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vß3 Integrin Are Coexpressed in the Human Endometrium during the Menstrual Cycle But Regulated Differentially
Department of Obstetrics and Gynecology (K.B.C.A.R., M.A.F., W.R.M., P.R.T., B.A.L.), Division of Reproductive Endocrinology and Infertility, University of North Carolina, Chapel Hill, North Carolina 27599; Kaiser Permanente (M.J.M.), Department of Obstetrics and Gynecology, Sacramento, California 95815-4807; and London Regional Cancer Centre and Department of Oncology (A.F.C.), University of Western Ontario, London, Ontario, Canada N6A 4L6
Address all correspondence and requests for reprints to: Dr. Bruce A. Lessey, Department of Obstetrics and Gynecology, The University of North Carolina at Chapel Hill, MacNider Building, CB 7570, Chapel Hill, North Carolina 27599. E-mail: lessey{at}med.unc.edu
Abstract
Osteopontin is an arginine-glycine-aspartic acid-containing acidic
glycoprotein component of the extracellular matrix that is postulated
to bind to integrin receptors at the cell surface to mediate cellular
adhesion and migration during embryo implantation. The primary aim of
this study was to examine the uterine expression of osteopontin
throughout the menstrual cycle in normal fertile controls sampled
prospectively based on urinary LH surge detection. Expression of
osteopontin was documented using Northern blot analysis, in
situ hybridization, and immunohistochemistry. Furthermore, the
temporal pattern of osteopontin expression was compared with that of
its receptor, the 
vß3 integrin. Using Ishikawa cells, a well
differentiated endometrial adenocarcinoma cell line, the in
vitro regulation of osteopontin and its receptor vß3
integrin was studied. By Northern blot analysis, osteopontin mRNA
appears during the early secretory phase, with maximal expression
occurring in mid to late secretory-phase endometrium. The in
situ hybridization analyses showed that osteopontin mRNA
specifically localized in epithelial cells within the endometrium.
Immunostaining of osteopontin was detected in the glandular secretions
and on the apical portions of surface (luminal) epithelium. The
patterns of expression of osteopontin by Northern blotting, in
situ hybridization, and immunohistochemistry are remarkably
similar to the pattern for the vß3 integrin. Despite these
similarities in distribution, in vitro studies
demonstrate that osteopontin and ß3 integrin subunit expression are
differentially regulated. The expression of osteopontin was primarily
induced in response to progesterone, whereas the ß3 integrin subunit
was up-regulated by epidermal growth factor or heparin-binding
epidermal growth factor. The differential regulation of these two
endometrial proteins suggests the existence of two separate pathways
regulating epithelial gene expression in human endometrium during the
window of implantation. In adhesion assays using Ishikawa cells,
vß3 but not vß5 or ß1 integrins appear to be the primary
receptors for osteopontin. These findings may better define the factors
that favor the development of a receptive endometrium.
THE ESTABLISHMENT OF pregnancy requires interaction between maternal and embryonic cells and involves the action of ovarian steroid hormones acting through their specific receptors (1). The endometrium plays a critical role in this process. During the implantation period, the endometrial glands are known to secrete certain peptides that help nourish and maintain the pregnancy. It is speculated, therefore, that these secretions support the conceptus during the early events of adhesion, migration, and placentation (2).
Osteopontin (OPN) is a 70-kDa secreted glycosylated phosphoprotein that was originally isolated from bone matrix (3). It is also found in a wide variety of tissues and bodily fluids, including milk, blood, and urine, uterus, placenta, kidney, leukocytes, smooth muscle cells, and some tumors (4, 5, 6, 7, 8). Young et al. (5) demonstrated that the expression of OPN is increased in secretory-phase endometrium and in the decidua of pregnant women. Recent studies have demonstrated the presence of OPN in uterine flushings from pregnant ewes between d 11 and 17 (9), a period that corresponds to the attachment phase of early implantation in this species. Furthermore, OPN mRNA in glands and protein secretions into the lumen, the expression of which is regulated by progesterone (10), have also been demonstrated.
The diverse distribution pattern of OPN suggests that it is multifunctional. Although its precise role in the endometrium remains poorly understood, OPN has been shown to promote adhesion, signaling, and migration in the immune response and in neoplasia (7, 11). Evidence is also emerging for a role of OPN and its receptor in angiogenesis and tissue remodeling (12, 13, 14, 15). OPN and its receptor have also been implicated in embryo attachment or signaling during the early stages of implantation (9, 16).
OPN can be classified as an adhesion protein (17).
Analysis of the amino acid sequence of OPN revealed the existence of a
conserved cell-binding arginine-glycine-aspartic acid (RGD) sequence,
which was shown to be involved in adherence to cell surface receptors
such as members of the integrin family (18). Mutation of
this site in OPN has been shown to disrupt both adhesion and migration
(19). OPN has been shown to bind to several different
integrin receptors, including vß1, vß3, vß5, 4ß1,
and 9ß1, through its RGD domain (20, 21, 22, 23).
Interestingly, the occupancy of OPN with these different receptors may
have distinct functional consequences. For example, in smooth muscle
cells, vß1, vß3, and vß5 mediate adhesion, whereas only
vß3 supports cell migration (22). In addition,
OPN-induced migration of breast cancer cells of high vs. low
malignancy has been shown to be mediated through different integrins
(24). Although OPN binds to various integrin receptors,
the vß3 integrin has been recognized as a primary receptor for
OPN, promoting cell-to-cell attachment and cell spreading via changes
in the cytoskeleton (25, 26). Moreover, a monoclonal
antibody (LM609) directed against the integrin vß3 has been
reported to block OPN-stimulated changes in osteoclast cytosolic
calcium, suggesting that OPN is the major ligand for vß3 integrin
in bone (25).
In the human endometrium, vß3 integrin is a well characterized
biomarker of uterine receptivity, appearing on endometrial epithelial
cells at the time of embryo attachment (27, 28, 29). Integrin
perturbation studies have suggesting that this integrin plays an
important role in the implantation process (30). The
vß3 integrin recognizes and binds to the RGD sequence present in
OPN and other ligands (18, 31). This integrin may
participate in cell-cell interactions, whereby both cells maintain cell
surface integrin receptors and bind to a common bridging ligand
(9, 32). Trophoblast and preimplantation embryos, like
endometrium, also express both vß3 (33, 34, 35) and OPN
(16) on the surface epithelium. Although previous reports
have placed both vß3 and vß5 on the apical surface
(35), we have found that only vß3 occupies this
location (36).
The present investigation was carried out to study the temporal and
spatial patterns of expression for OPN in endometrium of normal fertile
women throughout the menstrual cycle and to evaluate and compare both
its distribution and its regulation to its receptor, vß3 integrin.
Although the patterns of expression appear to be remarkably similar, we
describe for the first time the differential regulation of OPN compared
with it integrin receptor.
Materials and Methods
Study design
This was a prospective, randomized, blinded study in which endometrial specimens were obtained from normally cycling fertile women at various times during the menstrual cycle. All secretory-phase (n = 84) samples were obtained in cycles in which the mid cycle was identified by urinary LH surge (luteal d 0). Subjects were randomly assigned to undergo biopsy on luteal d 1–14. Thirty specimens were obtained in the early secretory phase (LH + 1 to LH + 5), 31 in the mid secretory phase (LH + 6 to LH + 10), and 23 in the late secretory phase (LH + 11 to LH + 14). Additional proliferative-phase samples (n = 8) were obtained from fertile women at the time of elective bilateral tubal ligation. One sample was obtained in the first trimester of pregnancy at the time of pregnancy termination. All cycling patients had delivered at least one live child in the past, and none had received exogenous hormones for the 60 d preceding the study.
Endometrial samples were obtained using a Pipelle-type suction catheter. Tissue was fixed in 10% buffered formalin and embedded in paraffin before sectioning for histological dating or immunohistochemistry. A portion of each specimen was immediately snap frozen in liquid nitrogen and stored at -80 C for subsequent Northern blot and/or in situ hybridization analysis. Collection of human material for this study was approved by the Committee for the Protection of Human Subjects at the University of North Carolina, Chapel Hill.
RNA isolation and Northern blot analysis
Total RNA was extracted from endometrial specimens obtained from
across the menstrual cycle using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH). RNA samples were stored at -70 C
until use after determining the concentrations by spectrophotometry at
260 nm. Total RNA (20 µg/lane) was glyoxylated, followed by
fractionated through 1.0% agarose gels, and blotted by capillary
action onto a nylon membranes. RNA was cross-linked to the membrane by
UV radiation (Stratalinker 1800, Stratagene, La Jolla, CA)
using 12 x 104 µJ radiation. The membrane
was prehybridized with NorthernMax-Gly hybridization solution
(Ambion, Inc., Austin, TX) at 42 C for 2 h. A cDNA
fragment (approximately 1.4 kb) encoding the full length OPN gene
served as the template for synthesis of a labeled DNA probe using a
random priming method (Random Primed DNA labeling kit, Roche Molecular Biochemicals, Indianapolis, IN). The membrane was
subsequently hybridized with [-32P]dCTP
(Amersham Pharmacia Biotech, Piscataway,
NJ)-labeled OPN probe for 16 h at 42 C. After
hybridization, the membrane was washed twice at room temperature for 15
min in 2x SSC and 0.1% SDS and twice for 15 min at 42 C with
0.1x SSC and 0.1% SDS (standard saline citrate; single strength: 0.15
M sodium chloride and 0.015 M sodium
citrate). Autoradiography was performed using Hyperfilm (Amersham Pharmacia Biotech) for 24–72 h at -70 C until the desired
exposure was obtained. The integrity and relative amount of RNA loaded
onto each lane were confirmed using glyceraldehyde phosphate
dehydrogenase (GAPDH) 32P-labeled cDNA probe as a
constitutively expressed marker.
Immunohistochemistry
The anti-OPN antibody (mAB 53) is a monoclonal mouse antibody
raised against the recombinant glutathione S-transferase
(GST)-human OPN fusion protein that has been characterized in detail
and shown by western blot analysis, ELISA, and immunohistochemistry to
efficiently and specifically detect human OPN (37, 38, 39).
The anti-vß3 integrin receptor monoclonal antibody (SSA6) has been
characterized in endometrium (27). Paraffin-embedded
sections (5 µm) were deparaffinized in xylene and rehydrated in
ethanol with increasing concentrations of water. Endogenous peroxidase
activity was quenched with 3% hydrogen peroxide for 30 min.
Nonspecific binding sites were blocked with 2% normal goat serum (NGS)
for 30 min at room temperature.
Each primary antibody was serially diluted in a solution of PBS containing 1% NGS and 0.1% sodium azide to optimize the appropriate concentrations to achieve maximum sensitivity and specificity. Tissue sections were incubated with primary antibody at 4 C overnight at the following dilutions: SSA6, 1:200; mAB 53, 1:16,000. Negative control sections were treated with nonimmune serum diluted in the same manner. After primary antibody incubation, sections were washed twice with PBS followed by treatment with 1% NGS for an additional 30 min. Subsequently, sections were washed with PBS and incubated with biotinylated goat antimouse secondary antibody (Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1:400 for 30 min at room temperature. After rinsing with PBS, the immunoreactive antigen was visualized using avidin-biotin peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Inc.) and 3,3'-diaminobenzidine as chromagen. Slides were counterstained with Mayer’s hematoxylin blue/toluidine blue followed by dehydration in a graded series of ethanols, cleared in xylene, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). The resulting staining was evaluated on a Nikon (Tokyo, Japan) microscope by a single blinded observer.
Assessments of staining intensity and distribution were made using the
semiquantitative HSCORE system. HSCORE was calculated using the
following equation: HSCORE = 
Pi (i + 1), where
i = intensity of staining with a value of 1, 2, or 3 (weak,
moderate, or strong, respectively) and Pi is the percentage of stained
epithelial cells for each intensity, varying from 0–100%. Low
intraobserver (r = 0.983; P < 0.0001) and
interobserver (r = 0.994; P < 0.0001) differences
for HSCORE in uterine tissues have been reported previously using this
technique (40).
Hybridization probes
The human osteopontin cDNA clone encompassing the entire coding sequence was linearized with XbaI and XhoI for the generation of antisense and sense (control) cRNA probes using T7 and T3 polymerases, respectively. A 683-bp cDNA fragment of human ß3 integrin subunit cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA) was used as a template for generating both antisense and sense riboprobes using appropriate T7/SP6 polymerases and restriction enzymes.
In situ hybridization
In situ hybridization was performed essentially as described previously (41). Briefly, frozen sections (10 µm) were mounted onto poly-L-lysine-coated slides and stored at -70 C until used. Slides upon removed from -70 C, placed on a slide warmer (37 C) for 2 min, and then fixed in 4% paraformaldehyde in PBS for 15 min at 4 C. After prehybridization, hybridization was carried out in a humidified chamber using 35S-labeled sense and antisense complementary RNA probes specific for OPN and the ß3 subunit of integrin for 4–5 h at 42 C. After hybridization, the coverslips were removed by washing in 4x SSC followed by incubation with 20 µg/ml RNase A for 30 min at 37 C. After a series of washes, the RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Exposure was carried out for 7–14 d at 4 C. The slides were counterstained with hematoxylin and eosin, dehydrated through a graded series of alcohol, cleared in xylene, and coverslipped. Representative dark and bright fields were photographed at x200 magnification on a microscope (Olympus Corp., Tokyo, Japan).
Cell culture
Ishikawa cells, a well differentiated endometrial adenocarcinoma cell line (generously provided by Dr. Richard Hochberg, Yale University Medical Center, New Haven, CT), were maintained in MEM (Life Technologies, Inc.-BRL, Gaithersburg, MD) supplemented with 5% FBS (HyClone Laboratories, Inc., Logan, UT), 1% penicillin-streptomycin (Life Technologies, Inc.-BRL), and 1% L-glutamine (Life Technologies, Inc.-BRL). Ishikawa cells plated onto 100-mm dishes (near to 70–80% density) were exposed to medium alone (control) or medium containing steroids, E2 17ß (10-8 M; Sigma Corp., St. Louis, MO) alone or in combination with progesterone (10-6 M; Sigma Corp.), or with 10 ng/ml heparin-binding epidermal growth factor (HB-EGF). The hormones were added from 1000-fold concentrated stocks prepared in absolute ethanol. The appropriate vehicle (ethanol) was added to control cultures in each experiment. In certain experiments, antagonists such as antiestrogen (ICI 182780, 10-7 M; Zeneca Pharmaceuticals, Wilmington, DE), antiprogestin (RU-486, 10-5 M; Sigma Corp.), and C225 (30 µg/ml) were also added to Ishikawa cells along with E, progesterone, and HB-EGF, respectively. The C225, a monoclonal antibody that neutralizes the EGF receptor, was generously provided by Dr. John Mendelsohn (M.D. Anderson Cancer Center, The University of Texas, Houston, TX). The treatment media consisted of phenol red-free MEM/Ham’s F-12 supplemented with 0.5% charcoal-stripped FBS, 1% penicillin-streptomycin, and 1% L-glutamine. The time of exposure ranged from 72–96 h.
Western blot analysis
Whole cell lysate proteins from both treated and untreated
Ishikawa cells were isolated as described (27).
Cells from 100-mm dishes were harvested using cell scrapers after
washing with cold PBS. The cell pellets obtained from the various
treatment groups were dissolved in RIPA buffer (1x PBS, 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS) supplemented with freshly
prepared protease inhibitor cocktail (Roche Molecular Biochemicals). Lysates were sheared three to four times through
a 21-gauge needle, transferred to a microcentrifuge tube, and then
incubated on ice for 45 min. Lysates were centrifuged at 15,000 x
g for 20 min at 4 C, and supernatants were collected as the
total cell lysates. Protein concentrations were measured using the
protein assay kit from Bio-Rad Laboratories, Inc.
(Hercules, CA). Aliquots of each sample containing 100 µg of total
protein were fractionated on a 10% polyacrylamide-SDS gel and
transferred to a nitrocellulose membrane. Membranes were blocked with
10% nonfat dry milk in Tris-buffered saline containing 0.2% Tween for
1 h at room temperature. After brief washes with PBS containing
0.4% Tween-20 (PBS-T), blots were incubated with 500 ng/ml monoclonal
anti-OPN antibody (MPIIIB101), which reacts
specifically with OPN (Developmental Studies Hybridoma Bank, Iowa City,
IA) or with a 1:2000 dilution of the anti-vß3 monoclonal antibody
SSA6 for overnight rocking at 4 C. After a PBS-T wash for 1 h, the
blots were incubated with a 1:5000 dilution of horseradish
peroxidase-conjugated antimouse IgG secondary antibody (Promega Corp., Madison, WI) for 1 h while rocking. After washing
with PBS-T twice for 30 min each, the immunoreactive protein complexes
were detected using an enhanced chemiluminescence detection kit
(Amersham Pharmacia Biotech) and exposed to
Kodak Biomax ML film for 1–5 min to achieve satisfactory
exposure.
Cell adhesion assay
An adhesion assay was performed as described previously
(42). GST, recombinant GST-fused mouse OPN (GST-mOPN), and
recombinant RGD-mutated OPN proteins were used. Briefly, OPN proteins
were diluted to a final concentrations of 10 µg/ml in PBS, and 100
µL of each protein was coated onto 96-well microtiter plates
overnight at 4 C. Wells were washed with PBS, and nonspecific binding
sites were blocked with 1% BSA for 1 h at 37 C. Ishikawa cells
were harvested from tissue culture dishes using trypsin-EDTA, washed,
and resuspended in MEM containing 1% BSA, L-glutamine, and
antibiotics. Cycloheximide was added to the cells at a concentration of
20 µg/ml to block the synthesis of secreted proteins that might
interfere with adhesion. For antibody inhibition, cells were
preincubated with blocking antibodies against
vß3 (LM609; Chemicon, Temecula, CA),
vß5 (PIF6; Life Technologies, Inc.-BRL), and ß1 integrin subunit (P4C10; Life Technologies, Inc.-BRL), alone or in combination, for 15 min at
37 C before plating onto 96-well plates. As a negative control, OPN
coating was omitted. Ishikawa cells (200,000 cells/100 µl)
preincubated with or without the various antibodies were added to wells
and allowed to incubate at 37 C for 1 h. After incubation, the
nonadherent cells were washed off with PBS by gentle aspiration.
Adherent cells were detected by measuring endogenous hexosaminidase
activity at 405 nm using Titertek Multiscan (Flow Laboratories Inc., McLean, VA) and
p-nitrophenol-N-acetyl-ß-D-glucosaminide
as substrate. In this enzymatic assay, absorbance values were found to
be directly proportional to the number of cells that attached to the
96-well plates. The data are presented as percentage of control. All
experiments were performed at least three times yielding similar
results.
Statistical analysis
Relative levels of OPN were estimated using the semiquantitative HSCORE, a numerical score ranging from 0 to 4. Comparisons between groups were made using ANOVA with Scheffé’s correction for multiple comparisons. Significance was based on a 95% confidence interval and a P value less than 0.05.
Results
The demographic characteristics of the subject population,
composed of 93 fertile women, are shown in Table 1
. Patients in the secretory phase were
randomized and assigned a specific luteal day to return for endometrial
biopsy based on the detection of the LH surge (luteal d 0). The mean
age between groups based on phase of the menstrual cycle was not
statistically different.
|
. OPN message was faintly detectable in
the both proliferative-phase and early secretory-phase endometrium.
However, expression was dramatically increased during the mid secretory
phase and sustained throughout the late secretory phase. GAPDH was used
as an internal RNA loading control. This pattern of OPN expression is
nearly identical to the pattern of ß3 integrin subunit expression,
with maximal appearance during the mid secretory phase (36, 43).
|
, OPN mRNA transcripts were observed in
a small percentage of cells in a scattered manner throughout the early
secretory-phase endometrium (Fig. 2C) but were strongly expressed in
the mid secretory-phase endometrium (Fig. 2D). The greatest expression
was in the glandular epithelium (inset). By comparison, ß3 integrin
subunit mRNA transcripts were also strongly expressed in the mid
secretory phase (Fig. 3D) but not in the
early secretory phase (Fig. 3C). No signals were observed for either
gene message in the proliferative phase of the cycle (data not shown).
No hybridization signals were evident when sense probe was used on
corresponding sections, indicating the specificity of the hybridization
probe (data not shown).
|
|
.
Representative photomicrographs of endometrial sections from different
stages of the menstrual cycle are shown in Fig. 4. Little if any immunoreactivity is seen
in the proliferative phase (A). In the early secretory phase, OPN can
be identified first in the glandular epithelial cells that are
beginning to show evidence of secretion formation (B and C). By the mid
secretory phase, strong immunostaining was observed on the apical pole
of the surface (luminal) epithelium and in the secretions of the
glandular epithelium (D and E). In pregnancy, OPN is strongly expressed
by the decidua (F). As shown in Fig. 5, using HSCORE assessment of immunohistochemical staining, OPN levels
were significantly higher in glands and lumen compared with stroma
during mid and late secretory phases (Table 1). The changes in OPN
expression during the early secretory phase were not significantly
different from the proliferative phase, but increased significantly in
mid and late secretory phase endometrium. Interestingly, like vß3,
OPN increases in secretory phase stroma, the rise reaching significance
by the late secretory phase. Staining appeared strong over individual
cells and likely represented infiltration of leukocytes during the
final days of the cycle before menstruation.
|
|
vß3 integrin
(27, 28), with little if any integrin expression found in
the proliferative phase. By the mid secretory phase, both glandular
(Fig. 4G) and luminal (Fig. 4H) cells stained strongly for this
integrin. Pseudodecidual stromal cells (Fig. 4I) and decidua (data not
shown) also stained for this integrin. It is striking that the surface
epithelium stained only on the apical projections and that this pattern
was similar to that of luminal OPN. Control sections, in which mouse
IgG replaced primary antibody for OPN, showed no immunoreactivity,
indicating the specificity of the immunostaining. One interpretation of
these findings is that OPN in glandular secretions enters the lumen and
binds to the apical surface epithelium that expresses the vß3
receptor (36).
To demonstrate whether vß3 integrin is involved in cell adhesion
to OPN, Ishikawa cells were allowed to attach to GST-OPN, RGD-mutated
OPN, and GST alone in the presence or absence of LM609 (anti-vß3),
PIF6 (anti-vß5), and P4C10 (anti-ß1 subunit) antibodies. There
was no binding observed when cells were plated on RGD-mutated OPN or
GST alone (data not shown), similar to negative controls. As shown in
Fig. 6, the adhesion of Ishikawa
cells to OPN was reduced only in the presence of function-blocking
antibodies against vß3. On the other hand, PIF6 (anti-vß5) or
P4C10 (anti-ß1 subunit) antibodies, either separately or together,
had negligible effects on the prevention of adhesion of Ishikawa cells
to OPN protein. These results suggest that vß3 is a major receptor
for OPN in this cell line. It also appears that other integrins may
participate in this binding, such as another v-containing integrin
that remains to be identified.
|
, OPN and ß3 were
differentially regulated by ovarian steroids and the EGF-like growth
factor HB-EGF. As previously reported (44), E2 and E2 plus
progesterone reduce the expression of the ß3 integrin subunit.
Inhibition of E2 by the antagonist ICI 182780 increased the relative
expression of ß3, whereas the antiprogestin RU-486 had little effect,
suggesting that the inhibitory influence rests primarily with E2. Like
EGF, HB-EGF dramatically increased ß3 subunit expression, an effect
that was mitigated by the EGF receptor-blocking antibody C225.
Expression of OPN, on the other hand, was dramatically increased by E2
plus progesterone, whereas E2 alone also appeared to antagonize its
expression. RU-486 reduced the stimulatory effect of progesterone on
OPN, whereas HB-EGF had little if any effect on its expression.
|
This study reports the coordinated expression of OPN and its
receptor vß3 integrin in the human endometrial epithelium across
the menstrual cycle. Our results demonstrate the increased expression
of both OPN and vß3 in mid secretory-phase endometrium around the
time that implantation occurs. Transcripts for OPN and vß3
integrin were localized to the glandular epithelium, but immunostaining
of these proteins appeared more prominently on the apical surfaces of
the luminal epithelium.
Because both OPN and vß3 are expressed by the glandular
epithelium, we used the Ishikawa cell line as an in vitro
epithelial cell culture model to study their regulation. This cell line
derives from the glandular epithelium of the endometrium
(45) and is known to possess receptors for E,
progesterone, and the growth factor receptor for EGF (44).
We have now shown that OPN expression is modulated by progesterone,
presumably acting through its specific nuclear receptor PR, whereas
vß3 integrin is modulated primarily by the growth factor EGF
(44) and now HB-EGF. Increased expression of OPN in mid
secretory-phase human endometrium and its localization to epithelial
cells is in agreement with earlier reports in human (5)
and other mammalian species, including sheep (46), baboon
(47), rabbit (48), and mouse
(49). It was recently demonstrated that OPN and its
receptor vß3 integrin are coexpressed in the secretory-phase
endometrium of sheep (9) and human decidua
(16). Furthermore, it was shown that OPN expression is
induced by progesterone in animal models (10, 48).
Although the functional significance of increased OPN expression in
secretory-phase endometrium remains to be understood, its temporal and
spatial patterns of expression suggest an important role during
implantation and placentation. However, it should be noted that OPN
null mutant mice remain fertile (50, 51). Implantation is
a complex process involving proliferation and tissue remodeling in
which adhesion molecules, cytokines, and growth factors are believed to
play critical roles (2, 52, 53). The redundancy of
extracellular matrix molecule expression during the secretory phase
suggests that the loss of a single protein may be compensated by other
ligands that bind a common receptor, thus ensuring a higher likelihood
of successful pregnancy.
OPN has been shown to mediate the adhesion and migration of various
cell types by interacting with different cell surface receptors of the
integrin family (22, 24, 54). Coexpression of vß3
integrin mRNA and protein with OPN, as shown in the present study,
suggests that OPN and the vß3 integrin receptor may play
complementary roles during the implantation process. The in
vitro adhesion studies reported here support this hypothesis and
suggest that OPN is an adhesive substrate in Ishikawa cells and that
these cells use vß3 integrin receptor in an RGD-dependent manner
to interact with OPN. The minimal participation of vß5 integrin
receptor in the adhesion of Ishikawa cells to OPN was unexpected
because this integrin is known to interact with OPN. We have previously
reported, however, that vß3, but not vß5, is expressed on the
apical pole of the luminal epithelium (36); no information
is available regarding vß5 in the Ishikawa cell line. The adhesion
of Ishikawa cells to OPN was undeterred even in the presence of
ß1-neutralizing antibody, indicating that ß1 integrins do not
mediate adhesion to OPN in this cell line. The involvement of the
vß3 integrin receptor in adhesion to OPN supports the hypothesis
that vß3-OPN binding occurs at the luminal surface during the time
of maximal endometrial receptivity. Previous studies have demonstrated
the coexpression of OPN and its receptor under certain injury and
tissue-remodeling conditions, such as during reendothelialization
(12) and neointima formation (54). It has
also been suggested that OPN binding to the vß3 integrin receptor
promotes angiogenesis (55), rapid phosphoinositide
turnover and inositol triphosphate production (56), and
increased intracellular calcium (57). It will be of
interest to examine this binding in subsets of patients with
infertility who fail to express this integrin (29). Would
such patients, for example, express glandular OPN but fail to exhibit
luminal binding of this extracellular ligand? Such studies are
currently under way.
The increased expression of OPN and vß3 integrin receptor in the
progesterone-dominant secretory phase of the menstrual cycle
underscores the role of progesterone in implantation. In the human
menstrual cycle, the mid cycle LH surge triggers ovulation and
subsequent formation of the corpus luteum. Without luteal-phase
progesterone, early pregnancy will fail (58). The in
vitro studies presented here clearly show that OPN expression is
induced by progesterone, levels of which peak during with mid secretory
phase. It has been documented that the 5' upstream flanking regions of
both the human and mouse OPN genes possess progesterone regulation
elements that enable this steroid to exert transcriptional control on
OPN expression (59, 60). Paradoxically, It should be noted
that OPN expression in glandular epithelium coincides with the putative
down-regulation of PR in the mid secretory-phase endometrium (61, 62). These results are consistent with observations in the
porcine and ovine uterus that suggest that epithelial PR
down-regulation is somehow required for the induction of endometrial
gland secretory function (63, 64). More recently, Mote
et al. (65) suggested that PR-A and PR-B
isoforms are differentially regulated around the time of implantation.
This study opens the door to the possibility that endometrial OPN is
regulated differentially by PR-B, consistent with recent observations
in PR-A-ablated mutant mice (66). Further studies will be
required to test this hypothesis in the human endometrium.
We have observed that endometrial vß3 integrin expression is
down-regulated by E2 and induced by EGF or EGF-like growth factors.
Blocking the EGF receptor reduces this stimulatory effect. The
differential regulation of OPN and its receptor vß3 integrin is
quite interesting given the nearly identical patterns of expression
during the luteal phase. Previous studies from our laboratory and
others have demonstrated that uterine stromal cells can mediate
epithelial cell function (67, 68, 69). It is possible that EGF
or EGF-like growth factors are stroma-derived paracrine factors that
regulate vß3 integrin expression during the menstrual cycle,
whereas epithelial OPN expression may be mediated directly by
progesterone. These data suggest that epithelial receptivity in the
human, and perhaps other mammalian species as well, is regulated by two
distinct pathways. The first is a paracrine effect of stromal cells on
epithelial gene expression, and the second involves a direct effect of
progesterone on endometrial epithelium. It will be important to better
understand such pathways, especially in infertile women with putative
defects in endometrial receptivity. In addition, differential
regulation of PR isoforms may provide new avenues for targeting the
endometrium for purposes of contraception and provide a deeper
understanding of the pathogenesis of endometrial cancers.
Acknowledgments
We thank Jinning Zhang for her immunohistochemical expertise.
Footnotes
This research was supported by the National Institute of Child Health and Human Development/NIH through cooperative agreement U54 HD-35041 (B.A.L.) as part of the Specialized Cooperative Centers Program in Reproduction Research, by the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (HD-34824 to B.A.L.), by an American College of Obstetricians and Gynecologists/Parke-Davis Research Award (to M.J.M.), by the Fogerty International Fellowship award (to K.B.C.A.R.), and by the Canadian Breast Cancer Research Initiative (Grant 1130 to A.F.C.).
Abbreviations: GAPDH, Glyceraldehyde phosphate dehydrogenase; GST, glutathione S-transferase; GST-mOPN, recombinant GST-fused mouse osteopontin; HB-EGF, heparin-binding epidermal growth factor; NGS, normal goat serum; OPN, osteopontin; PBS-T, PBS containing 0.4% Tween 20; RGD, arginine-glycine-aspartic acid; SSC, standard saline citrate.
Received December 19, 2000.
Accepted June 15, 2001.
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RGD-GST-OPN) or GST alone (data not shown). Each
data point represents the average of triplicate measurements.
Significant differences in binding are indicated by
asterisks.
105
kDa) appears to be induced primarily by HB-EGF and inhibited by E2. OPN
(