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Original Studies |
Medical Research Council Group in Fetal and Neonatal Health and Development, Departments of Physiology and Obstetrics and Gynecology (F.A.P, V.L.C., J.R.G.C.), University of Toronto, Toronto, Ontario, Canada; and Research Laboratories of Schering AG (K.C.), Berlin, Germany
Address all correspondence and requests for reprints to: Dr. Falguni A. Patel, 1 Kings College Circle, Medical Sciences Building, Room 3209, Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: fal.patel{at}utoronto.ca
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. PGDH messenger ribonucleic
acid was quantified by in situ hybridization and
computerized image analysis.
The basal output of 13,14-dihydro-15-keto-PGF2 was lower
in placenta or chorion collected at spontaneous labor than in that
obtained at elective cesarean section. Cortisol had a significant
dose-dependent inhibitory effect on PGDH activity in both placental and
chorion trophoblast cells and significantly decreased levels of PGDH
messenger ribonucleic acid. Responses were similar between tissues from
laboring and nonlaboring women. PGDH activity was increased by R5020
and medroxyprogesterone acetate and was inhibited by RU486,
onapristone, and trilostane. We conclude that cortisol inhibits PGDH
activity and expression and that progestagens increase PGDH activity in
human chorion and placenta.
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Introduction |
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) within the intrauterine
environment is critical in controlling the levels of bioactive PGs
reaching the myometrium and cervix, the proposed sites of PG
action. During late pregnancy, PG synthesis increases in the amnion, chorion, and decidua (3, 4). PG synthetic activity and levels of PGH2 synthase-2 messenger ribonucleic acid (mRNA) are elevated further in amnion and chorion at the time of labor (5, 6, 7, 8). However, several reports have indicated that the in vitro transfer of unmetabolized PGE2 across full thickness membranes is low and increases only marginally at the time of labor (9, 10, 11).
The lack of PG transfer is attributable partially to the
PG-catabolizing enzyme, NAD+-dependent
15-hydroxyprostaglandin dehydrogenase (PGDH), which is present at high
activity in placental syncytiotrophoblast and chorionic trophoblast
cells throughout gestation (4, 12, 13, 14). PGDH is responsible for the
initial inactivation of PGs, catalyzing the conversion of
PGE2 and PGF2 to their biologically inactive
15-keto derivatives. The chorion, interposed between amnion and
decidua, thus becomes an important site of PG metabolism during
pregnancy and has been described as a protective barrier to prevent the
passage of primary PGs synthesized within the amnion or chorion from
reaching the myometrium and stimulating the onset of preterm or term
delivery (15, 16). Clearly, the level of bioactive PGs in intrauterine
tissues reflects a balance between the synthesis and metabolism of PGs.
There is an increasing body of literature on factors affecting PG
synthesis (2, 7, 8), but little information on the regulation of the
metabolizing enzyme, PGDH.
Levels of mRNA encoding PGDH and PGDH activity are lower in chorion from patients at term spontaneous labor but not at term elective cesarean section and decreased further in tissue collected from patients at idiopathic preterm labor and at preterm labor with underlying infection (17). These differences were measured in chorion and were not found in placenta from the same patient groups. PGDH activity was also reduced significantly from chorion collected in the region of the lower uterine segment at active labor than at elective cesarean section compared to that in other areas of the uterus (18). These observations suggested that in a subgroup of 10–15% of patients with idiopathic preterm labor without infection, deficiency of PGDH might allow PG generated within amnion or chorion to pass unmetabolized to the underlying decidua and myometrium (19). In patients at preterm labor with infection, PGs generated within membranes would similarly be unmetabolized with loss of the chorionic barrier. We suggested that loss of PGDH in the lower segment chorion at term might allow PG generated in membranes to reach the cervix, to facilitate effacement and ripening (18).
In preterm labor with infection, loss of PGDH activity was correlated with loss of trophoblast cells (17). In idiopathic preterm labor, chorionic trophoblast cells are not destroyed, but factors concerned with regulation of PGDH are not well known. In fetal rat lung, dexamethasone (DEX) has been suggested to increase PGDH activity (20); however, renal PGDH activity in rats has been reported to decrease upon treatment with DEX (21, 22). During human pregnancy, cortisol increases PGH2 synthase-2 mRNA (23, 24) but the effect of cortisol on PGDH is unclear. In vivo (25, 26) and in vitro (27) studies have implicated progesterone as the stimulus to PGDH activity in lung, decidua, and myometrium, but any effect of progesterone on PGDH activity in chorion is unknown. We hypothesized that locally produced steroids in membranes and placenta would affect the activity of PGDH and that this would change at the time of labor. To examine this possibility we cultured human trophoblast cells from placenta and chorion collected from patients in the presence and absence of labor and treated these cells with cortisol and progesterone to determine any change in PGDH activity and/or expression. Because these cells also produce progesterone (28), we examined the possibility of autocrine/paracrine regulation of PGDH by cultured cells in the presence of trilostane, an inhibitor of 3ß-hydroxysteroid dehydrogenase enzyme (3ßHSD; pregnenolone to progesterone conversion), and in the presence of the progesterone receptor antagonists, onapristone and RU486.
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Materials and Methods |
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Trophoblast cells from placental cotyledons and chorio-decidual tissue were isolated and cultured using a modification of the technique described by Kliman et al. (29), as described previously (30). Briefly, human placentae (n = 32) and chorio-decidual tissue (n = 32) were obtained from uncomplicated normal term pregnancies after elective cesarean section or spontaneous vaginal delivery. Approximately 60 g cotyledon tissue were removed randomly from the maternal side of the placenta, pooled, and digested with 0.125% trypsin (Sigma Chemical Co., St. Louis, MO) and 0.02% deoxyribonuclease I (Sigma Chemical Co.) in DMEM (Life Technologies, Grand Island, NY) containing 0.1% BSA, 0.005% gentamicin, and 0.01% streptomycin three times for 30 min each time. The chorion with adherent decidua was peeled off amnion and digested three times for 60 min each time using the same digestion medium with the addition of 0.2% collagenase (Sigma Chemical Co., St. Louis, MO). The dispersed placental or chorio-decidual cells were filtered with a 200-µm nylon gauze and loaded onto a continuous Percoll (Sigma Chemical Co.) gradient (5–70% in 5% steps of 3 mL each), then centrifuged at 37 C and 1200 x g for 20 min to separate different cell types. Cytotrophoblast cells between the density markers of 1.049 and 1.062 g/mL were collected and plated in 24-well plates (Corning Costar Corp., Cambridge, MA) at a density of 106 cells/mL/well in DMEM culture medium containing 10% FCS (Life Technologies). Cells were also plated on eight-well chamber slides (Lab-Tek, Nunc, Inc., Naperville, IL) at a density of 0.3 x 106 cells/well. The cells were cultured for 3 days at 37 C in 5% CO2 and 95% air before experimentation.
Treatment of cells with steroids
After a 3-day incubation period, the cells were washed with
FCS-free culture medium (pH 7.4), then treated with fresh medium
containing progesterone, estradiol, cortisol, DEX,
mifepristone (RU486;
17ß-hydroxy-11ß-[4-dimethylaminophenyl]-17-[1-propynyl]-estra-4,10-dien-3-one),
medroxyprogesterone acetate (MPA), promegestone (R5020; a gift from Dr.
N. MacLusky, University of Toronto, Toronto, Canada), onapristone (ZK
98 299), trilostane
(4,5-epoxy-17-hydroxy-3-oxoandrostane-2-carbonitrile, a 3ßHSD
inhibitor synthesized at Schering AG, Berlin Germany; gift
from Dr. M. Novy, Oregon Health Sciences University, Portland
OR), or combinations of these compounds. Each treatment was performed
in duplicate or triplicate for each preparation of cells for 24 h.
The medium was then changed and replaced with fresh medium containing
PGF2 (100 ng/mL; 282 nmol/L) for 4 h without
steroids (31). The culture medium was collected and stored at -80 C
for later assessment, by RIA, of PGDH activity by measuring
13,14-dihydro-15-keto-PGF2 (PGFM), the stable metabolite
of PGF2 (32).
Immunohistochemical analysis
The purity of the cell preparation was assessed at the end of each experiment by immunohistochemistry (13). Representative wells were stained using an antibody to cytokeratin (Dako Corp., Santa Barbara, CA) at a dilution of 1:1000 and vimentin (Dako Corp.) at a dilution of 1:100. In addition, cells were stained for immunoreactive (ir)-PGDH using the avidin-biotin peroxidase method (Vector ABC, Vector Laboratories, Inc., Burlingame, CA). The polyclonal primary PGDH antibody was raised in rabbits against purified human placental type 1 PGDH (Cayman Laboratories, Ann Arbor, MI) and used at a dilution of 1:1000. Cells were counterstained with Carazzi’s hematoxylin, dehydrated, and mounted with Permount (Fisher Scientific, Fairlawn, NJ).
PGFM RIA
The activity of PGDH was assessed by measuring the PGFM content
in duplicate aliquots (10 and 50 µL) of culture medium using a
modification of the RIA technique described by Cornette et
al. (32). PGFM antiserum (200 µL; Oxford Biomedical, MI;
raised in rabbit), diluted 1:2000, and 100 µL [3H]PGFM
(10,000–15,000 cpm
13,14-dihydro-15-keto-[5,6,8,11,12,14-N-3H]PGF2;
Amersham, Arlington Heights, IL) were added to each tube.
All solutions were made in Tris-gelatin buffer (0.01 mol/L Tris, 0.14
mol/L NaCl, and 0.1% gelatin, pH 7.4). The combined within- and
between-assay coefficient of variation was 6.7 ± 2.9%
(±SEM; n = 32).
PG RIAs
Concentrations of PGE2 and PGF2 were
measured in culture medium collected after 24-h steroid treatment but
before the addition of 282 nmol/L PGF2. PGE2
concentrations were determined using a specific RIA described
previously (33). PGE2 polyclonal antibody (raised in
rabbits; gift from Dr. Tom Kennedy, University of Western Ontario,
London, Ontario, Canada) was used at a final dilution of 1:4000.
PGF2 concentrations were determined using a
[3H]PGF2 assay system obtained from
Amersham Life Science (Aylesbury, UK). Intraassay coefficients of
variation were 5.1 ± 1.3% and 3.9 ± 0.7%, respectively
(±SEM; n = 12)
In situ hybridization
In situ hybridization for PGDH mRNA was performed on placental and chorion trophoblast cells, plated and cultured in chamber slides in the presence of cortisol (n = 5 different placentae; n = 4 sets of fetal membranes) or progesterone (n = 1 placenta and 3 fetal membranes) or as a control (n = 6 placentae; n = 7 chorion). Cells were fixed and incubated overnight with radiolabeled PGDH oligonucleotide probe, washed, and exposed to x-ray film (Biomax, Eastman Kodak Co., Rochester, NY) together with 14C-labeled standards (American Radiochemical, St. Louis, MO). The autoradiographic films were developed using standard procedures (34) and analyzed by densitometry within the linear range using a computerized image analysis system (MCID 2,4, Imaging Research, Inc., St. Catharines, Canada). All values are expressed as relative optical density after subtraction of background values for absorbance. The sections were counterstained with Carazzi’s hematoxylin to permit identification of nuclei.
The oligonucleotide probe for PGDH was 45 bases long and was complementary to bases 659–704 of the human gene (35). It was made by solid phase synthesis using an Applied Biosystem DNA synthesizer (Foster City, CA) and was purified on an 8% polyacrylamide-8 mol/L urea preparative sequencing gel. A 45-mer sense probe (17) was prepared and used to determine the specificity of hybridization.
Statistical analysis
Results are presented as the mean ± SEM for the number of observations (different tissues) indicated. The effects of treatment on concentrations of PGFM in the culture medium were determined by one-way ANOVA corrected for repeated measures. The effects of treatments between cultured placental and chorion trophoblast cells and between labor and nonlabor groups were determined by two-way ANOVA corrected for repeated measures. Student-Newman-Keuls multiple range tests were used to assess the effects of different treatment doses. When treatment effects were not normally distributed with equal variances the Friedman repeated measures ANOVA on ranks, a nonparametric test, was used to determine the statistical significance of the data. Relative optical density determinations were analyzed by the Student’s t test. Statistical significance was set at P < 0.05. Calculations were performed using SigmaStat (Jandel Scientific Software, San Rafael, CA).
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Results |
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After 72 h of culture, placental trophoblast cells aggregate to form syncytial clumps that correspond to placental syncytiotrophoblast, whereas chorionic trophoblast cells either remained as single cells or formed clumps. Both placental (>90%) and chorion (>75–95%) trophoblast cell cultures were predominantly cytokeratin positive and vimentin negative, suggesting the presence of mainly trophoblast cells and few fibroblast or decidual cells. Both placental and chorion cultures were positive for ir-PGDH. Trypan blue exclusion staining also showed that the percent viability of cultured cells before and after treatment was greater than 95%.
Effects of cortisol, DEX, progesterone, and trilostane
on PGE2 and PGF2
output by trophoblast cells in placenta and chorion
The basal output of PGE2 and PGF2 was
higher in placenta than chorion. Neither progesterone nor
trilostane affected PG output. However, cortisol or DEX decreased PG
output significantly in placenta (P < 0.001) and
raised PGE2 and PGF2 output by chorion
trophoblast cells (Table 1
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Conversion of added PGF2 (282 nmol/L) to PGFM in
the absence of steroid treatment was significantly less in placental
syncytiotrophoblast and chorion trophoblast cells cultured after
spontaneous labor (placenta, 5.7 ± 1.8 ng/mL; chorion, 1.2
± 0.05 ng/mL) compared to those from nonlabor tissues (placenta,
11.0 ± 2.0 ng/mL; chorion, 14.1 ± 3.4 ng/mL; Fig. 1; n = 8 for each group;
P < 0.05, by Student’s t test). Basal PGFM
outputs were not significantly different between either placental
syncytiotrophoblast and chorion trophoblast cells obtained from
laboring patients or between the two cell types obtained from patients
in the absence of labor.
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Cortisol significantly inhibited PGF2 to PGFM
conversion in a dose-dependent manner in both placental
syncytiotrophoblast (n = 8) and chorion trophoblast cells (n
= 8; Fig. 2a). In placenta, PGFM
conversion was reduced by 78 ± 17.5% at 100 nmol/L cortisol in
the labor group (n = 4) and by 66 ± 14.1% in the nonlabor
group (n = 4). In chorion, PGFM conversion was reduced by 56
± 8.0% at 100 nmol/L cortisol in the labor group (n = 4) and by
44 ± 8.6% in the nonlabor group (n = 4; P
< 0.05; Fig. 2a). There was no statistically significant difference in
cortisol inhibition of PGFM formation between placenta and chorion or
between labor and nonlabor groups. ED30 values in placenta
were 5.8 ± 0.8 and 17.0 ± 20.3 nmol/L in the labor and
nonlabor groups, respectively. In chorion, ED30 values were
35.0 ± 9.2 and 15.0 ± 17.7 nmol/L in the labor and nonlabor
groups (both P > 0.05). Exogenous
progesterone (0–1 µmol/L) or estradiol (0–1 µmol/L)
had no significant effect on PGFM formation in this set of cultured
placental syncytiotrophoblast and chorion trophoblast cells collected
from either labor (n = 8) or nonlabor (n = 8) groups of
patients (Fig. 2a).
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; P < 0.05, by Student’s
t test). On the other hand, cells treated with
progesterone (1 µmol/L) showed a trend toward increasing
PGDH mRNA levels in both placenta (n = 1) and chorion (n =
3), but these differences were not significant (Fig. 2b). Effects of cortisol and RU486 on PGDH activity
Cultured placental and chorion trophoblast cells were treated with
cortisol (0–1 µmol/L), RU486 (0–1 µmol/L), and cortisol (0–1
µmol/L) in the presence of a fixed concentration (100 nmol/L)
of RU486 (n = 4; Fig. 3). In this set of cultures, cortisol (1
µmol/L) significantly decreased PGFM levels in placenta by 48% (mean
basal value of 12.3 ± 1.9 ng/mL) and in chorion by 51% (mean
basal value of 14.2 ± 8.9 ng/mL; P < 0.05).
Exogenous progesterone (100 nmol/L and 1 µmol/L) tended
to increase PGFM output in placenta, but not in chorion, in this group
of patients, although this was not significant. RU486, a
glucocorticoid/progestin antagonist, significantly inhibited PGFM
output in placenta and chorion in a dose-dependent fashion
(P < 0.05; Fig. 3).
However, the inhibitory effect of cortisol on PGF2 to
PGFM conversion was not affected by coincubation with RU486 in either
placental syncytiotrophoblast or chorion trophoblast cells.
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Human placental syncytiotrophoblast and chorionic trophoblast
cells (n = 4) were treated with progesterone (0–1
µmol/L), RU486 (0–1 µmol/L), and progesterone (0–1
µmol/L) in the presence of a fixed concentration (100 nmol/L)
of RU486 (Fig. 4a). As reported for the
previous set of cultures, RU486 significantly inhibited PGFM formation
in a dose-dependent manner (P < 0.05) in both placenta
and chorion. Exogenous progesterone had no statistically
significant effect on PGFM output in either placenta or chorion.
However, the inhibition by RU486 on PGFM formation was attenuated by
the addition of progesterone in both placental
syncytiotrophoblast (100 nmol/L and 1 µmol/L; both P
< 0.05) and chorion trophoblast (1 µmol/L; P <
0.05) cells (Fig. 4a).
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). MPA (1
µmol/L) stimulated PGFM formation in placenta by 33% (mean basal
value of 12.2 ± 2.4 ng/mL) and in chorion by 38% (mean basal
value of 22.6 ± 7.8 ng/mL; P < 0.05). Similarly,
R5020 (1 µmol/L) increased PGFM levels in placenta by 36% and in
chorion by 44% (P < 0.05). Cells were also treated
with MPA (0–1 µmol/L) and R5020 (0–1 µmol/L) in the presence of a
fixed amount of RU486 (100 nmol/L; Fig. 4b). Increasing concentrations
of both progestin analogs reversed the inhibition of PGFM formation
that occurred in the presence of RU486 alone.
Onapristone (1 µmol/L), a more specific progesterone
receptor antagonist than RU486, significantly decreased PGFM levels in
medium from placenta (n = 4) by 26% (mean basal value of
12.3 ± 2.4 ng/mL) and from chorion (n = 4) by 36% (mean
basal value of 21.5 ± 6.1 ng/mL; P < 0.05; Fig. 4c). The addition of increasing concentrations of exogenous
progesterone (0–1 µmol/L) in the presence of 100 nmol/L
onapristone reversed the inhibition of PGFM formation by onapristone in
both placental syncytiotrophoblast and chorion trophoblast cells.
Effects of progesterone and trilostane on PGDH activity
The output of progesterone decreased from a basal
value of 2.1 ± 0.9 to 0.3 ± 0.3 ng/mL in placenta and from
a basal value of 1.1 ± 0.3 to 0.2 ± 0.3 ng/mL in chorion
after the addition of 100 nmol/L trilostane. Treatment of placental and
chorion trophoblast cells with trilostane, a 3ßHSD inhibitor,
significantly inhibited PGF2 to PGFM conversion in a
dose-dependent manner in placenta (n = 4) by 30% (mean basal
value of 12.5 ± 2.6 ng/mL) and in chorion (n = 4) by 45%
(mean basal value of 19.4 ± 4.0 ng/mL; P < 0.05;
Fig. 5). The addition of increasing
concentrations of progesterone (0–1 µmol/L) in the
presence of 100 nmol/L trilostane stimulated PGDH activity back to
basal levels in both placenta and chorion (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by placental
cells was decreased in the presence of cortisol and DEX; however, the
output of PGE2 and PGF2 from chorion was
increased in the presence of cortisol and DEX. Therefore, cortisol (and
DEX) affected the basal outputs of PGE2 and
PGF2 by placenta and chorion differently (Table 1). This
observation suggests strongly that the similar patterns of cortisol
effects on PGDH activity and mRNA levels in these cell types is not a
result of any alteration in substrate concentrations during the 24-h
period before the addition of excess PGF2 to the cells.
Progesterone and trilostane had no effect on PG output in
both placenta and chorion. The net output of PGs at term reflects a
balance between synthesis and metabolism, and in the present study we
have focused on PG metabolism. At the present time we can only
speculate as to the relative importance of synthesis vs.
metabolism in human fetal membranes and placenta, in normal and
abnormal pregnancy in vivo.
Cultures of placental syncytiotrophoblast and chorion trophoblast cells
showed considerable variation in their metabolism of added
PGF2 in the absence of any steroid treatment. Overall,
there was significantly less PGFM formation in tissues after
spontaneous labor compared to that in elective cesarean section
tissues. This supports the suggestion of a decrease in PG metabolism at
the onset of labor, consistent with earlier findings of lower PGDH mRNA
levels and PGDH activity at term spontaneous labor than at term
elective cesarean section (19). This finding also supports the use of
cultured trophoblast cells as an appropriate model in which to study
changes related to the onset of labor. The cells appear to have
retained different characteristics in vitro over an
incubation period of 5 days. Although we have immunostained cultured
cells for PGDH, we have not in this study determined the PGDH protein
content of the cells.
Previous reports concerning the effects of corticosteroids on PGDH
activity have been conflicting. Erman et al. (22) reported
that renal PGDH activity in rats treated with DEX for 2 weeks was
reduced by 57%; however, Xun et al. (36) reported that PGDH
activity in HEL cells was optimally induced by DEX, and Moore et
al. (37) have shown an increase in the tissue activity of PGDH in
rat lung and kidney after treatment with prednisolone. Recently,
Brennand et al. (38) using explants of human amnion and
chorion disks obtained from membranes of patients at spontaneous labor
and elective cesarean section reported that DEX had no effect on PG
metabolism. Similarly, PG metabolism by isolated cells from human
chorion laeve obtained at term by elective cesarean section was not
affected by cortisol or DEX (39). In contrast, we found a significant
dose-dependent inhibition of PGDH activity and a significant decrease
in PGDH mRNA by in situ hybridization after treatment of
both placental and chorion trophoblast cells with cortisol. One
explanation for this discrepancy may be that the basal output of PGs in
cell culture systems is generally well below the
Km of the enzyme. This makes it difficult to
measure changes in metabolite concentrations at low substrate
availability. Studies on the substrate specificity of the placental
PGDH enzyme have shown that the Km for various
PGs is in the micromole per L range (40). In our study we followed 24-h
steroid treatment of cultured trophoblast cells by incubation with
PGF2 at 282 nmol/L. Although this is still less than the
Km for the enzyme, it is a much higher
concentration than the basal PG levels measured in previous cell
culture studies, and this may facilitate measurement of PG
metabolite.
In nonprimate mammals, a decline in the maternal progesterone concentration is associated with the onset of labor. In contrast, humans and other primates undergo spontaneous labor even though maternal peripheral plasma progesterone concentrations continue to rise. It is possible that locally produced steroids within the placenta and fetal membranes may influence the initiation of labor in women in an autocrine/paracrine manner without a demonstrable fall in the systemic hormone concentration. We found that the addition of exogenous progesterone to the trophoblast cells had no effect on PG metabolism, in accord with a previous report (38). Several studies have shown that progesterone stimulates PGDH activity in various species and cell types (25, 26, 27, 36). However, one early study suggested that progesterone inhibited PGDH activity in human term placenta (41), but this effect was at very high steroid concentrations (32 µmol/L).
Mifepristone (RU486), a synthetic steroid with both antiglucocorticoid
and antiprogestin actions, has been shown previously to decrease PGDH
activity in guinea pig myometrium and chorion (42). In addition, women
pretreated with RU486 in early pregnancy had reduced PGDH activity in
decidua (43). Recent studies showed that the metabolism of added
PGE2 to PGEM was significantly reduced with RU486 treatment
in spontaneous labor tissue only (38). We found that the addition of
RU486 also significantly reduced PGDH activity in both cultured
placental syncytiotrophoblast and chorion trophoblast cells. Unlike
Brennand et al. (38), however, we found a reduction in
PGF2 metabolism after RU486 treatment in both
spontaneous labor tissue and elective cesarean section tissue. It is
possible that this may be due to differences in the tissue culture
method. We also found that onapristone (ZK 98299), a specific synthetic
antiprogestin, significantly inhibited PGDH activity in these cells.
Furthermore, the addition of exogenous progesterone at
high concentrations reversed the inhibitory effect of onapristone.
Human trophoblast cells isolated from term placentae and chorion tissue
contain the enzyme 3ßHSD, which is necessary to synthesize
progesterone from pregnenolone (44). Therefore, we suggest
that the inhibitory effect of RU486 and onapristone on PGDH activity in
placental and chorion cells results from antagonism of endogenous
progesterone produced by these cells, from substrates
taken up during the preincubation period.
In a separate series of experiments we found that the inhibition of PGDH by RU486 was reversed by coincubation with progesterone at high concentrations. Addition of cortisol in the presence of RU486 did not affect the inhibition of PGDH activity seen with cortisol alone. RU486 has previously been shown to have both glucocorticoid antagonistic and agonist actions in humans and nonhuman primates (45, 46, 47, 48, 49, 50, 51). These reports suggest that when ambient glucocorticoid levels are low, RU486 can display significant glucocorticoid agonist effects. It is unclear whether RU486 in this cell culture system is acting directly on PGDH as a glucocorticoid agonist, or as an antiprogestin to the effects of endogenous progesterone produced by the cells.
We found that MPA and promegestone (R5020), two stable synthetic progestins, significantly increased PGDH activity in both placenta and chorion. In addition, treatment of cells with trilostane (an inhibitor of 3ßHSD), resulting in a reduction in endogenous progesterone output, significantly decreased PGDH activity in a dose-dependent manner. The addition of increasing concentrations of exogenous progesterone reversed the inhibitory effect of trilostane. These results support strongly the hypothesis that endogenous progesterone may be exerting a stimulatory effect on 15-hydroxyprostaglandin dehydrogenase activity in these cells. This effect could not be enhanced by the addition of exogenous progesterone, but could be overcome by the antiprogestins RU486 and onapristone.
Estrogen has been shown to increase PGDH activity in rat decidual and myometrial tissues (25), although others reported that estradiol decreased PGDH activity by 50% in the rat kidney (52, 53). Endometrium from women who had been treated with the antiestrogen clomiphene at an early stage of the menstrual cycle showed high PG production and extensive inactivation by PGDH compared to those seen in the secretory phase of the cycle, suggesting that estradiol inhibits PGDH (54). However, we found no effect on PGDH activity in response to exogenous estradiol in our cultured cells. Similarly, Myatt et al. (27) have shown that estradiol has no effect on PG metabolism over a period of 120 h in cultured human placental cells. Interestingly, they were able to show a significant increase in PG metabolism with the combination of estradiol plus progesterone after a lag period of 24 h. This might also reflect a stimulatory effect of estradiol on progesterone receptor activity. In our studies, however, the addition of varying ratios of estradiol and progesterone had no effect on PGDH activity, but we did not pretreat the cells with estradiol before the addition of progesterone.
In summary, this study has shown that PG metabolism in cultured trophoblast cells from chorio-decidua and placenta is decreased in the presence of labor, suggesting that these cells may retain in vivo characteristics during in vitro culture. We have shown that in trophoblast tissue, glucocorticoids down-regulate PGDH activity and expression and that the mode of delivery, spontaneous vaginal delivery vs. cesarean section, does not appear to alter cortisol-induced inhibition of PGDH. PGDH activity was increased in the presence of the stable progestagen analogues R5020 and MPA and was inhibited by RU486, onapristone, and trilostane. Therefore, progestagens increase PGDH activity, an effect seen with exogenous progesterone only after inhibition or antagonism of endogenously produced steroid. We speculate that in vivo PGDH activity and expression may reflect a balance between the opposing effects of cortisol and progesterone on enzyme activity and expression. Further studies on the interaction of cortisol and progesterone and elucidation of the receptor types involved are required to determine the precise molecular mechanism(s) involved in the regulation of PG metabolism by steroids in placenta and fetal membranes of patients at term and preterm labor.
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Acknowledgments |
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Footnotes |
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Received April 29, 1998.
Revised September 11, 1998.
Accepted October 13, 1998.
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References |
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metabolites by
radioimmunoassay. Prostaglandins. 25:155–164.
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J. F. Johnstone, A. D. Bocking, E. Unlugedik, and J. R.G. Challis The Effects of Chorioamnionitis and Betamethasone on 11{beta}, Hydroxysteroid Dehydrogenase Types 1 and 2 and the Glucocorticoid Receptor in Preterm Human Placenta Reproductive Sciences, May 1, 2005; 12(4): 238 - 245. [Abstract] [PDF]
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 R. F. Johnson, C. M. Mitchell, V. Clifton, and T. Zakar Regulation of 15-Hydroxyprostaglandin Dehydrogenase (PGDH) Gene Activity, Messenger Ribonucleic Acid Processing, and Protein Abundance in the Human Chorion in Late Gestation and Labor J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5639 - 5648. [Abstract] [Full Text] [PDF]
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M. Lappas, M. Permezel, H. M. Georgiou, and G. E. Rice Regulation of Phospholipase Isozymes by Nuclear Factor-{kappa}B in Human Gestational Tissues in Vitro J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2365 - 2372. [Abstract] [Full Text] [PDF]
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N. Mirazi, N. Alfaidy, R. Martin, and J. R. G. Challis Effects of Dexamethasone and Sulfasalazine on Prostaglandin E2 Output by Human Placental Cells In Vitro Reproductive Sciences, January 1, 2004; 11(1): 22 - 26. [Abstract] [PDF]
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N. Alfaidy, W. Li, T. MacIntosh, K. Yang, and J. Challis Late Gestation Increase in 11{beta}-Hydroxysteroid Dehydrogenase 1 Expression in Human Fetal Membranes: A Novel Intrauterine Source of Cortisol J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 5033 - 5038. [Abstract] [Full Text] [PDF]
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F. A. Patel, J. W. Funder, and J. R. G. Challis Mechanism of Cortisol/Progesterone Antagonism in the Regulation of 15-Hydroxyprostaglandin Dehydrogenase Activity and Messenger Ribonucleic Acid Levels in Human Chorion and Placental Trophoblast Cells at Term J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2922 - 2933. [Abstract] [Full Text] [PDF]
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K. J. McKeown and J. R. G. Challis Regulation of 15-Hydroxy Prostaglandin Dehydrogenase by Corticotrophin-Releasing Hormone through a Calcium-Dependent Pathway in Human Chorion Trophoblast Cells J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1737 - 1741. [Abstract] [Full Text] [PDF]
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 A. I. Ivanov, A. C. Scheck, and A. A. Romanovsky Expression of genes controlling transport and catabolism of prostaglandin E2 in lipopolysaccharide fever Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2003; 284(3): R698 - R706. [Abstract] [Full Text] [PDF]
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 E. Hirsch and R. Muhle Intrauterine Bacterial Inoculation Induces Labor in the Mouse by Mechanisms Other than Progesterone Withdrawal Biol Reprod, October 1, 2002; 67(4): 1337 - 1341. [Abstract] [Full Text] [PDF]
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D. Giannoulias, F. A. Patel, A. C. Holloway, S. J. Lye, H. H. Tai, and J. R. G. Challis Differential Changes in 15-Hydroxyprostaglandin Dehydrogenase and Prostaglandin H Synthase (Types I and II) in Human Pregnant Myometrium J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1345 - 1352. [Abstract] [Full Text] [PDF]
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F. A. Patel and J. R. G. Challis Cortisol/Progesterone Antagonism in Regulation of 15-Hydroxysteroid Dehydrogenase Activity and mRNA Levels in Human Chorion and Placental Trophoblast Cells at Term J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 700 - 708. [Abstract] [Full Text] [PDF]
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N. Alfaidy, Z. G. Xiong, L. Myatt, Stephen. J. Lye, J. F. MacDonald, and J. R. G. Challis Prostaglandin F2{alpha} Potentiates Cortisol Production by Stimulating 11{beta}-Hydroxysteroid Dehydrogenase 1: A Novel Feedback Loop That May Contribute to Human Labor J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5585 - 5592. [Abstract] [Full Text] [PDF]
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W.L. Whittle, F.A. Patel, N. Alfaidy, A.C. Holloway, M. Fraser, S. Gyomorey, S.J. Lye, W. Gibb, and J.R.G. Challis Glucocorticoid Regulation of Human and Ovine Parturition: The Relationship Between Fetal Hypothalamic-Pituitary-Adrenal Axis Activation and Intrauterine Prostaglandin Production Biol Reprod, April 1, 2001; 64(4): 1019 - 1032. [Abstract] [Full Text]
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E. Schoof, M. Girstl, W. Frobenius, M. Kirschbaum, H. G. Dörr, W. Rascher, and J. Dötsch Decreased Gene Expression of 11{beta}-Hydroxysteroid Dehydrogenase Type 2 and 15-Hydroxyprostaglandin Dehydrogenase in Human Placenta of Patients with Preeclampsia J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1313 - 1317. [Abstract] [Full Text]
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 J. R.G. Challis, S. G. Matthews, W. Gibb, and S. J. Lye Endocrine and Paracrine Regulation of Birth at Term and Preterm Endocr. Rev., October 1, 2000; 21(5): 514 - 550. [Abstract] [Full Text]
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 W. L. Whittle, A. C. Holloway, S. J. Lye, W. Gibb, and J. R. G. Challis Prostaglandin Production at the Onset of Ovine Parturition Is Regulated by Both Estrogen-Independent and Estrogen-Dependent Pathways Endocrinology, October 1, 2000; 141(10): 3783 - 3791. [Abstract] [Full Text] [PDF]
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W. X. Wu, X. H. Ma, G. C. S. Smith, C. A. Mecenas, S. V. Koenen, and P. W. Nathanielsz Prostaglandin dehydrogenase mRNA in baboon intrauterine tissues in late gestation and spontaneous labor Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2000; 279(3): R1082 - R1090. [Abstract] [Full Text] [PDF]
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 F. C. Denison, S. C. Riley, C. L. Elliott, R. W. Kelly, A. A. Calder, and H. O.D. Critchley The effect of mifepristone administration on leukocyte populations, matrix metalloproteinases and inflammatory mediators in the first trimester cervix Mol. Hum. Reprod., June 1, 2000; 6(6): 541 - 548. [Abstract] [Full Text] [PDF]
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 N. Hanna, I. Hanna, M. Hleb, E. Wagner, J. Dougherty, D. Balkundi, J. Padbury, and S. Sharma Gestational Age-Dependent Expression of IL-10 and Its Receptor in Human Placental Tissues and Isolated Cytotrophoblasts J. Immunol., June 1, 2000; 164(11): 5721 - 5728. [Abstract] [Full Text] [PDF]
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K. J. Greenland, I. Jantke, S. Jenatschke, K. E. Bracken, C. Vinson, and B. Gellersen The Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase Gene Promoter Is Controlled by Ets and Activating Protein-1 Transcription Factors and Progesterone Endocrinology, February 1, 2000; 141(2): 581 - 597. [Abstract] [Full Text] [PDF]
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Interleukin-10 Modifies the Effects of Interleukin-1{beta} and Tumor Necrosis Factor-{alpha} on the Activity and Expression of Prostaglandin H Synthase-2 and the NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase in Cultured Term Human Villous Trophoblast and Chorion Trophoblast Cells J. Clin. Endocrinol. Metab., December 1, 1999; 84(12): 4645 - 4651. [Abstract] [Full Text]
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F. A. Patel, K. Sun, and J. R. G. Challis Local Modulation by 11{beta}-Hydroxysteroid Dehydrogenase of Glucocorticoid Effects on the Activity of 15-Hydroxyprostaglandin Dehydrogenase in Human Chorion and Placental Trophoblast Cells J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 395 - 400. [Abstract] [Full Text]
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), estradiol (
), and cortisol (•) on PGF2
), RU486 (•), and cortisol in the presence of a fixed
concentration (100 nmol/L) of RU486 (
), RU486 (
) on
PGFM formation in cultured term placental syncytiotrophoblast and
chorion trophoblast cells. The mean ± SEM are shown
(n = 4). *, P < 0.05 vs.
basal. C, The effects of progesterone (