Endocrinology Vol. 138, No. 9 3638-3644
Copyright © 1997 by The Endocrine Society
Induction of Cyclooxygenase-2 and Prostaglandin F2
Receptor Expression by Interleukin-1ß in Cultured Human Granulosa-Luteal Cells1
Kirsi Narko,
Olli Ritvos and
Ari Ristimäki
Departments of Obstetrics and Gynecology and Clinical Chemistry
(K.N., A.R.) and the Haartman Institute, Department of Bacteriology and
Immunology (O.R., A.R), University of Helsinki, Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Ari Ristimäki, Research Laboratory, Department of Obstetrics and Gynecology, University of Helsinki, Haartmaninkatu 2, SF-00290 Helsinki, Finland
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Abstract
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Prostanoids are important regulators of ovarian function, especially
during ovulation and luteolysis. Cyclooxygenase (Cox) is the
rate-limiting enzyme in conversion of arachidonic acid to prostanoids.
We have examined the expression and regulation of the inducible Cox
isoform (Cox-2) and of the receptor for PGF2
(FP) in
human granulosa cells obtained from women undergoing oocyte retrieval
for in vitro fertilization. Freshly isolated granulosa
cells express Cox-2 and FP receptor messenger RNAs (mRNAs). FP receptor
mRNA is also expressed in cultured human granulosa-luteal (GL) cells,
but Cox-2 transcripts are expressed only upon induction.
Interleukin-1ß (IL-1ß) elevated Cox-2 mRNA steady state levels in a
concentration-dependent manner, and kinetic studies showed that Cox-2
mRNA levels were already induced at the 2 h point and returned to
the basal level after incubation for 24 h. The protein synthesis
inhibitor, cycloheximide, induced Cox-2 mRNA expression and potentiated
the effect of IL-1ß. Degradation of Cox-2 mRNA was inhibited by
IL-1ß, which suggests regulation at the posttranscriptional level.
IL-1ß also induced the expression of Cox-2 protein, as detected by
immunofluorescence staining using Cox-2-specific polyclonal antibodies.
Further, IL-1ß-induced synthesis of prostanoids was blocked by a
Cox-2-specific inhibitor, NS-398. In addition, hCG induced Cox-2 mRNA
expression and potentiated the effect of IL-1ß. However, in contrast
to the rapid and transient effect of IL-1ß on Cox-2 mRNA, the effect
of hCG followed slower kinetics. We have previously shown that hCG
induces expression of human FP receptor mRNA in cultured human GL
cells. We now show that IL-1ß induces FP receptor mRNA in a time- and
concentration-dependent manner. Our data suggest that Cox-2 and FP
receptor are coexpressed in freshly isolated human granulosa cells and
that their expression is up-regulated by IL-1ß and hCG in cultured
human GL cells.
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Introduction
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CYCLOOXYGENASE (Cox) is the rate-limiting
enzyme in the conversion of arachidonic acid to prostanoids (1). Two
Cox isoforms exist, Cox-1 and Cox-2. Cox-1 is expressed constitutively
and ubiquitously, and it is thought to be responsible for the synthesis
of prostanoids necessary for physiological functions, such as platelet
aggregation, vasodilatation in the kidney, and cytoprotection of the
stomach. In contrast, Cox-2 is usually low under basal conditions, but
it is highly induced by tumor promoters, growth factors, and
proinflammatory cytokines in several cell types in vitro,
and its expression is elevated in chronic inflammatory diseases and in
colon tumors in vivo (1, 2). Gonadotropins have also been
shown to modulate the expression of Cox-2 in rat and bovine
preovulatory follicles (3, 4, 5, 6). In the rat and bovine ovary, Cox-2 is
expressed in the granulosa cells, and it has been suggested to be
responsible for the increased synthesis of prostanoids during
ovulation, which leads to increased blood flow, vascular permeability,
and protease production and eventually to the rupture of the follicular
wall (4). This is supported by the antiovulatory effect of nonsteroidal
antiinflammatory drugs, such as aspirin and indomethacin, in several
species (7, 8, 9, 10), including rhesus monkeys (11) and humans (12).
Further, targeted deletion of the Cox-2 gene has been shown to reduce
the frequency of ovulation in mice (13), whereas Cox-1 knock-out mice
had normal ovarian function (14). However, it is not known which Cox
isoforms are expressed in the human ovary.
Most prostanoids exert their actions through specific cell surface
receptors, and the response to prostanoids could potentially be
modulated by regulation of expression of the respective receptor.
PGF2
is a prostanoid that triggers regression of corpus
luteum (15). It acts via the PGF2
receptor (FP), which
is a putative seven-transmembrane domain receptor that signals through
G proteins that activate phospholipase C, which leads to formation of
inositol trisphosphate and a subsequent increase in cytosolic free
calcium (16, 17). Human FP receptor has been cloned from uterine (16)
and placental tissues (17). We have recently shown that the expression
of FP receptor is induced by hCG and down-regulated by phorbol
12-myristate 13-acetate (PMA) in human granulosa-luteal (GL) cells
(18). Further, the expression of FP receptor is induced by LH and
decreased by PMA and PGF2
in ovine corpus luteum (19)
and is induced by hCG and forskolin in cultured bovine granulosa cells
(6).
Interleukin-1 (IL-1) is an important mediator of inflammation (20).
Several lines of evidence suggest that IL-1 may also have a regulatory
role in the ovary: 1) IL-1ß is synthesized in ovarian stroma by
macrophages (21); 2) IL-1-like activity is present in human (22) and
porcine (23) follicular fluid; and 3) synthesis and expression of
IL-1ß, its receptor, and receptor antagonist have been localized in
human ovarian granulosa cells (24). Finally, IL-1ß induces ovulation
in perfused rat (25, 26) and rabbit (27) ovaries. The proovulatory
effect of IL-1ß may be facilitated by prostanoids, as IL-1ß has
been shown to stimulate PG production in rat whole ovarian dispersates
(28) and preovulatory follicles (22) and in cultured human granulosa
cells (29, 30).
The objective of the present study was to examine the expression of Cox
enzymes in freshly isolated human granulosa cells and to investigate
whether Cox-2 and FP receptor expression can be regulated by IL-1ß in
cultured human GL cells.
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Materials and Methods
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Cell culture
Human granulosa cells were obtained from women undergoing
hormone treatment for in vitro fertilization. Approval for
the study was obtained from the committee of ethics of the Department
of Obstetrics and Gynecology, University of Helsinki. The granulosa
cells from three or four patients were pooled, dispersed with
hyaluronidase (Sigma Chemical Co., St. Louis, MO), and separated from
red blood cells by centrifugation through Ficoll-Paque (Pharmacia,
Uppsala, Sweden). Cells were seeded in 35-mm six-well dishes (Costar,
Cambridge, MA) at a density of 25 x 105 cells/well.
The cells were maintained in DMEM supplemented with 10% FCS, 2
mM L-glutamine, and antibiotics (Life
Technologies, Grand Island, NY). The cells were grown at 37 C in 5%
CO2, and the culture medium was changed every other day.
All experiments were conducted in DMEM containing 2.5% FCS with or
without human recombinant IL-1ß (0.110 ng/ml) obtained from R&D
Systems (Abingdon, UK) or hCG (30100 ng/ml) from the National Hormone
and Pituitary Program (Rockville, MD). 8-Bromo-cAMP (1 nM),
cycloheximide (10 µg/ml), and actinomycin D (10 µg/ml) were
purchased from Sigma, and NS-398 (15 µM) was obtained
from Cayman Chemical Co. (Ann Arbor, MI).
RNA isolation and Northern and dot blot hybridization analyses
Cytoplasmic RNA was isolated from cultured granulosa cells using
the Nonidet P-40 lysis procedure (31) or TriZol reagent (Life
Technologies). Total RNA from freshly isolated granulosa cells was
extracted using the guanidine isothiocyanate-cesium chloride method
(32). For Northern blot analysis, 1020 µg RNA were first denatured
in 1 M glyoxal, 50% dimethylsulfoxide, and 10
mM phosphate buffer at 50 C for 60 min and then
electrophoresed through 1.2% agarose gel. The RNA was transferred to
Hybond-N nylon membranes (Amersham International, Aylesbury, UK). For
dot blots, 12 µg RNA were denatured in 7.5% formaldehyde and
6 x SSC (1 x SSC = 0.15 M NaCl and 0.015
M sodium citrate, pH 7.0) at 60 C for 30 min and then
spotted onto nylon membranes using a 96-well Minifold device
(Schleicher and Schuell, Dassel, Germany). All blots were baked for
1 h at 80 C and cross-linked under UV light for 6 min (Reprostar
II UV, Camag, Muttenz, Switzerland). Human Cox-1, Cox-2, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNAs
(cDNAs) (33) were labeled using [
-32P]deoxy-CTP
(DuPont-New England Nuclear, Boston, MA) and the Prime-a-Gene kit
(Promega, Madison, WI). Linear amplification labeling was performed as
previously described (18, 34) to obtain a single stranded human FP
receptor cDNA probe. Probes were purified with nick columns (Pharmacia)
and used at 1 x 106 cpm/ml. Hybridizations were
performed at 42 C for 16 h in solution containing 50% formamide,
6 x SSC, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA, 100
µg/ml herring sperm DNA, 100 µg/ml yeast RNA, and 0.5% SDS.
Membranes were washed three times at 50 C for 15 min each time in
0.1 x SSC and 0.1% SDS. Dot blots were quantitated using a
Fujifilm IP-Reader Bio-Imaging Analyzer BAS 1500 (Fuji Photo Co.,
Tokyo, Japan) and MacBas software supplied by the manufacturer.
Northern blots were visualized by autoradiography.
Reverse transcriptase-PCR (RT-PCR)
Total RNA (1 µg) was converted to cDNA and PCR amplified with
Cox-1, Cox-2, and GAPDH sense and antisense primers as described
previously (33). Cox-1 and Cox-2 were amplified for 35 cycles, and
GAPDH was amplified for 30 cycles of denaturation at 94 C for 1 min,
annealing at 60 C for 1 min, and extension at 72 C for 1 min. Amplified
products were electrophoresed through 1% agarose gel and visualized by
ethidium bromide staining.
Assay of 6-keto-PGF1
and measurement of
DNA content of cell cultures
GL cells were incubated with arachidonic acid (10
µM; Sigma) for 15 min. Conversion of arachidonic acid to
prostacyclin (PGI2) was evaluated by measuring the
production of its stable hydrolysis product,
6-keto-PGF1
, from cell culture medium by RIA,
employing a specific antibody (35) and a tritiated
6-keto-PFG1
(Amersham). DNA concentrations in cell
cultures were measured using a slightly modified method originally
described by Rymaszewski et al. (36). Briefly, the cells
were lysed in alkaline EDTA and neutralized with
KH2PO4, followed by the addition of
fluorochrome bisbenzimidazole (Hoechst 33258, Pharmacia) and
measurement of fluorescence by DyNA Quant 200, as suggested by the
manufacturer (Hoefer Pharmacia Biotech, San Francisco, CA).
Immunofluorescence staining
For immunofluorescence, GL cells were grown on chamber slides
(Nunc, Naperville, IL) for 6 days and then treated with or without
IL-1ß (10 ng/ml) for 4 h. The cells were fixed with ice-cold
acetone for 10 min, and the chambers were washed three times for 10 min
each time in PBS (HyClone, Northumberland, UK). Nonspecific binding of
antibody was blocked with 1% BSA in PBS for 15 min. The samples were
incubated with human Cox-2 polyclonal antibodies (Cayman Chemical Co.)
at room temperature for 30 min and then washed with PBS three times for
10 min each time. The secondary antibody, fluorescein
isothiocyanate-conjugated goat antirabbit IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA), was added and incubated for 30 min, and
the samples were then washed with PBS three times for 10 min each time.
As a negative control, the cells received the same treatment with the
exception of the primary antibody.
Statistical analysis
Statistical significance for a single comparison was calculated
using Students t test. For multiple comparison, the
t test was used only if one-way ANOVA indicated a
significant difference. All results are shown as the mean ±
SEM, and P < 0.05 was selected as the
statistically significant value. All experiments were repeated at least
three times, and each experiment consisted of three separate
observations.
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Results
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Cox-2 messenger RNA (mRNA) is expressed in human granulosa
cells
Freshly isolated human granulosa cells expressed Cox-2 mRNA, but
the signal was undetectable or very weak in cultured GL cells, as
detected by Northern blot hybridization (Fig. 1A
) or RT-PCR (Fig. 1B
). In Northern blot
analysis, 4.6- and 2.8-kb transcripts were detected. The transcript
sizes are consistent with our earlier results obtained in human lung
fibroblasts, and they are likely to represent alternatively
polyadenylated Cox-2 isoforms (37). Cox-1 mRNA was detected in
uncultured and cultured cells by RT-PCR (Fig. 1B
), but not by Northern
blot analysis. FP receptor mRNA was detected by Northern blot
hybridization in both freshly isolated cells and cultured GL cells
(Fig. 1A
).

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Figure 1. Detection of Cox-2 and FP receptor mRNA from human
granulosa cells. A, Cytoplasmic RNA from two separate pools of freshly
isolated human granulosa cells (lanes 1 and 2) and from one pool of
cultured (lane 3) human granulosa cells was analyzed by Northern blot
hybridization using Cox-2 and FP receptor probes and the probe for
GAPDH as a loading control. The 4.6- and 2.8-kilobase Cox-2 transcripts
are depicted by arrows. B, mRNA was isolated from three
individual pools of human granulosa cells before culture (lanes 13)
and after 6 days in culture (lanes 46). Cytoplasmic RNA was reverse
transcribed and PCR amplified with primers for human Cox-1, Cox-2, and
GAPDH (see Materials and Methods for details).
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IL-1ß induces Cox-2 mRNA expression in cultured human GL
cells
To investigate the effect of IL-1ß on Cox-2 mRNA expression, GL
cells were incubated with IL-1ß (10 ng/ml) for 248 h. Kinetic
studies showed that Cox-2 mRNA levels were already induced at the
2 h point and had returned to the basal level after incubation for
24 h (Fig. 2A
). As shown in Fig. 2B
, the effect of IL-1ß was concentration dependent. To examine whether
the cell culture stage has an effect on the induction of Cox-2 mRNA, GL
cells were treated with IL-1ß (10 ng/ml) or hCG (30 ng/ml) for 8
h at days 2, 5, and 7 after initiation of the cultures. As shown in
Fig. 3A
, IL-1ß induced Cox-2 mRNA at
all stages of culture. This was in contrast to the results obtained
with hCG, which did not initially (on day 2) have an effect on Cox-2
mRNA expression. Interestingly, hCG potentiated the effect of IL-1ß
on Cox-2 expression (Fig. 3B
). Incubation of GL cells for 2 h with
cycloheximide, an inhibitor of protein synthesis, resulted in an
approximately 4-fold induction of Cox-2 mRNA steady state levels.
Cycloheximide also potentiated the effects of IL-1ß, hCG, and
8-bromo-cAMP (Fig. 4
). IL-1ß did not
modulate Cox-1 mRNA expression, as detected by RT-PCR (not shown).

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Figure 2. Time- and concentration-dependent effect of
IL-1ß on Cox-2 mRNA expression. A, Human GL cells were cultured for 4
days and incubated with or without IL-1ß (10 ng/ml) for 248 h. The
mRNAs were analyzed by Northern blot hybridization. B, Human GL cells
were cultured for 4 days and incubated for 2 h with different
concentrations (010 ng/ml) of IL-1ß. The values of dot blot
analyses are shown as the ratios of Cox-2 mRNA and GAPDH mRNA (x1000)
calculated from arbitrary densitometric units (mean ±
SEM of three observations). Asterisks
indicate significant (P < 0.05) differences
between IL-1ß-treated cultures and the control.
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Figure 3. Effects of IL-1ß and hCG on Cox-2 mRNA
expression at different stages of culture. A, Human GL cells were
cultured for 2, 5, and 7 days. The cultures were incubated with or
without IL-1ß (10 ng/ml) or hCG (30 ng/ml) for 8 h. B, Human GL
cells were cultured for 5 days and incubated with or without IL-1ß
(10 ng/ml), hCG (30 ng/ml), or IL-1ß and hCG in combination for
8 h. Cytoplasmic RNA was analyzed by dot blot hybridization. The
values shown represent the ratio of Cox-2 mRNA and GAPDH mRNA
calculated from arbitrary densitometric units (mean ±
SEM of three observations). Asterisks
indicate significant (P < 0.05) differences
between treated cultures and controls.
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Figure 4. Effect of cycloheximide on Cox-2 mRNA expression.
Human GL cells were cultured for 6 days and incubated with hCG (30
ng/ml), 8-bromo-cAMP (1 mM), and IL-1ß (10 ng/ml) alone
or in combination with cycloheximide (10 µg/ml) for 2 h.
Cytoplasmic RNA was analyzed by dot blot hybridization. The values
represent the ratio of Cox-2 mRNA and GAPDH mRNA (x1000) calculated
from arbitrary densitometric units (mean ± SEM of
three observations). Asterisks indicate a significant
(P < 0.05) difference between treated cultures and
the control.
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Effect of IL-1ß on degradation of Cox-2 transcripts
To investigate whether IL-1ß has an effect on the stability of
Cox-2 mRNA, GL cells were first incubated with cycloheximide for 2
h, after which the cells were washed, and actinomycin D was added to
inhibit any further transcription. Cox-2 message decayed with a
half-life of approximately 1.7 h (Fig. 5A
), and IL-1ß was able to inhibit
degradation of the Cox-2 transcript (Fig. 5B
).

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Figure 5. Effect of IL-1ß on degradation of Cox-2
mRNA. A, Human GL cells were cultured for 6 days, preincubated with
cycloheximide for 2 h, and then incubated with actinomycin D (10
µg/ml) for 04 h. Cytoplasmic RNA was analyzed by dot blot
hybridization. Cox-2 mRNA values were first normalized by GAPDH mRNA,
and the values shown are the mRNA remaining (mean ±
SEM of three observations) compared with that at the 0
h point. B, Human GL cells were preincubated with cycloheximide for
2 h and then incubated with actinomycin D (control) alone or with
IL-1ß for 4 h. Dot blot analyses were performed as described in
A. Asterisks indicate a significant
(P < 0.05) difference between treated cultures and
the control.
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IL-1ß induces Cox-2 protein expression and enzyme activity
We next examined expression of Cox-2 protein by immunofluorescence
in cultured GL cells. Cox-2 was present in IL-1ß-treated GL cells,
but not in untreated cells (Fig. 6
). We
also examined the effect of a Cox-2-specific inhibitor, NS-398, on
IL-1ß-induced prostanoid production in GL cells by measuring the
concentrations of 6-keto-PGF1
, a stable metabolite of
prostacyclin. IL-1ß increased 6-keto-PGF1
concentrations in the cell culture medium approximately 2-fold (Fig. 7A
), and this induction was blocked by
NS-398 (Fig. 7B
).

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Figure 6. Immunofluorescence staining of Cox-2 in
IL-1ß-treated human GL cells. Human GL cells were subjected to
immunofluorescence staining using Cox-2 polyclonal antibodies and
fluorescein isothiocyanate-conjugated goat antirabbit IgG as described
in Materials and Methods. A, Nontreated cells stained
with Cox-2 antibody. B, IL-1ß-treated (10 ng/ml for 4 h) GL
cells stained with Cox-2 antibody. C, IL-1ß-treated cells without
primary antibody. Magnification, x400.
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Kinetics of hCG-induced Cox-2 mRNA expression
Induction of Cox-2 mRNA by hCG was slow and sustained. Increased
Cox-2 mRNA levels were detected after incubation for 8 h, and
transcript levels remained elevated for at least 72 h (Fig. 8A
). 8-Bromo-cAMP induced Cox-2
expression with similar kinetics as hCG (data not shown).

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Figure 8. Time-dependent effect of hCG on Cox-2 mRNA
expression. Human GL cells were cultured for 4 days and treated with or
without hCG (100ng/ml) for 272 h. The mRNAs were analyzed by Northern
blot hybridization.
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IL-1ß induces FP receptor mRNA
IL-1ß induced steady state levels of FP receptor mRNA. The
induction peaked at 24 h (Fig. 9A
)
and was concentration dependent (Fig. 9B
). IL-1ß together with hCG
resulted in 2-fold induction compared with that produced by IL-1ß or
hCG alone (Fig. 9B
).

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Figure 9. Effect of IL-1ß on FP receptor mRNA expression.
A, Human GL cells were cultured for 6 days and incubated with or
without IL-1ß (10 ng/ml) for 248 h. B, Human GL cells were cultured
for 6 days and incubated with or without IL-1ß (010 ng/ml), hCG (30
ng/ml), or IL-1ß and hCG in combination for 24 h. Cytoplasmic
RNA was analyzed by Northern blot hybridization.
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 |
Discussion
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In this report we show that Cox-2 mRNAs are expressed in
freshly isolated human granulosa cells and that Cox-2 transcripts and
protein are expressed after induction by IL-1ß in human GL cells.
Using RT-PCR we detected Cox-1 message, but the expression was not
regulated by IL-1ß. However, Cox-1 mRNA was not detected by Northern
blot analysis, indicating that only low levels of Cox-1 mRNA are
present in isolated human granulosa cells and in GL cell cultures.
Cox-1 is constitutively expressed in thecal cells in the rat ovary
(38), but the cellular origin of Cox-1 expression in human follicular
aspirates was not studied further.
Wong et al. (39) and Sirois et al. (40)
previously reported that IL-1ß does not induce the expression of
endogenous Cox-2 or activity of a transfected Cox-2 promoter construct
in rat granulosa cells. However, induction of Cox-2 mRNA has been
detected by RT-PCR in rat preovulatory granulosa cells (41). Further,
IL-1ß has been shown to stimulate synthesis of prostanoids in rat
whole ovarian dispersates (28) and preovulatory follicles (21), and the
synthesis of PGE2 and PGF2
has been shown to
be stimulated by IL-1ß in cultured human granulosa cells (29, 30). In
our experiments IL-1ß treatment resulted in a rapid and transient
induction of Cox-2 mRNA in cultured human GL cells. Expression of Cox-2
protein was also stimulated by IL-1ß in these cells. In addition,
IL-1ß-induced conversion of arachidonic acid to a prostanoid, which
was inhibited by the Cox-2-specific inhibitor, NS-398. This suggests
that elevated expression of Cox-2 enzyme is important in
IL-1ß-induced prostanoid production in human ovarian granulosa
cells.
Inhibition of protein synthesis induces expression of Cox-2 mRNA
in several cell types (33, 42). However, cycloheximide did not induce
Cox-2 mRNA expression in rat granulosa cells (43), and it decreased the
induction of Cox-2 mRNA stimulated by gonadotropins (44). In the
present study cycloheximide induced the expression of Cox-2 mRNA and
potentiated the effects of IL-1ß, hCG, and 8-bromo-cAMP, indicating
that induction of Cox-2 mRNA expression is not dependent on protein
synthesis in human GL cells. The mechanism by which cycloheximide
induces Cox-2 mRNA is not known, but it is possible that it inhibits
the synthesis of proteins responsible for the rapid degradation of
Cox-2 transcripts. IL-1
and IL-1ß have been previously shown to
induce Cox-2 mRNA in fibroblasts and endothelial cells by inducing
transcription and preventing degradation of the Cox-2 mRNA (33, 37).
IL-1ß also stabilizes Cox-2 message in mesangial cells (45).
Induction of Cox-2 mRNA by IL-1ß in human GL cells seems to depend at
least partially on posttranscriptional stabilization of Cox-2
transcript.
Cox-2 mRNA and protein have been shown to be rapidly and
transiently induced by ovulatory concentrations of gonadotropins via
cAMP-dependent protein kinase A (PKA) pathway (3, 43, 46). In rat
granulosa cells Cox-2 mRNA levels peak after 4 h and disappear
after incubation for 6 h. Induction of Cox-2 by hCG in bovine
granulosa cells from preovulatory follicles follows slower kinetics and
is sustained (5, 6), which was also the case in our experiments with
human GL cells. We detected similar kinetics in induction of Cox-2 mRNA
with 8-bromo-cAMP as with hCG, suggesting that Cox-2 is also induced
via PKA pathway in human GL cells. Cox-2 is also induced by other
signaling pathways, including protein kinase C (PKC) and tyrosine
kinases, in rat granulosa cells (47). Involvement of the PKC pathway in
the induction of Cox-2 in human granulosa cells is supported by the
findings that PMA increases prostacyclin production (48) and that PMA
up-regulates Cox-2 mRNA levels in human GL cells (our unpublished
observation). In our study, Cox-2 expression was stimulated by hCG only
when the GL cells were cultured for more than 2 days. A similar lag
period in responsiveness of other parameters to hCG has been observed
in previous studies using this cell culture model (18, 34). This may
reflect the down-regulation of LH/hCG receptors or their signaling
pathways in response to hormone treatment received by the granulosa
cell donors. Interestingly, simultaneous administration of hCG and
IL-1ß resulted in synergistic induction of Cox-2 mRNA in GL
cells.
Our group has previously shown that the FP receptor is induced after
incubation for 2448 h with hCG in human GL cells (18). We now show
that IL-1ß induces FP receptor mRNA expression in GL cells.
Interestingly, the induction followed slower kinetics than that of
Cox-2, suggesting that the mechanism of induction may be different.
However, as FP receptor Cox-2 mRNAs were induced with similar kinetics
by hCG, these transcripts may be coregulated via the PKA pathway in
human GL cells.
Our data show that IL-1ß-induced production of prostanoids is
dependent on Cox-2 expression in cultured human GL cells. We also show
that FP receptor mRNA is regulated by IL-1ß in human GL cells. To
better define the biological significance of the present findings,
additional studies are in progress to further characterize the
role of prostanoid-forming enzymes and PG receptors in the human
ovary.
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Acknowledgments
|
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We thank Prof. Olavi Ylikorkala and Dr. Ulf-Håkan Stenman for
their support. We also thank Ritva Javanainen, Kaija Antila, and
Marjatta Vallas for their excellent technical assistance. The personnel
of the Felicitas In Vitro Fertilization Clinic, the Finnish Population
Council In Vitro Fertilization Laboratory, and the In Vitro
Fertilization Unit at the Departments of Obstetrics and Gynecology,
University of Helsinki, are kindly acknowledged for their
collaboration.
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Footnotes
|
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1 This work was supported by the Academy of Finland, Helsinki
University Central Hospital research funds, and the Novo Nordisk
Foundation. 
Received March 17, 1997.
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