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Original Studies |
, Is Present in the Vascular Endothelium of the Human and Nonhuman Primate Endometrium1
Department of Obstetrics and Gynecology, University of Edinburgh (H.O.D.C., T.A.H.), and Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology (K.W., M.R.M., P.T.K.S.), Edinburgh, United Kingdom EH3 9ET; and Oregon Regional Primate Research Center (R.M.B., N.R.N., O.D.S.), Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Prof. Hilary Critchley, Department of Obstetrics and Gynecology, University of Edinburgh, Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: hilary.critchley{at}ed.ac.uk
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in full
thickness endometrial samples (from the superficial to the basal zone)
obtained from both women and rhesus macaques. Immunohistochemical
localization with specific antibodies revealed that ER and ERß
were both expressed in nuclei of the glands and stroma. Consistent with
previous studies, expression of ER declined in the glands and stroma
of the functionalis during the secretory phase. The luminal epithelium
also displayed positive immunoreactivity for ERß. Expression of ERß
declined in glandular cell nuclei, but not stroma, within the
functionalis during the late secretory phase. Levels of expression of
ER and ERß in all cellular compartments remained unchanged in the
basalis. Both receptor subtypes were detected on Western blots using
proteins extracted from uterine samples obtained throughout the
menstrual cycle.
There was a striking contrast between the pattern of expression of
ER and ERß in the vascular endothelium and the perivascular cells
surrounding endometrial blood vessels; only ERß was present in the
endothelial cell population, although both forms of ER were expressed
in perivascular cells. We conclude that estrogen action(s) within the
vascular endothelium in the endometrium may be mediated via direct
binding to the ERß isoform and that these cells could therefore be a
target for agonists or antagonists that selectively target the ß form
of the ER.
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)
(5) protein. Reports have been consistent in describing a
decline in ER immunoreactivity in the superficial layer in both
glands and stroma during the secretory phase, although the decline in
glandular immunoreactivity is delayed compared with that in the stroma.
No significant change in ER immunoreactivity has been observed in
the basal endometrium (4). ERs have been reported in
nonpregnant uterine vascular smooth muscle, but not in endothelium
(6). ER expression is negligible in the glands and
stromal cells of early pregnancy decidua (7).
A second ER, now usually known as ERß, was first cloned from a rat
prostate complementary DNA library in 1996 (8). Homologs
have now been identified in many species, including human (9, 10), mouse (11), and marmoset monkey (accession no.
Y09372). The function of ERß in the uterus remains unknown
(12). All members of the steroid receptor family share a
common arrangement of five structure-function domains, denoted A–F,
which have been identified by experiments in which receptor domains
have been exchanged (13), and ERß is no exception. Like
ER and other members of the steroid hormone receptor family, the
human (h) ERß gene has been shown to be encoded by eight exons
(9), with the highest levels of homology between ER and
ERß present in the DNA-binding (C) and ligand-binding (E)
domains.
PCR analysis of ER messenger ribonucleic acid (mRNA) levels in the rat
has suggested that ERß mRNA is less abundant than ER in uterine
tissue extracts (14) a result confirmed and extended by
Wang et al. (15), who examined levels of mRNAs
in control and steroid-treated rat uteri using solution hybridization.
Both ER and ERß proteins have been immunolocalized to rat uterine
cell nuclei (15, 16). The functional importance of ER
for female fertility has been highlighted by studies using mice in
which the function of ER was disrupted (ERKO) (17); the
loss of ER expression in the uteri of these mice is associated with
the lack of estrogen responsiveness (18). In contrast,
young female mice expressing ERß lacking the DNA-binding domain
(ßERKO) (19) are fertile.
Rey and colleagues (20) reported that levels of ERß mRNA
in endometrial samples obtained from infertile patients across the
menstrual cycle did not change. In situ hybridization with
biotin-labeled oligonucleotide probes on hysterectomy specimens showed
recently that ERß mRNA was present in glandular epithelial cells,
stromal cells, and the smooth muscle of the uterine wall at every stage
of the menstrual cycle (21), with predominant expression
of ERß in glandular epithelial cells. However, this study made no
mention of mRNA expression in endometrial endothelial cells.
Brandenberger and co-workers (22) used a semiquantitative
RT-PCR assay to compare the relative expression of ER and ERß in
stromal cells derived from normal endometrium and from endometriosis
and found that the relative expression of the two subtypes differed
between these two cell populations. In both normal endometrium and
endometriomas, levels of ER mRNA are reported to be higher than
those of ERß (21, 22). These workers did not discuss the
vascular endothelium. In the uterus of the cynomolgus macaque
(23), ERß mRNA expression was higher in endometrium than
myometrium and was greater in glandular epithelium than stromal cells.
Unfortunately, this study did not assess possible changes during the
menstrual cycle, nor did it report whether endometrial blood vessels
expressed ERß. Immunohistochemical studies of ERß proteins in human
endometrium are limited and have not included a detailed examination of
ERß expression in all the cellular components of human endometrium
(24, 25).
Studies in vitro have demonstrated that although both ER
and ERß bind E2 with equal affinity
(14), these receptors may have differential responses to
some estrogen agonists and antagonists (26, 27, 28). When
expressed in the same cell, ER and ERß have the capacity to form
homo- or heterodimers (29, 30). Studies in mammary tissues
of the rat have suggested that one role of ERß may be to antagonize
ER-mediated actions in epithelial cells (31), a
function supported by data from in vitro cell transfections
(32) but not yet confirmed in vivo.
As a first step toward addressing the relative roles of the two ER
isoforms in mediating estrogen actions within the uterus, we used
hormonally well characterized, full thickness endometrial samples (from
the superficial endometrium to the basal/myometrial junction) from the
endometrium of both women and rhesus macaques for immunocytochemical
and Western blotting analysis with antibodies specific for ER and
ERß. Our results show for the first time that endometrial vascular
endothelial cells express ERß, but not ER, which suggests that any
direct actions of E2 on the vascular endothelium
of the uterus are mediated predominantly by ERß.
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Materials and Methods |
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Endometrial tissue was collected from 32 women (median age, 44 yr; range, 27–51) undergoing hysterectomy. Written informed consent was provided by all subjects, and ethical approval for tissue collection was granted by the Lothian research ethics committee. All women reported regular menstrual cycles (25–35 days) and had not received exogenous hormones or used an intrauterine device in the 3 months before inclusion in the study. After the uterus was removed, a wedge of tissue from the lumen to the muscular myometrial layer that included superficial and basal endometrium as well as myometrium was taken. These samples were fixed overnight at 4 C in 4% paraformaldehyde, rinsed, and stored in 70% ethanol before routine processing into paraffin wax using an 18-h cycle on a TP1050 machine (Leica Corp., UK). Some tissue samples were frozen in liquid nitrogen for subsequent extraction and Western analysis.
The stage of the menstrual cycle was consistent with the patient’s
reported last menstrual period and histological dating using the
criteria of Noyes et al. (33). Cases with
severe uterine pathology, e.g. polyps or fibroids, were
excluded. Serum samples taken at the time of hysterectomy were used for
determination of circulating P and E2 levels by
RIA as described previously (34). All were consistent with
the designated cycle stage based on morphological criteria, and
circulating P concentrations were significantly lower in the late
secretory phase compared with the early and midsecretory phases (see
Table 1
). Endometrial samples were
classified as early (n = 3), mid (n = 8), or late (n =
4) proliferative and early (n = 7), mid (n = 4), or late
(n = 6) secretory.
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Six adult female rhesus macaques (Macaca mulatta) were ovariectomized and treated sequentially with E2 and P to create artificial menstrual cycles as described previously (35). Specifically, each macaque first received a sc implant of a 3-cm SILASTIC brand capsule (Dow Corning Corp., Midland, MI) packed with crystalline E2 (Sigma, St. Louis, MO) to stimulate the development of an artificial proliferative phase endometrium. After 14 days, a 6-cm SILASTIC brand capsule packed with crystalline P (Sigma) was implanted sc, and both implants remained in place for 14 days to stimulate an artificial secretory phase endometrium. Uteri were removed by hysterectomy after 14 days of E2 (n = 3; proliferative phase) and after 14 days of E2 plus P (n = 3; secretory phase) treatment, and full thickness wedges were made of the endometrium from the luminal surface to the myometrium. Tissue samples were microwaved, fresh-frozen, and prepared for immunocytochemistry as previously described (36). In each case serum was harvested at the time of tissue collection, and concentrations of E2 and P were determined by RIA and found to be within physiological ranges as previously reported (37). Animal care during these studies was provided by the veterinary staff of the Division of Animal Resources, Oregon Regional Primate Research Center, in accordance with the NIH policy for the care and use of laboratory animals.
ER antibodies
A peptide chosen within the hinge D domain (P4,
CAGKAKRSGGHAPRVREL) of human ERß (9, 10) was
synthesized, conjugated, and used for immunization as described
previously (38, 39). The polyclonal antiserum was further
purified by precipitation with caprylic acid and binding to the
recombinant unconjugated peptide immobilized on a Sulfolink column
(Pierce Chemical Co., Rockford, IL) (39). The
anti-hER antibody used was a mouse monoclonal (clone 1D5), obtained
from BioGenex Laboratories, Inc. (San Ramon, CA), or
DAKO Corp. (Cambridge, UK).
The specificity of the antisera was tested in several ways. First, the
ability to cross-react with recombinant hER or hERß proteins
(PanVera, Madison, WI) was determined using Western blotting (see
below) (39). Second, antibodies were incubated separately
with either of the recombinant proteins or with the immunizing peptide
before immunohistochemistry. Third, the secondary antibodies were
checked by incubation of selected tissue sections without the addition
of the primary antibodies or with the inclusion of preimmune serum. For
positive controls, we used sections of human ovary that we had
previously shown to express ERß (39).
Western blotting
Recombinant human ERß corresponding to the short,
approximately 53-kDa form of the receptor (ßs), which was synthesized
from a complementary DNA (10) lacking the first potential
start site for translation (40), and recombinant hER
were both obtained from PanVera. Gel analysis and blotting were carried
out as described in Saunders et al. (39).
Briefly, proteins were extracted from frozen biopsy specimens by rapid
homogenization of tissue in denaturing/loading buffer [50 mmol/L
Tris-HCl (pH 6.8), 100 mmol/L dithiothreitol, 2% SDS, 0.1%
bromophenol blue, and 10% glycerol; all from Sigma].
Recombinant proteins (0.5 µg/lane), tissue extracts (400 µg total
protein), and prestained protein molecular weight markers
(Bio-Rad Laboratories, Inc., Richmond, CA) were separated
on denaturing minigels containing an acrylamide gradient from 4–20%
(wt/vol) polyacrylamide (Novex, San Diego, CA). Membranes
were incubated overnight with the primary antibodies: sheep polyclonal
anti-hERß P4 (code S40; 1:2000) or mouse monoclonal anti-hER
(code1D5; 1:100). All of the antibodies were diluted in Tris-buffered
saline containing 0.05% Tween-20 with either 5% normal donkey serum
(anti-hER) or 5% normal rabbit serum (anti-hERß). Bound
antibodies were detected with the appropriate second antibodies (rabbit
antisheep Ig or rabbit antimouse Ig) and the enhanced chemiluminescence
visualization system (Amersham Pharmacia Biotech,
Aylesbury, UK) according to the manufacturer’s instructions.
Immunocytochemistry on cryosections (Macaca endometrium)
Immunohistochemistry on cryosections was performed as described
recently (36). Briefly, samples of fresh tissue were
microwave stabilized with an Amana Radarrange Touchmatic Oven (Amana,
IA) for 7 s in 0.5 mL Hanks’ Balanced Salt Solution (Life Technologies, Inc., Grand Island, NY), then chilled on ice in
10% sucrose dissolved in 0.1 mol/L phosphate-buffered saline (PBS;
Sigma), mounted in Tissue-Tek II OCT (Miles, Inc.,
Elkhart, IN), and frozen in liquid propane. Cryosections (5 µm) were
thaw-mounted on SuperFrost Plus slides (Fisher Scientific,
Pittsburgh, PA), placed on ice at 4 C, and microwaved again for 2
s. Microwave-treated sections were fixed (0.2% picric acid and 2%
paraformaldehyde in PBS) for 10 min and thoroughly rinsed. Nonspecific
binding was blocked by incubation for 20 min at room temperature in
either nonimmune horse serum for 1D5 or nonimmune rabbit serum for
anti-hERß. Slides were then incubated overnight at 4 C with mouse
monoclonal anti-ER (1D5, BioGenex Laboratories, Inc.,
San Ramon, CA) at a dilution of 1:50 in PBS containing BSA and gelatin
or with polyclonal sheep anti-ERß P4 at a dilution of 1:500 in the
same vehicle. The primary antibody was reacted for 60 min at room
temperature with either biotinylated horse antimouse IgG (for 1D5) or
rabbit antisheep IgG (for polyclonal anti-ERß) as second antibody and
was detected with an avidin-biotin peroxidase kit (Vector Laboratories, Inc., Burlingame CA) as described previously
(36).
Immunohistochemistry on paraffin sections
Tissue sections were dewaxed, rehydrated, and subjected to
antigen retrieval (41) either in a microwave oven, for
ER and PR, on a high setting twice for 5 min each time in 0.01 mol/L
sodium citrate buffer (pH 6) or, for ERß, in a pressure cooker
(42) containing 0.05 mol/L glycine and 0.01%
ethylenediamine tetraacetate (pH 3.5) for 7 min at setting 2 (Tefal,
Nottingham, UK). After cooling for 20 min, the slides were washed in
PBS for ER or PR or in 0.05 mol/L Tris-buffered saline (TBS) for
ERß. Endogenous peroxidase activity was quenched by immersion in 3%
hydrogen peroxide (Merck & Co., Inc., Poole, UK) in
dH2O for 10 min for ER and PR or in 3%
H2O2 in methanol for 30 min
for ERß at room temperature. Nonspecific binding of the primary
antibodies was blocked by incubating the sections for 20–30 min at
room temperature in nonimmune horse serum (Vectastain,
Vector Laboratories, Inc., Peterborough, UK) for ER and
PR or in a 1:5 dilution of nonimmune rabbit serum (Diagnostics
Scotland, UK) in TBS containing 5% BSA (Sigma)
for ERß.
For ER, the sections were incubated at 37 C for 60 min with a 1:400
dilution of 1D5 made up in PBS. After washing, the slides were
incubated in biotinylated horse antimouse secondary antibody
(Vectastain) in normal horse serum for 60 min at room
temperature, reacted with the avidin-biotin peroxidase complex
(Vectastain Elite) for 60 min at room temperature, and
visualized with substrate and chromagen 3,3'-diaminobenzidine (DAB;
Vector Laboratories, Inc.). Negative controls were
performed by replacing the primary antibody with mouse IgG at a matched
concentration.
For ERß, the sections were incubated overnight at 4 C at a 1:800 dilution of anti-hERß P4 antibody made up in normal rabbit serum containing BSA. After washing, the slides were incubated at room temperature for 60 min in a 1:500 dilution of biotinylated rabbit antisheep antibody (Vector Laboratories, Inc.) in normal rabbit serum containing BSA. After an additional TBS wash, the sheep anti-ERß-treated slides were incubated for 60 min at room temperature in an avidin-biotin peroxidase solution (DAKO Corp.) before both sets of slides were incubated in DAB (DAKO Corp.). The negative control step involved incubation of prediluted antibody with an excess of the unconjugated form of the peptide used for immunization overnight at 4 C.
For PR, sections were incubated with primary antibody mouse monoclonal antibody (NCL-PgR, Novocastra Laboratories Ltd., Newcastle, UK) at a dilution of 1:40 for 60 min at 37 C. After a TBS wash, the slides were incubated with a biotinylated horse antimouse IgG antibody (Vector Laboratories, Inc.) followed by avidin-biotin peroxidase complex (Vectastain Elite) both for 60 min at room temperature. Staining was visualized by the addition of DAB (DAKO Corp.). After rinsing, all sections were counterstained in Harris’s hematoxylin (Pioneer Research Chemicals Ltd., Colchester, UK). A negative control was performed by replacing the primary antibody with mouse IgG at a matched antibody concentration.
Scoring of immunoreactivity
The immunostaining intensity of epitopes in all human tissue sections was assessed in a semiquantitative manner on a four-point scale: 0 = no immunostaining, 1 = mild immunostaining, 2 = moderate immunostaining, and 3 = intense immunostaining. All human tissue sections were scored blind by two observers. We had previously validated (43) this scoring system in a subset of tissue sections in which immunoreactivity was measured with a computerized image analysis system, and a strong correlation between quantitative data derived from image analysis and subjective scores by a trained observer was obtained. Statistical analysis was carried out using the Kruskal-Wallis test.
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Results |
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On Western blots (Fig. 1)
proteins that were recognized by anti-hER (Fig. 1A) and anti-hERß
(Fig. 1B) antibodies were extracted from human endometrial biopsies
obtained during both the secretory and proliferative stages of the
cycle. The commercial anti-ER antibody bound to recombinant ER
(Fig. 1A, lane ), but not to recombinant ERß (lane ßs), and
likewise, the polyclonal anti-hERß antiserum did not bind to
recombinant ER (Fig. 1B, lane ), but, as expected
(39), recognized recombinant human ERß (lane labeled
ßs). Both ER and ERß were detected in all samples of endometrium
regardless of the stage of the cycle at which they were obtained. Two
sizes of ERß protein were recognized by the polyclonal antibody (Fig. 1B); these corresponded in size to the long, approximately 59-kDa
(9, 40), and the short, 53-kDa (10), forms of
the protein formed by the use of alternative start sites within the
mRNA. Both forms contained the peptide used to generate the antibody,
and therefore both would be detected in immunocytochemical
preparations. The Western analysis was repeated several times on whole
tissue extracts obtained at different stages of the cycle, and although
the amounts of ERß protein detected varied between samples, there was
no stage of the cycle at which total protein levels were consistently
different from those at other stages.
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, A and D). In
cryosections of macaque endometrium, staining with a monoclonal
anti-ER antibody was abolished by preincubation of the antibody with
recombinant ER protein (Fig. 2B), but not with recombinant ERß
(Fig. 2C). Likewise, immunostaining using anti-ERß (Fig. 2D) was
abolished when it was preabsorbed with recombinant ERß (Fig. 2E), but
not when it was preincubated with recombinant ER (Fig. 2F).
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ER and ERß were immunolocalized to cell nuclei in each of the
cellular compartments within the functionalis and basalis (Figs. 3 and 4).
Both ER (Figs. 3A and 4A) and ERß (Figs. 3C and 4C) were
homogeneously immunolocalized in the nuclei of glandular (including
luminal) epithelium and stromal cells of superficial and basal
endometrium in the proliferative phase. Consistent with previous
reports (4), the intensity of ER immunostaining was
lower in glandular and stromal cells of the functionalis in the late
secretory phase (Fig. 3B) compared with that in the proliferative phase
(Fig. 3A); however, expression was maintained within the basalis (Fig. 4, B compared with A).
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) declined in the late secretory phase (after 10–12 days of
exposure to elevated P; see Table 1) in the glandular epithelium.
Analysis of immunostaining scores in the glandular epithelium confirmed
that there was a significant difference (P = 0.028)
among the four stages of the cycle. As shown in Fig. 5 (upper panel), scores were
lower in the late secretory phase. The intensity of the staining was
unchanged in the basalis zone (Fig. 5, lower panel).
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and 4, arrows), but
not ER (Figs. 3 and 4, A and B, arrowheads). This
difference in expression of ER, ERß, and PR is clearly seen at
higher magnification (Fig. 6). The
intensity of immunostaining for ERß in endothelial cells showed
little change throughout the proliferative and secretory phases of the
menstrual cycle (Figs. 2 and 3, and 6, respectively). It is notable
that in contrast to the selective pattern of expression of receptors in
endothelial cells, ER (Fig. 6B), ERß (Fig. 6A), and PR (Fig. 6C)
were all detected in the nuclei of the perivascular cells
(arrowheads).
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In the macaque endometrium during hormonally controlled cycles,
the pattern of ERß staining was essentially identical to that seen in
the human endometrium. The nuclei of glandular and stromal cells were
strongly stained after 2 weeks of E2 treatment,
and this staining was moderately suppressed by two subsequent weeks of
E2 plus P treatment. However, in the basalis
glands, nuclear staining of ERß did not differ greatly under
E2 vs. E2 plus P
treatment. These results were very similar to those seen in the human
endometrium and are therefore not shown as a separate figure. As in the
human endometrium, there was strong staining for ERß, but not ER,
in the nuclei of the endothelium of all endometrial blood vessels under
all hormonal conditions, whereas the perivascular cells were positive
for both ER (Fig. 7, A and C) and
ERß (Fig. 7, B and D).
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has been localized to perivascular smooth
muscle cells in nonpregnant endometrium (1, 6). The
expression of ERß in endometrial endothelial cells will be of
particular importance to furthering our understanding of how estrogens
affect mechanisms underlying vascular permeability and vascular growth
during the menstrual cycle.
The observations in human and macaque are consistent with the recent
report from Wang et al. (15), who conducted a
detailed study comparing the expression, distribution, and regulation
of ER and ERß in the rat uterus. It is notable that in the rat
they also observed that endothelial cells in endometrial and myometrial
vessels contained detectable ERß nuclear immunoreactivity, but not
ER. These researchers suggested that the two ER isoforms may play
different roles with regard to vascular effects of estrogen in the rat
uterus.
There are no previous reports on localization of ERß protein in human endometrial vascular endothelium. Fujimoto et al. (24) described localization of ERß protein in the glandular nuclei of eutopic endometrium collected at the time of surgical treatment of endometriosis, but made no specific mention of ERß immunoreactivity in the vasculature. Taylor and Al-Azzawi (25) reported detection of ERß protein in a wide range of human tissues with commercial polyclonal antibodies raised against peptides in the N- and C-terminal regions of the receptor. In their report ERß was localized to cells within the stroma and luminal epithelial cells. These researchers commented that glandular cells lacked detectable ERß, but this might be a reflection of the fact that the endometrial tissue in their report was collected during the late luteal phase. These findings therefore appear consistent with our own data obtained with the polyclonal P4 antibody, which showed a decline in glandular ERß immunoreactivity in the late luteal/premenstrual phase, when P concentrations have declined after a period of elevated circulating P concentrations.
In view of the observation that the only sex steroid receptor immunolocalized in endometrial endothelial cells during the cycle is ERß, we propose that ERß may prove to be the steroid receptor responsible for regulation of steroid-mediated effects in the human endometrial vasculature. In our studies of the ovary (39) we saw no evidence for ERß in the ovarian vascular endothelium, and in the rhesus macaque, neither the ovarian or oviductal vasculature has any detectable ERß in the endothelium (Brenner, R. M., unpublished). Consequently, the expression of ERß by the endometrial vasculature appears to be unique. The importance of E2 (and P) in regulating the vasculature and process of angiogenesis in the uterus is well recognized. For example, vascular endothelial growth factor has been shown to be estrogen responsive in the endometrium (44). In other body systems it has been suggested that ERß mediates some of the direct effects of E2 on the vasculature. For example, ERß mRNA is up-regulated in endothelial and luminal smooth muscle cells after balloon injury (45).
Data on the role(s) of the and ß ER receptor subtypes in
regulating responses to estrogens within the vasculature and
endometrium have been obtained by studying mice in which ER and
ERß have been genetically modified (17, 19). Studies in
the ER-knockout mouse (ERKO) have shown that the receptor is
essential for E2-induced PR down-regulation in
uterine epithelium and that this effect is mediated by ER expressed
in the stroma (46). New studies on the uteri of mice in
which the ß receptors lack a functional DNA-binding domain (ßERKO)
reported that the expression of PR was up-regulated in the stroma
compartment, but was not down-regulated in epithelium in response to
E2, leading to the suggestion that ERß may
modulate the effects of ER in these cell types (12).
Studies of cells surrounding the vasculature have been limited to the
use of a cardiovascular injury model (47, 48). For
example, in ERKO mice (47) similar effects were observed
after exposure to E2 in both wild-type and ERKO
animals, prompting the suggestion that vascular protective effects
might be mediated by ERß. However, very similar results were obtained
using the ßERKO mice (48), and the current thinking is
that in the vasculature of the cardiovascular system either of the two
known receptors is sufficient to protect against injury.
Studies in vitro using cell lines transfected with ER- or
ERß-containing constructs have been used to investigate both the
relative affinities of the two subtypes for selected ligands as well as
their ability to induce transcription of reporter constructs (14, 49, 50). Although both receptor types bind
E2 with high affinity, some phytoestrogens
(e.g. genistein) compete more strongly with
E2 for binding to ERß than ER
(50). ER modulators known to produce distinct biological
effects in vivo, such as 4-hydroxytamoxifen and raloxifine,
induced distinct conformational changes in ER and ERß
(51); novel ligands that function selectively for one of
the receptors have also been identified (28). In the
HEC-1-A uterus-derived cell line, raloxifine, faslodex and tamoxifen
acted as agonists when ERß was cotransfected with a reporter
construct containing the collagenase promoter, but only tamoxifen was
agonistic when ER was transfected in place of ERß
(52). Detailed studies by Hall and McDonnell
(32) of the functional domains within the receptor have
shown that whereas both the AF-1 and AF-2 domains in the A/B and F
domains of the protein are essential for full activity of ER, the
AF-1 domain of ERß contains a repressor domain. It is notable that
ERß, but not ER, will bind DNA in a ligand-independent manner
(32). When ER and ERß are coexpressed they are able
to form homo- or heterodimers (29, 30), and it appears
that ERß may form heterodimers in preference to homodimers when ER
is present. One function of ERß may be to modulate ER
transcriptional activity by competing for binding to DNA at low
concentrations of ligand (32). Based on the results from
the current study, the widespread expression of endometrial ER and
ERß make it likely that in the presence of saturating levels of
ligand, most of the ERß will exist in a heterodimer with ER, and
gene transcription will be activated.
However, there are two exceptions to this rule: first, in endothelial
cells, where we have failed to detect expression of ER protein, and
second, during the late secretory phase, when expression of ER
declined dramatically in both stroma and glands of the superficial
layer whereas ERß declined much more in the glands than in the
stroma. In both of these cases ERß/ERß homodimers would be favored,
and based on the data quoted above, this situation would result in
different degrees of activation or repression of genes compared with
other cell types where heterodimers dominate.
The data presented support the hypothesis that estrogen action(s) within the vascular endothelium of the endometrium is mediated via binding to the ERß isoform. Angiogenesis and vascular remodeling are crucial components of several reproductive processes, and perturbed angiogenesis has been implicated in disorders of menstruation and disrupted implantation (44). Manipulation of estrogen-mediated local endometrial mechanisms may be a novel approach to the management of these reproductive disorders. A number of antiestrogens (partial estrogen antagonists, tamoxifen, and pure estrogen antagonists) have been reported to be effective inhibitors of angiogenesis. Interestingly, the angiostatic activity is unaltered in the presence of excess estrogen, thereby implicating alternative mechanisms for the inhibition of estrogen action (53). Schatz et al. (54) recently described a method for the specific isolation of endometrial endothelial cells using a specific lectin to prepare cells that had the characteristics of endothelial cells in culture. A detailed analysis of the cell-specific effects of natural and synthetic estrogens on the function of endometrial endothelial cells isolated using this or a similar method is an essential first step in devising regimens to regulate specific cell function within the endometrium. The results presented in the current paper suggest that endometrial endothelial cells may be a novel target for agonists or antagonists that selectively target the ß form of the ER (28).
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Acknowledgments |
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Footnotes |
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Received August 31, 2000.
Revised November 6, 2000.
Accepted November 20, 2000.
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H. O. D. Critchley, T. A. Henderson, R. W. Kelly, G. S. Scobie, L. R. Evans, N. P. Groome, and P. T. K. Saunders Wild-Type Estrogen Receptor (ER{beta}1) and the Splice Variant (ER{beta}cx/{beta}2) Are Both Expressed within the Human Endometrium throughout the Normal Menstrual Cycle J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5265 - 5273. [Abstract] [Full Text] [PDF]
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V Speirs, G P Skliris, S E Burdall, and P J Carder Distinct expression patterns of ER{alpha} and ER{beta} in normal human mammary gland J. Clin. Pathol., May 1, 2002; 55(5): 371 - 374. [Abstract] [Full Text] [PDF]
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N. R. Nayak and R. M. Brenner Vascular Proliferation and Vascular Endothelial Growth Factor Expression in the Rhesus Macaque Endometrium J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1845 - 1855. [Abstract] [Full Text] [PDF]
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A. Christow, X. Sun, and K. Gemzell-Danielsson Effect of mifepristone and levonorgestrel on expression of steroid receptors in the human Fallopian tube Mol. Hum. Reprod., April 1, 2002; 8(4): 333 - 340. [Abstract] [Full Text] [PDF]
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C. M. Klinge, S. C. Jernigan, and K. E. Risinger The Agonist Activity of Tamoxifen Is Inhibited by the Short Heterodimer Partner Orphan Nuclear Receptor in Human Endometrial Cancer Cells Endocrinology, March 1, 2002; 143(3): 853 - 867. [Abstract] [Full Text] [PDF]
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S. Hague, I.Z. MacKenzie, R. Bicknell, and M.C.P. Rees In-vivo angiogenesis and progestogens Hum. Reprod., March 1, 2002; 17(3): 786 - 793. [Abstract] [Full Text] [PDF]
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 R. Soares, J. S. Reis-Filho, F. Gartner, F. C. Schmitt, L. Iruela-Arispa, and M. D. Graubert Vascular Endothelial Growth Factor, Transforming Growth Factor-{alpha}, and Estrogen Receptors: Possible Cross-Talks and Interactions Am. J. Pathol., January 1, 2002; 160(1): 381 - 383. [Full Text] [PDF]
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D. Stygar, H. Wang, Y. S. Vladic, G. Ekman, H. Eriksson, and L. Sahlin Co-localization of oestrogen receptor {beta} and leukocyte markers in the human cervix Mol. Hum. Reprod., September 1, 2001; 7(9): 881 - 886. [Abstract] [Full Text] [PDF]
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