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J. Biol. Chem., Vol. 277, Issue 48, 46447-46455, November 29, 2002
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From the Departments of
Received for publication, April 12, 2002, and in revised form, September 6, 2002
Previous studies indicated that calcitonin
(CT), a peptide hormone involved in calcium homeostasis, is transiently
expressed in the receptive rat and human endometrial epithelia within
the window of implantation. Attenuation of uterine CT expression using antisense methods severely impaired implantation in the rat. The molecular pathway of CT in the pregnant uterus, however, remains unknown. In the present study, we investigated the cellular events following the binding of CT to its membrane receptors in human endometrial epithelial cell line Ishikawa. We observed that CT treatment triggers a transient rise in intracellular calcium in these
cells. Most interestingly, CT treatment also led to the disappearance
of E-cadherin, a critical cell adhesion molecule, from cell-cell
contact sites. Blockade of intracellular calcium release by BAPTA-AM
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl) prevented the CT-induced disappearance of E-cadherin. Our studies further revealed that CT treatment markedly down-regulates the level of E-cadherin mRNA in Ishikawa cells. We
also examined whether CT influences the expression of E-cadherin mRNA in intact rat uterine tissue during implantation. In pregnant rats, high levels of E-cadherin mRNA were expressed during the first 3 days of gestation when the CT mRNA in uterine epithelial cells is undetectable. Concomitant with a transient burst of CT expression during days 4-5 of pregnancy, the level of E-cadherin mRNA declined sharply. Furthermore, administration of exogenous CT
to animals on day 2 of pregnancy led to a premature suppression of
E-cadherin mRNA level on day 3, indicating a direct link between elevated levels of uterine CT and the down-regulation of E-cadherin expression in the surface epithelium. Collectively, our results are
consistent with the hypothesis that CT-induced reduction in E-cadherin
expression may remodel the adherens junctions between epithelial cells,
and this change in epithelial cell phenotype might be a critical event
during the implantation of the blastocyst.
Implantation involves complex and progressively intimate
interactions between the blastocyst and the uterine epithelium (1, 2).
In humans and rodents, the blastocyst initially adheres to and
penetrates the uterine epithelium and subsequently invades the uterine
stroma (1, 2). At the initial stages of implantation, the uterine
epithelium undergoes pronounced changes in cell proliferation and
remodeling, which prepares it to be "receptive" to invasion by the
embryo (1-6). The specific modifications leading to the acquisition of
the receptive state of the uterine epithelium are regulated by a
complex interplay of a variety of effectors including steroid hormones,
growth factors, and cytokines (2, 7-9). However, the precise nature of
the molecular mechanisms through which these effectors promote uterine
receptivity remains unclear.
Our previous studies identified calcitonin
(CT),1 a peptide hormone
known to regulate calcium homeostasis in bone and kidney cells, as a
potential regulator of implantation (10, 11). The expression of CT was
transiently induced in rat endometrium epithelium precisely at the
onset of implantation (10, 11). Our studies also showed that the
expression of CT is induced in the human endometrial epithelium
specifically during the mid-secretory phase (days 19-24) of the
menstrual cycle, which closely overlaps with the putative window of
implantation (12). Most importantly, suppression of the steady-state
level of the CT mRNAs in the preimplantation rat uterus by
antisense ODNs resulted in a dramatic reduction in the number of
implanted embryos (13). These results suggested strongly that a
transient surge of CT expression in the preimplantation rat uterus is
crucial for blastocyst implantation. The precise function of CT in rat
and human endometrium during implantation, however, remains unknown.
To gain an understanding of the function of CT during implantation, we
investigated the regulation of cellular functions by this hormone in a
human endometrial epithelial cell line Ishikawa. We observed that
binding of CT to its cell surface receptor in Ishikawa cells leads to a
transient rise in intracellular calcium, which in turn suppresses the
expression of calcium-dependent cell adhesion glycoprotein,
E-cadherin, at cell-cell contact sites. Consistent with this in
vitro observation, we found that administration of exogenous CT
down-regulates E-cadherin expression in the endometrial epithelium of
pregnant rats without altering the expression of ZO-1, a marker of
tight junctions. Based on these results, we favor the hypothesis that
uterine CT plays a critical role during implantation by modulating
adherens junctions. We postulate that the hormone-induced
down-regulation of E-cadherin expression results in the reorganization
of the adherens junctions between epithelial cells, thereby
facilitating implantation of the blastocyst.
Reagents--
Salmon CT (sCT), calcitonin gene-related peptide
(CGRP), and CGRP antagonist 8-37 were purchased from Peninsula
Laboratories (Belmont, CA). Antibodies against E-cadherin (human) and
ZO-1 (rat) were purchased from Zymed Laboratories Inc.
(Burlingame, CA). An antibody against E-cadherin (rat) was
purchased from BD Transduction Company (Lexington, KY).
Animals--
All experiments involving animals were conducted
according to NIH standards for the care and use of experimental
animals. Virgin female rats (Sprague-Dawley, from Charles River,
Wilmington, MA; 60-75 days of age), in proestrus, were mated with
adult males. The different stages of the cycle in the nonpregnant rats
were ascertained by examining vaginal smears. The presence of a vaginal plug after mating was designated as day 1 of pregnancy. The animals were killed at various stages of gestation and the uteri collected. In
some experiments, animals were injected intravenously or
intramuscularly with 100 µg of sCT as described under "Results."
The rats were killed 24 h after the final injection.
CT Binding Assay--
Previous studies have shown that T47D
cells harbor abundant CT receptors. Two forms of human CT receptors
have been cloned from expression cDNA library generated from these
cells (14). We have therefore used T47D as control cells.
Ishikawa and T47D cells were grown in calcium containing Dulbecco's
modified Eagle's medium (Invitrogen) and 10% fetal bovine serum. 0.5 ml of culture medium containing Ishikawa or T47D cells was placed in
test tubes. Each tube received 50,000 cpm of
125I-sCT (Peninsula Laboratories). Unlabeled sCT was
added to the tubes to produce a cold displacement curve. The tubes were
incubated overnight with gentle agitation at 4 °C, washed twice with
2 ml of culture medium, and counted in a Measurement of Intracellular Calcium--
Intracellular calcium
was monitored in Ishikawa cells by fluorescence microscopy. 1,000 Ishikawa cells/well were cultured in 96-well plates overnight and
loaded with 10 µM fluo-3 acetoxymethyl ester (fluo-3 AM)
at 37 °C for 1 h. The attached cells were rinsed three times
with medium and cultured in 100 µl of fresh medium. Some of the cells
were preloaded for 1 h with 10 µM BAPTA-AM in addition to fluo-3 AM. Fluorescent images were generated by excitation of fluo-3 at 450-490 nm with a mercury lamp and detecting the light
emitted at 525 nm. A GenIISys image intensifier (DAGE-MTI, Inc.,
Michigan City, IN) was used to enhance the signal. All images were
video-taped every 5 s using a Panasonic AG-7350 video recorder. The video camera (CCD 72, DAGE-MTI, Inc.) was set to reverse the image
so that a computer-based image analysis system (MCID M4, Image
Research, St. Catherine's, Ontario, Canada) could be utilized to
determine the fluorescence intensity.
[Ca2+]i was estimated using
Equation 1,
Immunofluorescence--
Ishikawa cells were grown in
calcium-containing Dulbecco's modified Eagle's medium to 60-70%
confluence. Cells were then treated with either 10 nM/100
nM CT or vehicle. 24 h after treatment with CT, cell
surface distribution of E-cadherin was examined by immunofluorescence using a monoclonal antibody that specifically recognizes E-cadherin. For certain experiments cells were also treated with 100 nM
CGRP or CGRP antagonist 8-37 for 24 h. In other experiments, cells were pretreated with 33.3 µM BAPTA-AM for 1 h before
the addition of CT. The experiments were repeated at least three times.
Northern Blot Analysis--
For Northern analysis 20 µg of
total RNA was separated by formaldehyde-agarose gel electrophoresis and
transferred to Duralon membrane (Stratagene). After transfer, the
membranes were baked at 80 °C for 2 h. Blots were prehybridized
in 50 mM NaPO4, pH 6.5, 5× SSC, 5× Denhardt's, 50%
formamide, 0.1% SDS, and 100 µg/ml salmon sperm DNA for 4 h at
42 °C. Hybridization was carried out overnight in the same buffer
containing 106 cpm/ml 32P-labeled E-cadherin
cDNA fragment. The filters were washed twice for 15 min in 1× SSC,
0.1% SDS at room temperature, then twice for 20 min in 0.2× SSC,
0.1% SDS at 55 °C, and the filters were exposed to x-ray films for
24-72 h. The intensities of signals on the autoradiogram were
estimated by densitometric scanning. To correct for RNA loading, the
obtained signals were normalized with respect to the glyceraldehyde
3-phosphate dehydrogenase (GAPDH) signal in the same blot. For this the
filters were stripped of the radioactive probe by washing for 10 min in
0.5% SDS at 95 °C. The blots were then reprobed with a
32P-labeled GAPDH probe as described above.
In Situ Hybridization--
Uterine tissues from pregnant animals
were collected and frozen. Tissues were fixed in 4% paraformaldehyde
at 4 °C. Cryostat sections were cut at 8 µm and attached to
3-aminopropyltriethoxysilane (Sigma)-coated slides. In situ
hybridization was performed with digoxigenin (DIG)-labeled sense
or antisense RNA probes complementary to E-cadherin gene. DIG-labeled
RNA probes were synthesized from E-cadherin cDNA using T3 or T7 RNA
polymerase and DIG-labeled nucleotides according to manufacturer's
specifications (Roche Molecular Biochemicals). Prehybridization was
carried out in a damp chamber at 37 °C for 60 min in hybridization
buffer (50% formamide, 5× SSC, 2% blocking reagent, 0.02% SDS,
0.1% N-laurylsarcosine). Hybridization was carried out at
42 °C overnight in a damp humidified chamber. To develop the
substrate, sections were washed sequentially in 2× SSC, 1× SSC, and
0.1× SSC for 15 min in each buffer at 37 °C. Sections were then
incubated with anti-DIG alkaline phosphatase-conjugated antibody.
Excess antibody was washed away, and the color substrate nitro blue
tetrazolium salt and 5-bromo-4-chloro-3-indoyl phosphate was added.
Slides were allowed to develop in the dark, and the color was
visualized under light microscopy until maximum levels of staining were
achieved. The reaction was stopped, and the slides were counterstained
in Nuclear Fast Red for 5 min. The slides were washed in water,
dehydrated, and coverslipped. Control incubations utilized a
DIG-labeled RNA sense strand and were performed under identical conditions.
Immunohistochemistry--
Polyclonal antibodies against rat
E-cadherin and ZO-1 were diluted 1:1,000 for immunohistochemistry.
Frozen uteri were sectioned at 7 µm, mounted on slides, and then
fixed in 5% formaldehyde in phosphate-buffered saline. Sections were
washed in phosphate-buffered saline for 20 min and then incubated in a
blocking solution containing 10% normal rabbit serum for 10 min before
incubation in primary antibody overnight at 4 °C. Immunostaining was
performed using a streptavidin-biotin kit for rabbit primary antibody
(Zymed Laboratories Inc.). Red deposits indicate the
sites of immunostaining.
Reverse Transcription (RT)-PCR--
Total RNA was subjected to
RT-PCR using a Stratascript kit. Briefly, the RNA samples were mixed
with oligo(dT) primer, incubated at 65 °C for 5 min, and annealed at
room temperature. First strand cDNA was synthesized using Moloney
murine leukemia virus reverse transcriptase at 37 °C, and the
reaction was stopped by heating the tubes at 95 °C for 5 min. PCR
was then performed in a 100-µl total volume using 35 ng of CT;
E-cadherin; and GAPDH-specific primers; 200 µM each dATP,
dGTP, dCTP, and dTTP; 1.5 mM Mg2+; and 0.5 µl
of Taq polymerase (PerkinElmer Life Sciences). The conditions for PCR were: 94 °C for 30 s for 1 cycle followed by 94 °C for 30 s, 65 °C for 30 s, and 68 °C for 2 min
for 25 cycles. PCR products were electrophoresed on agarose gels and
processed for Southern blot analysis.
Total RNA was prepared from Ishikawa cells as well as from T47D breast
cancer cells. The T47D cells are known to express abundant CTRs and
served as a positive control (14). The RNA samples were reverse
transcribed and amplified by PCR using CTR-specific primers that flank
the region of the insert containing 16 amino acids. The PCR products
were then subjected to Southern blot analysis employing a radiolabeled
CTR cDNA fragment as a probe.
Southern Blot Analysis--
PCR products (2 µl each) were run
on 1% agarose gel. After electrophoresis, the gel was transferred to
Duralon membrane (Stratagene). The membrane was prehybridized in 6×
SSC, 5× Denhardt's, 0.5% SDS, and 100 µg/ml salmon sperm DNA for
2 h at 68 °C. Hybridization was performed in the same buffer
containing 106 cpm/ml 32P-labeled cDNA
fragments of CT, E-cadherin, and GAPDH overnight at 68 °C. The
membrane was washed with 2× SSC and 0.1% SDS for 15 min at room
temperature, in 0.1× SSC containing 0.5% SDS at 68 °C for 45 min
and exposed to x-ray film for 8 h.
SDS-PAGE and Immunoblot Analysis of Uterine Extracts--
Uteri
were collected from rats on days 1, 4, and 6 of pregnancy, homogenized,
and extracted in chilled buffer containing 0.5% Triton X-100, 25 mM KCl, 120 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.1 mM dithiothreitol, 0.015 mM pepstatin A, 0.085 mM bestatin, and 0.011 mM E-64 in 10 mM Tris-HCl, pH 7.5. The extract
was centrifuged, and the supernatant was loaded (50-80 µg of total
protein in each lane) onto a 7.5% SDS-polyacrylamide gel. The gel was
then blotted onto a polyvinylidene difluoride membrane, which was
transferred to a solution of 5% dried nonfat milk in TBS (0.1 M Tris-buffered saline, pH 7.5) and incubated overnight
with primary antibody (rabbit anti-E-cadherin polyclonal antibody or
rabbit anti-ZO-1 polyclonal antibody). After washes, the membrane was
probed with secondary antibody coupled to horseradish peroxidase,
washed extensively before incubation in enhanced chemiluminescence
reagent, and exposed to autoradiographic film.
Treatment of Animals with ODNs--
Pregnant rats were treated
with sense and antisense ODNs targeted to CT mRNA as described
previously (13). Briefly, rats were deeply anesthetized, and an
incision was made in the lower abdomen. The sense or antisense ODNs
were mixed with DOTAP (Roche Molecular Biochemicals) and 20% F127
pluronic gel (Sigma). The solution was maintained in liquid form at
4 °C before injection. 20 µg of this ODN solution was taken in
prechilled syringes and injected into each uterine horn on day 2 of
pregnancy. After surgery, the animals were returned to their cages.
48 h after the surgery, the animals were killed, uteri were
collected, and mRNAs were isolated for Northern blot analysis. The
blot was hybridized with 32P-labeled probes containing CT,
E-cadherin, and GAPDH cDNA sequences.
Ishikawa Cells Contain Abundant Cell Surface Receptors for
CT--
To investigate the regulation of cellular functions by CT and
its receptor, human endometrial adenocarcinoma (glandular origin) Ishikawa cells present a convenient model system. These cells, like
normal preimplantation uterine epithelial cells, harbor estrogen and
progesterone receptors and retain a response to these steroid hormones.
Because the responsiveness of a target cell to CT is dependent upon the
presence of the cognate receptors on the cell surface, we first
ascertained that CTRs are indeed expressed in Ishikawa cells. The human
CTR is expressed in two molecular forms, which differ slightly in size
(14). The larger isoform contains a 16-amino acid insert that the
shorter form lacks. We initially analyzed the expression of CTR
mRNAs in Ishikawa cells by RT-PCR. Because previous reports
indicated that T47D cells harbor abundant functional CTRs (14), these
cells were used as positive control cells. As shown in Fig.
1, significant amounts of the two
isoforms of CTR mRNAs were detected in Ishikawa cells (lane
2), and the levels of these mRNAs were comparable with those
in T47D cells (lane 1).
We also determined the number of CT binding sites in Ishikawa cells by
using radiolabeled CT. The results of the Scatchard analysis are shown
in Table I. The dissociation constants
for 125I-CT binding in T47D and Ishikawa cells were
statistically equivalent. The number of CT binding sites in T47D and
Ishikawa cells were also similar. T47D cells exhibited 3.76 × 104 CT binding sites/cell, whereas the Ishikawa cells
harbored 2.78 × 104 CT binding sites/cell. These
results indicated that Ishikawa cells are likely to be responsive to
CT.
Binding of CT to Its Cell Surface Receptor Increases Intracellular
Calcium in Ishikawa Cells--
Binding of CT to CTRs is known to
elevate intracellular calcium levels in a variety of cell types,
including bone and kidney cells (16, 17). We therefore investigated the
effect of CT on intracellular calcium levels in Ishikawa cells using
fluorescence microscopy. In this experiment, the cells were plated at a
density of 104 cells/well in a 96-well plate and loaded
with the fluorescent calcium indicator fluo-3 AM. Some of the cells
were preloaded with BAPTA-AM, an intracellular calcium chelator. 10 cells in each well were monitored to determine the average fluorescence intensity for calculation of
[Ca2+]i. As shown in Fig.
2 (top panel), no
intracellular calcium elevation was observed when cells were treated
with vehicle alone. Treatment of the cells with 3 or 5 nM
CT led to a marginal rise in intracellular calcium (Fig. 2,
middle panel, left and center images,
respectively). Addition of 10 nM CT, however, elicited a
sharp rise in intracellular calcium level within 5 s (Fig. 2, middle panel, right image). This rise in
intracellular calcium level was blocked when cells were preloaded with
BAPTA-AM (Fig. 2, bottom panel), indicating that the
increased fluorescence of fluo-3 was specific for calcium. Taken
together, these results demonstrated that Ishikawa cells respond to CT
through receptor-mediated calcium signaling.
CT Treatment Triggers the Disappearance of E-cadherin Protein from
Cell-Cell Contact Sites--
An alteration in cellular calcium is
known to control adhesiveness and polarity of epithelial cells (18).
Our observation that CT alters the level of intracellular calcium in
Ishikawa cells raised the possibility that such alterations may
influence cellular adhesiveness by changing the expression or
triggering redistribution of critical cell adhesion molecules or
junctional complexes. E-cadherin is a cell surface glycoprotein that
mediates calcium-dependent cell-cell adhesion and is
believed to be critical for the establishment and maintenance of
adherens junctions in epithelial cells (19, 20). We therefore
investigated whether treatment with CT altered expression or
distribution of E-cadherin in Ishikawa cells.
Ishikawa cells grown in a calcium-containing medium to 60-70%
confluence were treated with either 100 nM CT or vehicle.
24 h after treatment with CT, the cell surface distribution of
E-cadherin was examined by immunofluorescence using a monoclonal
antibody that specifically recognizes E-cadherin. E-cadherin was
clearly localized at points of cell-cell contact in the untreated
Ishikawa cells (Fig. 3A,
panels B and C, and Fig. 3B,
panel A). Upon treatment with CT, E-cadherin immunostaining
at the cell surface was reduced dramatically after 24 h (Fig.
3A, panels b and c, and Fig.
3B, panel B). We also performed a time course to
monitor the decline in E-cadherin after 2, 6, 12, and 24 h of CT
treatment. Our studies detected a marked decline in the level of
E-cadherin in response to CT only after 24 h of hormone treatment
(data not shown).
We next examined the role of CTRs in the CT-induced disappearance of
E-cadherin from contact sites of Ishikawa cells. We observed that when
the cells were treated with another peptide hormone, CGRP, which does
not bind to CTRs, E-cadherin expression at the contact sites of
epithelial cells remained unaffected (Fig. 3B, panel
C). Interestingly, when cells were treated with a modified CGRP
(8-37), which binds to CTRs and functions as an agonist (40), E-cadherin expression at the points of cell-cell contact declined (Fig.
3B, panel D), indicating that the effect of CT is
dependent on CTR-mediated signaling.
Calcium Signaling Is Critical for CT Regulation of E-cadherin
Expression--
We next tested the hypothesis that the CT-induced rise
in intracellular calcium is a critical upstream event for the observed loss of E-cadherin from the cell-cell junctions. We therefore treated
the cells with BAPTA-AM, which effectively chelates intracellular calcium. We found that in the presence of BAPTA-AM, CT failed to induce
a loss of E-cadherin from the cell-cell contact sites (Fig.
3B, panel E). These results suggested that this
effect is indeed triggered by an intracellular calcium spike induced by CT. Collectively, these data are consistent with the view that CT acts
through its cell surface receptor to initiate calcium signaling, which,
through a cascade of events, eventually alters the expression of
E-cadherin in epithelial cells.
CT Down-regulates E-cadherin mRNA in Ishikawa Cells--
We
next investigated whether the CT-induced change in expression of
E-cadherin in Ishikawa cells is caused by a down-regulation of
E-cadherin mRNA. Ishikawa cells were grown to 60-70% confluence and treated with CT or vehicle. 24 h after treatment, cells were harvested to isolate mRNA for Northern blot analysis. Our results showed that significant amounts of E-cadherin mRNA were present in
Ishikawa cells treated with vehicle alone (Fig.
4, lanes 1 and 2).
The addition of CT to Ishikawa cells led to a marked decline in the
level of E-cadherin mRNA (Fig. 4, lanes 3 and
4). The relative levels of expression of E-cadherin mRNA
in Ishikawa cells treated with or without CT were estimated by
densitometric scanning followed by normalization with respect to the
control GAPDH mRNA signal. By our estimate, the magnitude of
E-cadherin mRNA reduction in Ishikawa cells upon CT administration
was greater than 60%. The calcium signaling induced by CT is therefore
transduced by an unknown second messenger pathway(s) to down-regulate
expression of E-cadherin gene in Ishikawa cells.
E-cadherin mRNA and Protein Decline in Rat Uterine Epithelium
at the Onset of Implantation--
Our previous studies showed that CT
is transiently induced in rat endometrial epithelium within the
implantation window (10). Because CT induced down-regulation of
E-cadherin expression in our in vitro cell culture studies,
we sought to determine whether it exerts a similar effect on E-cadherin
expression in the uterine tissue in vivo.
As a first step toward this goal, we analyzed the profile of E-cadherin
mRNA expression in the uterus during early pregnancy. RNA was
isolated from the uteri of animals on days 1-6 of pregnancy and
subjected to RT-PCR analysis using both CT- and E-cadherin-specific primers. Our results revealed a significant amount of E-cadherin mRNA expression in the uterus during days 1 and 2 of pregnancy when
CT mRNA expression remained undetectable or low (Fig.
5). As the CT mRNA level started to
rise on day 3 of gestation, we noted a progressive decline in the
E-cadherin mRNA level. Concomitant with the high levels of CT
mRNA in rat uterus on days 4 and 5 of pregnancy, there was a sharp
decline in the level of E-cadherin mRNA on these days. As the CT
mRNA level dropped on day 6, we observed that E-cadherin mRNA
expression returned to a level that was ~50% of that on day 1 of
gestation.
We also performed immunoblot and immunohistochemical analyses to
monitor the expression of E-cadherin protein in rat uterus on days 1, 4, and 6 of pregnancy. Fig. 6A
shows immunoblotting of equal amounts of uterine extracts obtained from
rats on days 1, 4, and 6 of pregnancy, employing antibodies against
E-cadherin (top row) or tight junction protein ZO-1
(bottom row). A robust E-cadherin signal was observed in the
endometrial extracts of day 1 pregnant rats (lane D1). The
intensity of this signal declined considerably on day 4 of gestation
(lane D4) and then recovered slightly on day 6 (lane
D6). The level of ZO-1, however, did not alter significantly among
days 1, 4, and 6 of pregnancy (Fig. 6A). Consistent with the
immunoblot analysis, we found an intense staining for E-cadherin, both
at the lateral and apical junctions between the epithelial cells, on
day 1 of pregnancy (Fig. 6B, panel A). However,
on day 4 of gestation, there was an overall decline in the E-cadherin
staining in the epithelium. Almost no E-cadherin staining was observed
at the lateral interfaces, and very little staining was found at the
apical junctions. On day 6 of gestation, the E-cadherin staining
increased predominantly in the apical junctions compared with day 4 (Fig. 6B, panels B and C).
Collectively, these results indicate that the level of E-cadherin at
the cell-cell junctions in the uterine epithelium declines at the onset
of implantation.
Inhibition of CT Expression in Preimplantation Uterus by Antisense
ODN Results in Up-regulation of E-cadherin--
We have demonstrated
previously that administration of antisense ODN targeted against CT
mRNA led to an inhibition of CT gene expression in the
preimplantation rat uterus (13). We therefore investigated whether the
suppression of CT mRNA expression upon antisense ODN treatment
influenced E-cadherin level during early pregnancy. As described
previously (13), we administered either the antisense or the
corresponding sense CT ODNs into both uterine horns of rats on the
afternoon of day 2 of pregnancy. The animals were killed on day 4 of
gestation, uteri were collected, and mRNAs were isolated for
Northern blot analysis. The blot was hybridized with
32P-labeled probes containing CT, E-cadherin, and GAPDH
cDNA sequences. The results of these experiments are shown in Fig.
7. As shown in the top row of
the figure, the uterine horns that were treated with antisense ODNs
exhibited significantly reduced CT mRNAs on day 4 compared with the
horns that were injected with the same doses of corresponding sense
ODNs (compare lanes 1 and 3 with lanes
2 and 4, respectively). Interestingly, the uterine
horns that exhibited a reduced level of CT expression upon
administration of antisense ODN showed a higher level of E-cadherin
compared with the horns that were treated with sense ODNs (Fig. 7,
middle row, compare lanes 1 and 3 with
lanes 2 and 4, respectively). Hybridization of
the blot with GAPDH probe indicated no significant alteration in the
intensity of this signal in the antisense or sense ODN-treated uterus
(Fig. 7, bottom row). These results suggested that in the
pregnant rat uterus CT and E-cadherin expression exhibit a reciprocal
relationship and are likely to be functionally linked.
Administration of Exogenous CT Leads to Precocious Down-regulation
of E-cadherin during Early Pregnancy--
We reasoned that if the
transient expression of CT and the down-regulation of E-cadherin in the
surface epithelium are functionally linked events, administration of
exogenous CT to pregnant rats would be expected to lead to a further
decline in the level of E-cadherin in the uterus. Specifically, we
examined whether premature elevation of CT levels in the pregnant rat
uterus leads to a concomitant reduction in the level of E-cadherin in
the epithelial cells. Animals on day 2 of pregnancy, when the level of
endogenous CT level is extremely low and the level of E-cadherin is
high, were injected intravenously or intramuscularly with CT or
vehicle. 24 h after administration of the peptide hormone, uterine
mRNA was isolated and subjected to Northern blot analysis using a
radiolabeled fragment of E-cadherin cDNA as a probe. Fig.
8 shows that compared with animals
treated with vehicle only, the level of uterine E-cadherin mRNA on
day 3 of pregnancy was markedly reduced after administration of CT
(compare lanes 1 and 2; 3 and
4).
We also performed in situ hybridization and
immunohistochemistry to confirm further the down-regulation of
E-cadherin mRNA and protein in uterine epithelium in response to
exogenous CT. In situ hybridization with antisense
E-cadherin RNA probe revealed an intense signal of E-cadherin mRNA
in the surface epithelial cells of vehicle-treated (control) uteri of
day 3 pregnant animals (Fig.
9A, panel A).
Control uterine sections hybridized with the corresponding sense RNA
probe of equal length did not exhibit any signal demonstrating the
specificity of the hybridization reaction (Fig. 9A,
panel C). A remarkable decline in the level of E-cadherin
mRNA was observed in the luminal epithelium of day 3 pregnant
animals after exogenous administration of CT on day 2 of pregnancy
(Fig. 9A, panel B).
Essentially similar results were obtained by immunohistochemical
analysis. As shown in Fig. 9B, a marked E-cadherin-specific staining was observed in the luminal epithelium of uterine sections of
animals treated with vehicle only on day 2 and killed on day 3 of
pregnancy (panel a). However, a dramatic reduction in the level of E-cadherin was observed in the epithelial cells of uterus following administration of CT (panel b). The specificity of
this down-regulation of E-cadherin in response to CT was demonstrated by the fact that the expression of the tight junction protein ZO-1 in
the uterine epithelial cells was unaffected upon administration of CT
(Fig. 9C). Consistent with these results, immunoblot
analysis of uterine extracts obtained from day 2 pregnant rats treated with CT showed a dramatic decline in the level of E-cadherin compared with vehicle-treated controls 24 h after treatment (Fig.
9D, top row, compare lanes 1 and
3 with lanes 2 and 4, respectively). The level of ZO-1 in the uterine extracts, however, did not change significantly upon administration of CT (Fig. 9D,
bottom row, compare lanes 1 and 3 with
lanes 2 and 4, respectively). Taken together,
these studies strongly support our hypothesis that uterine CT functions
during implantation by specifically remodeling the adherens
junctions in the endometrial epithelium.
This study addresses the mechanism of action of CT as a paracrine
or autocrine effector in the uterus during implantation. Our previous
studies revealed that the uterine epithelium of species as diverse as
rat and human transiently produces CT within the window of implantation
(10, 12). Targeted suppression of CT mRNAs in the preimplantation
uterus upon administration of antisense ODNs led to a block of
implantation (13). These results prompted us to speculate that CT is
secreted into the uterine lumen at the time of implantation, and its
principal function in the preimplantation uterus is to regulate
blastocyst implantation in a paracrine manner. Consistent with this
prediction, we detected significant amounts of CT in the luminal
secretions of rat uterus collected on days 4-5 of pregnancy,
indicating that this hormone is secreted at or near the implantation
bed (11). It was speculated that the secreted CT would bind to the CTRs
of surface epithelial cells and modulate their function at the time of
implantation. Here, we describe that CT indeed alters cell adhesiveness
in the surface epithelium by reprogramming the expression of
E-cadherin, a critical adhesion molecule.
CT functions through its cell surface receptor, CTR, which belongs to a
class of seven transmembrane G-protein-coupled receptors (21). Previous
studies have shown that binding of CT to CTRs on the target cell leads
to a rise in intracellular calcium and/or cAMP, which in turn regulate
cellular functions (16, 17, 22). Depending upon the target cell, the
CTRs are known to couple to multiple heterotrimeric G proteins, leading
to the activation of adenyl cyclase and/or phospholipase C (16,
17, 22). Signaling through both cAMP and intracellular calcium are
important in CT action in osteoclasts, chondrocytes, and renal cells.
In brain, however, CT apparently causes no change of adenylate cyclase
activity and there is some evidence that calcium acts as a second
messenger in this tissue (23-25). Likewise, in hepatocytes CT-induced
adenylate cyclase activity has not been shown, but CT, even at a very
low concentration, is capable of increasing intracellular calcium levels (26). Although we do not know whether binding of CT to its
receptor activates cAMP production in Ishikawa cells, we did observe a
rapid elevation in intracellular calcium in response to this hormone,
presumably via phospholipase C activation. It is likely that inositol
phosphate, which is produced after the activation of phospholipase C by
CT, stimulates release of calcium from intracellular stores.
As intracellular calcium level rises in response to CT, one or more
calcium-sensitive signal transduction cascades are likely activated,
leading to regulation of cellular functions. Three well established
calcium-sensitive pathways involve protein kinase C,
calmodulin-dependent kinase, and mitogen-activated protein kinase. In each of these pathways, the kinase is activated either directly or indirectly in response to calcium. The activated kinase phosphorylates the transcription factors that recognize specific cis-acting elements in the control regions of a specific
network of target genes. Alternatively, calcium-dependent
kinases can influence assembly or disassembly of junctional complexes
through direct phosphorylation or dephosphorylation of undefined
protein targets.
Our study shows that a CT-induced upsurge in intracellular calcium
suppresses the level of E-cadherin mRNA in Ishikawa cells. This
effect is manifested in the loss of E-cadherin protein from cell-cell
contact sites in in vitro cell cultures as well as in pregnant uterine epithelium at the time of implantation. E-cadherin, a
transmembrane linker protein of the intercellular adherens junctions, plays a key role in the maintenance of the adhesive and polarized phenotype of uterine and other epithelial cells (19, 20, 37). Cell-cell
adhesion results from head-to-head contact between cadherin homodimers
in adjacent cell membranes. It is important to note that many previous
studies hinted at a role of calcium in reprogramming of cell-cell
interactions in the surface epithelium during implantation, although
the precise molecular basis of this process eluded us (27-29). The
present study, for the first time, clearly links changes in calcium
homeostasis to specific reorganization of a master cell adhesion
molecule in the surface epithelium at the time of implantation.
A number of recent studies have reported down-regulation of E-cadherin
expression during the acquisition of metastatic potential at late
stages of epithelial tumor progression (30-33). In majority of these
cases the decline in the level of E-cadherin has been attributed to a
transcriptional event in which trans-acting regulators, such as
multi-zinc finger proteins snail and SIP1, bind directly to E-cadherin promoter and repress RNA synthesis (30-32). However, down-regulation of E-cadherin is also known to occur via endocytosis after ubiquitination of the E-cadherin complex (33). Our studies in
Ishikawa cells and in intact rat uterine epithelium seem to suggest
that down-regulation of E-cadherin in response to CT is a
transcriptional event. It is likely that a CT-induced cascade modulates
the activity of transcription factors that regulate the E-cadherin
promoter. The chain of events is initiated by binding of CT to its
receptor, resulting in a surge of intracellular calcium. The blockade
of the elevation in intracellular calcium by BAPTA-AM prevented
down-regulation of E-cadherin, indicating that signaling downstream of
calcium release is critical for this effect. The elevated intracellular
calcium activates one or more kinase signaling pathways, leading to the
phosphorylation of a transcription factor, which then regulates
E-cadherin gene expression. We, however, cannot rule out the
possibility that post-transcriptional/translational modifications in
response to CT also contribute to the down-regulation of E-cadherin mRNA.
The identity of the kinase cascade(s) that is activated by CT-induced
calcium signaling and is operative in down-regulating E-cadherin
expression in the endometrial epithelial cells remains unknown.
Interestingly, a recent study has shown that CTRs can activate parallel
independent signaling pathways in the same target cell (34). In
cultured cells stably expressing the CTRs, CT induced time- and
concentration-dependent increases in mitogen-activated protein kinase phosphorylation and activation. The study further showed
that besides increasing mitogen-activated protein kinase phosphorylation, CT also induces the activation of other signaling effectors, including protein kinase C. Thus, multiple signaling pathways are operative downstream of an increase in intracellular calcium for full CTR-dependent cellular response. Taken
together, these studies point to the complexity of CT/CTR-mediated
signaling pathway in a target cell.
CT-induced down-regulation of E-cadherin in the pregnant uterus may
have important implications for the process of implantation. Biochemically distinct junctional complexes hold the uterine luminal epithelial cells together. Whereas tight junctions occur between apical
surfaces, adherens junctions at basolateral membranes mediate stable
cell-cell adhesion. A remodeling of various junctional complexes in the
uterine epithelial cells during implantation is well documented
(35-39). It is thought that as the uterus enters the receptive phase
under the influence of steroid hormones, the epithelial cells acquire
adhesiveness for trophoblast. The molecular basis of this switch from a
nonadhesive state to an adhesive state is not clear. It is, however,
conceivable that during acquisition of adhesiveness, alterations in
both apical and basolateral junctional complexes in the surface
epithelium take place in a concerted way, and multiple steroid
hormone-induced factors regulate this process. CT, which specifically
alters the adherens junctions without affecting the ZO-1 expression at
the apical interfaces, is clearly one of these factors. Most
interestingly, we have shown previously that the expression of CT in
the receptive uterus is induced by progesterone. CT, therefore,
represents one of the molecular links between steroid hormone action
and the functional reprogramming of epithelial cell adhesiveness during implantation.
A CT-induced decline in the level of E-cadherin would disrupt lateral
adhesiveness between the surface epithelial cells. This scenario may
have functional consequences for the process of implantation in rodents
in which the trophoblast invasion occurs via a "displacement" mechanism (2). The trophectoderm cells of the embryo need to penetrate between the intact luminal epithelial cells of the uterus. One can, therefore, envision that the down-regulation of E-cadherin at
the time of implantation may lead to a plastic epithelium in which the
intercellular space can be penetrated more easily by invading
trophoblast cells. After penetration of the trophoblast cells between
the lateral surfaces of the epithelium, the cells of the lining
epithelium are displaced and appear to undergo apoptosis. Although
apoptosis is restricted to the cells adjacent to the embryo, the
reduction in E-cadherin occurs throughout the epithelium. The
disruption of cell-cell contact, which Glasser (6) suggested to be the
initial signal that ultimately leads to the loss of uterine epithelial
integrity and sloughing of cells during implantation, could in part be
the result of the CT-mediated down-regulation of E-cadherin in
epithelial cells. Interestingly, our previous studies have shown that
addition of CT to embryos cultured in vitro advances their
development (15). One can, therefore, envision a role for CT in
coordinating the developmental programs of both embryo and uterus
during implantation. In response to this hormone, the trophoblast cells
are ready to invade as the epithelium becomes receptive and the
junctions begin to open up. Future development of an in
vitro model system to study the interactions between trophoblast
and uterine epithelial cells would allow us to test this hypothesis rigorously.
We thank Michelle Macaraig for expert
technical assistance, and we are grateful for the advice and help of
Dr. Li-Ji Zhu in the immunostaining and immunofluorescence studies.
*
This work was supported in part by National Institutes of
Health Grants R01 HD-34527 and R01 HD-39291 (to I. C. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Veterinary
Biosciences, University of Illinois at Urbana-Champaign, 3641 VMBSB,
2001 S. Lincoln, Urbana, IL 61802. Tel.: 217-333-7986; Fax:
217-244-1652; E-mail: ibagchi@uiuc.edu.
Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M203555200
The abbreviations used are:
CT, calcitonin;
BAPTA-AM
1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-acetoxymethyl ester;
CGRP, calcitonin gene-related peptide;
CTR(s), calcitonin receptor(s);
DIG, digoxigenin;
fluo-3 AM, fluo-3
acetoxymethyl ester;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
RT, reverse transcription;
sCT, salmon calcitonin;
ODN, oligodeoxynucleotide.
Calcitonin Down-regulates E-cadherin Expression in Rodent Uterine
Epithelium during Implantation*
,
, and Veterinary Biosciences and
¶ Molecular and Integrative Physiology, University of Illinois at
Urbana-Champaign, Urbana, Illinois 61802 and the
§ Department of Obstetrics and Gynecology, C. S. Mott
Center for Human Growth and Development, Wayne State University School
of Medicine, Detroit, Michigan 48201
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-counter. The results were subjected to Scatchard analysis to determine the dissociation constant
(Kd) and number of binding sites/cell.
where Kd is the dissociation constant for
[Ca2+]i (316 nM),
F is the fluorescence intensity, Fmin
is the background fluorescence, and Fmax is the
maximal fluorescence obtained by equilibrating cytoplasmic and
extracellular Ca2+ using 5 µM ionomycin. 10 cells in each well were monitored to determine the average fluorescence
intensity for calculation of [Ca2+]i.
![[<UP>Ca<SUP>2+</SUP></UP>]<SUB>i</SUB>=K<SUB>d</SUB> (F−F<SUB><UP>min</UP></SUB>)/(F<SUB><UP>max</UP></SUB>−F)](/content/vol277/issue48/fulltext/46447/img001.gif)
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
[in a new window]
Fig. 1.
Expression of CTRs in Ishikawa cells.
Total RNA (0.2 µg) was isolated from T47D (lane 1) or
Ishikawa (lane 2) cells and subjected to RT-PCR using CTR-
(30 cycles) or GAPDH- (15 cycles) specific primers. The authenticity of
the amplified products was confirmed by Southern blotting using
32P-labeled CTR or GAPDH probes. The experiment was
repeated twice for reproducibility. The results of a representative
experiment are shown.
125I-sCT binding to Ishikawa cells
View larger version (28K):
[in a new window]
Fig. 2.
Profile of CT-induced calcium influx in
Ishikawa cells. Cells preloaded with fluo-3 AM were treated with
vehicle (top panel, all three images), CT
(middle panel, left image, 3 nM;
center image, 5 nM; and right image,
10 nM), or after pretreatment of BAPTA-AM (bottom
panel, all three images). Fluorescent images were
generated by excitation of fluo-3 at 450-490 nm with a mercury lamp
and detecting the light emitted at 525 nm.
View larger version (47K):
[in a new window]
Fig. 3.
A: changes in E-cadherin in Ishikawa
cells with or without CT treatment. Phase contrast images of
semiconfluent cultures of Ishikawa cells without (panel A)
or with CT treatment for 24 h (panel a) are shown.
Panels B, b, C, and c show
expression of E-cadherin by immunofluorescence. Ishikawa cells were
treated without (panels B and C) or with CT
(panels b and c) for 24 h. Magnification:
panels A, a, B, and b,
×40; panels C and c, ×100. B:
CT-induced change in E-cadherin expression is dependent upon
CTR-mediated intracellular calcium signaling. Analysis by
immunofluorescence of the expression of E-cadherin in Ishikawa cells
treated with vehicle (panel A), CT (panel B),
CGRP (panel C), CGRP (8-37) (panel D), or
BAPTA-AM prior to the addition of CT (panel E) is
shown.
View larger version (47K):
[in a new window]
Fig. 4.
CT down-regulates E-cadherin mRNA in
Ishikawa cells. Northern blot analysis of E-cadherin expression in
cells untreated (lanes 1 and 2) or treated with
CT (lanes 3 and 4) for 24 h is shown. 20 µg of total RNA was loaded per lane. GAPDH was used as an
internal control.
View larger version (37K):
[in a new window]
Fig. 5.
Profile of expression of CT and E-cadherin
mRNA in rat uterus between days 1 and 6 of early
pregnancy. Total RNAs (1 µg) were reverse transcribed
using oligo(dT) primer and RNase H reverse transcriptase. The first
strand cDNA products were amplified using either CT-specific
primers (top row) or E-cadherin primers (bottom
row).
View larger version (80K):
[in a new window]
Fig. 6.
Expression of E-cadherin in rat uterus
by immunoblotting and immunocytochemistry. A:
E-cadherin (upper row) and ZO-1 (lower row) were
detected in uterine extracts of rats on days 1 (lane 1), 4 (lane 2), and 6 (lane 3) of pregnancy by
immunoblotting. B: immunocytochemistry was performed
employing polyclonal rabbit anti-E-cadherin using sections from day 1 (panel A), day 4 (panel B), and day 6 (panel C) pregnant rat uterus as described under
"Materials and Methods." Red deposits indicate sites of
specific immunoreactivity.
View larger version (19K):
[in a new window]
Fig. 7.
Effects of antisense or sense CT ODNs on CT
and E-cadherin mRNA expression in pregnant rat uterus. Rats on
day 2 (afternoon) of pregnancy were injected in both uterine horns with
either antisense or the corresponding sense CT ODNs. The uteri were
collected 48 h after treatment. Total RNAs (20 µg/lane) were prepared from uteri collected from
ODN-treated animals and analyzed by Northern blotting. S and
As represent samples from sense and antisense ODN-treated
animals, respectively. The top row shows the pattern of
signals obtained after hybridization with a 32P-labeled CT
probe. The middle and bottom rows show the same
blot after hybridization with 32P-labeled E-cadherin and
GAPDH probes, respectively.
View larger version (54K):
[in a new window]
Fig. 8.
Administration of exogenous CT leads to a
down-regulation of E-cadherin mRNA in the uteri of pregnant
rats. RNA (20 µg/lane) was subjected to Northern blot
analysis and hybridized with a 32P-labeled E-cadherin
(upper panel) or GAPDH (lower panel) probe.
Lanes 1 and 3 represent RNA from uteri of animals
injected with vehicle only; lane 2, RNA from uteri of
animals injected intravenously with CT; lane 4, RNA from
uteri of animals injected intramuscularly with CT. All the injections
were performed on day 2 of pregnancy, and the uteri were isolated
24 h later on day 3 of gestation.
View larger version (52K):
[in a new window]
Fig. 9.
Expression of E-cadherin and ZO-1 in the
uterus of animals treated with CT during early pregnancy.
A: in situ hybridization analysis of uterine
sections from pregnant (day 3) rats treated with vehicle (panel
A) or CT (panel B) on day 2 of pregnancy. Hybridization
was performed employing a cRNA probe specific for E-cadherin.
Panel C represents hybridization of the CT-treated uterine
section with a sense E-cadherin probe. B:
immunohistochemistry was performed employing polyclonal rabbit
anti-E-cadherin using uterine sections from day 3 pregnant rats
injected with vehicle (panel a) or CT (panel b)
on day 2 of pregnancy. C: immunohistochemical analysis of
ZO-1 in the uterine sections of rats injected with vehicle (panel
A) or CT (panel B). D: immunoblot detection
of E-cadherin (upper row) and ZO-1 (lower row) in
uterine extracts of pregnant rats treated with vehicle (lanes
1 and 3) or CT (lanes 2 and 4).
The animals were injected on day 2 of gestation, and the uteri were
collected on day 3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by National Institutes of Health Grants R01-DK-50257
and U54-HD-13541.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Psychoyos, A.
(1973)
in
Handbook of Physiology
(Greep, R. O.
, and Astwood, E. G., eds)
, pp. 187-215, American Physiological Society, Washington, D. C.
2.
Carson, D. D.,
Bagchi, I.,
Dey, S. K.,
Enders, A. C.,
Fazleabas, A. T.,
Lessey, B. A.,
and Yoshinaga, K.
(2000)
Dev. Biol.
223,
217-237[CrossRef][Medline]
[Order article via Infotrieve]
3.
Schlafke, S.,
and Enders, A. C.
(1975)
Biol. Reprod.
12,
41-65[CrossRef][Medline]
[Order article via Infotrieve]
4.
Yoshinaga, K.
(1988)
Ann. N. Y. Acad. Sci.
541,
424-431[Medline]
[Order article via Infotrieve]
5.
Parr, M. B.,
and Parr, E. L.
(1989)
in
Biology of the Uterus
(Wynn, R. M.
, and Jollie, W. P., eds)
, pp. 233-277, Plenum Publishing Corp., New York
6.
Glasser, S. R.
(1990)
in
Trophoblast Invasion and Endometrial Receptivity: Novel Aspects of the Cell Biology of Embryo Implantation
(Denker, H. W.
, and Aplin, J. D., eds)
, pp. 377-416, Plenum Publishing Corp., New York
7.
Psychoyos, A. J.
(1976)
Reprod. Fertil. Suppl.
25,
17-28
8.
Psychoyos, A.
(1986)
Ann. N. Y. Acad. Sci.
476,
36-42[Medline]
[Order article via Infotrieve]
9.
Sharkey, A.
(1998)
J. Reprod. Fertil.
3,
52-61
10.
Ding, Y. Q.,
Zhu, L. J.,
Bagchi, M. K.,
and Bagchi, I. C.
(1994)
Endocrinology
135,
2265-2274[Abstract]
11.
Zhu, L.-Z.,
Cullinan-Bove, K.,
Polihronis, M.,
Bagchi, M. K.,
and Bagchi, I. C.
(1998)
Endocrinology
139,
3923-3934 12.
Kumar, S.,
Zhu, L. Z.,
Polihronis, M.,
Cameron, S. T.,
Baird, D. T.,
Schatz, F.,
Dua, A.,
Ying, Y. K.,
Bagchi, M. K.,
and Bagchi, I. C.
(1998)
J. Clin. Endocrinol. Metab.
83,
4443-4450 13.
Zhu, L. J.,
Bagchi, M. K.,
and Bagchi, I. C.
(1998)
Endocrinology
139,
330-339 14.
Kuestner, R. E.,
Elrod, R. D.,
Grant, F. J.,
Hagen, F. S.,
Kuijper, J. L.,
Matthewes, S. L.,
O'Hara, P. J.,
Sheppard, P. O.,
Stroop, S. D.,
Thompson, D. L.,
Whitmore, T. E.,
Findlay, D. M.,
Houssami, S.,
Sexton, P. M.,
and Moore, E. E.
(1994)
Mol. Pharmacol.
46,
246-255[Abstract]
15.
Wang, J.,
Rout, U. K.,
Bagchi, I. C.,
and Armant, D. R.
(1998)
Development
125,
4293-4302[Abstract]
16.
Chabre, O.,
Conkin, B. R.,
Lin, H. Y.,
Lodish, H. F.,
Wilson, E.,
Ives, H. E.,
Catanzariti, L.,
Hemmings, B. A.,
and Bourne, H. R.
(1992)
Mol. Endocrinol.
6,
551-556 17.
Force, T.,
Bonventre, I. V.,
Flannery, M. R.,
Gorn, A. H.,
Yamin, M.,
and Goldring, S. R.
(1992)
Am. J. Physiol.
262,
F1110-F1115 18.
Gumbiner, B. M.,
Stevenson, B.,
and Grimaldi, A.
(1988)
J. Cell Biol.
107,
1575-1587 19.
Gumbiner, B. M.
(1996)
Cell
84,
345-357[CrossRef][Medline]
[Order article via Infotrieve]
20.
Huber, O.,
Bierkamp, C.,
and Kemler, R.
(1996)
Curr. Opin. Cell Biol.
8,
685-691[CrossRef][Medline]
[Order article via Infotrieve]
21.
Lin, H. Y.,
Harris, T. L.,
Flannery, M. S.,
Aruffo, A.,
Kaji, E. H.,
Gorn, A.,
Kolakowski, L. F.,
Lodish, H. F.,
and Goldring, S. R.
(1991)
Science
254,
1022-1024 22.
Heersche, J. N. M.,
Marcus, R.,
and Aurbach, G. D.
(1974)
Endocrinology
94,
241-247 23.
Rizzo, A. J.,
and Goltzman, D.
(1981)
Endocrinology
108,
1672-1677 24.
Twery, M. J.,
Seitz, P. K.,
Nickols, G. A.,
Cooper, C.W.,
Gallagher, J. P.,
and Orlowski, R. C.
(1988)
Eur. J. Pharmacol.
155,
285-292[CrossRef][Medline]
[Order article via Infotrieve]
25.
Koida, M.,
Yamamoto, Y.,
Nakamuta, H.,
Matsuo, J.,
Okamoto, M.,
Morimoto, T.,
Seyler, J. K.,
and Orlowski, R. C.
(1982)
Jpn. J. Pharmacol.
32,
981-986[Medline]
[Order article via Infotrieve]
26.
Yamaguchi, M.,
and Yoshida, H.
(1985)
Acta Endocrinol.
110,
239-243
27.
Geisert, R. D.,
Renegar, R. H.,
Thatcher, W. W.,
Roberts, R. M.,
and Bazer, F. W.
(1982)
Biol. Reprod.
27,
925-939[CrossRef][Medline]
[Order article via Infotrieve]
28.
Geisert, R. D.,
Thatcher, W. W.,
Roberts, R. M.,
and Bazer, F. W.
(1982)
Biol. Reprod.
27,
957-965[CrossRef][Medline]
[Order article via Infotrieve]
29.
Geisert, R. D.,
Zavy, M. T.,
Moffatt, R. J.,
Blair, R. M.,
and Yellin, T.
(1990)
J. Reprod. Fertil. Suppl.
40,
293-305[Medline]
[Order article via Infotrieve]
30.
Battle, E.,
Sancho, E.,
Franci, C.,
Dominguez, D.,
Monhar, M.,
Baulida, J.,
and de Herreros, A. G.
(2000)
Nat. Cell Biol.
2,
84-89[CrossRef][Medline]
[Order article via Infotrieve]
31.
Cano, A.,
Perez-Moreno, M. A.,
Rodrigo, I.,
Locascio, A.,
Blanco, M. J.,
del Barrio, M. G.,
Portillo, F.,
and Nieto, M. A.
(2000)
Nat. Cell Biol.
2,
76-83[CrossRef][Medline]
[Order article via Infotrieve]
32.
Comijin, J.,
Berx, G.,
Vermassen, P.,
Verschueren, K.,
Grunsven, L.,
Bruyneel, E.,
Mareel, M.,
Huylebroeck, D.,
and van Roy, F.
(2001)
Mol. Cell
7,
1267-1278[CrossRef][Medline]
[Order article via Infotrieve]
33.
Fujita, Y.,
Krause, G.,
Scheffner, M.,
Zechner, D.,
Leddy, H. E.,
Behrens, J.,
Sommer, T.,
and Birchmeier, W.
(2002)
Nat Cell Biol.
4,
222-231[CrossRef][Medline]
[Order article via Infotrieve]
34.
Chen, Y.,
Shyu, J. F.,
Santhanagopal, A.,
Inoue, D.,
David, J. P.,
Dixon, S. J.,
Horne, W. C.,
and Baron, R.
(1998)
J. Biol. Chem.
273,
19809-19816 35.
Glasser, S.,
and Mulholland, J.
(1993)
Microsc. Res. Tech.
25,
106-120[CrossRef][Medline]
[Order article via Infotrieve]
36.
Denker, H. W.
(1993)
J. Exp. Zool.
266,
541-558[CrossRef][Medline]
[Order article via Infotrieve]
37.
Thie, M.,
Harrach-Ruprecht, B.,
Sauer, H.,
Fuchs, P.,
Albers, A.,
and Denker, H. W.
(1995)
Eur J. Cell Biol.
66,
180-191[Medline]
[Order article via Infotrieve]
38.
Thie, M.,
Fuchs, P.,
Butz, S.,
Sieckmann, F.,
Hoschutzky, H.,
Kemler, R.,
and Denker, H. W.
(1996)
Eur. J. Cell Biol.
70,
221-232[Medline]
[Order article via Infotrieve]
39.
Illingworth, I. M.,
Kiszka, I.,
Bagley, S.,
Ireland, G. W.,
Garrod, D. R.,
and Kimber, S. J.
(2000)
Biol. Reprod.
63,
1764-1773 40.
Chiba, T.,
Yamaguchi, A.,
Yamatani, T.,
Nakamura, A.,
Morishita, T.,
Inui, T.,
Fukase, M.,
Noda, T.,
and Fujita, T.
(1989)
Am. J. Physiol.
256,
E331-E335
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 S. Kumar, A. Brudney, Y.-P. Cheon, A. T. Fazleabas, and I. C. Bagchi Progesterone Induces Calcitonin Expression in the Baboon Endometrium Within the Window of Uterine Receptivity Biol Reprod, April 1, 2003; 68(4): 1318 - 1323. [Abstract] [Full Text] [PDF]
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