VCAM-1 expression in vascular endothelial cells is suggested to be a molecular mechanism of the antiatherogenic action of progesterone.
In the present study, MPA, a synthetic progestin,
was different from progesterone in that it had no effect on
TNF-–induced VCAM-1 expression. A recent
observation18 has also shown
the differential effect of MPA from progesterone. In that study,
progesterone plus 17ß-estradiol protected coronary vasospasm
in ovariectomized rhesus monkeys, whereas MPA plus 17ß-estradiol
failed to protect. Such contrasting effects of progesterone and MPA are
clinically important issues, inasmuch as MPA is now a widely used
progestin in the regimen of hormone replacement therapy. In this
relation, several studies have demonstrated unfavorable effects of MPA
on lipid
metabolism41 42 43
and
atherosclerosis.44 45
The present study showed that TNF-–induced VCAM-1
mRNA accumulation was also reduced by progesterone. Thus, the
progesterone-induced repression of VCAM-1 is indicated to occur at
pretranslational levels. Because there is no evidence that progesterone
response elements exist on the VCAM-1 gene
promoter,31 32 it
is unlikely that progesterone-bound PgR directly interacts with the
5'-flanking region of the VCAM-1 gene. Rather, progesterone exerts its
effect by an indirect mechanism. TNF-–induced transcription of the
VCAM-1 gene in vascular endothelial cells is known to
be dependent, at least in part, on the activation of NF-B
transcription
factors.31 32 In
our experiments, progesterone as well as R5020 clearly suppressed
TNF-–activated NF-B binding to its specific DNA binding
elements. In contrast, MPA had no inhibition of the NF-B binding.
Thus, the differential effects of these progestins on the NF-B
binding are correlated with those on VCAM-1 expression. Therefore, the
progesterone-induced inhibition of VCAM-1 expression is mediated at
least via interference with the NF-B activity. The present study
has demonstrated that in HUVECs TNF--activated NF-B
proteins consist mainly of p65 (RelA) and p50 (NFKB1).
Immunocytochemical analysis revealed that progesterone did not
block TNF-–stimulated translocation of NF-B p65 protein from
cytoplasms to the nuclei, indicating that the progesterone exerts its
effect mainly at steps after the nuclear translocation of NF-B.
Thus, progesterone is suggested to demonstrate its
inhibitory effect on VCAM-1 expression by transrepressing
the NF-B activity. Such a different mechanism from PgR-mediated
transactivation of the target genes might be why the effective
concentration of progesterone appears to be higher.
It is undefined how progesterone and R5020, but not MPA,
inhibit NF-B binding to its specific DNA binding elements of the
VCAM-1 gene promoter. Much evidence has accumulated indicating that
several coactivators, such as cAMP response
element–binding protein/p300, steroid receptor
coactivator-1/nuclear receptor coactivator-1,
and transcription intermediary factor 2/glucocorticoid
receptor–interacting protein 1/nuclear receptor
coactivator-2, are involved in the signaling of various
nuclear receptors, including
PgR.46 Among these
coactivators, cAMP response element–binding protein/p300
and steroid receptor coactivator-1 have been shown to
interact with NF-B, enhancing its transcriptional
activity.47 48 49
Thus, it is possible that progesterone- or R5020-bound PgR, but not
MPA-bound PgR, may interfere with NF-B activity by squelching such
coactivators. Another possibility is that PgR directly
interacts with NF-B in a ligand-specific manner, followed by the
reduction of NF-B transcriptional activity. In this context,
cotransfection experiments showed that the concomitant expression of
PgR and NF-B results in mutual antagonism of the transcriptional
activities between PgR and
NF-B.50 51 In
the present study, combined treatment with 17ß-estradiol and
progesterone did not demonstrate any additive inhibition on VCAM-1
expression, suggesting that estrogen receptors can exhibit an
inhibitory effect on VCAM-1 expression by sharing the same
molecular targets as PgR. In addition, the present study showed
that RU486 and ZK98299 failed to antagonize the progesterone-mediated
inhibition of VCAM-1 expression. These results are not
surprising, inasmuch as these "antagonists" have been
demonstrated to repress NF-B–evoked transcriptional activity as the
"agonist" in transfection
systems.50
In conclusion, we have shown that progesterone, but not MPA,
reduces TNF-–enhanced VCAM-1 expression in vascular
endothelial cells. The effect of progesterone may be
mediated via the inhibition of binding of NF-B to the specific
sequence of the VCAM-1 gene promoter. To our knowledge, this is the
first study to demonstrate that progesterone inhibits the gene
expression in which NF-B is involved. In view of these observations,
we believe that the inhibitory effect on VCAM-1 expression
in vascular endothelial cells should be added to the
lists of biological actions of progesterone on vascular
walls.
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Acknowledgments |
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This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan. We thank Keiko Tsujii for her excellent secretarial assistance in preparing the manuscript.
Received March 27, 2000; accepted November 1, 2000.
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