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Production in Endometrial Epithelial Cells in Vitro: Role of Cyclooxygenase-21
Département d’Ontogénie et Reproduction (E.A., P.D., M.A.F.), Centre de Recherches du Centre Hospitalier de l’Université Laval, Centre de Recherche en Biologie de la Reproduction, Ste-Foy, Québec G1V 4G2, Canada; and Département d’Obstétrique et Gynécologie (M.A.F.), Université Laval, Québec G1V 4G2, Canada
Address all correspondence and requests for reprints to: Dr. M. A. Fortier, Ontogénie et Reproduction, Centre de Recherche du Centre Hospitalier, de l’Université Laval, 2705 Boulevard Laurier, Sainte-Foy, Québec G1V 4G2, Canada. E-mail: mafortier{at}crchul.ulaval.ca
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Abstract |
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is produced by
endometrial cells, in response to oxytocin (OT). The mechanism by which
OT induces the release of PGF2 remains to be defined. We
have used 13 different cultures of bovine epithelial endometrial cells
to study the effect of OT on the regulation of PGF2 and
to identify the possible involvement of cyclooxygenases (COXs). OT
induced a dose-dependent increase of both inositol phosphates (IPs) and
[Ca2+]i concentration in epithelial cells
labeled with [3H]-myoinositol or loaded with fura-2
(using a fluorescent microscope imaging system), respectively. OT
induced a dose-dependent increase of both PGF2
production and COX-2 gene expression (as demonstrated by RT-PCR and
Northern blots). PGF2 production was increased from
13.3 ± 2.0 to 166.8 ± 22.5 ng/ml (P <
0.0001). On the other hand, COX-2/ß-actin mRNA gene expression (as
determined by densitometric analysis) was increased 5.1 ±
0.7-fold (P < 0.001) with OT (10-7
M) treatment, compared with control. Addition of
indomethacin (1 µM) and a specific COX-2 inhibitor
(NS-398, 1 µM) blocked the OT-induced PGF2
production. COX-1 and phospholipase A2 mRNA were expressed
at steady-state levels, but no effect of OT was detected on their
regulation. Combined to OT, 10 µg/ml of recombinant ovine
interferon-tau (roIFN-
) was able to decrease significantly
(P < 0.0001) the dose-dependent increase of
PGF2 production. Furthermore, partial bovine COX-1 (777
pb) and COX-2 (449 bp) cDNAs were cloned and sequenced. An homology of
83% and 97% was found in relation with rat and sheep, for COX-1,
respectively. COX-2 was found to bear 84%, 86%, and 87% of homology
in relation to rat, guinea pig, and human, respectively. Collectively,
these results demonstrate, for the first time, that COX-2 is involved
in the mechanism by which OT regulates PGF2 production
in the endometrium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PGF2), of uterine origin, is
responsible for luteolysis and the return to the estrous cycle. The
identification of the uterus as the source of luteolytic
PGF2 has been submitted to strict requirements, which
have been met in several species but not in primates. In primates,
PGF2 seems also to be responsible for luteolysis, but
its origin is likely within the ovary itself (2). In contrast with
PGF2, PGE2 may have a luteoprotective action
that is either luteotrophic or antiluteolytic (3, 4). PGE2
also is required for changes in vascular permeability that occur in the
endometrium before implantation in rodents (5, 6). Inhibitors of
prostaglandin synthesis, such as indomethacin, delay or inhibit these
changes and prevent implantation from occurring (7). In species such as
ruminants, in which PGF2 of uterine origin is
luteolytic, local recognition of the presence of a viable embryo is
necessary to prevent the release of PGF2 and ensure
establishment of pregnancy (8). The production of PGF2
by the uterus is responsible for the luteolytic signal during the
estrous cycle, and the administration of PGF2 is able to
terminate pregnancy in several species (9). In ruminants, the pulsatile
release of oxytocin (OT) by the neurohypophysis stimulates the
production of uterine PGF2. The ability of OT to
stimulate PGF2 release is higher at the time of
luteolysis (10). The initial released PGF2 triggers the
release of additional OT from the corpus luteum by a positive feedback
loop (11), and OT binds to endometrial OT receptors (OTR) to stimulate
PGF2 secretion (12, 13). We and others have previously
demonstrated that PGF2 is regulated by OT in
vitro (14, 15, 16). Oxytocin has been shown to activate the
inositol-specific phospholipase C in various cell systems (17, 18),
generating two second messengers: inositol triphosphate (which
increases intracellular calcium) and diacylglycerol (DAG) (which
activates protein kinase C) (19). Calcium in human decidual cells (20)
or the activation of protein kinase C in human amnion cells (17, 21)
and bovine endometrial cells (15) have been shown to increase PG
production.
In ruminants, large amounts of interferon (IFN)- are produced by the
trophoblast during the preimplantation period (22, 23) and serve as the
embryonic signal, released at the time of maternal recognition of
pregnancy, to prevent the luteolytic process from occurring. Recently,
we have demonstrated that roIFN- stimulated PGE2
production and COX-2 gene expression in the bovine endometrium in
vitro (24, 25), and others have shown that in the ovine, it
inhibits estrogen receptor necessary for induction of OT receptor
before luteolysis (26). When no viable embryo is present, a net
increase in PGF2 production is observed in response to
OT in vivo. We suggest that OT induces gene expression of
cyclooxygenase (COX) and/or phospholipase A2 (27) to effect
increased PGF2 production.
The COX protein is a rate-limiting enzyme involved in the biosynthesis
of PGs using arachidonic acid (AA) as its principal substrate (28, 29).
Two genes (COX-1 and COX-2) (30) encode this enzyme. The constitutive
isoform, COX-1, is expressed in all tissues (31) and most, if not all,
nucleated cells. On the other hand, the inducible form, COX-2, is
present only after induction by a variety of factors (such as CG,
cytokines, and tumor promoters) (32, 33, 34, 35). Other reports demonstrated
that COX-2 also was expressed during the implantation period in the rat
endometrium (36, 37). Phospholipase A2 also is a
rate-limiting enzyme for PGs production, because it acts directly on
phospholipids (phosphatidyl choline) to release AA, necessary for PGs
synthesis (1). A recent study, using sheep uterine tissues,
demonstrated that COX-2 was highly, but transiently, expressed from
days 12–15 of the estrous cycle in vivo (38). In the same
study, COX-2 protein was expressed for an extended period in pregnant
animals. These results in vivo would support the results
obtained in vitro in our laboratory, where roIFN- induced
an up-regulation of COX-2 mRNA and PGE2 production (24, 25). Consistent with results obtained in vivo, the present
study was conducted in vitro, to determine the mechanism by
which OT regulates PGF2 production in endometrial
epithelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
used in the enzyme immunoassay (EIA), were purchased from Cayman
Chemical Co. (Ann Arbor, MI). All reagents used for the RT-PCR
(MgCl2, DTT, MMLV-RT, Taq polymerase, and
respective buffers) were purchased from Gibco BRL (Burlington, ON).
Indomethacin was purchased from Sigma (St. Louis, MO) and specific
COX-2 inhibitor (NS-398) from Biomol (Plymouth Meeting, PA).
Production of recombinant ovine (ro)IFN- and antiviral activity
assay
The roIFN- was provided by Dr. Fuller Bazer. It was produced
and purified as described previously by Ott et al. (39), and
antiviral activity of IFN- was determined as described by Pontzer
et al. (40). In the present study, the dose used was 10
µg/ml. The concentration of roIFN- used is in the physiological
range reported for this IFN to produce antiproliferative effects
in vitro (41) and PGE2 regulation in
vitro (24). Further, it has been shown that secretion of oIFN-
increases to about 10,000 ng/h on day 16 for sheep conceptuses (42),
and intrauterine injections of 100 µg/day on days 11–15 delay
luteolysis (39). The antiviral activity of roIFN- was 1 x
108 U/mg protein.
Isolation of endometrial cells and culture
Bovine uteri were collected at the slaughterhouse within 15 min
of death, and the physiological status of the tissue was estimated by
examination of ovarian morphology (43). Uteri were transported to the
tissue culture laboratory and dissected under a laminar flow hood. In
this study, a total of 13 early-cycle uteri (days 1–5) were used: 4
for COXs experiments (Northern blots and RT-PCR), 3 for inhibitor
experiments, 3 for roIFN- experiments, and 3 for IP measurements.
Endometrial epithelial cells were cultured in 6-well plates (for RNA
extraction) or 24-well plates (inhibitor assays and roIFN-
experiments), as described previously (16). Medium (RPMI-1640 + 10%
FBS-DC depleted of steroids by dextran-charcoal extraction) was changed
every 2 days until the cells were used. Confluency of epithelial cells,
isolated from endometrium in the beginning (days 1–5) of estrous
cycle, occurs after 6–7 days in culture.
Experimental protocol
After the cells reached confluency, the medium was replaced with
2.0 ml of fresh serum-free RPMI-1640 containing increasing doses of OT.
One plate was used for each concentration tested. The OT vehicle
(water) was added to control plates. Cells were then incubated at 37 C
in an atmosphere of 5% CO2: 95% air for 24 h. For
all experiments, at the end of the incubation period, culture medium
was recovered for PG measurements and stored at -20 C until further
processing. Cells were recovered for RNA extraction or for DNA
measurement (inhibitor assays and roIFN- experiments).
COX inhibitors assays and roIFN- experiments
After the cells reached confluency, the medium was replaced with
1.0 ml of fresh serum-free RPMI-1640 containing OT (10-7
M), combined or not with indomethacin (1 µM)
or NS-398 (1 µM) in triplicates for 24 h at 37 C in
an atmosphere of 5% CO2: 95% air. Using three different
epithelial cell cultures, different doses of OT were added combined or
not with roIFN- (10 µg/ml) for 24 h. The OT vehicle (water)
was added to control wells. At the end of the incubation period,
culture medium was recovered for PG measurement and stored at -20 C
until further processing. The plates were rinsed with ethanol, and DNA
content was determined for each well by 3,5 diamino benzoic acid (DABA)
fluorescence, according to Fiszer-Szafarz et al. (44).
RT-PCR
Total RNA (400 ng) from each OT dose and controls for each cell
type were used for preparation of first-strand cDNA by RT. The RNA
samples were incubated with 0.5 µg oligo (deoxythymidine) primers at
65 C for 10 min in a final vol of 10 µl and then put on ice. Samples
were then incubated 60 min at 37 C in a reaction mixture containing
buffer (1X), 10 mM DTT, 1.25 mM deoxynucleotide
triphosphate (dNTP), and 200 U Muloney murine leukemia virus RT
(MMLV-RT) in a final vol of 20 µl. After incubation, reaction volumes
were brought up to 65 µl. A negative control was performed at the
same time, using the same reaction mixture, substituting water for the
MMLV-RT, to ensure absence of any contaminating genomic DNA in the RNA
template.
Expression of the COX-1 gene was determined by amplification of a 777-bp region of the sheep (45) COX-1 cDNA sequence (370–1144). Amplification was carried out using the antisense downstream sequence 5'-TCC AAC CTT ATC CCC AGC C-3' and the sense upstream sequence 5'-CAT GGC GAT GCG GTT GC-3'. For COX-2, expression of the gene was determined by amplification of a 449-bp region of the human (30) COX-2 cDNA sequence (411–858), and the sequences of the primers were 5'-TCC AGA TCA CAT TTG ATT GAC A-3' and 5'-TCT TTG ACT GTG GGA GGA TAC A-3'. Primers sequences were chosen from homologous nucleotide sequences of published human (30) and rat (46) COX-1 cDNAs and sheep (45) and rat (46) COX-2 cDNAs. After amplification, COX-1 and COX-2 cDNAs were cloned in pCR 2.1 plasmid using a InvitroGene cloning kit. The cDNA were sequenced by the sequencing service (Laval University, Québec) using a dideoxy PCR technique. The bovine COX-1 and COX-2 cDNAs were submitted to the GenBank database (accession numbers AF004943 and AF004944, respectively).
As a positive control for each RNA preparation, a ß-actin sequence was also amplified simultaneously in adjacent tubes. A 458-bp fragment was amplified using bovine ß-actin cDNA primers 5'-GAG GAT CTT CAT GAG GTA GTC TGT CAG GTC-3' and 5'-CAA CTG GGA CGA CAT GGA GAA GAT CTG GCA-3'. All other conditions were identical to those for COX cDNA amplification. All primers were chosen with the aid of the OLIGO 4.01 primer analysis software (National Biosciences, Inc., Plymouth, MN). Each reaction contained 5 µl of RT template or negative control, 1.5 mM MgCl2, 1x Buffer, 0.2 mM dNTPs, 10 µM of each primer, and 1.5 U Taq polymerase in a final volume of 50 µl. The PCR cycling conditions chosen were 30 sec at 94 C, 30 sec at 55 C, and 30 sec at 72 C for 30 cycles, followed by a 10-min extension at 72 C. Reaction products were analyzed on 0.7% agarose gels. Bands were visualized by ethidium bromide staining. Each experiment was performed four times using different epithelial cell preparations.
RNA isolation and Northern blots
Total RNA was prepared according to Chomczynski and Sacchi (47).
Briefly, after homogenizing the cells in 4 M guanidium
thiocyanate, RNA was extracted with phenol/chloroform-isoamyl alcohol
(42:1), precipitated with isopropanol and washed with 70% ethanol.
Total RNA (20 µg) was loaded on a 1% agarose-2.2 M
formaldehyde gel and transferred to nylon membrane (Quiagen,
Chatsworth, CA). After 4 h of prehybridization in a 50% formamide
solution at 42 C, the membrane was sequentially hybridized overnight at
42 C in the same solution, to which was added a
[32P]dCTP-labeled cDNA probe corresponding to bovine
COX-1 and COX-2 (sequences determined above), human PLA2
mRNA, and [32P]dCTP-labeled ß-actin human cDNA. The
[32P]-labeled DNA probes were produced by random priming
protocol using a T7 Quickprime kit (Pharmacia Biotech, Baie
D’Urfé, Québec). Membranes were washed and exposed to
x-ray film with an intensifying screen at -80 C for 1 (ß-actin) to
24 h (COX-1, COX-2, and PLA2). Membranes were stripped
between hybridizations with a boiling 0.1% SSC, 0.5% SDS solution.
Autoradiograms were quantified by densitometry using Whole Band
Analysis (WBA) software on BioImage. Each experiment was performed four
times using different epithelial cell cultures.
Measurement of [Ca2+]i
Endometrial cells were grown on 25-mm glass coverslips placed in
6-well plates. At confluency, cells were loaded for 30 min at 22 C with
2 µM Fura-2/AM (from a 1 mM stock solution in
DMSO) in 1 ml RPMI 1640 culture medium. After the loading period, cells
were rinsed with Hanks’ buffered saline solution (HBS), containing
(mM): NaCl, 130; KCl, 3.5; CaCl2, 1.1; MgCl2,
0.1; NaHCO3, 5; HEPES, 20; supplemented with 1% glucose.
Hydrolysis of Fura-2-loaded cells was continued for 30–60 min at 22 C
in the same HBS buffer. All these manipulations were conducted in the
dark. Before use, the cells were washed three times with HBS buffer.
The coverslips were mounted on a 2-ml chamber and placed over an
Axiovert 100 HD/DIC-TV inverted epifluorescence microscope (Zeiss,
Germany). The light source was generated by a 100-W mercury-xenon lamp
(Hamamatsu Photonics, Japan), and the two wavelengths (340 and 380 nm)
were provided by two monochromators. Fluorescent images were collected
by an intensified charge-coupled device camera (Hamamatsu Photonics,
Model Sony XC-75). A commercial image analysis software (Metafluor
v2.0, Universal Imaging Corp., West Chester, PA) was used for data
acquisition, monitoring, and image analysis. Stimulation of cells was
achieved by adding 10 µl HBS, containing OT (10-7
M final concentration), near the recording site.
Total inositol-phosphates measurement
When cells reached confluency, 24 h before stimulation with
OT, medium was replaced with TCM-199 (Gibco BRL, Life Technologies,
Inc.) containing 10% FBS-DC and 1 µci/ml myo-3H-inositol
(Amersham, Oakville, Ontario, Canada). Inositol-phosphates were
measured, as described previously (with a slight modification) (48).
Medium was aspirated, and cells were incubated for 15 min at 37 C in
the presence of Kreb’s solution gased with 5% CO2, 95%
O2 and containing 20 mM lithium chloride
(LiCl). The enzymatic assay was conducted at 37 C by adding OT
(10-7 M) for 10 min, and the reaction was
stopped by addition of an equal volume of ethanol/HCl 0.02 N. Plates
were stored at -20 C until further processing. All samples were
lyophilized and resuspended in 1 ml of water and eluted on Dowex
columns. To remove free 3H-inositol, the columns were
rinsed with H2O and 6 ml of 5 mM sodium
tetraborate/60 mM ammonium formate (NH4COOH).
Then, 6 ml of 1 M NH4COOH/0.1 M
HCOOH were added to remove total 3H-inositol-phosphates.
Samples were recuperated separately in scintillation vials and counted
with a ß counter in 10 ml scintillation liquid. The results were
expressed in percent of control.
Enzyme immunoassays (EIA) of prostaglandins
For PGE2 and PGF2 measurements, an
enzyme immunoassay technique (EIA) was used, which used
acetylcholinesterase-linked PG tracers, as described previously (16).
We have used fully characterized rabbit anti-PGE2 (49) and
sheep anti-PGF2 (Bio Quant, Ann Arbor, MI). The inter-
and intraassay coefficients of variation (n = 12) were 16% and
10%, respectively.
Statistical analysis
Data were analyzed by ANOVA using super ANOVA software (ABACUS
Concepts, Inc., Berkeley, CA). Sources of variation included effects
caused by treatments and their interactions. The effect of the
different dose-responses was determined using orthogonal
contrasts.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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regulation by OT in epithelial cells
(13.3 ± 2.0 vs. 1.8 ± 0.5 ng/ml for
PGE2) is the major prostaglandin produced in nonstimulated
epithelial cells (Fig. 1
). OT increased
(P < 0.0001) PGF2 production up to
166.8 ± 22.5 ng/ml in a dose-dependent manner, whereas
PGE2 production also was stimulated in a dose-dependent
manner, but proportionately at a lower level (23.3 ± 3.6 ng/ml at
the highest dose used).
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). Because IP
production is thought to mediate mobilization of
[Ca2+]i, we next evaluated, using a
fluorescent imaging system, the effects of OT on
[Ca2+]i concentrations (Fig. 3). OT (10-7 M)
caused a rapid increase in [Ca2+]i in
epithelial cells. This increase was rapid in onset, with a spike-type
increase achieved within 5 sec of stimulation, followed by a gradual
reduction to near basal levels.
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production described above is effected,
steady-state levels of the mRNAs for COX-1, COX-2, and PLA2
were determined (Fig. 4). In epithelial
cells, a 2.8-kb transcript was detected by hybridization with the COX-1
cDNA probe. The abundance of this transcript was not altered by OT
treatment consistent with its housekeeping function. However, after
exposure to OT, the 4.1-kb COX-2-associated mRNA was increased in a
dose-dependent fashion (P < 0.001), as determined by
densitometric analysis, and was increased 508 ± 71%, compared
with control. Under unstimulated conditions, COX-2 transcript was
slightly detectable. The apparent increase of the 3.4-kb transcript
associated with PLA2 mRNA was not significant
(P > 0.1). Similarly, COX-1 expression was not
affected. OT had no effect on ß-actin mRNA gene expression used as
control.
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). As for Northern blot analysis,
ß-actin was used as a control, and a 349-bp band was observed in all
samples, and no variation was observed. Results demonstrate that COX-1
is present in epithelial cells and is not regulated by OT. On the other
hand, OT induced a dose-dependent increase of COX-2 mRNA in epithelial
cells. These fragments were cloned respectively in pCR 2.1 plasmid,
sequenced, and used as cDNA probes for Northern blots. Figure 6 shows the sequences of both bovine
COX-1 and COX-2 mRNA. An homology of 83% and 97% was found in
relation with rat (46) and sheep (45) for COX-1, respectively. On the
other hand, COX-2 was found to bear 84%, 86%, and 87% of homology in
relation to rat (46), guinea pig (51), and human (30),
respectively.
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production production (Fig. 7),
as described previously in Fig. 1
. Addition of indomethacin (1
µM) alone inhibited significantly (P <
0.0001) PGF2 synthesis, compared with control (1903
± 390 vs. 342 ± 35 pg/µg DNA). Combined with OT,
indomethacin inhibited the stimulation of PGF2
production (P < 0.0001). Addition of a specific COX-2
inhibitor (NS-398, 1 µM) also blocked the effect of OT on
PGF2 production (P < 0.0001) but had no
effect on basal PGF2 production.
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on OT-induced PGF2
production on PGF2 production
induced by OT is shown in Fig. 8. As
demonstrated in Fig. 1, OT stimulated PGF2 production in
a dose-dependent manner. However, in the presence of roIFN- (10
µg/ml), the stimulation of PGF2 production was
decreased significantly (P < 0.0001), at
10-7, to 10-5 M of OT.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(52, 53). The source of PGF2 is from
the endometrium in vivo. Thus, cultured epithelia have been
used to study the effect of OT on the regulation of prostaglandins. The
binding of OT to its receptor, coupled to a G protein, activates a
phospholipase C, which in turn, activates phosphoinositide turnover and
generates IP3 (54) and DAG. IP3 is known to
cause mobilization of intracellular Ca2+
([Ca2+]i) stores, whereas DAG can activate a
protein kinase C (PKC). The first part of this study was to confirm
that OT could regulate IPs and Ca2+ in cultured endometrial
epithelial cells. A recent study, using gonadotrope-derived T3 cell
line, demonstrated that OT was able to induce IP production and
increase cytosolic Ca2+ (55). Using a fluorescent
microscope imaging system, we were able to demonstrate a direct effect
of OT on [Ca2+]i mobilization only at the
epithelial level. The results obtained in the present study were
comparable with results obtained with the T3 cell line, suggesting
that in the endometrium, OT acts also on a G protein-coupled receptor.
We have previously demonstrated that activation of PKC is necessary for
OT response (15), and now we demonstrate that IPs and Ca2+
are second messengers for OT action in the regulation of
PGF2 production.
In terms of PGs production, our previous results (15, 16) are in
agreement with the literature suggesting that OT response is limited to
epithelial cells of the endometrium, inducing a 12.5-fold increase of
PGF2 production. However, the intracellular mechanisms
involved in the regulation of PGF2 in the endometrium
are poorly understood. In ovine, a recent study has proposed a possible
role of phospholipase A2 (PLA2) in the OT-induced
PGF2 production (27). The exclusive role of
PLA2 in the process is unlikely because, in that study, an
inhibitor and a stimulator of PLA2 activity were used to
characterize the effect of OT. Stimulation of PLA2 with
fluoride induced the release of AA in all cases, but an inhibition of
the PGF2 production was reached only at the highest dose
of aristolochic acid (10-4 M) used as a
PLA2 inhibitor. Furthermore, in the present study, the
results demonstrate that OT had no significant effect on the regulation
of PLA2 gene expression. It still is possible, however,
that OT may stimulate the activity of the PLA2 protein.
However, to support our findings, a more recent study showed that COX-2
was expressed during days 12–15 during the estrous cycle in the ewe
uterus, showing at least that PLA2 is not the only factor
regulated around the time of luteolysis (38).
In the biosynthetic pathway of PG production, COX is a rate-limiting
suicide enzyme, which uses AA as a substrate (56). The results in the
present study demonstrate the first evidence that COX-2 is expressed in
bovine endometrium and that OT up-regulates COX-2 mRNA gene expression.
The constitutive form of COX (COX-1) did not vary with OT treatment but
seems to be more abundant than COX-2, even at its maximally stimulated
level. The abundance of the COX-1 transcript is not surprising, given
the suicidal nature of the catalytic process and its housekeeping
function (56). The results of the present study, therefore, would
suggest regulation of COX-2, not only at the transcription but also at
the translation level, and this is supported by results obtained
by Charpigny et al. (38) in the ovine in vivo.
Another study in the ovine demonstrated that mRNA for PGHS
(prostaglandin synthase or COX) did not vary during the cycle (57).
However, the mRNA detected was 2.8 Kb and probably corresponds to the
constitutive COX-1, as demonstrated in the present study. The presence
of a specific inhibitor of COX-2 (NS-398) was able to block the
OT-induced PGF2 production, which supports the
hypothesis that COX-2 is necessary for the stimulation of
PGF2 production. In epithelial cells, under unstimulated
conditions, the major PG produced is PGF2. Thus, by
inducing COX-2 gene expression, the resulting OT effect is a net
increase of PGF2 production in these cells. This
preferential production of PGF2 in response to OT may be
simply an amplification of the conditions prevailing at the basal
level. The present study was conducted only in epithelial endometrial
cells because we have shown that OT response was not present in stromal
cells (16, 24, 25). Similarly, in a preliminary experiment, we have
found that OT receptors (OTR) were present in epithelial cells but not
in stromal cells or in the circular or longitudinal cells of the
myometrium in vitro (data not shown). In epithelial cells,
we previously have demonstrated that IFN- was able to change the
primary PG produced from PGF2 to PGE2, also
via an up-regulation of COX-2 gene expression (24, 25). Therefore, in
these cells, IFN- probably acts also farther down in the PGs
biosynthesis cascade. On the contrary, OT induced a stimulation of both
PGE2 and PGF2, but the ratio is maintained
in favor of PGF2. In the present study, addition of
IFN- was able to reduce the effect of OT on PGF2
production. The mechanism responsible for this inhibition by IFN- is
not known, but it may involve a down-regulation of OTR in
vitro, as demonstrated by others in the ovine in vivo
(26). A recent study published by Charpigny et al. (38)
strongly supports the present results and those obtained with IFN-
(25). They have demonstrated in vivo that the COX-2 protein
was transiently expressed in high concentrations close to the time when
OT is released to induce luteolysis. During early pregnancy, COX-2 also
was expressed, indicating that PGs are necessary for establishment of
pregnancy. We have previously reported that PGE2 production
and COX-2 gene expression were regulated by roIFN-. Because
PGE2 possesses antiluteolytic and luteotrophic properties,
it can be proposed that the IFN- regulation of PGE2 and
COX-2 is a mechanism necessary for establishment of pregnancy. However,
in the absence of a conceptus signal, OT stimulate PGF2
production via COX-2.
In conclusion, the present results demonstrate, using an
integrated in vitro system, that OT stimulation, which
stimulates PGF2 production, also: 1) stimulates the
generation of inositol phosphates; 2) induces the release of
[Ca2+]i; and 3) up-regulates COX-2 gene
expression. These results in vitro support and complement
the results obtained in vivo by Charpigny et al.
(38), suggesting that COX-2 may be a key enzyme in the regulation of
PGF2 production by OT during luteolysis.
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Acknowledgments |
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, Dr. Thomas G. Kennedy for generously donating
the PGE2 antiserum for the ELISA technique, and Lise
Lacouline for her help in PG measurements. |
Footnotes |
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Received May 22, 1997.
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