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and ß in Rat Decidua Cells: Cell-Specific Expression and Differential Regulation by Steroid Hormones and Prolactin1
Department of Physiology and Biophysics, University of Illinois College of Medicine (C.T., S.D., A.P.-T., S.F.-G., G.B.G., G.G.), Chicago, Illinois 60612; and Department of Physiology, Faculty of Medicine, University of Manitoba (R.P.C.S.), Winnipeg, Manitoba, Canada R3E 0W3
Address all correspondence and requests for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. E-mail: ggibori{at}uic.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. In this
investigation, we analyzed the expression of decidual ER and ERß,
studied their regulation by PRL and steroid hormones and examined the
ability of decidual ERß to transduce the estradiol signal to the
progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot
analysis showed that both ER species are coexpressed in the decidua
during pseudopregnancy. Interestingly, these genes were preferentially
found in a cell population localized in the antimesometrial site of the
uterus where blastocyst implantation takes place. Using decidual cells
in primary culture obtained from pseudopregnant rats and a
decidua-derived cell line (GG-AD), we show a differential regulation of
ER and ERß by PRL and ovarian steroid hormones. Whereas PRL,
estradiol, and progesterone all increased ERß messenger RNA (mRNA)
expression in a dose-dependent manner, only PRL up-regulated the mRNA
levels of ER. Estradiol had no effect on ER expression, whereas
progesterone markedly decreased its mRNA levels. Interestingly,
progesterone, which up-regulates the ability of PRL to signal to a
PRL-regulated gene in mammary-gland derived cells, prevented PRL
stimulation of decidual ER and had no synergistic effect on ERß
expression. The use of GG-AD cells, which express only ERß, allowed
us to demonstrate that this receptor subtype is functional and
transduces estradiol signal to the progesterone receptor. In summary,
the results of this investigation have revealed that ERß is expressed
in addition to ER in the rat decidua, and that the expression of
both ERs are cell specific and differentially regulated by PRL and
steroids. One salient finding of this investigation is that
progesterone down-regulates ER, but concomitantly increases the
expression of a functional ERß that mediates estradiol up-regulation
of the decidual progesterone receptor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein (9, 17, 18), but no evidence exists as to
whether the rat decidua expresses ERß as well.
ER and ERß are distinct gene products, but have high homology,
particularly in the DNA-binding domain (>90% amino acid identity) and
the C-terminal ligand-binding domain (55% homology) (19).
In addition, both ERs have similar binding affinity for physiological
ligands and estrogenic substances (20). Differences in the
distribution and relative expression of ER and ERß isoforms in
various rat tissues have been observed (20), although the
ovaries and uterus express both receptors.
The rat decidua is formed by two major cell populations localized in
mesometrial or antimesometrial sites of the uterus (21).
The attachment of the blastocyst, an event that is dependent on
estradiol in the progesterone-primed uterus of the rat, occurs in the
antimesometrium, which develops into the decidua capsularis in the
pregnant rats. The mesometrial cells, which form the decidua basalis in
the pregnant animal, are the site of trophoblast invasion. Expression
of ER has been previously shown to be localized to this cell type
(7, 8, 9, 16, 17, 18).
Two recent investigations using ER knockout (ERKO) mice have
demonstrated that progesterone alone is enough to induce stromal cell
decidualization in response to artificial stimulation of the uterus
(22, 23). However, although Curtis et al.
(22) found that decidualization is still dependent on
estrogen in the wild-type animal, confirming the results reported
previously by other groups (24, 25, 26, 27), Paria et
al. (23) showed that progesterone alone is able to
induce a decidual response in both wild-type and ERKO mice uteri. As no
ERß messenger RNA (mRNA) could be detected in the uterus of either
wild-type or ERKO mice (28), these studies suggest that
neither ER nor ERß is necessary for decidualization. Because one
role of estrogen in uterine decidualization was thought to be the
induction of the progesterone receptor (PR), it is not clear how the PR
is induced in ERKO mice. Indeed, decidualization of the endometrial
stromal cells involves a 4- to 5-fold increase in the level of PR in
ERKO mice (23). However, the situation seems to be
different in the rat, as ERß mRNA was shown to be expressed in the
uterus (20). This prompted us to examine whether the rat
decidua expresses the ERß gene. The results of this investigation
have revealed for the first time that ERß, in addition to ER, is
expressed in the rat decidua. We have also shown a developmental and
subcellular localization of both ERs, a differential regulation of
ER and ERß by PRL and steroids, and the ability of decidual ERß
to transduce the estradiol signal for the up-regulation of the
progesterone receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]dGTP were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL); the oligonucleotides
used as primers in the RT-PCR analysis were obtained from Life Technologies, Inc. (Grand Island, NY); tissue culture medium
(RPMI 1640), antibiotic-antimycotic solution, nonessential amino acids,
and sodium pyruvate were purchased from Mediatech (Washington DC); FBS
was purchased from HyClone Laboratories, Inc. (Logan, UT);
trypsin-EDTA was obtained from Life Technologies, Inc.;
17ß-estradiol was obtained from Steraloids, Inc. (Wilton, NH);
progesterone, phenylmethylsulfonylfluoride, leupeptin, pepstatin
A, aprotinin, and all other reagent grade chemicals were purchased from
Sigma (St. Louis, MO); ovine PRL (oPRL; PRL-18; 30 IU/mg),
the polyclonal ER-715 antibody and the ER-715 antigenic peptide were
gifts from the NIDDK, NIH (Bethesda, MD); the polyclonal ERß antibody
was purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY); and the antigenic peptide was kindly provided by Dr. N.
Ben Jonathan (University of Cincinnati, Cincinnati, OH).
Animal model
Pseudopregnancy was induced in Holtzman female rats by mating
them with vasectomized males at the Harlan facilities (Harlan Sprague Dawley, Inc., Madison, WI). The day a vaginal plug was
found was designated day 1 of pseudopregnancy. Rats were kept under
controlled conditions of light (14 h/day; lights on, 0500–1900 h) and
temperature (22-24 C), with free access to standard rat chow and
water. All experiments were conducted in accordance with the principles
and procedures of the NIH Guide for the Care and Use of Laboratory
Animals and were approved by the institutional animal care and use
committee. Decidualization of uterine endometrium was induced by
scratching the antimesometrial surface of both uterine horns with a
hooked needle on day 5 of pseudopregnancy under ether anesthesia. For
the developmental studies, rats were used at various stages of
pseudopregnancy from days 9–13. Decidualized uterine horns were
isolated and washed thoroughly in ice-cold PBS to remove excess blood.
The antimesometrial and mesometrial regions were separated as described
by Martel et al. (16). All tissue was frozen in
liquid nitrogen and stored at -80 C until processed for RNA or protein
preparation.
Primary cell culture
Total decidual tissue obtained from three to five rats on day 9
of pseudopregnancy were minced and incubated for 1 h with 50 U/ml
collagenase, 2.4 U/ml dispase, and 200 U/ml deoxyribonuclease in a
water-jacketed cell stir (Wheaten Scientific, Millville, NJ) at 37 C
under mild agitation. At the end of the incubation, dispersed cells
were filtered and centrifuged at 200 x g for 10 min.
Cells were gently resuspended in RPMI 1640 medium containing 2 x
antibiotic-antimycotic solution (200 U/ml penicillin G, 0.5 µg/ml
amphotericin B, and 200 µg/ml streptomycin), 1 x nonessential
amino acids, 1 mM sodium pyruvate, 0.45%
D-glucose, and 10% FBS. Cells (1.2–1.5 x
106) were seeded in six-well plates and incubated
at 37 C in a 95% air-5% CO2 humidified
atmosphere. Cells were allowed to attach for 3–4 h, washed, and then
treated for 12 h with different hormones in RPMI 1640 phenol-red
free medium supplemented with 1% charcoal dextran-stripped FBS. After
treatment, cells were washed twice with ice-cold PBS and stored at -80
C until RNA extraction.
Although progesterone is needed for the in vitro differentiation of stromal cells into decidual cells, no progesterone is required for maintaining decidual cells in vitro (29, 30, 31, 32). Our decidual cells cultured in FBS-DCC retained all of the characteristics of decidual cells and never dedifferentiated.
GG-AD cell culture
The temperature-sensitive GG-AD cells derived from rat
antimesometrial decidual cells (33) and the GG-AD cells
stably transfected with the long form of the PRL receptor
(5) were grown at 33 C in RPMI 1640 medium supplemented
with 10% FBS, 2 x antibiotic-antimycotic solution (200 U/ml
penicillin G, 0.5 µg/ml amphotericin B, and 200 µg/ml
streptomycin), 1 x nonessential amino acids, 1 mM
sodium pyruvate, and 0.45% D-glucose in a 95% air-5%
CO2 humidified atmosphere. These cells have
retained morphological characteristics of antimesometrial cells; they
are polynucleated, large, and have a cytoplasm filled with lipids
droplets. They also express the same mRNA as antimesometrial cells,
such as activin ßA and decidual PRL-related protein (dPRP).
Before each experiment, cells were cultured for 24 h at 39 C to allow cell differentiation. Cells were then treated with either oPRL (0.01–10 µg/ml) or estradiol (0.01–100 ng/ml) for 24 h in RPMI 1640 phenol-red free medium supplemented with 1% charcoal-dextran-treated FBS. At the end of the incubation, the cells were washed with PBS and stored at -80 C for RNA or protein isolation.
RNA isolation and RT-PCR analysis
Total RNA from frozen decidual tissue was purified using
TriReagent (Sigma) according to the manufacturer’s
instructions, whereas total RNA from primary decidual cells was
isolated by a one-step guanidinium-thiocyanate-phenol-chloroform
extraction procedure (34).
For mRNA analysis by RT-PCR, oligonucleotide primer pairs were based on
the sequences of the rat ER gene (35)
(5'-AATTCTGACAATCGACGCCAG-3' and 5'-GTGCTTCAACATTCTCCCTCCTC-3'), the
rat ERß gene (19) (5'-GTCCTGCTGTGATGAACTAC-3' and
5'-CCCTCTTTGCGTTTGGACTA-3'), and the rat progesterone receptor gene
(5'-CCCACAGGAGTTTGTCAAGCTC-3' and 5'-TAACTTCAGACATCATTTCCGG-3')
(36). Primers for either L19
(5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-CGTTCACCTTGATGAGCCCATT-3')
(37) or S16 (5'-TCCAAGGGTCCGCTGCAGTC-3' and
5'-CGTTCACCTTGATGAGCCCATT-3') (38) ribosomal proteins mRNA
were included to normalize the data. The predicted sizes of the
PCR-amplified products were 344, 285, and 325 bp for ER, ERß, and
progesterone receptor, respectively. One to 2 µg total RNA were
reverse transcribed at 42 C using the Advantage TM RT for PCR kit
(CLONTECH Laboratories, Inc., Palo Alto, CA). The 20-µl
reaction mixture containing 20 pmol random hexamer primer, 10 pmol
oligo(deoxythymidine)18 primer, 1 x
reaction buffer, 0.5 mM dNTP, 20 U RNase, and 200 U Moloney
murine leukemia virus reverse transcriptase was diluted to 100 µl by
adding diethylpyrocarbonate-treated water at the end of the RT
reaction. The complementary DNA (cDNA) was either used immediately for
PCR or stored at -20 C until use. For PCR amplification, a mixture
containing oligonucleotide primers (20 pmol),
[-32P]dCTP (2 µCi of 3000 Ci/mmol), dNTP,
and Taq polymerase (0.8 U) was added to 2–10 µl cDNA. The
total volume was increased to 40 µl with 1 x PCR buffer, and
the samples were overlaid with 50 µl mineral oil. For PCR
amplification of the gene products, cDNA was amplified for five cycles
with high annealing temperature (annealing temperature of the primer
plus 4 C) to increase specificity, and then amplified for 20–30 cycles
using 94 C for denaturing, 62-65 C for annealing depending on the
primer, and 71 C for extension in a Robocycler Gradient 40
(Stratagene, La Jolla, CA). The conditions were such that
amplification of the product was in the exponential phase, and the
assay was linear with respect to the amount of input cDNA. Reaction
products were electrophoresed on an 8% polyacrylamide nondenaturing
gel. After autoradiography, data were quantified using a
Molecular Dynamics, Inc. PhosphorImager and ImageQuant
version 3 software (Molecular Dynamics, Inc., Sunnyvale,
CA).
Molecular cloning of the rat decidual ERß by PCR
Total RNA obtained from decidua day 13 pseudopregnant rats was
isolated, and RT-PCR was performed using oligonucleotides primers as
described above for RNA isolation and RT-PCR analysis. The predicted
size of the PCR product was 285 bp. The PCR product was electrophoresed
on a 0.7% agarose gel. Only one band was detected by ethidium bromide
at the expected size (285 bp). The cDNA fragment was extracted from the
agarose gel, purified, and subcloned into the pGEM-T Easy vector
(Promega Corp., Madison, WI). DH5-competent cells
(Life Technologies, Inc.) were then transformed with this
vector. Three clones were selected and sent to the DNA Sequencing
Facility of the University of Chicago for DNA sequencing.
Northern blot analysis
Poly(A)+ mRNA (10 µg) obtained from
decidual tissue day 12 pseudopregnancy was isolated by the
oligo(deoxythymidine)-cellulose method using an Ambion, Inc., isolation kit (Austin, TX). RNA was fractionated through a
1% agarose gel containing 0.74 M formaldehyde and
transferred to a GeneScreen nylon membrane, NEN Research Products (Boston, MA) by overnight capillary blotting with
10 x sodium chloride-sodium citrate buffer (SSC buffer; 1.5
M sodium chloride and 150 mM sodium citrate, pH
7.0). Membranes were backed at 80 C under vacuum for 2 h. A 750-bp
ERß fragment kindly provided by Dr. Park Sarge was used to synthesize
the -32P-labeled riboprobe with SP6 RNA
polymerase as outlined by the vendor (Promega Corp.). RNA
blot hybridization with the complementary RNA (cRNA) ERß probe was
performed at 42 C in 50% deionized formamide, 4 x SET [600
mM sodium chloride, 80 mM Tris (pH 7.8), and 4
mM EDTA], 0.2% polyvinylpyrrolidone, Ficoll, BSA, and 8%
dextran sulfate. The final oligonucleotide probe concentration was
2 x 107 cpm/ml. Blots were hybridized for
24 h, then washed with 1 x SSC (containing 0.1% SDS) at 25
C for 15 min, followed by 0.2 x SSC (containing 0.1% SDS) at 42
C for 15 min and finally 0.2 x SSC (containing 0.1% SDS) at 55 C
for 15 min. The resultant blots were exposed to Kodak
X-OMAT film (Eastman Kodak Co.) using intensifying screens
at -80 C.
Immunocytochemistry
Primary decidual cells obtained from day 9 pseudopregnant rats
were grown for 24 h in RPMI 1640 phenol red-free medium
supplemented with 1% charcoal-dextran-treated FBS on sterile
coverslips (13-mm diameter) in four-well plates (Nunc, Naperville, IL).
At the end of the incubation, the cells were washed twice in PBS and
fixed for 10 min in PBS-4% paraformaldehyde solution at room
temperature. The cells were then washed three times in Tris-buffered
saline (TBS; pH 7.6) and permeabilized for 15 min at room temperature
in TBS-10% BSA, 0.1% Triton X-100, and 0.2% Tween-20 solution. After
one more wash in TBS, the cells were incubated overnight at 4 C with
either a polyclonal antibody to ER (1:100 final dilution; ER-715,
provided by the NIDDK) or a polyclonal antibody to ERß (10 µg/ml
final dilution; Upstate Biotechnology, Inc., Lake Placid,
NY) in TBS-1% BSA. Control cells were treated with TBS-1% BSA alone.
The cells were exposed for 3 h at room temperature to a
TRITC-conjugated antirabbit IgG (1:200 final dilution). The
coverglasses were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA) containing a counterstain
for DNA (DAPI-4',6-diamino-2-phenylindole) and observed with a
Carl Zeiss LSM 510 laser confocal microscope (Oberkochen,
Germany) equipped with a 40x water immersion objective lens (NA 1.2).
The ER polyclonal antibody (ER-715) was raised against a peptide
corresponding to a 15-amino acid sequence lying in the hinge region of
the rat ER. The antirat ERß was raised against a synthetic peptide
representing amino acids 54–71 of rat ERß. It recognizes
specifically ERß and does not detect ER protein.
Immunoblot analysis
To isolate decidual protein, tissue was homogenized in 2 ml
ice-cold homogenization buffer containing 25 mM Tris-HCl
(pH 7.4), 2 mM MgCl2, 1
mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1
mM dithiothreitol, 1 µM leupeptin, 1
µM pepstatin A, and 1 µg/ml aprotinin in a glass
homogenizer.
To obtain protein from cultured cells, cells were washed at least twice
in cold PBS after treatment and were scraped off the culture dishes
using a rubber policeman. The cells were then disrupted with a
26.5-gauge needle/1-cc syringe in homogenization buffer. The protein
concentration of the homogenates was determined as described by
Bradford (39). Fifty to 100 µg of protein were mixed
with an equal volume of 2 x Laemmli buffer and heated for 5 min
at 100 C. Equivalent amounts of protein were separated through 12%
SDS-PAGE gels under reducing conditions in electrophoresis buffer [25
mM Tris-HCl (pH 8.3), 192 mM glycine, and 0.1%
SDS] according to the method of Laemmli (40). Proteins
were then electrotransferred to nitrocellulose membranes (0.2 µm;
Protan, Schleicher & Schuell, Inc., Keene, NH) in cold
transfer buffer [20 mM Tris-HCl (pH 8.3), 192
mM glycine, and 20% methanol]. Immunoblotting was
performed by first blocking nonspecific binding sites with 5% nonfat
milk in TBS buffer [20 mM Tris-HCl (pH 7.6) and 137
mM NaCl] containing 0.1% Tween-20. Blots were then
incubated overnight with either ERß antibody at a dilution of 2
µg/ml or ER antibody at a dilution of 1:750. The membranes were
then washed with TBS-Tween 0.1% and incubated with a secondary
antibody linked to horseradish-peroxidase-labeled antirabbit IgG
(Sigma) for 2 h. After extensive washing, blots were
developed using an enhanced chemiluminescence Western blotting system
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and
exposed for 1–5 min to x-ray film (Biomax MR, Kodak). The
specificity of the stained protein band was verified by saturating the
antisera with excess antigenic peptide (2 and 1.25 µg/ml,
respectively, for ERß and ER) overnight before exposure to a
blotted membrane. The molecular sizes of the immunoreactive bands were
estimated by comigration of prestained SDS-PAGE mol wt standards
(Benchmark, Life Technologies, Inc.).
Statistical analysis
Data were examined by one-way ANOVA, followed by Duncan’s
multiple range test. When appropriate, Student’s t test was
used. P < 0.05 was accepted as statistically
different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß in decidual cells in primary
culture and/or
ERß, primary cells obtained from total decidual tissue of day 9
pseudopregnant rats were cultured for 24 h as described in
Materials and Methods and were subjected to
immunocytochemical analysis with either ER or ERß antibodies.
Although no fluorescence was observed in control cells incubated
without primary antibody (Fig. 1A
),
immunoreactive ER was detected in both cytoplasm and nuclei of
decidual cells (Fig. 1B). Immunoreactive ERß was observed as
punctuate regions only within the nuclei (Fig. 1C). To confirm the
nuclear localization of ER and ERß proteins, we have performed
optical sections in the z-axis of the cells. Immunostaining
of both ER (Fig. 1D) and ERß (Fig. 1E) was clearly located within
the nuclei.
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and ERß mRNA in the rat
decidua and ERß expression is tissue
specific and developmentally regulated, decidual tissue were collected
on different days of pseudopregnancy and separated into mesometrial and
antimesometrial decidua as previously described by Martel et
al. (16). Total RNA was isolated and subjected to
RT-PCR with L19 as an internal control. The results shown in Fig. 2 revealed that both forms of the ER mRNA
are expressed in the rat decidua and that ERß mRNA is preferentially
expressed in the antimesometrial tissue, with little expression in the
mesometrial decidua throughout pseudopregnancy (Fig. 2A). ER mRNA is
also mainly found in the antimesometrial decidua on days 10 and 11 of
pseudopregnancy, but becomes expressed in both antimesometrial and
mesometrial decidual tissue later in pseudopregnancy (Fig. 2B).
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and
the product amplified by PCR in Fig. 2 was ERß, RNA was obtained from
decidual tissue of day 13 pseudopregnant rats and was amplified by
RT-PCR. The PCR product was directly cloned into the pGEM-T Easy
vector. Three clones were selected for DNA sequencing. As shown in Fig. 3A, the three clones were similar and
identical to the rat ERß sequence previously described in the
prostate (19). In all three clones, one base was shown to
be different from the original published sequence: the A165 was
substituted by a G. Northern blot analysis in Fig. 3B showed that the
cRNA probe synthesized, as described in Materials and
Methods, specifically hybridized to decidual ERß mRNA. As
previously described in other tissues (41, 42), several
transcripts were observed for ERß in the rat decidua.
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and ERß mRNA expression by PRL, rat placental
lactogen I (rPL-I), and steroids in decidual cells in primary
culture, the expression of both ER (upper panel) and ERß
(lower panel) mRNA was stimulated by PRL treatment. As
rPL-I, a trophoblast-produced protein, also binds to the PRL
receptor, we examined the effect of this hormone on ER mRNA expression.
The results shown in Fig. 3B indicate that within 12 h of culture,
rPL-I stimulates both ER and ERß mRNA expression.
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(9, 18) regulation by steroids have led to
contradictory results. Therefore, we examined the regulation of
decidual ER and ERß by estradiol and progesterone. Estradiol had
no significant effect on ER mRNA levels, but up-regulated those of
ERß (Fig. 5A). The smallest dose of
estradiol (0.1 ng/ml) was highly effective, and no further stimulation
was seen at higher doses. Of great interest was the finding that
progesterone regulates the expression of ER and ERß in an opposite
manner (Fig. 5B). Whereas progesterone caused a dose-related
down-regulation of ER mRNA expression, it simultaneously induced an
increase in the expression of ERß.
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and
ERß expression. Progesterone or PRL alone caused an increase in
ERß, but no synergism was observed when both hormones were added to
decidual cells in primary culture (Fig. 6B). As shown in Fig. 6A, ER was
up-regulated by PRL and down-regulated by progesterone; however, the
inhibitory effect of progesterone on ER was not affected by the
addition of PRL to the culture, whereas the stimulatory effect of PRL
was totally obliterated.
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and ERß species. RNA and
protein isolated from GG-AD cells and from decidual tissue were
analyzed by RT-PCR (Fig. 7A) and Western
analysis (Fig. 7B). Decidual tissue from day 9 pseudopregnant rats was
used as a control. Whereas mRNA species for both forms of ER were
readily detectable in the rat decidual tissue, only the mRNA encoding
ERß could be detected in the GG-AD cells (Fig. 7A). The basal level
of ERß mRNA expression varied between cell cultures. However, this
variation was lower than the regulations observed in Fig. 8, when GG-AD cells were treated with PRL
or estradiol. Western analysis, using a polyclonal antibody raised
against ERß (Fig. 7B), revealed that GG-AD cells express an
immunoreactive ERß protein that migrates at the same molecular mass
(61 kDa) as the protein detected in the decidua. No signal could be
detected in either GG-AD or decidual tissue when the antiserum was
preabsorbed with an excess of the antigenic peptide, demonstrating the
specificity of the reaction. No ER protein could be detected in
these cells.
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. The results
showed that similar to its effect on primary cells (Fig. 3B), PRL
up-regulates, in a dose-dependent manner, ERß mRNA levels in GG-AD
cells stably transfected with the long form of the PRL receptor (Fig. 8A). In contrast, estradiol regulation of ERß in GG-AD cells differed
from that in decidual cells in primary culture and clearly showed a
biphasic effect (Fig. 8B). At lower concentrations, estradiol
stimulated ERß mRNA levels as previously observed in primary cells.
In contrast, at higher concentrations estradiol induced a marked
inhibition of ERß expression, suggesting that the presence of ER
may prevent estradiol-induced inhibition of ERß.
Biological activity of ERß in GG-AD cells
To determine whether estradiol can transduce its signal through
ERß in the decidua-derived GG-AD cells, we studied the effects of
different doses of 17ß-estradiol on the expression of the PR, a well
known estrogen target gene. The results depicted in Fig. 9 clearly indicate that estradiol is able
to markedly increase the expression of PR in these cells, which express
only ERß. Interestingly, high doses of estradiol (1 ng/ml) had no
stimulatory effect, probably because these levels of estradiol cause a
marked down-regulation in the level of ERß expression (Fig. 8B),
rendering the cells less sensitive to estradiol action.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, is expressed in the rat decidua. The molecular
cloning of our PCR product confirmed that the ERß mRNA detected in
the decidua is identical to the rat ERß previously cloned in the
prostate (19). The expression of ERß in the rat
decidua contrasts with reports from the mouse decidua, where very low,
if any, expression of ERß could be detected (48). Such a
discrepancy in the expression pattern of ERß between rat and mouse
was also described in the pituitary (49).
Previous studies have shown the presence of estrogen-binding sites in
the decidua of rats (7, 8, 9, 10, 11, 12, 13, 14, 15, 16). This binding activity is
probably due to both ER subtypes, which, as shown herein, are largely
confined to the antimesometrial cells where blastocyst apposition, a
process that depends upon estradiol and progesterone, takes place. The
cell specific localization of ER mRNA found in this investigation
confirmed previous studies that showed higher levels of
estradiol-binding sites in the larger polyploid antimesometrial cells
than in the smaller mesometrial cells (14). Interestingly,
the same observations were described in humans, in whom ER
immunostaining has been detected in greater amounts in the decidua
capsularis than in the decidua parietalis (50). The high
expression of the two ER isoforms in this specific cell population may
be due to decidual PRL. In the rat, the antimesometrial decidual cells
are known to express members of the PRL family such as PRL-like
proteins (51, 52) and dPRP (53), which
have no PRL-like activity, and PRL (43), which binds to
the PRL receptor in decidual cells and affects their function (5, 44). The role of decidual PRL in pregnancy is not yet
understood. However, the results of this investigation suggest that one
important action of this hormone is to act locally to up-regulate the
expression of both ER and ERß, rendering the decidua more
responsive to estradiol. rPL-I, secreted by the trophoblast later in
pregnancy (54), is also able to up-regulate decidual ER
expression, suggesting a trophoblast-decidual cell interaction in the
expression of this steroid receptor.
PRL appears to be a key up-regulator of the ER in many reproductive tissues, such as the ovarian corpus luteum (45) and the mammary gland (55). We have shown that PRL increases mRNA and protein levels of both ER subtypes in the corpus luteum and in a luteal-derived cell line and that this PRL up-regulation of ER is a prerequisite for estradiol stimulation of steroidogenesis and corpus luteum hypertrophy (45). PRL was also shown previously to increase the levels of estradiol-binding sites in the mammary gland (55) and to enhance estradiol stimulation of growth in breast tumor-derived cells (56). Whether this effect is due to PRL up-regulation of gene expression of either one or both ER subtypes remains to be investigated.
Whereas PRL up-regulates in concert and in a dose-related manner both
ER and ERß mRNA expression, estradiol up-regulation of ER is
subtype specific. Estradiol has no detectable effect on ER
expression at any dose used, but increases ERß mRNA at low doses in
both decidual cells in primary culture and GG-AD cells. In contrast,
high doses of estradiol caused a severe down-regulation of ERß mRNA
expression in GG-AD cells. This inhibitory effect was not seen in
decidual cells in primary culture. As the GG-AD cells express only
ERß, whereas decidual cells in primary culture coexpress both
subtypes, it is possible that ER in the primary cell culture system
may prevent the deleterious effect of high doses of estradiol on ERß
mRNA.
Of great interest was our finding that progesterone, a steroid
considered to inhibit ER expression in the uterus (57, 58)
and widely used to prevent estradiol-induced endometrial cell
proliferation, has opposite effects on the two ER subtypes in the
decidua; whereas progesterone decreases ER expression in a
dose-related manner, it concomitantly up-regulates that of ERß. This
finding may be of physiological importance and may explain at least in
part why some progesterone target cells can remain responsive to
estradiol despite progesterone treatment (59, 60). The
differential effect of progesterone on the two ER subtypes may also be
the reason why the ovaries express high levels of ERß and little
ER, as this gland is subjected to very high levels of locally
produced progesterone. As both PRL and progesterone up-regulate ERß,
whereas they have opposite effects on ER and because progesterone
was shown to enhance the ability of PRL to signal to a PRL-regulated
gene in mammary-gland tumor derived cells (48), we
examined the combined effects of PRL and progesterone on ER expression.
We showed that in the decidua, progesterone does not stimulate
PRL-mediated regulation of either ER or ERß. Quite to the
contrary, PRL stimulation of ER mRNA expression was totally
prevented by progesterone, which has, by itself, a strong inhibitory
effect on this receptor subtype. In addition, whereas both progesterone
and PRL each stimulated ERß mRNA expression, they had no synergistic
effect when added together to the cell culture, suggesting that both
hormones may be acting on ERß expression by a similar mechanism. The
mechanism by which PRL and progesterone signal to ER and ERß genes
in decidual cells remains to be investigated.
The finding that the rat decidua expresses ERß in addition to ER
led us to examine whether this receptor subtype transduces an estradiol
signal to the PR gene. In ERKO mice, the PR is up-regulated in uterine
stroma after the induction of decidualization, and the decidua is fully
progesterone responsive in terms of gene regulation and morphological
changes, suggesting that ER is not necessary for PR expression or
function leading to decidualization (22, 23). As estradiol
plays a key role in the induction of the PR, it is not clear whether in
mice ERß is responsible for such an effect. It is clear, however,
that in rat decidual cells, estradiol causes a marked stimulation of PR
mRNA expression through ERß. The development of the decidua-derived
cell line (GG-AD), which expresses only ERß, has allowed us to
establish that this receptor type is functional, stimulating gene
expression. Decidual ERß was shown to transduce a strong estradiol
inhibition of IL-6 and gp130 expression (5). This
estradiol effect prevents the decidual production and action of IL-6
(5), a cytokine known to be detrimental to the normal
progress of pregnancy. The present investigation suggests that ERß
may also play an important role in sensitizing the uterus to
progesterone action by inducing the PR, a process critical for the
initiation and maintenance of the decidual reaction. An interesting
finding of this investigation is the fact that, in contrast to
physiological concentrations, high levels of estradiol totally prevent
the expression of PR in GG-AD cells, probably by down-regulating ERß
and preventing ER signaling. This finding may explain at least in part
why high levels of estradiol cause abortion and collapse of decidual
tissue.
In conclusion, the results of this investigation have revealed for the
first time that the rat decidua expresses both ER and ERß genes
and that these genes are preferentially expressed in a cell population
localized in the antimesometrial site of the uterus that forms the
decidua capsularis in pregnant rats. The results also indicate that the
two ER subtypes are differentially regulated by PRL and ovarian
steroids, and that ERß can transduce the estradiol signal to the PR
gene in decidual cells.
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Acknowledgments |
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Footnotes |
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Received March 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology 138:863–870 knockout mouse uterus. Proc Natl Acad Sci
USA 96:3646–3651-deficient
mice. Endocrinology 140:2704–2710 (ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621 and ß in the rat corpus luteum of
pregnancy: regulation by prolactin and placental lactogens.
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3),
and the right panels show the densitometric analysis
(mean ± SEM of values expressed as a percentage of
the control, which was considered 100%). *, P <
0.05 compared with vehicle-treated controls (by one-way ANOVA, followed
by Duncan’s multiple range test).
