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Division of Endocrinology, Long Beach Veteran Affairs Medical Center, Long Beach, California 90822; and Departments of Medicine (A.P., M.R., R.-M.H., E.B., E.R.L.) and Pharmacology (A.K.M., B.A.P., E.R.L.), University of California, Irvine, Irvine, California 92717
Address all correspondence and requests for reprints to: Ellis R. Levin M.D., Medical Service (111-I), Long Beach Veterans’ Hospital, Long Beach, California 90822. E-mail: elevin{at}pop.long-beach.va.gov
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The mechanisms by which the female sex hormones could affect the development of vascular disease are only partially defined and are mainly correlative. E stimulates the production of HDL, lowers the serum concentration of LDL cholesterol and decreases LP(a) levels by 50% (3, 4, 6). The effects of progesterone are less clear. It has been estimated that the ability of estrogen to effect a favorable lipid status accounts for 25–50% of its "protective" effect (7). Therefore, other mechanisms must be involved (8). One hypothesis to explain the beneficial effects of sex hormone action on the vasculature is that E and P inhibit the mitogenic action of growth factors, which are secreted from vascular cells or macrophages recruited to the early inflammatory lesion of atherosclerosis. The growth factors stimulate the proliferation of vascular smooth muscle cells, an early and important event in the pathogenesis of atherosclerosis (9). One such hormone, endothelin-1 (ET-1) is a powerful vasoconstrictor and mitogen for VSMC, acting through specific transmembrane receptors (10, 11). ET-1 has been strongly implicated in the development of acute and chronic vascular diseases, including atherosclerosis and cardiac hypertrophy (12, 13). Therefore, sex steroids could serve a protective function by inhibiting the pathophysiologic effects of ET-1. Based upon the demonstration of functional estrogen receptors in human VSMC (14), an interaction between E and ET-1 is tenable.
We sought to determine if E or P could suppress the stimulation of vascular smooth muscle cell proliferation, induced by ET-1 or the complex but relevant mitogen, serum. We also determined the effects of E and P on several important cell signaling events that potentially transmit the growth stimulus to the nucleus, resulting in VSMC proliferation.
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Materials and Methods |
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25 cm) were cannulated and
flushed with 50 ml of sterile DMEM, containing
antibiotics/antimycotics. The veins were incubated intraluminally with
0.1% collagenase, type II, (Boehringer Mannheim, IN) in DMEM, in a
water bath at 37 C for 30 min, then flushed to remove endothelial
cells. After this digestion, the veins were again loaded with
collagenase in DMEM for 2 h at 37 C. The vessels were then flushed
with 50 ml of the DMEM and the cells obtained were pelleted and
resuspended in DMEM with 20% FBS, containing antibiotic/antimycotic
mixture and cultured onto culture dishes coated with rat tail collagen.
The uniform growth of smooth muscle cells was identified by their
typical hill and valley growth pattern and morphology, and by
immunostaining for smooth muscle cell specific 
-actin. No factor
VIII staining cells were identified. hUVSMC were generally used at
passage 3 for the experiments. For thymidine experiments, the cells
were plated in 24-well culture dishes containing DMEM and 10% FBS. For
immediate early gene expression and kinase studies, cells were plated
in 100-mm plates.
Northern analysis
Total RNA was extracted from the cells at various time points
after incubating the cells with serum, ET-1, 17ß-estradiol, or P or
combinations of these substances for up to 2 h. RNA was extracted
using the Tri-Reagant (Molecular Research Center, Cincinnati, OH), and
20 µg from each experimental condition was denatured and separated on
a 1.2% agarose gel containing 7.4% formaldehyde, then transferred to
nitrocellulose. The RNA was then prehybridized overnight at 42 C in the
presence of 50% deionized formamide, 5x SSC, 5x Denhardt’s
solution, 25 mM sodium phosphate buffer (pH 6.8), 0.1%
SDS, and 100 µg/ml of salmon sperm DNA. The blots were then
hybridized for 12–18 h at 42 C in hybridization solution
(prehybridization solution with 10% dextran sulfate added) containing
32P-labeled, antisense c-fos cRNA [PSP 65c-fos
1A (rat), kindly provided by Dr. Tom Curran (16), or
32P-labeled, antisense c-myc cRNA (American Type
Culture Collection, Rockville, MD), using techniques previously
described by us (17). The membrane was then washed at 55 C with 1
x SSC plus 0.1% SDS, then subjected to autoradiography for 2–3 days
at -70 C temperature, and hybridization bands were quantified by laser
densitometry. Sense probes produced no hybridization. RNA loading was
determined according to hybridization of the RNA with a GAPDH
complementary DNA (cDNA) probe (American Type Culture Collection,
Rockville, MD) and densitometric values were normalized for loading.
The location of 28 and 18s rRNA in the samples is noted on the
figures.
3H-thymidine incorporation
Subconfluent hUVSMC were transfected with dominant negative
mitogen-activated protein (MAP)-kinase constructs (or control) as
described below. Cells were synchronized for 24 h in serum-free
media. All cells were then incubated for 20 h in the absence or
presence of ET-1 or serum. In some conditions, the MAP kinase kinase
(MEK) inhibitor, PD 98059, 20 µM, was added to the
incubation mixture of nontransfected cells, 1 h before the
mitogen. This was then followed later by the addition of 0.5 µCi of
[3H]-thymidine for an additional 4 h, as previously
described (18). Cells were then washed in cold HBSS, incubated for 10
min with 10% TCA at 4 C to precipitate the nuclear incorporated
thymidine, washed two additional times with HBSS, lysed with 0.2
N NaOH overnight, and the lysates were counted in a liquid
scintillation ß-counter.
Cell number
hUVSMC were cultured at 105 cells/well in six-well
plates in DMEM without serum and synchronized for 24 h, then
cultured with each experimental condition over 3 days or in DMEM
without serum (control). Experimental conditions included 3% serum or
ET-1,100 nM, and in the presence or absence of various
concentrations of E or P and estrogen antagonists, tamoxifen, 1
µM, or ICI 182,780, 1 µM (Dr. Alan
Wakeling, Zeneca Pharmaceuticals, Alderley Park, UK). At the end of the
third day, the cells were briefly trypsinized, resuspended for single
cell suspension, and counted by hemocytometer and coulter counter in
duplicate determinations per well, quadruplicate wells per condition.
Trypan blue exclusion indicated that approximately 93–97% of the
cells in all wells were still viable at the end of these experiments,
and so the data were not adjusted for viability. Cell counting
experiments were repeated three times in total. In parallel studies,
cells exposed to steroid still excluded trypan blue and showed the
usual morphology 24 h after steroid withdrawal.
MAP-K and MEK studies
For MAP-K or MEK activity, hUVSMC were synchronized by serum
deprivation overnight. The cells were then cultured in 3% serum, or
ET-1, 100 nM, with or without E or P for 7 min, or were
treated with no added substance (control kinase expression), or
steroids alone. Plates were washed, lysed, and the supernatant was
frozen. MAP-K activity was assessed as directed against myelin basic
protein in an in vitro kinase assay, as we have previously
described (19). The phosphorlyated MBP was separated by SDS-PAGE, the
gel was fixed, stained for MAP-K protein visualization, and
autoradiography ensued. MEK activity was similarly assessed (19)
against K52R, a mutant MAP-K, which does not autophosphorylate but
which serves as a substrate for MEK (20, 21) (provided by Dr. Michael
Weber, University of Virgina).
Dominant negative MAP kinase
To determine whether MAP-K activation was necessary for ET-1 or
serum-induced proliferation, the VSMC were transiently transfected with
either 0.2 µg DNA of the Y185F dominant negative MAP-K construct
(22), or the empty vector (pCMV5) as a control, using 1 µl
lipofectamine (GIBCO-BRL-Life Sciences, Grand Island, NY). The
transfected cells were incubated at 37 C for 5 h, then switched to
DMEM with 10% FBS and allowed to recover for 24 h. Serum was then
removed for 24 h to synchronize the cells, and then MAP-kinase or
thymidine studies were carried out as described above. Transfection
efficiency was determined by cotransfecting the Y185F construct with
pGreen Lantern (GIBCO-BRL). The number of cells which expressed the
green fluorescent protein by appearing yellow-green under an inverted
fluorescent microscope with excitation filter 24 h after
transfection, was counted. To corroborate the data with Y185F, ET or
serum-stimulated cells were incubated with PD 98059, a specific
inhibitor of MEK activity (23), and thymidine studies were carried out
as described above.
Estrogen-BSA studies
We incubated the cells for 10 min with fluorescein
isothiocyanate (FITC)-conjugated estradiol (E)-BSA, and performed
direct immunofluorescence labeling to determine which pool of estrogen
receptors was bound (labeled). The FITC conjugated E-BSA compound has
previously been shown to label a membrane estrogen receptor (ER) in
several cell types (24, 25). Competition of labeling by the FITC-E-BSA,
with tamoxifen, 17-ß estradiol, 0.1 µM, or an antibody
(H-222) to the ligand binding domain of the ER (provided by Geoffrey
Green, University of Chicago) (17) was carried out. Cells were also
permeabilized with DTT to allow the labeling the nuclear pool of ER. We
also incubated the cells in the presence of the E-BSA compound and
compared the effects on MAP-kinase activation and thymidine
incorporation to estradiol alone.
Statistics
Data from at least three thymidine studies or cell counting
experiments were combined and then analyzed by calculating a mean and
SE for each treatment or group. Data from the conditions
were compared for significant overall differences by ANOVA; Fisher’s
protected least square difference (FPLSD) was used to precisely compare
the different experimental treatments when significant F values (at a
level of P < 0.05) were found by ANOVA. RNA
comparisons were quantified by laser densitometry of autoradiographs,
and data were normalized for RNA loading by creating a ratio of the
density of the experimental RNA hybridized with the c-fos or
c-myc probe, divided by the same amount of RNA hybridized
with a GAPDH cDNA probe. A ratio was then established by comparing
normalized experimental RNA to normalized RNA from control cells. This
resulted in values expressing the relative densities of the
experimental conditions compared with the control. The MAP kinase and
MEK studies were repeated a total of four times, and the densitometries
from each condition were combined and statistically analyzed as
above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). E caused a 64
± 2% reversal of serum-stimulated cell proliferation at 10
nM E, and a 23 ± 2% inhibition at 1 nM
steroid, whereas P caused a 62 ± 4 and 18 ± 1% inhibition
at these concentrations. As a more defined mitogen, ET-1 caused a
44 ± 2% increase in cell number, significantly reversed 70
± 2 and 32 ± 2% by E at 10 and 1 nM, or 75 ±
3 and 23 ± 1% by P at the same concentrations (Fig. 1B). P alone
had a small but significant effect on basal cell number. In more
limited studies, testosterone (100 nM) had no effect on
basal cell number or ET-1 or serum-induced proliferation (data not
shown), indicating the specificity of E or P action.
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). Tamoxifen was comparable
in effect to ICI 182,780, which is considered to be a more pure E
antagonist and which caused a 65 ± 5 and 69 ± 6% reversal
of the effects of E on serum and ET-1. Tamoxifen had no effect on the
P-related inhibition (Fig. 1C), as was also found for ICI (data not
shown). Combining E and P at 10-10 M each, or
at 10-9 M P plus 10-10
M E, was neither additive nor synergistic compared with the
individual sex steroid (data not shown). The hUVSMC were cultured in
media containing phenol red, which has weak estrogenic actions (26),
but data from cells cultured in the absence of phenol red were only
4–12% different across all conditions from cells cultured in parallel
with phenol red and so all data were combined.
MAP and MEK kinases
ET-1 and serum each significantly stimulated MAP-K (erk) activity
in the human UVSMC. ET-1 caused a 4.32 ± 0.5-fold enhancement,
whereas 3% serum caused a significant 4.33 ± 0.6-fold increase
above basal MAP-K (erk) activity (P < 0.05 compared
with control). E and P were approximately equipotent in inhibiting the
stimulation of MAP-K by either mitogen. E (10 nM) caused a
77 ± 10% reduction of ET-1-stimulated MAP-K activity, and a
48 ± 12% reduction at 1 nM, whereas P caused
reductions of 72 ± 11 and 48 ± 13%, at these
concentrations (Fig. 2A, top, and 2C)
(P < 0.05). The steroids also inhibited
serum-stimulated MAP-K by 79 ± 10 and 46 ± 8% for E and
76 ± 11 and 45 ± 13% for P, 10 nM and 1
nM, respectively, all statistically significant compared
with mitogen alone. The above data reflect four combined experiments
(Fig. 2C), whereas the gel figure (2A) is a representative study.
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). E and P reversed the ET-stimulation of MEK by
84 ± 14 and 34 ± 10%, and by 83 ± 7 and 35 ±
10%, in concentration-related fashion, based upon combined data from
four experiments (Fig. 2C). E and P, at 10 and 1 nM,
inhibited serum-stimulated MEK activity by 84 ± 6 and 36 ±
9%, and 87 ± 4 and 35 ± 7%, respectively. The inhibitions
were statistically significant for each of the sex steroids, at both
concentrations. Our data also suggests that the sex steroids act
upstream of MEK because mitogen-activated MEK activity results from the
stimulated activity of upstream kinases such as c-raf, which
can activate MEK (28).
The importance of MAP-K for the ability of ET-1 and serum to promote
vascular smooth muscle proliferation has not been established. To do
this, we transiently transfected the hUVSMC with a MAP-K construct
(Y185F), where the critical tyrosine185 has been mutated to
phenylalanine. This construct has been shown to significantly inhibit
wild-type MAP-K activity (22). We found that the Y185F construct
inhibited basal MAP-kinase activity by approximately 30% and the ET-1
stimulated MAP-K activity by 60% (Fig. 3). Similar
results were seen with serum (data not shown). The pCMV5 vector
(control empty vector) had no effect on ET-1 stimulated MAP-K activity.
The transfection efficiency was approximately 78%, determined by
counting the number of cells that expressed the green lantern protein
under fluorescent microscopy, after being cotransfected with this
protein expression vector and Y185F. When Y185F transfected cells were
subjected to thymidine incorporation, this construct (but not pCMV5)
inhibited the proliferative effects of ET-1 or serum by 68 ± 10
and 38 ± 12%, respectively (Table 1). Serum is a
complex mitogen and probably signals through multiple pathways to enact
the growth program in the nucleus, including MAP-K. E and P probably
inhibit serum-stimulated proliferation, at least in part, through
inhibition of the MAP-K cascade.
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). MEK inhibition again revealed the necessity of
the MAP-K cascade for growth factor-induced VSMC proliferation and a
greater dependence on this pathway by ET, compared with serum. These
results support the idea that E and P inhibit VSMC proliferation via
negative regulation of this signaling cascade.
Estradiol-BSA studies
Because the effects of estradiol to inhibit stimulated-MAP-K
occurred rapidly (by 7 min), we asked whether this might be a
nongenomic effect of E, attributable perhaps to estrogen binding to a
nonnuclear pool of ER. This might include a putative membrane ER,
demonstrated on a variety of cell membranes (24, 25). We first showed
that the FITC-conjugated E-BSA labeled a membrane pool of putative ER,
and was essentially excluded from entering the cell after a 7-min
incubation (Fig. 4A). Although some differential
magnitude of labeling of cells occurred, clear and often strong
labeling was seen in about 75% of cells. Binding of E-BSA to a
putative membrane ER was completely competed off by unlabeled E (E),
tamoxifen (t), and also by an antibody to the ER (ab), which is very
unlikely to enter the cell, especially after 7 min If we first
permeabilized the cells (p), the labeling of ER by the FITC-conjugated
E-BSA compound was entirely nuclear. This indicates that the E-BSA
compound did not gain access to the intact cell interior and provides
us a tool to assess the possible contribution of the membrane ER to
antiproliferation in this model. It has been shown that the E-BSA
compound is at most 10% as potent as estrogen in binding the membrane
ER (24, 25), probably because the bulky BSA protein limits access of E
to the receptor sites. Our differential labeling of whole cells
supports this observation.
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, B and C). E, 10 nM, and the E-BSA
compound, 100 nM, each inhibited ET-stimulated MAP-K by
71 ± 4 and 60 ± 5%, respectively. The inhibition by E or
E-BSA, respectively, was reversed 58% and 49% by the ER antibody, and
comparably by tamoxifen (Fig. 4B). BSA alone, or added to E or to ET-1,
had no effects on MAP-K activity. Regarding DNA synthesis, E, 10
nM, caused a 60 ± 4% decrease in ET-1 stimulated
thymidine incorporation, and a 44 ± 3% decrease in
serum-stimulated thymidine incorporation, whereas E-BSA, 100
nM, caused 41 ± 3 and 40 ± 2% decreases,
respectively (Fig. 4C). Additionally, E or E-BSA inhibition of
ET-stimulated thymidine incorporation was reversed approximately 70%
by tamoxifen, and E or E-BSA inhibition of this ET-1 effect was also
reversed 40% and 35%, respectively, by the ER antibody. The ER
antibody also reversed the inhibition by E or E-BSA of
serum-stimulated thymidine incorporation, by 35% and 34%
(Fig. 4C). BSA or the ERab alone had no effect on proliferation.
Inhibition of mitogen-stimulated MAP-K by the cell impermeable E-BSA,
or E2, leading to inhibition of proliferation, suggests
that E may be acting, in part, through binding to a membrane ER.
c-fos and c-myc expression
The c-fos and possibly the c-myc gene can be
regulated by MAP-K (29), and because these two immediate early
genes/transcription factors are implicated in UVSMC proliferation, we
determined the effects of E and P on mitogen-stimulated fos
and myc expression. ET-1 or serum caused a rapid and strong
induction of c-fos, approximately 10.6 ± 0.4 and
8.4 ± 0.3-fold above control at 60 min, which returned to basal
levels by 120 min (Fig 5A). Based upon combined data
from four separate experiments, ET-1-stimulated c-fos
expression was maximally inhibited 78 ± 8% by 10 nM
E and 83 ± 5% by 10 nM P (Fig. 5C). E or P, 10
nM, also inhibited serum-stimulated fos by
90 ± 6 and 88 ± 7%, respectively. The inhibition was also
significant at 1 nM steroids. The induction of
c-myc by ET-1 or serum was significantly induced after 30
min (Fig. 5B), by about 3.5-fold and 6.6-fold above control,
respectively (Fig. 5C). Again, E or P, 10 nM, significantly
inhibited c-myc expression 85 ± 5% and 80 ± 6%
for ET-stimulated, and 89 ± 5% and 88 ± 10% for
serum-stimulated myc expression. The inhibition was also
significant at 1 nM E (Fig. 5C).
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1
nM), and to serum levels of E or P measured in women during
the reproductive years or after hormonal replacement therapy (1–10
nM) (reviewed in 30 . In fact, tissue levels of these
steroids may actually be even greater (31). The antiproliferative
effects correlated with the ability of either E or P to inhibit MAP-K
and MEK activity and to reverse the mitogenic stimulation of the
production of transcription factors, c-fos and
c-myc. We found that activation of the MAP-K enzyme is
important for the ability of ET-1 or serum to enhance VSMC
proliferation. Therefore, inhibition of this pathway by the sex
steroids is also likely to mediate their antiproliferative actions.
c-fos and c-myc are important for the in
vivo and in vitro proliferation of VSMC and other cell
types (32, 33, 34, 35), and inhibition by the steroids of these downstream
targets of MAP-K action provides an additional likely mechanism. Our in vitro results support epidemiologic and prospective studies that show that E is protective against the development of atherosclerosis and cardiovascular disease (1, 2, 3, 4, 5) and provides a potential novel mechanism by which E may act. E is also a direct vasodilator (36), favorably alters the lipoprotein profile (3, 37) and the oxidation of LDL, suppresses intimal proliferation, arterial wall matrix production (reviewed in 37 , and participates in blood vessel formation or remodeling (38). Estrogen receptors (ER) are present on VSMC of human coronary arteries, and one study showed that atherosclerotic coronary vessels in women generally lack ER (39). A recent human study by Rosano et al. (40) suggests that E can inhibit an acute mechanism of coronary vasoconstriction, perhaps mediated through antagonizing the action of a vasoconstricting peptide, such as endothelin.
The ability of progesterone to protect against the development of cardiovascular disease is much less clear, with evidence favoring a beneficial effect of P, in the setting of E, but other data indicating that P antagonizes the favorable lipid effects of E (8, 37, 41, 42, 43). Our results indicate that P is equipotent to E as an antigrowth factor for the cultured VSMC. As noted, we found that testosterone had no effect on mitogen-stimulated cell number, indicating the specificity of E and P action.
We also have begun to identify a cellular mechanism by which sex steroids could influence this early and important event in the pathogenesis of atherosclerosis (9). Vascular growth factors stimulate the proliferation of VSMC after generating an intracellular signal following the binding of specific transmembrane receptors (44, 45). This propagates a cytoplasmic signal that often leads to the activation of tyrosine and threonine/serine protein kinases and associated proteins, which in turn transmits the growth signal to the nucleus and activates cell cycle specific kinase activity (46, 47, 48). Although several signaling mechanisms span the cytoplasmic-nuclear transition, the MAP-K cascade is felt to be an important pathway by which growth factors, such as PDGF, EGF, and bFGF, stimulate cell proliferation (49). MAP-K activity has been detected in arteries and vascular smooth muscle (48, 50), where ET acts as a mitogen for VSMC. ET signals in many different cells through several pathways that impinge on the nucleus, including the MAP-K (erk) (19), and c-Jun kinase pathways (51). However, it was previously undetermined in any cell which pathway is important for the mitogenic action of ET. Our data, which is derived from two separate but complementary approaches (dom-neg MAP-K and MEK inhibitor) supports the importance of the MAP-K (erk) cascade as mediating in large part, the growth signal transmitted to the nuclear growth program in response to ET-1 in the VSMC. Further, both serum and ET-stimulated MEK and MAP-K (erk) activity can be significantly reversed by either E or P. However, the greater potency of E compared with P, particularly in reversing the ET-1 stimulation of erk activity, suggests that P may also be working through additional signaling or direct transcriptional inhibitory pathways to limit VSMC proliferation. The ability of the sex steroids to inhibit MEK stimulated MAP-K is probably related to inhibition of the activity of upstream kinases, such as c-raf or MEK-K but needs to be defined. Because serum contains many growth promoting factors, it is likely that serum induces VSMC proliferation through several signaling mechanisms, ultimately triggering the nuclear growth program. Nevertheless, our results indicate that at least part of the serum-induced growth stimulus is through the erk cascade.
Target substrates for MAP-K include proteins that transactivate the genes encoding Fos and Myc (29). This leads us to speculate that MAP-K stimulates the transcription of c-fos (and perhaps c-myc) in these cells, for instance, by phosphorylating regulatory proteins such as Elk-1 (52), that bind the serum response element and modulate the transcription of c-fos (53). MAP-K can also phosphorylate the Myc protein and alter its ability to transactivate growth regulatory genes (29), which are likely to be critical to the growth program. We found that the stimulation of c-fos and c-myc gene expression by our two mitogenic stimuli is reversed by the sex steroids. Although we propose that the ability of E and P to inhibit mitogen-stimulated c-fos and c-myc is through the inhibition of MAP-K, there is also a nonclassical estrogen-response element (ERE) that is present on the promoter of the c-fos gene (54). This element directly mediates positive transactivation of this gene when E acts as a mitogen in reproductive tissues, but it is unknown whether inhibitory effects of E could be mediated through this element, as in our context. We found that E and P had no significant effects on basal c-fos (or c-myc) gene expression.
An additional novel finding is that ER or PR modulates MAP-K (erk) activation by the cytoplasmic protein, MEK. Erk translocates to the nucleus where it could interact with ER or PR, but MEK is clearly a cytoplasmic protein. The concept that nuclear receptor proteins can affect cytoplasmic cell signaling is newly emerging. Recently, it has been shown that vitamin D, presumably acting through its nuclear receptor, can activate protein kinase C (PKC) and the MAP-K pathway in cells where vitamin D is a mitogen (55). It is currently unknown whether E or P can modulate PKC activity. We report here the first evidence of negative modulation of MEK and MAP-K by ER and PR, and these interactions could provide a model to examine steroid signaling through this kinase cascade.
One important qualification, however, is that there is immunocytochemical evidence in VSMC that ER can be found in the cytoplasm, although the ligated receptor is predominantly perinuclear in location (56). However, there are limitations in the sensitivity of immunocytochemistry to identify other small pools of receptors. In several cell types, ER is present on cell membranes (57), and E has nongenomic and extremely rapid effects on intracellular calcium (58). This suggests that a membrane ER may be relevant for some actions of E, but no known cell biological action mediated by these receptors has yet been reported. In fact, there is very little information available about the molecular and structural nature of these receptors, except that antibodies raised against the nuclear ER do identify the membrane ER (25), suggesting some shared structural epitopes. Recently, a second ER has been cloned in the rat and has particularly strong homology in the ligand binding domain to the established ER (59). It is unknown whether this receptor is present in human tissues or in the cell membrane.
Therefore, it appears that ERs exist in several forms and locations. We found here that an estrogen compound that labels a putative ER pool on the cell membrane of the VSMC is excluded from entering the cell and can inhibit ET-1 or serum-activation of MAP-K. The labeling of the putative membrane ER was competed off by E, tamoxifen, or an ER antibody. Tamoxifen and the ER antibody significantly reversed the inhibitory effects of E-BSA on MAP-K and thymidine incorporation, linking these events. Importantly, the ER antibody comparably reversed the inhibitory action of E alone or E-BSA on mitogen-stimulated MAP-K. Because the antibody does not enter the cell, especially in 7 min, this indicates that E is likely to be acting through a putative membrane ER to inhibit erk activation. Comparable reversal of all of the E effects were seen with the ER antibody (excluded from the cell) or tamoxifen (can act at any ER). We propose that E may have both genomic and nongenomic effects to inhibit VSMC proliferation and that the membrane ER is a candidate to mediate the early signaling events that underlie this action.
Our finding that E can act in vitro to suppress VSMC proliferation supports several previous in vivo studies, where E administration was found to suppress intimal hyperplasia of the aorta in several animal models (60, 61). It was speculated in each of these studies that E might be inhibiting VSMC proliferation. Sullivan et al. showed that physiological replacement of estradiol significantly inhibits vascular smooth muscle proliferation in the mouse carotid media following balloon injury (62). E inhibits thymidine incorporation into segments of pig coronary artery (63) or in mixed cultures from the media of rabbit aorta in response to hyperlipemic serum (64). Few studies examining comparable effects of P in vascular models exist.
In summary, physiologic concentrations of estrogen and progesterone can inhibit the proliferation of cultured human vascular smooth muscle cells, as induced by serum or ET-1. E or P inhibition of mitogen-activated MEK and MAP-K (erk) activity and the stimulation of c-fos and c-myc expression provides a potential pathway by which sex hormones can limit growth factor-induced VSMC proliferation, an early and important step in the pathogenesis of atherosclerosis. These in vitro data support the use of sex steroids to inhibit the development of vascular diseases which are dependent on vascular smooth muscle proliferation.
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Footnotes |
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Received September 26, 1996.
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References |
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gene expression in cultured
bovine aortic endothelial cells. Mol Cell Endocrinol 103:65–71[CrossRef][Medline]
and macrophage colony-stimulating factor
in human aortic medial smooth muscle cells. J Clin Invest 95:1133–1139
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R. K. Dubey, E. K. Jackson, D. G. Gillespie, M. Rosselli, F. Barchiesi, A. Krust, H. Keller, L. C. Zacharia, and B. Imthurn Cytochromes 1A1/1B1- and Catechol-O-Methyltransferase-Derived Metabolites Mediate Estradiol-Induced Antimitogenesis in Human Cardiac Fibroblast J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 247 - 255. [Abstract] [Full Text] [PDF]
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 M. Maggiolini, A. Vivacqua, G. Fasanella, A. G. Recchia, D. Sisci, V. Pezzi, D. Montanaro, A. M. Musti, D. Picard, and S. Ando The G Protein-coupled Receptor GPR30 Mediates c-fos Up-regulation by 17{beta}-Estradiol and Phytoestrogens in Breast Cancer Cells J. Biol. Chem., June 25, 2004; 279(26): 27008 - 27016. [Abstract] [Full Text] [PDF]
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S. Chen and D. G. Gardner Suppression of WEE1 and Stimulation of CDC25A Correlates with Endothelin-dependent Proliferation of Rat Aortic Smooth Muscle Cells J. Biol. Chem., April 2, 2004; 279(14): 13755 - 13763. [Abstract] [Full Text] [PDF]
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S. Ling, A. Dai, R. J. Dilley, M. Jones, E. Simpson, P. A. Komesaroff, and K. Sudhir Endogenous Estrogen Deficiency Reduces Proliferation and Enhances Apoptosis-Related Death in Vascular Smooth Muscle Cells: Insights From the Aromatase-Knockout Mouse Circulation, February 3, 2004; 109(4): 537 - 543. [Abstract] [Full Text] [PDF]
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D.-H. Kang, E. S. Yu, K.-I. Yoon, and R. Johnson The Impact of Gender on Progression of Renal Disease: Potential Role of Estrogen-Mediated Vascular Endothelial Growth Factor Regulation and Vascular Protection Am. J. Pathol., February 1, 2004; 164(2): 679 - 688. [Abstract] [Full Text] [PDF]
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J. Abramowitz, C. Dai, K. K. Hirschi, R. I. Dmitrieva, P. A. Doris, L. Liu, and J. C. Allen Ouabain- and Marinobufagenin-Induced Proliferation of Human Umbilical Vein Smooth Muscle Cells and a Rat Vascular Smooth Muscle Cell Line, A7r5 Circulation, December 16, 2003; 108(24): 3048 - 3053. [Abstract] [Full Text] [PDF]
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