BIOLOGY OF REPRODUCTION 68, 457–464 (2003)
Published online before print 17 October 2002.
DOI 10.1095/biolreprod.102.007625
Signal Transduction Pathways Activated by Chorionic Gonadotropin in the Primate
Endometrial Epithelial Cells1
Santha Srisuparp,3 Zuzana Strakova,3 Allison Brudney,3 Sutapa Mukherjee,4 Scott Reierstad,4
Mary Hunzicker-Dunn,4 and Asgerally T. Fazleabas2,3
Department of Obstetrics and Gynecology,3 University of Illinois at Chicago, Chicago, Illinois 60212-7313
Cell and Molecular Biology,4 Northwestern University Medical School, Chicago, Illinois 60611
ABSTRACT
trium. The series of these interactions are limited to a specific window of receptivity during the menstrual cycle [2,
3].
Chorionic gonadotropin (CG), a glycoprotein hormone,
is the major embryonic signal in primates. During implantation and pregnancy, CG is primarily secreted by the syncytiotrophoblast. Our previous in vivo studies have shown
that CG, when infused into the baboon’s uterine cavity,
modulated the receptive endometrium during the implantation window [4]. These effects included a plaque response
in the luminal epithelium, an induction of a-smooth muscle
actin (a-SMA) in the subepithelial stromal fibroblasts and
an increase in glycodelin expression and secretion in the
glandular epithelium. In the human, in vivo infusion of CG
into the uterine cavity during the luteal phase also regulates
the secretion of several cytokines and growth factors into
the uterine fluid [5].
The CG/LH receptor (CG R) is a member of the subfamily of glycoprotein hormone receptors within the superfamily of G protein-coupled receptor (GPCR)/seventransmembrane domain receptors [6]. Extragonadal CG R
has been reported in both reproductive and several nonreproductive organs in human [7–10]. The structural and
functional properties of these extragonadal CG R, especially in the endometrium, are still controversial [11, 12].
However, these receptors are functional based on the in
vivo studies in baboons [4] and human and in vitro studies
with cells obtained from the human endometrium [13].
In gonadal tissues, binding of CG to its receptor generates signal transduction through the associated G proteins.
The classical responses are an increase in cAMP and consequent activation of protein kinase A (PKA) upon activation of the adenylyl cyclase (AC) pathway and an increase in intracellular calcium through inositol triphosphate
(IP3)/phospholipase A2 (PLA2) pathway. The possible activation of protein kinase C (PKC) through diacylglycerol
(DAG) has also been suggested [14].
The major cell types of the uterine endometrium of the
baboon undergo marked cellular changes in response to CG
during the window of uterine receptivity. The luminal epithelial cells undergo endoreplication and form epithelial
plaques, while the glandular epithelial cells increase their
secretory activity [4]. Therefore, the objective of this study
was to explore the signal transduction cascade activated by
CG in the epithelial cells that in turn may modulate the
mitogenic and secretory responses in the uterine epithelium.
Successful implantation requires synergism between the developing embryo and the receptive endometrium. In the baboon,
infusion of chorionic gonadotropin (CG) modulates both morphology and physiology of the epithelial and stromal cells of the
receptive endometrium. This study explored the signal transduction pathways activated by CG in endometrial epithelial cells
from baboon (BE) and human (HES). Incubations of BE and HES
cells with CG did not significantly alter adenylyl cyclase activity
or increase intracellular cAMP when compared with Chinese
hamster ovarian cells stably transfected with the full-length human CG/luteinizing hormone (LH) receptor (CHO-LH cells).
However, in BE and HES cells, CG induced the phosphorylation
of several proteins, among them, extracellular signal-regulated
protein kinases 1 and 2 (ERK 1/2). Phosphorylation of ERK 1/2
in uterine epithelial cells was protein kinase A (PKA) independent. This novel signaling pathway is functional because, in response to CG stimulation, prostaglandin E 2 (PGE 2) was released
into the media and increased significantly 2 h following CG
stimulation. CG-stimulated PGE 2 synthesis in epithelial cells was
inhibited by a specific mitogen-activated protein kinase (MEK 1/
2) inhibitor, PD 98059. In conclusion, immediate signal transduction pathways induced by CG in endometrial epithelial cells
are cAMP independent and stimulate phosphorylation of ERK 1/
2 via a MEK 1/2 pathway, leading to an increase in PGE 2 release
as the possible result of cyclooxygenase-2 activation.
human chorionic gonadotropin, signal transduction, uterus
INTRODUCTION
Implantation in the primate is a complex spatiotemporal
interaction between the blastocyst and the receptive endometrium. Cytokines, growth factors, hormones, extracellular matrix proteins and related enzymes, and cell-cell adhesion molecules and their corresponding receptors are involved during blastocyst apposition, attachment, and invasion [1]. In addition, interactions between epithelial and
stromal cells, epithelial cells and blood vessels, and stromal
cells and lymphocytes also occur to facilitate the ability of
the genotypically different embryo to invade the endomeThese studies were done as part of the National Cooperative Program for
Markers of Uterine Receptivity and Blastocyst Implantation funded by
NIH HD 29964.
2
Correspondence: Asgerally T. Fazleabas, The University of Illinois at Chicago, Department of Obstetrics and Gynecology, 820 South Wood Street
(M/C 808), Chicago, IL 60612-7313. FAX: 312 996 4238;
e-mail: [email protected]
1
MATERIALS AND METHODS
Materials
Received: 27 May 2002.
First decision: 11 June 2002.
Accepted: 22 August 2002.
Q 2003 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
Recombinant human chorionic gonadotropin CG was obtained from
Dr. A.F. Parlow (National Hormone and Pituitary Program, Harbor-UCLA
457
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SRISUPARP ET AL.
Medical Center, Torrance, CA). The 125I-labeled CG (4674 Ci/mmol) was
purchased from Perkin Elmer Life Sciences (Boston, MA). A phosphotyrosine-RC20 antibody (catalog no. E120H) was from Transduction Laboratories, Inc. (Lexington, KY). Monoclonal phospho-extracellular signalregulated protein kinases 1 and 2 (ERK 1/2) (Thr202/Tyr204) E10 antibody (catalog no. 9106), polyclonal ERK 1/2 (catalog no. 9102), and polyclonal phospho-cAMP regulatory element-binding protein (CREB)
(Ser133, cat no. 9191) antibodies were from Cell Signaling Technology,
Inc. (Beverly, MA). Monoclonal a-tubulin clone B-5-1-2 (catalog no.
T5168) antibody, IBMX (3-isobutyl-1-methylxanthine), and forskolin (7acetoxy-1a, 6b, 9a-trihydroxy-8, 13-epoxy-labd-14-en-11-one) were from
Sigma Chemical Company (St. Louis, MO). Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad Laboratories (Hercules,
CA). Enhanced chemiluminescence kit and prostaglandin E 2 (PGE 2) enzyme immunoassay kit were from Amersham Life Sciences (Arlington
Heights, IL). PD 98059 (29-amino-39-methoxyflavone), H-89 {N-[2-((pbromocinnamyl)amino)ethyl9-5-isoquinolinesulfonamide, 2HCl}, and
cAMP enzyme immunoassay kit were from Biomol Research Laboratories,
Inc. (Plymouth Meeting, PA). TriReagent was from Molecular Research
Center (Cincinnati, OH). All cell culture supplies were obtained from Gibco BRL (Gaithersburg, MD). Other reagents of cell culture grade were
purchased from Fisher Scientific (Itasca, IL), Sigma, or Boehringer-Mannheim (Indianapolis, IN).
Isolation of Baboon Endometrial Epithelial (BE) Cells
Baboon endometrial (BE) tissue was obtained from adult female baboons (Papio anubis) following ovarian hyperstimulation (n 5 2) or during the midluteal phase of the menstrual cycle between Days 9 and 12
postovulation (PO) (n 5 4). The endometrial tissues were obtained either
by endometrectomy or hysterectomy. All animal studies were approved by
the Animal Care Committee at the University of Illinois at Chicago. Epithelial cells were isolated from baboon endometrial tissues as previously
described [15], with the following modifications. Briefly, the endometrial
tissues were cut into 1–3-mm3 pieces and digested with 0.5% collagenase
and 0.02% deoxynuclease in calcium- and magnesium-free Hanks balanced salt solution (HBSS) in a shaking water bath at 378C for 1 h. After
centrifugation (200 3 g, 5 min), the pellet was resuspended with 5 ml of
HBSS and passed through a 95-micron pore-size nylon mesh. The retentate
was redigested with 0.5% collagenase, 0.02% deoxynuclease, 0.1% pronase, and 0.2% hyaluronidase in HBSS in a shaking water bath at 378C
for another 30 min. Following centrifugation at 200 3 g for 5 min, the
supernatant was collected. The pellet was resuspended with 5 ml of HBSS
and passed through a 95-micron pore-size nylon mesh. The retentate from
this step was mixed with the collected supernatant and digested in a shaking water bath at 378C for another 30 min. The filtrate was passed through
a 20-micron pore-size nylon mesh twice. At each time, the retentate, which
contained enriched glandular epithelial cells, was resuspended in HBSS
and collected. Following the last digestion, the mixture was passed through
a 95-micron pore-size nylon mesh. The filtrate was passed through a 20micron pore-size nylon mesh twice. Each time the retentate, containing
enriched glandular epithelial cells, was collected and resuspended in
HBSS. The final solutions were mixed and centrifuged at 200 3 g for 5
min. The cell pellet was resuspended in culture medium, which consisted
of Dulbecco Modified Eagle Medium (DMEM) with D-valine, 0.1 mM
sodium pyruvate, penicillin-streptomycin; 10 000 units-10 mg/ml (P/S),
and 10% heat-inactivated and charcoal-stripped fetal bovine serum
(SFBS), and the cells were incubated for 24 h at 378C in a humidified
atmosphere of 5% CO2–95% air. Medium was changed after 24 h and then
every other day. BE cells were cultured for 5–7 days prior to the initiation
of the experiments. The purity of the attached cells was determined by
immunocytochemistry using an antibody against cytokeratin (DAKO, Carpenteria, CA) to confirm the epithelial phenotype and an antibody against
vimentin (Zymed, San Francisco, CA) to confirm the contaminating stromal fibroblast phenotype. The purity of the glandular epithelial cell preparation used in these studies was .90%.
Cell Lines
A human endometrial epithelial cell line (HES) was used for comparative studies. These cells were isolated from a proliferative, noncancerous
endometrium at hysterectomy and spontaneously immortalized during culture [16]. They have similar immunocharacteristics to primary epithelial
cells from the baboon. They also express estrogen and progesterone receptors [17]. For these studies, they were cultured in DMEM, 0.1 mM
sodium pyruvate, P/S, and 10% SFBS. Medium was changed every other
day. The cells were subcultured every 4–5 days.
CHO-LH cells [18] were used as positive controls. They were cultured
in Minimum Essential Medium a medium (a-MEM), 0.1 mM sodium
pyruvate, P/S, geneticin (G418 sulfate, 600 mg/ml), and 10% SFBS. Medium was changed every other day. The cells were subcultured every 4–
5 days.
The presence of CG/LH receptors on each of the cell types used in
these studies was confirmed by reverse transcriptase and polymerase chain
reaction (RT-PCR). The specific primers were designed against sequences
in exon 11 that code for the transmembrane domain and between exon 5
and 8 for the extracellular domain. Amplification of the extracellular domain in BE and HES cells required nested PCR of the initially amplified
product.
Preparation of Cell Membranes and Ligand Binding
Baboon corpora lutea (CL) were obtained on Day 10 postovulation.
CHO-LH and HES cells were grown to confluence in the appropriate media. Membrane preparations were made by homogenizing the CL and
scrapped cells using a dounce homogenizer in a buffer containing 50 mM
Tris-HCl (pH 5 7.4), 0.1 mM PMSF, and 0.25 M sucrose. The homogenates were centrifuged for 80 min at 100 000 3 g. For the affinity crosslinking experiments, 125 mg of membrane protein was incubated overnight
at 48C with 1 3 106 cpm 125I-labeled CG. An additional set of membrane
preparations was preincubated with 25 nM CG for 30 min before the
addition of the radiolabeled CG. The membranes were affinity cross-linked
by incubating the samples with 0.1 mM of disuccinimidyl suberate for 30
min at 48C [19]. Following extensive washing, each sample was resuspended in Laemmli buffer, boiled for 5 min, and then separated on a 10%
SDS-PAGE gel. The gel was either dried or blotted onto nitrocellulose
membranes and exposed to Kodak Biomax film for 7–21 days.
Preparation of Cells
For all in vitro experiments in this study, the cells were prepared according to the following scheme. Each cell type was cultured in its particular culture medium plus 10% SFBS until the cells were 60%–70%
confluent. The medium was then changed to 2% SFBS until the cells were
80%–100% confluent (80%–90% confluent for BE cells). To prevent the
confounding effects of SFBS on cell signaling studies, the cells were
rinsed twice with serum-free medium and were maintained in this medium
for 14 h in 5% CO2:95% air at 378C. After 14 h, the cells were rinsed
twice with serum-free medium and stabilized in this medium for another
4 h prior to the initiation of the experiments. In the experiments with PD
98059, a specific mitogen-activated protein kinase (MEK 1/2) inhibitor
[20], the cells were pretreated with this inhibitor for 1 h prior to stimulation with CG. In the experiments with H-89, the PKA inhibitor, the cells
were pretreated with this inhibitor for 30 min prior to stimulation with
CG. Inhibitors were added at the concentrations indicated in the figure
captions. CG was used at a concentration of 10 nM (bioactivity 5 1.8 IU/
ml; NIH/NHPP) in all experiments. This concentration approximates the
measurable levels of CG in the baboon blastocyst culture medium [21].
Measurement of Intracellular cAMP Levels
HES and CHO-LH cells were grown on 12-well tissue culture plates.
BE cells were grown on 24-well tissue culture plates. Krebs-Ringer solution was equilibrated in 5% CO2 for 24 h prior to the initiation of the
experiments. The cells were washed twice with Krebs-Ringer solution and
equilibrated in this solution for 30 min at 378C. The medium was then
aspirated and Krebs-Ringer with 0.5 M IBMX was added for another 15
min. The cells were then treated with CG (10 nM) for 5, 15, 30, and 60
min. In addition, a dose-response effect was also determined by treating
the cells with either 10 or 100 nM CG for 15 min. Forskolin (10 M), the
adenylyl cyclase (AC) activator, was used as a positive control at the 15min time point. At the end of the incubation, the cells were lysed with 0.1
M HCl and stored at 2208C. Intracellular cAMP concentrations were determined by using a cAMP enzyme immunoassay kit.
Measurement of Adenylyl Cyclase Activity
Membranes from HES cells were prepared by homogenizing cells in
10 mM Tris-HCl, pH 7.0, 1.0 mM EDTA, and 27% sucrose, with a glassglass Dounce homogenizer, followed by centrifugation at 1000 3 g for 5
min to remove nuclei and cell debris and then at 10 000 3 g for 30 min.
The final pellet was resuspended in 10 mM Tris HCl and aliquots frozen
at 2708C [22]. AC activity in ;30 mg membrane protein was measured
in a 10-min reaction at 308C in the presence of 25 mM 1,3 bis-
SIGNAL TRANSDUCTION BY CHORIONIC GONADOTROPIN IN EPITHELIAL CELLS
459
[tris(hydroxymethyl)-methylamino] propane (pH 7.2), 0.4 mM EDTA, 1
mM EGTA, 0.2 mg/ml creatine phosphokinase, 20 mM phosphocreatine,
5 mM MgCl2, 100 mM GTP, 1 mM ATP, and 1 mM [3H]cAMP (;20 000
cpm]), [a-32P]ATP (;10 mCi, 100–200 cpm/pmol), 10 mg/ml BSA, 10
mg/ml CG, or 100 mM forskolin. The reaction was stopped and [32P]cAMP
was purified and quantified [22–24].
Preparation of Cell Lysates and Immunodetection
HES and CHO-LH cells were grown on 100-mm2 tissue culture dishes.
BE cells were grown on 60-mm2 tissue culture dishes due to the limited
availability of primary cells. Following each of the respective treatments,
the cells were rinsed twice with ice-cold PBS, pH 7.4, and lysed on ice
with 600 ml of lysis buffer (200 ml for BE cells) as previously described
[25]. Protein concentration was determined using the Bradford assay. Cell
lysate proteins (15–50 mg) were separated by 8% SDS-PAGE under reducing conditions. The separated proteins were transferred into polyvinylidene difluoride (PVDF) membranes. The immunodetection procedures
for each antibody followed the protocols provided by the manufacturers.
Immunocomplexes were visualized by enhanced chemiluminescence.
Measurement of PGE 2 Levels
HES cells were grown on 12-well tissue culture plates in triplicate and
subjected to various treatments. Following each of the respective treatments, the medium was collected and stored at 2708C. The PGE 2 concentration in the cell culture medium was determined by using a PGE 2
enzyme immunoassay kit from Amersham Life Sciences. The sensitivity
of the assay was 2.5 pg/ml of medium.
Statistical Analyses
One-way analysis of variance was used to test the null hypothesis of
group differences, followed by a two-tailed Student t-test for pairwise
comparisons. Each experiment was repeated three times in triplicate and
a P value of ,0.05 was considered significant.
RESULTS
Presence of CG/LH Receptors in Endometrial
Epithelial Cells
In the initial study, the presence of the CG/LH receptors
in HES and CHO-LH cells were confirmed by ligand affinity cross-linking studies. Baboon corpus luteum (CL) membranes were used as a positive control. In baboon CL and
CHO-LH cells, the predominant affinity cross-linked band
had an approximate molecular weight of 80 kDa (Fig. 1,
arrow, lanes 1 and 2). HES cells also showed a band of
similar molecular weight, albeit at a lower intensity (Fig.
1, lane 3). The specificity of the ligand receptor complex
was confirmed by competition with unlabeled ligand (Fig.
1, lanes 4–6). Semiquantitative RT-PCR analysis and Western blot analysis of RNA and membrane extracts from
CHO-LH and HES cells also confirmed that both mRNA
and protein for the LH/CG receptor were in much lower
amounts in the HES cells in comparison with CL and CHOLH cells (data not shown).
CG Does Not Induce cAMP Production or the
Phosphorylation of CREB in Endometrial Epithelial Cells
The classical signal transduction pathway induced by
CG is the activation of the AC-cAMP-PKA pathway. We
chose to determine if CG could activate this pathway in
endometrial epithelial cells. Intracellular cAMP concentrations were measured following CG stimulation for 5, 15,
30, and 60 min (Fig. 2, A–C). Stimulation of BE (Fig. 2A)
and HES (Fig. 2B) cells with CG (10 nM) showed no significant increase in cAMP at any of the observed times.
Similar responses from BE, HES, and CHO-LH cells were
observed when the concentration of CG was increased to
FIG. 1. The presence of CG R in endometrial epithelial cells. Membranes from baboon CL (lanes 1, 4), CHO-LH (lanes 2, 5), and HES (lanes
3, 6) cells were incubated with 125I-CG in the absence (lanes 1–3) or
presence of 25 nM unlabeled CG (lanes 4–6). Note the distinct radioactive bands at ;80 kDa (upper arrow), which are most prominent in the
CL and CHO-LH cells. Binding is also evident in HES cells (lane 3). Incubation with unlabeled ligand inhibited the binding of the 125I-labeled
CG (lanes 4–6). Lower bands (arrow) represent free 125I-CG.
100 nM and treatment time was 15 min (Fig. 3). Only
CHO-LH cells (Fig. 2C) showed significant increases in
cAMP production in response to CG stimulation at all observed times, with the highest response seen at 5 min. However, the AC enzymes of the three cell types were functional because forskolin (10 M), a direct activator of AC,
induced robust increases in cAMP production (Fig. 2, A–
C) at 15 min. Consistent with the cellular cAMP data, forskolin but not CG activated AC in a HES cell membrane
preparation (Fig. 2D).
Activation of the AC-cAMP-PKA pathway results in
several cellular biological responses. One of these is the
phosphorylation of the nuclear transcription factor cAMP
regulatory element-binding protein (CREB). As a readout
of a typical cAMP response, we therefore determined
whether CG stimulated CREB phosphorylation in BE,
HES, and CHO-LH cells (Fig. 4). Phosphorylation of
CREB was not evident in BE cells. In HES cells, stimulation with CG did not induce an increase in the baseline
levels of p-CREB. In contrast, CREB phosphorylation was
significantly increased in CHO-LH cells in response to 10
nM CG (Fig. 4). These results further substantiate that CG
action in the endometrial epithelial cells does not involve
immediate activation of the cAMP/PKA pathway.
CG Induces Phosphorylation of Proteins in HES Cells
Because classical activation of the AC-cAMP-PKA pathway was not evident following CG stimulation in both BE
and HES cells, we chose to determine what other signal
transduction pathways were activated by CG. Due to the
limited availability of BE cells, we used HES cells as the
primary cell model. Western blot analysis of HES cell lysates using a phosphotyrosine antibody revealed phosphorylation of several proteins (approximately 38, 42, 54, 73,
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SRISUPARP ET AL.
FIG. 2. Production of intracellular cAMP
and AC activity following CG stimulation.
Confluent BE (A), HES (B), and CHO-LH
cells (C) were treated with CG (10 nM) for
5, 15, 30, and 60 min and forskolin (10
nM) for 15 min. The concentrations of increased intracellular cAMP were determined in cell lysates. Data are expressed
as the mean SEM of three different experiments done in triplicate. Significant differences (P , 0.05) between groups are designated by different lowercase letters. Note
that only CHO-LH cells responded to CG
stimulation by increasing cAMP production. Forskolin increased cAMP in all three
cell types. D) Cell membrane proteins (30
mg) prepared from HES cells were treated
with 10 mg/ml BSA or CG, as indicated, or
100 mM forskolin, and the AC activity was
measured as described in Materials and
Methods. In contrast with CG, forskolin
stimulated adenylyl cyclase activity. Results are means 6 SEM of quadruplicate
determinations from a single assay.
116, 158 kDa) following stimulation with CG (10 nM) for
10 and 20 min (Fig. 5).
CG Activates PKA-Independent Phosphorylation
of ERK 1/2 in Endometrial Epithelial Cells
FIG. 3. Dose response of effects of CG on cAMP production. Confluent
BE, HES, and CHO-LH cells were treated with 10 and 100 nM CG or 10
nM forskolin for 15 min. Intracellular cAMP levels were determined in
cell lysates as described in Figure 2. Note that even treatment with 100
nM CG did not appreciably increase intracellular cAMP in either BE or
HES cells. In addition, in CHO-LH cells, the 100 nM concentration did
not significantly increase intracellular cAMP when compared with the 10
nM dose. Significant differences (P , 0.05) between groups are designated by different lowercase letters.
It is well established that the activation of various
GPCRs can lead to the activation of the MAPK pathway
and consequent cellular responses. The ability of the CG/
LH receptor to signal via the MAPK/ERK pathway was
previously reported in a heterologous cell model [26] and
in porcine granulosa cells [27]. We therefore sought to determine whether CG also activated the MAPK pathway in
HES cells because the major phosphorylated proteins evident in response to CG stimulation had a molecular weight
of approximately 42 kDa, which corresponds to the ERK
subfamily of MAPK. Using a specific antibody against the
phosphorylated forms of ERK1/2, our results demonstrate
that ERK 1/2 were rapidly phosphorylated in response to
CG stimulation in comparison with controls (Fig. 6A). In
HES cells, this response was MEK dependent because incubation with PD 98059 inhibited the phosphorylation of
ERK 1/2 in both a time- and dose-response manner (Fig.
6B). In addition, this response was PKA independent because H-89, the PKA inhibitor [28], did not inhibit phosphorylation of ERK 1/2 in HES cells, in contrast with its
effects in CHO-LH cells (Fig. 7, A and B).
SIGNAL TRANSDUCTION BY CHORIONIC GONADOTROPIN IN EPITHELIAL CELLS
461
FIG. 4. CREB phosphorylation in response to CG stimulation. Confluent
BE, HES, and CHO-LH cells were treated with CG (10 nM) for 10 and
20 min (C 5 control). Proteins (25 mg) from cell lysates were separated
on 8% SDS-PAGE and transferred to PVDF membranes. Membranes were
probed with a phospho CREB antibody (top panel) and reprobed with an
tubulin antibody as a loading control (bottom panel). Note that only
CHO-LH cells showed an increase in the phosphorylation of CREB following CG stimulation.
CG Induces PGE 2 Production in Endometrial
Epithelial Cells
The implantation process in several species is similar to
an inflammatory response. This response is characterized
by prominent endometrial stromal edema due to an increase
in vascular permeability. Cyclooxygenase-2 (COX-2) and
PGE 2 have been shown to be the important mediators of
this event [29]. In the baboon, COX-2 is expressed in the
glandular and luminal epithelial cells during the luteal
phase [30], and its expression is maintained in the epithelial
cells following the infusion of CG into the uterine lumen.
Because COX-2 is the rate-limiting enzyme in prostaglandin biosynthesis, we measured the PGE 2 concentrations in
the culture media of the HES cells following CG stimulation. Within 2 h, a significant increase in PGE 2 was detectable in response to CG. However, PD 98059 inhibited
CG-stimulated PGE 2 production (Fig. 8). These results suggest that activation of ERK 1/2 by CG activates a cascade
of events that ultimately increases PGE 2 synthesis by endometrial epithelial cells.
FIG. 6. PD 98059 inhibits CG-induced ERK 1/2 activation. A) Confluent
BE, HES, and CHO-LH cells were treated for 10 and 20 min with CG (10
nM) (C 5 control). Protein (25 mg) from cell lysates were separated on
8% SDS-PAGE and transferred to PVDF membrane. Membranes were
probed with a phospho ERK 1/2 antibody (top panel) and reprobed with
an tubulin antibody (bottom panel). All cell types showed the presence
of the phosphorylated ERK 1/2 after CG stimulation. B) Confluent HES
cells were pretreated for 1 h with PD 98059, a specific MEK 1/2 inhibitor.
After pretreatment, CG (10 nM) was added for 5, 10, and 20 min. Protein
(50 mg) from cell lysates were separated on 8% SDS-PAGE and transferred
to PVDF membranes. Membranes were probed with a phospho ERK 1/2
antibody (top panel) and reprobed with an ERK 1/2 antibody (bottom
panel). Note the inhibitory effect of PD 98059 on the phosphorylation of
ERK 1/2 following CG stimulation at 5, 10, and 20 min. This effect was
also dose dependent.
DISCUSSION
Successful implantation requires a reciprocal dialogue
between the embryo and the receptive endometrium. In ro-
FIG. 5. CG induces phosphorylation of proteins in HES cells. Confluent
HES cells were treated with CG (10 nM) for 10 and 20 min. Protein (15
mg) from cell lysates were separated on 8% SDS-PAGE and transferred to
PVDF membranes. Membranes were probed with a phosphotyrosine antibody. Molecular weights of protein standards (kDa) are shown on the
right of the panel. Several phosphorylated bands are evident in the CGtreated cells in comparison with control. Note the enhanced signal in the
42-kDa range (arrow).
FIG. 7. CG induces ERK 1/2 phosphorylation through a PKA independent pathway. Confluent HES and CHO-LH cells were pretreated for 30
min with H-89 (100 nM, 1 and 10 M), a PKA inhibitor. After pretreatment,
CG (10 nM) was added for 10 min. Protein (50 g) from cell lysates were
separated on 8% SDS-PAGE and transferred to PVDF membranes. Membranes were probed with a phospho ERK 1/2 antibody (top panels of A
and B) and reprobed with an tubulin antibody (bottom panels of A and
B). Note the lack of inhibitory effects of H-89 (10 M) on the phosphorylation of ERK 1/2 after CG stimulation in HES cells (A) compared to the
CHO-LH cells (B).
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SRISUPARP ET AL.
FIG. 8. CG induces an increase in PGE 2 production. Confluent HES cells
were pretreated for 1 h with PD 98059 (50 mM). After pretreatment, CG
(10 nM) was added for 2, 4, and 8 h. The PGE 2 concentrations in cell
culture media were analyzed by ELISA assay. Data are expressed as the
mean 6 SEM of three different experiments done in triplicate. Significant
differences (P , 0.05) in each group are designated by different lowercase
letters. Note an increase in the PGE 2 production within 2 h of CG stimulation but not at longer time points. This effect was also inhibited by PD
98059.
dents [31] and large domestic animals [32], embryonic signals activate an essential cascade of events in the uterine
endometrium that is crucial for the establishment of pregnancy. In our previous in vivo studies, we demonstrated
that a similar interaction exists in primates and that CG
modulates the receptive endometrium [4].
In gonadal tissues, both CG and LH signal via the CG
R to stimulate the AC-cAMP-PKA pathway [33]. The presence of this classic pathway was confirmed with our results
using CHO-LH cells. Our results, however, indicate that, in
epithelial cells of endometrial origin (human and baboon),
CG does not activate the AC-cAMP-PKA pathway. Instead,
the stimulation with CG leads to the phosphorylation of
ERK 1/2. This suggests that CG may activate a different
set of GPCR signal transduction pathways in endometrial
epithelial cells. The lack of stimulation of the AC-cAMPPKA pathway in endometrial epithelial cells could be due
to the presence of different AC isoforms, an increase in
phosphodiesterase activity, deficient receptor coupling to
Gs, or competition for overlapping effector sites with other
G proteins [34]. However, data in HES cell membranes
showing that CG does not promote AC activation suggests
that the inability of CG to raise cAMP levels is not attributable to increases in phosphodiesterase activity or inhibition of cAMP production. Identification of these alternate
signaling pathways is currently under investigation.
Our results also showed that CG can rapidly induce
phosphorylation of ERK 1/2 both in CHO-LH and endometrial epithelial cells. Phosphorylation of ERK 1/2 in
CHO-LH cells is PKA dependent, consistent with results in
porcine granulosa cells [27] and rat granulosa cell lines [35]
treated with LH. Crosstalk between ERK 1/2 and cAMP
has also been shown to be present in other cell types [36].
In contrast with CHO-LH cells, phosphorylation of ERK 1/
2 in HES cells is PKA independent. This novel result further confirms our data that CG does not activate AC to
elevate cAMP and suggests that unique signal transduction
pathways are activated by CG in the endometrial epithelial
cells. Knowledge regarding the upstream events and the
role of ERK 1/2 in CG actions in endometrial epithelial
cells is absent. The ERK kinases function as integrators of
mitogenic and other signals originating from distinct classes
of cell surface receptors, such as receptor tyrosine kinases
and GPCRs [37]. Binding of diverse extracellular stimuli
to GPCR can activate these kinases via several signal transduction pathways [38]. Phosphorylation of ERK 1/2 regulates processes in the cytoplasm, nucleus, cell membrane,
and cytoskeleton. The changes in cytoskeletal proteins are
also evident in the baboon uterus at the time of uterine
receptivity and implantation and are modulated by CG [4,
39]. In addition, phosphorylated ERK 1/2 are capable of
inducing cellular proliferation and differentiation. This in
turn may contribute to the formation of epithelial plaques
in the luminal epithelium and the increased secretory activity of glandular epithelial cells in response to the in vivo
infusion of CG [4].
In HES cells, the ERK 1/2 pathway activated by CG
increases PGE 2 production possibly through the regulation
of COX-2 activity. COX-2 and PGE 2 play important roles
in several aspects of implantation in primates and other
species [29, 40]. In vitro studies also suggest that CG regulates endometrial blood flow by increasing the secretion
of the vasoactive PGE 2 [13]. In addition, PGE 2 released
from the epithelial cells may act in an autocrine manner
through its cognate receptor and cause the upregulation of
COX-2 [41]. A similar pathway where PGE 2 could induce
the upregulation of COX-2 through a cAMP-dependent
pathway has been shown to be present in an endometrial
adenocarcinoma cell line, HEC-1B cells [42]. Thus, the increase in PGE 2 in epithelial cells in response to CG stimulation may be critical to prepare the endometrium for blastocyst implantation.
Our results, however, differ from two other studies that
showed that CG could activate the AC pathway in the endometrial epithelial cells, resulting in the increase of COX2 expression and PGE 2 production [13, 43]. One possibility
for this discrepancy is the type of cell line used. Previous
studies [43] utilized HEC-1B cells derived from the endometrial adenocarcinoma cells, which may have a greater
LH/CG receptor density compared with normal endometrium [44, 45]. In addition, these studies were done in the
presence of steroids. Estrogen and progesterone have been
shown to induce the AC and its activator, Gs, in the endometrium [46]. Another possibility for this discrepancy is
the differences in cAMP measurements both in terms of
time and the source of cAMP [13]. The patterns of immediate signal transduction induced by CG in BE and HES
cells in the current study are similar to those seen in CHOLH cells, which demonstrated rapid response both in intracellular cAMP production and phosphorylation of ERK 1/
2. Conceivably, over an extended period of time, PGE 2
generated in response to CG could increase intracellular
cAMP, leading to an increase in COX-2 mRNA via the
PKA pathway [36].
In summary, our results show that CG acts directly on
primate epithelial endometrial cells in a cAMP- and PKAindependent manner to stimulate ERK 1/2 phosphorylation.
This novel signal transduction pathway is functional and
leads to an increase in COX-2 mRNA and PGE 2 production. We can only speculate that this alternative signal transduction pathway may be mediated by an alternate spliced
form of the CG R in a manner similar to that of the FSH
receptor [47]. Alternatively and perhaps more likely, this
cAMP/PKA-independent pathway may prevent rapid receptor desensitization in the presence of high concentrations
of ligand that would be present during implantation and
SIGNAL TRANSDUCTION BY CHORIONIC GONADOTROPIN IN EPITHELIAL CELLS
early embryonic development, assuring that the CG is able
to modulate the endometrial receptor to develop an appropriately receptive endometrium that is essential for embryo
implantation.
20.
21.
ACKNOWLEDGMENTS
We thank Dr. Douglas Kniss (Ohio State University, Columbus, OH)
for his kind gift of the HES cells and Dr. Judith L. Luborsky (Rush Medical College, Chicago, IL) for her kind gift of the CHO-LH cells.
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