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Potentiates Cortisol Production by Stimulating 11ß-Hydroxysteroid Dehydrogenase 1: A Novel Feedback Loop That May Contribute to Human Labor
CIHR Group in Fetal and Neonatal Health and Development (N.A., S.J.L., J.R.G.C.) and Departments of Physiology (N.A., Z.G.X., S.J.L., J.F.M., J.R.G.C.) and Obstetrics and Gynecology (S.J.L., J.R.G.C.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Obstetrics and Gynecology, University of Cincinnati, Cincinnati College of Medicine (L.M.), Cincinnati, Ohio; and Mt. Sinai Hospital (S.J.L., J.R.G.C.), Samuel Lunenfeld Research Institute, CIHR Group in Development and Fetal Health, Toronto, Ontario M5G 1X5, Canada
Address all correspondence and requests for reprints to: Dr. Nadia Alfaidy, Department of Physiology, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada.
Abstract
In human pregnancy, cortisol and PGs are involved in the onset of
labor and play an important role in the mechanisms leading to
parturition. Recent studies have shown that at term, cortisol increases
PG synthesis and decreases PG metabolism in chorion trophoblast
(CT) cells. In CT, 11 ß-hydroxysteroid oxidase type 1 (11
ß-HSD1) converts biologically inactive cortisone to cortisol to
regulate cortisol availability. In the present study, we have
investigated whether 11 ß-HSD1 activity could be influenced by PGs.
We have shown that in CT, PGF2
rapidly increased 11
ß-HSD1 reductase activity in a dose-dependent manner via the
PGF2 receptor, localized in the fetal membranes.
PGF2 stimulated 11 ß-HSD1 activity through increased
intracellular calcium mobilization, activation of PKC, and the
phosphorylation of the 11 ß-HSD enzyme. We propose that within CT
there is a novel feed forward loop by which PGF2 acts to
promote cortisol production from cortisone through increases in
11ß-HSD1, and this in turn leads to further net PG output for the
onset of labor and birth.
LATE HUMAN GESTATION is associated with an
increase in the synthesis of bioactive glucocorticoids by the fetus and
PGs, in particular PGE2 and
PGF2, by intrauterine tissues. Both cortisol
and PGs are required for the processes leading to birth. The increase
in fetal glucocorticoids helps to promote lung maturation, required by
the fetus for extrauterine survival (1, 2). PGs have been
shown to stimulate myometrial contractility, regulate cervical
ripening, and precipitate membrane rupture at term. Alterations in
synthesis of glucocorticoids and PGs may predispose to preterm birth,
the major problem in obstetrical practice. Increased PG output results
from elevated expression and activity of prostaglandin H2 synthase type
2 (PGHS-2) in amnion and chorion (3, 4, 5, 6) and reduced mRNA
and activity of the PG catabolizing enzyme, 15-hydroxyprostaglandin
dehydrogenase (PGDH), particularly in preterm labor. Regulation of PG
output is multifactorial, involving cytokines, growth factors, and
steroids, including cortisol. An inhibitory effect of cortisol on PGDH
activity in chorion and a stimulatory effect of cortisol on PGHS-2 in
amnion has recently been reported (7, 8), suggesting an
interaction between PGs and glucocorticoid. However, the source
of that cortisol is unclear; potentially it could be derived from the
mother or fetus or by local formation in the fetal membranes
(9).
Cortisol and its biologically inactive metabolite cortisone are interconverted through the enzyme 11 ß-hydroxysteroid dehydrogenase (11 ß-HSD). Two distinct isozymes of 11 ß-HSD, known as 11 ß-HSD1 and 11 ß-HSD2, have been characterized and cloned (10). 11 ß-HSD1 possesses both oxidase (cortisol to cortisone) and reductase (cortisone to cortisol) activities. This enzyme has a higher affinity for cortisone than for cortisol (11, 12, 13) and operates predominantly in a reductase direction. In contrast, 11 ß-HSD2, under physiological conditions exhibits only oxidase activity and has a much higher affinity for glucocorticoids. 11 ß-HSD1 is expressed in human placental intermediate trophoblast cells and vascular endothelium and is expressed abundantly in chorion trophoblast (CT) cells and decidua (14). 11 ß-HSD2 has been localized to placental syncytiotrophoblast (15). The high affinity of placental 11 ß-HSD2 for cortisol makes it more suited to regulate the amount of maternal glucocorticoid passing across the placenta into the fetal circulation. Glucocorticoids reaching the fetus from maternal circulation facilitate fetal organ maturation (16), but excessive amounts may result in fetal growth restriction and program adult-onset disease (17).
There is an increasing body of literature concerned with regulation of
11 ß-HSD2 activity and expression. The enzyme is up-regulated by
cyclic AMP and its activators such as forskolin in the kidney and
placenta (18, 19). The enzyme is down-regulated by
progesterone and nitric oxide (20). Previous studies have
failed to demonstrate regulation of 11 ß-HSD1 activity or expression
by these factors (18, 19). Recently it has been suggested
that the products of arachidonic acid metabolism may alter 11 ß-HSD2
activity (21). We have established that cortisol formed
from cortisone within the fetal membranes can promote enzyme activities
leading to increased formation of biologically active prostaglandins at
the time of labor (22, 23). These PGs might then
contribute to rupture of the fetal membranes and stimulation of uterine
contractility. Therefore, we hypothesized that
PGF2, generated within fetal membranes, might
stimulate 11 ß-HSD1 activity in CT cells in a manner that would
increase the local formation of cortisol from cortisone. Our results
demonstrate, for the first time, a novel interaction between
PGF2 and 11 ß-HSD1 in human fetal membranes
that describes a positive feed-forward loop, which should result in
increased output of glucocorticoid and prostaglandin at the time of
labor.
Materials and Methods
Chorionic trophoblast cell culture
Trophoblast cells from choriodecidual tissue were isolated and cultured using a modification of the technique described by Kliman et al. (24). Briefly, human placentae were obtained from uncomplicated term pregnancies (38–40 wk, n = 27) after elective cesarean section delivery. Patient consent and ethical approval was obtained before each placenta was collected, in agreement with the regulations of Mount Sinai Hospital and the University of Toronto. The chorion with adherent decidua was peeled off the amnion and digested three times for 60 min each with 0.125% trypsin (Sigma, St. Louis, MO) and 0.02% deoxyribonuclease I (Sigma), and 0.2% collagenase (Sigma) in DMEM (Life Technologies, Inc., Grand Island, NY) containing 10% FCS (Life Technologies, Inc.; Burlington, Ontario, Canada). The dispersed choriodecidual cells were filtered through a 200-µM nylon gauze and loaded onto a continuous Percoll (Sigma) gradient (5–70% in 5% steps of 3 ml each) and then centrifuged at 1,200x g for 20 min at room temperature 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. Cells were also plated in 6-well plates at 106 cells/ml for immunoprecipitation, in 35-mm Petri dishes (106 cells/ml) containing 1 glass coverslip for calcium imaging, or in 8-well chamber slides (Lab-Tek, Nalge Nunc, Burlington, Ontario, Canada) (0.3 x 106 cells/well) for immunostaining. The cells were cultured for 3 d at 37 C in 5% CO2 and 95% air before experimental treatments. Under these conditions, the chorionic trophoblast cells formed small clumps or remained as single cells. Purity of the cell preparation was assessed at the end of the experiment by histochemical staining for cytokeratin, an epithelial cell lineage marker (DAKO Corp., Glostrup, Denmark) or vimentin, a mesenchymal cell lineage marker (DAKO Corp.) (25); cells were counterstained with Carrazzi’s hematoxylin.
Immunohistochemistry for PGF2 receptor (FP
receptor)
Trophoblast cells. After 72 h, representative cultures were fixed with 4% paraformaldehyde and stored in 90% ethanol for further analysis. Cells were rehydrated with serial increasing dilutions of ethanol ending with two washes with PBS. Endogenous peroxidase activity was quenched by pretreatment with 0.3% hydrogen peroxide in PBS. Cells were then washed in PBS and incubated with 10% normal goat serum that served as a blocking agent for nonspecific binding. Immunoreactive FP receptor was detected with a rabbit polyclonal antibody (1:500) raised against the first extracellular loop of human FP receptor (amino acids 7–19: sequence SKQLVSPAAALLA). The tissues were incubated at 4 C for 18 h. FP receptor antibody binding was identified by the avidin-biotin-peroxidase technique using a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). Cells were washed, incubated with biotinylated secondary antibody for 60 min, washed again, and incubated with the ABC (avidin biotin-peroxidase complex) for 60 min. After a final washing, the immunoreactive proteins were visualized after the addition of 3,3'diaminobenzidine (Sigma) for 2 min. Cells were counterstained with hematoxylin, dehydrated in graded ethanol, cleared, and cover slips applied. To test the specificity of the antibody, cells were also stained with primary antibody preabsorbed with excess of immunizing peptide (1:100 dilution).
Fetal membranes. Fetal membranes with adherent decidua were rapidly separated from the placentae, rolled, and fixed by immersion in 4% paraformaldehyde and 0.2% glutaraldehyde in 70 mM phosphate buffer (pH 7.0) at 4 C overnight. Fixed tissues were then washed with PBS and stored in 90% ethanol before embedding them in paraffin wax. The paraffin blocks were sectioned at 5 µm for immunohistochemistry. Tissue sections were deparaffinized in xylene, rehydrated, and washed in PBS. Staining was performed according to the same protocol described above except that the primary antibody used was 1:1000 dilution and the immunizing peptide at 1:200.
Preparation of 3H-cortisone
We prepared 3H-cortisone by oxidizing 3H-cortisol (specific activity: 64 Ci/mmol, Amersham Pharmacia Biotech, Buckinghamshire, UK) with chromium trioxide as described previously (26) and purifying it by thin-layer chromatography (TLC: chloroform, ethanol; 95:5 vol/vol) before it was used in the enzyme activity experiments.
Determination of 11 ß-HSD1 activity
Trophoblast cells from placenta or fetal membranes were
cultured for 72 h. Then the media was replaced with FCS-free DMEM
(pH 7.4) and incubated for 1 h. 11 ß-HSD1 reductase activity
(cortisone to cortisol) was measured using 1-µM cortisone
containing 100,000 cpm 3H-cortisone as substrate
and oxidase activity was measured using 1 µM
cortisol containing 100,0000 cpm
3H-cortisol. The activity assays were performed
in the absence or presence of PGF2
(Oxford Biomedical Research, Cederlaine, Hornby,
Ontario, Canada). Fluprostenol, an FP receptor agonist, was also used
to determine whether any PGF2 effect was
mediated through the FP receptor. To investigate the role of calcium
(Ca2+) in this interaction, cells were incubated
with A23187 (Ca2+ ionophore). To determine the
source of mobilized calcium in CT cells for the
PGF2 effect on 11 ß-HSD1 activity, we
incubated the cells in the presence of EGTA; extracellular calcium
chelator; or BAPTA-am, an intracellular calcium chelator. To further
examine the second messenger of the PGF2, we
incubated cells with either PMA
(10-8–10-6
M), a PKC activator, or calphostin
(10-7 M), a specific inhibitor for
PKC. Each treatment was performed in triplicate for each preparation of
cells (n = three to six separate experiments; see
Results). Media were collected after 30-min incubations at
37 C, except in the experiments designed to examine the time course of
the PGF2 effect on 11 ß-HSD1 activity. Cell
viability before and after drug treatment was examined by trypan blue
exclusion.
To measure the conversion of 3H-cortisone to 3H-cortisol (reductase activity) or 3H-cortisol to 3H-cortisone (oxidase activity), a mixture of cortisol and cortisone (40 µg each) was added to the collected medium to allow the subsequent localization of the steroids during purification by TLC (27). Steroids in the media and cells were extracted with ethyl acetate (3 ml). The extract from the medium was dried under air, reconstituted with ethyl acetate (100 µl), and applied to a TLC plate (silica gel GF, Fisher Scientific, Pittsburgh, PA). Cortisol and cortisone were separated in the solvent system chloroform/ethanol (95/5, vol/vol). Steroids were visualized under UV light, scraped off the plate, and extracted with ethyl acetate. The solvent was dried, scintillation fluid added, and the radioactivity was counted in a liquid scintillation counter. The recovery rate of 3H-cortisone or 3H-cortisol was measured by adding 3H-cortisone or 3H-cortisol to culture media and processing the samples in an identical fashion. The mean recovery of 3H-cortisone was 88.3% ± 4.3% and 90% ± 3.8% for 3H-cortisol. In all cases, the background conversion, estimated from radioactivity in duplicate blank wells not containing cells, was subtracted from that in the experimental wells before analysis. Activity was expressed as femtomoles of cortisol or cortisone produced per 106 cells per 30 min (fmoles/106 cells per 30 min).
Calcium imaging
Ratio imaging of intracellular free Ca2+
was performed using the acetoxymethyl ester of the fluorescent
indicator Fura-2 (Molecular Probes, Inc., Eugene, OR) as
described previously (28). Washed CT cells were incubated
for 45 min at room temperature with Fura-2 (5 µM). The
cells on the cover slips were rinsed, placed in a perfusion chamber on
an inverted fluorescence microscope (Diophot 300,
Nikon, Melville, NY), illuminated with a 75-W Xenon
lamp, and viewed using a 40x fluorimmersion objective. For Fura-2
excitation, the shutter and filter wheel were controlled by Axon
Imaging Workbench software (AIW 2.1, Axon Instruments, Foster City, CA) to provide sequential
illumination at two alternating wavelengths, 340 and 380 nm.
Fluorescence of Fura-2 was detected at an emission wavelength of 510
nm. Consecutive video frames were acquired using an intensified change
coupled device camera (PTI IC-110, Monmouth, NJ). Digitized
images were obtained by averaging four consecutive frames using an
image-processing board also controlled by AIW 2.1 software.
Fluorescence emission ratios following excitation at 340 and 380 nm
were calculated by dividing averaged pixel values in circumscribed
regions of individual responding cells in the field of view. The values
were corrected for background fluorescence obtained by imaging a field
with no cells. Treatment with PGF2 at the
final concentration of 10-6 M was
performed at room temperature.
Immunoprecipitation
Phosphorylation and immunoprecipitation of 11 ß-HSD1.
Chorionic trophoblast cells (3 x 106
cells/well, 6-well plate) were washed twice with PBS and incubated for
90 min at 37 C in phosphate-free DMEM containing 0.4 mCi/ml
[32P] orthophosphate. Cells were then washed
with phosphate-free media and stimulated for 10 min with
PGF2 or the PMA, as described in the figure
legend. After rapidly rinsing twice with cold PBS, the cells were lysed
with cold RIPA lysis buffer (100 mM NaCl, 50 mM
NaF, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1
mM EDTA, 1 mM EGTA, 0.1 mM PMSF,
0.1 mg/ml aprotinin, 1 mM orthovanadate, and 50
mM Tris HCl, pH 7.5) for 20 min at 4 C. The total cell
lysates were collected with a cell scraper, vortexed vigorously, and
centrifuged (1,500x g for 20 min at 4 C). The precleared
cell extracts were transferred to new tubes. The protein content of the
supernatant was measured by the Bradford method using a protein assay
kit (Bio-Rad Laboratories, Inc., Mississauga,
Ontario, Canada) with BSA as standard. Immunoprecipitation was then
performed as described below:
11 ß-HSD1 immunoprecipitation. Cell extracts (200 µg protein) were incubated with an 11 ß-HSD1 antibody (5 µg/ml), a generous gift from Dr. K. Yang (29), for 2 h with continuous rotation at 4 C. Protein A-conjugated agarose beads (30 µl/ml) were then added, and the samples were incubated for an additional 2 h with continuous rotation. The immunocomplexed beads were collected by centrifugation and washed three times with RIPA lysis buffer. 11 ß-HSD1 was eluted from the beads with Laemmli buffer (0.5 M Tris HCL, 10% SDS, 20% glycerol 0.2% bromo phenol blue, and 10% ß-mercaptoethanol) and finally resuspended in 40 µl Laemmli buffer containing 10% ß-mercaptoethanol for subsequent gel electrophoresis using a 12% polyacrylamide gel. The gel was dried and autoradiography performed. Immunoblots with multiple exposures were quantified using Duo Scan transparency Scanner and NIH Image 6.1 software to determine the relative abundance of phosphorylated 11 ß-HSD1. Immunoprecipitation was conducted in four independent experiments.
Statistical analysis
All data are shown as mean ± SEM for the number of experiments with different chorion specimens from different patients. A t test or one-way ANOVA test were used to assess statistical differences. The effects of treatment on 11 ß-HSD1 activity were determined by one-way ANOVA, followed by post hoc multiple comparisons (t, Newman-Keuls, or Tukey’s HSD) method. Relative optical density determinations were analyzed by t test. Statistical significance was set at P < 0.05. Calculations were performed using Sigma Stat (Jandel Scientific Software, San Rafael, CA).
Results
Characterization of cell type
After 72 h of culture, chorionic trophoblasts appeared as rounded cells that either remained as single cells or formed small clumps. The cultured cells were 85% ± 3% (n = 24) cytokeratin positive and vimentin negative, suggesting the presence of mainly trophoblast cells and few fibroblast or decidual cells. Cell viability, assessed by trypan staining, was 95% before and after treatment.
FP receptor expression
Immunoreactive FP (ir-FP) receptor protein was identified in both
fetal membranes and CT cells (Fig. 1
).
Ir-FP receptor was localized to the amnion epithelial layer, the CT
cells, and extensively within the decidual stromal cell layer of the
fetal membranes (Fig. 1A). Purified CT cells after 72 h in culture
continued to exhibit intense ir-FP receptor expression (Fig. 1C).
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) and CT cells (Fig. 1D), thus confirming to the specificity of the
immunostaining response.
Effects of PGF2 on 11ß-HSD1 reductase activity
To examine the time course of PGF2
action, 11ß-HSD1 reductase activity was measured after different
incubation times (15–60 min) in the absence or presence of
PGF2 (10-6
M). Results are shown in Fig. 2. At each time point,
PGF2 significantly increased 11ß-HSD1
reductase activity (Fig. 2A); the difference was statistically
significant after 15 min. Therefore, all further determinations were
performed after 30-min incubation.
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. There was a dose-dependent stimulation
of cortisone to cortisol conversion. in the presence of increasing
concentrations of PGF2 from
10-10 M to the maximal effect at
10-6 M (Fig. 2B). We also
investigated the effect of PGE2 on 11ß-HSD1
activity under the same conditions. PGE2 (1
µM) significantly increased cortisol production by
45 ± 4% (n = 5, data not shown).
Effects of PGF2 on 11ß-HSD1 oxidase activity
11ß-HSD1 has both reductase and oxidase activity with the former
predominant in the CT cells (14). To examine whether
PGF2 has the same effect on both reactions, we
incubated CT cells in the presence of 1 µM cortisone or
cortisol in the absence or presence of PGF2 1
µM for 30 min. Results are shown in Fig. 3. PGF2
stimulated reductase activity with no enhancement of the low level of
oxidase activity present in CT cells.
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Although PGF2 is a potent FP agonist, it
is not very selective, having appreciable agonist activity at
PGE2 and thromboxane receptors. However,
fluprostenol, a PGF2 analog, has been
demonstrated to be equipotent with PGF2 at the
FP receptor but with much reduced agonist activity at other prostanoid
receptors (30, 31). To determine whether the
PGF2 effect on 11ß-HSD1 activity is mediated
through the FP expressed in chorion trophoblast cells, we examined the
effect of fluprostenol
(10-12–10-6
M) on 11ß-HSD1 activity (Fig. 4). Fluprostenol stimulated
11ß-HSD1 activity in a dose-dependent fashion indicating that the
effect of PGF2 on 11ß-HSD1 depends on
interaction of the hormone with FP receptor.
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on calcium release in chorion
trophoblast cells
It has been well documented in many other systems that
PGF2 increases intracellular calcium on
activation of FP receptor (32, 33, 34).
PGF2 (1 µM) increased the
intracellular calcium concentration of CT cells in less than 2 min. The
ratio 340/380 was increased from 0.44 to 0.56 following application of
PGF2 (Fig. 5).
Because the effect of PGF2 via FP receptor is
mediated by an increase in intracellular calcium, we examined whether
A23187, a calcium ionophore, was able to reproduce the effect of
PGF2. Incubation (30 min) with A23187
(10-8–10-6
M) resulted in a significant (100%) increase of 11ß-HSD1
activity (Fig. 6). Activation of FP
receptor is followed by an increase in intracellular concentration of
Ca2+. To determine whether intra- or extracelluar
calcium was mobilized to activate 11ß-HSD1, we measured the enzyme
activity in the absence or presence of EGTA (2 mM), an
extracellular calcium chelator, or BAPTA-am (1 mM), an
intracellular calcium chelator. The two chelators have been used
separately in two independent sets of experiments. Results are given in
(Fig. 7, A and B). Neither EGTA nor BAPTA
alone affected the basal activity of 11ß-HSD1. BAPTA
(10-6 M) but not EGTA in the
presence of PGF2 significantly decreased
11ß-HSD1 activity, compared with the effect of
PGF2 alone. These results strongly suggested
that PGF2 increased 11ß-HSD1 activity in CT
cells by mobilizing intracellular calcium stores.
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effects by PKC
In many other systems, it has been shown that
PGF2 stimulates PKC
(35, 36, 37). To determine whether 11ß-HSD1
activation by PGF2 involved the activation of
PKC, the effect of the PKC activator, PMA, was examined (Fig. 8A). PMA
(10-8–10-6
M) for 30 min stimulated 11ß-HSD1 activity by 40%, thus
reproducing the effect observed with PGF2.
Conversely, calphostin C, a PKC inhibitor, prevented the
PGF2-induced increase in 11ß-HSD1 activity
(Fig. 8B). Calphostin C (10-7 M) was
preincubated with the cells beginning 15 min before addition of
3H-cortisone (1 µM) in the absence
or presence of PGF2 (10-8
M). There was no effect of calphostin C alone on the basal
activity of 11ß-HSD1; however, coincubation of calphostin C with
PGF2 significantly decreased the effect of
PGF2.
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To examine further PGF2-dependent
11ß-HSD1 activation, in vitro phosphorylation was assayed.
A representative autoradiogram of phosphorylated 11ß-HSD1 that was
immunoprecipitated and then resolved by gel electrophoresis is shown in
Fig. 9A. The amount of phosphorylated
11ß-HSD1 was determined by densitometry of the band at 34 kDa.
11ß-HSD1 phosphorylation was increased 2.5-fold with
PGF2 (1 µM), and
2-fold with PMA (200 nM) (Fig. 9B).
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The present study demonstrates the potential for local production
of cortisol from cortisone in human chorionic tissue at term and shows
that PGF2 increases 11 ß-HSD1 reductase
activity in the CT cells. This action was associated with and dependent
on increased free calcium availability and PKC activation. The
PGF2 effect occurred within a few minutes. A
similar rapid effect of PGF2 has been reported
for other effects of the hormone in other systems, including the effect
of PGF2 on the contraction and proliferation
of glomerular mesangial cells and the stimulatory effect on protein
kinase signaling in bovine luteal cells (32, 38). The dose
dependence of the PGF2-induced increase in 11
ß-HSD1 (10-10–10-8
M) is consistent with physiological levels of
PGF2 in human placenta at term
(39). Therefore, the present data support the hypothesis
that under physiological conditions, locally produced
PGF2 may activate 11 ß-HSD1 reductase
activity in the fetal membranes, specifically the CT cells, in which
11ß-HSD1 and the FP receptor are colocalized. Our results demonstrate
for the first time a novel interaction between PGs and glucocorticoid
metabolism in CT cells that should create and then accelerate a local
feed-forward loop with the progression of labor.
In human intrauterine tissues, 11 ß-HSD1 is expressed in placental
intermediate trophoblast cells, the vascular endothelium, the chorionic
trophoblast cells, and the decidua (14). Our CT cell
preparation was predominantly cytokeratin positive, confirming their
origin from cells of epithelial lineage. The effect of
PGF2 to stimulate 11 ß-HSD1 is likely a
specific effect on CT cells, although we cannot exclude the possibility
that a portion of this effect may be mediated via decidual cells in our
preparation. Decidual cells have both 11 ß-HSD1 oxidase and reductase
activity, with predominantly oxidative activity (14, 40),
and express the FP receptor. However, a major contamination of the CT
preparation by decidual cells is unlikely because there was very little
oxidase activity in our chorion preparations that was not enhanced on
exposure to PGF2.
The increase in cortisol concentrations at term may result partly from an autocrine/paracrine regulation of 11 ß-HSD1 and 11 ß-HSD2 activities in the fetal membranes and placenta, respectively. In human choriocarcinoma JEG3 cells, it has recently been shown that PGs and leukotrienes decrease 11 ß-HSD2 activity by 40% (21). We found the same inhibitory effect of PGE2 on 11 ß-HSD2 activity in primary human placental trophoblast culture (N. Alfaidy et al.). A decrease in 11 ß-HSD2 activity was earlier reported in both human and guinea pig placenta near term (41, 42, 43). The inhibitory effect of PGs on 11ß-HSD2 dehydrogenase activity in the placenta leads to an increase in the rate of transplacental transfer of cortisol between the mother and fetus. In addition, the stimulatory effect of PGs on 11 ß-HSD1 reductase activity in fetal membranes also leads to an increase in locally formed cortisol derived from either maternal or fetal cortisone. This coordinated regulation of the 11 ß-HSD enzyme complex by PGs suggests a local positive feed-forward loop between cortisol and PG production at term in the human intrauterine tissue.
Primary PGs (PGE2 and
PGF2) act through specific receptors. There
are at least four subtypes of PGE2 receptors
(EP1, EP2, EP3, and EP4) and two subtypes of
PGF2 receptors (FPa and FPb). In this study we
showed for the first time that FP receptor is expressed in both fetal
membranes and purified CT cells. The specific subtype expression of FPa
and FPb and the expression of PGE2 receptor
subtypes have yet to be investigated in both human placenta and fetal
membranes. Although this report is focused on effects of
PGF2, we do not wish to imply that PGs of
other series are unimportant in this response. In preliminary studies
we found that PGE2 also increased chorionic 11
ß-HSD1 activity, but the mechanism of this effect remains to be
explored.
Several convergent arguments suggest that the FP receptor mediates the
observed PGF2 effect. Fluprostenol, which acts
exclusively at the FP receptor, reproduces the
PGF2-induced increase in 11 ß-HSD1 activity.
In the present study, we showed that FP receptor-mediated 11 ß-HSD1
activation was associated with a rapid elevation (within 1 min) of free
intracellular calcium concentration in CTs. The increase in free
calcium appears to be of intracellular origin because BAPTA but not
EGTA significantly attenuated the PGF2 effect.
Calcium, the effector of the FP pathway, mimics
PGF2 action, as demonstrated by the increase
in 11 ß-HSD1 activity in the presence of A23187. Our studies also
demonstrate that in vitro, PGF2
phosphorylates and stimulates 11 ß-HSD1 activity. A similar level of
11 ß-HSD1 phosphorylation was also obtained after PMA treatment,
suggesting PKC activation. Moreover, calphostin C significantly reduced
the PGF2 effect.
11 ß-HSD1 belongs to the short-chain alcohol dehydrogenase superfamily (46, 47). The active site consists of an invariant YXXXK motif and usually contains a serine and/or threonine residue at Y+1 and Y+3, potential sites of phosphorylation. A recent study (48) has shown that serines at the 11 ß-HSD1 active site play an important role in determining the rate of catalysis. The amino acid sequence of human 11 ß-HSD1 deduced from its cDNA sequence contains a potential PKC phosphorylation site TAR at position [64.66] and SIR at the position [196.198]. The data reported in the present study suggest that 11 ß-HSD1 is phosphorylated on PKC activation and that its activity may be regulated by phosphorylation-dephosphorylation. Further studies on the phosphorylation mechanism of 11 ß-HSD1 may enhance our understanding of the regulation of this enzyme.
Earlier studies have reported that the fetal membranes, in particular
the chorion layer, may provide an extraadrenal source of cortisol at
term in human pregnancy (49). In the present study, we
have described a mechanism for the stimulation of chorionic cortisol
production from circulating inactive precursors through activation of
11 ß-HSD1. We suggest that in the fetal membranes, 11 ß-HSD1 may be
pivotal to the integrated cellular regulation between glucocorticoid
and PGs. 11 ß-HSD1 is discretely colocalized with PGHS-2 and PGDH,
consistent with paracrine/autocrine regulation between glucocorticoids
and PGs. We speculate that this novel pathway, through which
PGF2 stimulates 11 ß-HSD1 activity in human
chorion trophoblasts to generate biologically active cortisol, could
have more general applicability in these tissues. In turn, cortisol
stimulates PG synthesis and decreases PG metabolism. This feed-forward
loop in the human fetal membranes may contribute to the mechanisms of
human parturition and potentially to the major clinical condition of
preterm birth.
Footnotes
Abbreviations: 11 ß-HSD1, 11 ß-Hydroxysteroid oxidase type
1; Ca2+, calcium; CT, chorion trophoblast; FP receptor, PG
F2 receptor; ir-FP, immunoreactive FP receptor; PGDH,
15-hydroxyprostaglandin dehydrogenase; PGHS-2, prostaglandin H2
synthase type 2; TLC, thin-layer chromatography.
Received December 15, 2000.
Accepted July 23, 2001.
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  A. E. Michael and A. T. Papageorghiou Potential significance of physiological and pharmacological glucocorticoids in early pregnancy Hum. Reprod. Update, September 1, 2008; 14(5): 497 - 517. [Abstract] [Full Text] [PDF]
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 C Chandras, T E Harris, A L. Bernal, D R E Abayasekara, and A E Michael PTGER1 and PTGER2 receptors mediate regulation of progesterone synthesis and type 1 11{beta}-hydroxysteroid dehydrogenase activity by prostaglandin E2 in human granulosa lutein cells J. Endocrinol., September 1, 2007; 194(3): 595 - 602. [Abstract] [Full Text] [PDF]
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H.-Y. Lee, T. J Acosta, M. Tanikawa, R. Sakumoto, J. Komiyama, Y. Tasaki, M. Piskula, D. J Skarzynski, M. Tetsuka, and K. Okuda The role of glucocorticoid in the regulation of prostaglandin biosynthesis in non-pregnant bovine endometrium J. Endocrinol., April 1, 2007; 193(1): 127 - 135. [Abstract] [Full Text] [PDF]
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 B. S. Knight, C. E. Pennell, R. Shah, and S. J. Lye Strain Differences in the Impact of Dietary Restriction on Fetal Growth and Pregnancy in Mice Reproductive Sciences, January 1, 2007; 14(1): 81 - 90. [Abstract] [PDF]
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 K. C. Jonas, C. Chandras, D. R. E. Abayasekara, and A. E. Michael Role for Prostaglandins in the Regulation of Type 1 11{beta}-Hydroxysteroid Dehydrogenase in Human Granulosa-Lutein Cells Endocrinology, December 1, 2006; 147(12): 5865 - 5872. [Abstract] [Full Text] [PDF]
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 D. B. Zaragoza, R. R. Wilson, B. F. Mitchell, and D. M. Olson The Interleukin 1beta-Induced Expression of Human Prostaglandin F2alpha Receptor Messenger RNA in Human Myometrial-Derived ULTR Cells Requires the Transcription Factor, NFkappaB Biol Reprod, November 1, 2006; 75(5): 697 - 704. [Abstract] [Full Text] [PDF]
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P. L. Grigsby, S. R. Sooranna, B. Adu-Amankwa, B. Pitzer, D. E. Brockman, M. R. Johnson, and L. Myatt Regional Expression of Prostaglandin E2 and F2alpha Receptors in Human Myometrium, Amnion, and Choriodecidua with Advancing Gestation and Labor Biol Reprod, August 1, 2006; 75(2): 297 - 305. [Abstract] [Full Text] [PDF]
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 D. J. Hosfield, Y. Wu, R. J. Skene, M. Hilgers, A. Jennings, G. P. Snell, and K. Aertgeerts Conformational Flexibility in Crystal Structures of Human 11{beta}-Hydroxysteroid Dehydrogenase Type I Provide Insights into Glucocorticoid Interconversion and Enzyme Regulation J. Biol. Chem., February 11, 2005; 280(6): 4639 - 4648. [Abstract] [Full Text] [PDF]
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 J. W. Tomlinson, E. A. Walker, I. J. Bujalska, N. Draper, G. G. Lavery, M. S. Cooper, M. Hewison, and P. M. Stewart 11{beta}-Hydroxysteroid Dehydrogenase Type 1: A Tissue-Specific Regulator of Glucocorticoid Response Endocr. Rev., October 1, 2004; 25(5): 831 - 866. [Abstract] [Full Text] [PDF]
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K. Sun and L. Myatt Enhancement of Glucocorticoid-Induced 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Expression by Proinflammatory Cytokines in Cultured Human Amnion Fibroblasts Endocrinology, December 1, 2003; 144(12): 5568 - 5577. [Abstract] [Full Text] [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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L. M. Thurston, K. C. Jonas, D. R. E. Abayasekara, and A. E. Michael Ovarian Modulators of 11{beta}-Hydroxysteroid Dehydrogenase (11{beta}HSD) Activity in Follicular Fluid from Bovine and Porcine Large Antral Follicles and Spontaneous Ovarian Cysts Biol Reprod, June 1, 2003; 68(6): 2157 - 2163. [Abstract] [Full Text] [PDF]
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