help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, F. A.
Right arrow Articles by Challis, J. R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, F. A.
Right arrow Articles by Challis, J. R. G.
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2922-2933
Copyright © 2003 by The Endocrine Society

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

Falguni A. Patel, John W. Funder and John R. G. Challis

Canadian Institutes for Health Research Group in Fetal and Neonatal Health and Development, Departments of Physiology and Obstetrics and Gynecology, University of Toronto (F.A.P., J.R.G.C.), Toronto, Ontario, Canada M5S 1A8; and Prince Henry’s Institute for Medical Research (J.W.F.), Clayton, Victoria, 8008 Australia

Address all correspondence and requests for reprints to: Dr. Falguni A. Patel, Department of Physiology, Faculty of Medicine, University of Toronto, 1 Kings College Circle, Medical Sciences Building, Room 3344, Toronto, Ontario, Canada M5S 1A8. E-mail: fal.patel{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin dehydrogenase (PGDH) metabolizes prostaglandins (PGs) to render them inactive. We reported previously that cortisol (F) decreases and progesterone (P4) maintains PGDH activity/expression in human chorion and placenta. Furthermore, we have shown that F and P4 compete for regulation of PGDH. We hypothesized that P4 maintains PGDH activity through interaction with the glucocorticoid receptor (GR) and that elevations in F compete with P4 at the GR, resulting in a decrease in PGDH at term. By immunohistochemistry and Western blotting analysis, we localized immunoreactive GR and progesterone receptor (PR) to chorion and placental trophoblast cells. We treated chorion and placental trophoblast cells in culture with F, dexamethasone (DEX), ß-methasone, P4, trilostane (a 3ß-hydroxysteroid dehydrogenase inhibitor), medroxyprogesterone acetate (MPA), and/or 21-hydroxy-6,19-oxidopregn-4-ene-3,20-dione (21OH-6OP; a GR antagonist). By RIA and Northern blotting analysis, all glucocorticoids (GCs) decreased PGDH activity/expression. Coincubation with 21OH-6OP reversed GC inhibition of PGDH; MPA, but not P4, treatment stimulated PGDH activity. Trilostane inhibited PGDH activity, and coincubation with P4 or MPA reversed trilostane inhibition of PGDH. Treatment with trilostane, P4, 21OH-6OP, or MPA plus 21OH-6OP reversed P4 and MPA up-regulation of PGDH activity. Our findings suggest that F inhibition and P4 stimulation of PGDH may be mediated by PR, but also via the GR, in chorion and placenta.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) HAVE been linked with a number of parturition events, including stimulation of myometrial contractility (1, 2, 3) and regulation of cervical effacement and dilatation (4, 5). Prostaglandin dehydrogenase (PGDH) is the enzyme that catalyzes the initial conversion of PGE2 and PGF2{alpha} to their biologically inactive 15-keto derivatives (6). PGDH activity and mRNA levels are lower in chorion from patients at term and preterm spontaneous labor compared with those in chorion from term elective cesarean section (7, 8, 9). We have shown previously that PGDH activity and gene expression in chorion and placental trophoblast cells from term pregnancies are maintained by progesterone (P4) and inhibited by cortisol (F) (10). Furthermore, we have shown that treatment of chorion and placental trophoblast cells with equimolar concentrations of P4 and F in the presence of trilostane, an inhibitor of cellular 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity, attenuated F-induced inhibition of PGDH activity and mRNA expression (11), demonstrating the local and antagonistic roles that these steroids play in regulating PGDH.

Steroid hormones mediate changes in gene expression by binding to high affinity receptors, which, in turn, regulate transcription by binding to hormone response elements located within promoters of hormone-inducible genes (12, 13). Within the steroid receptor superfamily, progesterone receptor (PR) and glucocorticoid (GC) receptor (GR) share regions of high homology, particularly within the DNA-binding domain (14). They can also bind to and enhance transcription from a common consensus sequence, the GC response element (GRE) (15, 16).

There has been some controversy as to whether PR are expressed by human intrauterine tissues at term. Several studies indicate that amnion, chorion, and placenta at term have no detectable levels of PR, whereas very low levels of PR were detected in decidua and myometrium (17, 18, 19, 20, 21, 22). In contrast, other studies have detected low levels of PR in human term amnion, chorion, decidua, myometrium, and placenta at term (23, 24, 25, 26, 27, 28, 29). GR have also been localized to human amnion epithelial cells, amnion mesenchymal cells, chorion, decidua, endometrium, and myometrium at term (30, 31, 32, 33). Although no labor-related changes in these steroid receptor concentrations have been reported, one group has demonstrated increased nuclear GR compared with cytosolic GR after preterm labor (33).

All of the steroids we used in previous experiments are able to bind to GR, in the absence of PR, with varying affinities (34). The affinity of GR for F (Kd) at 37 C is approximately 30 nM, which falls within the normal range for plasma concentrations of free hormone (35). P4 at physiological concentrations has been shown to be capable of binding to the GR (36) despite its affinity for GR being 25–50% that of F (37); F at physiological concentrations does not bind to PR (36, 38). GCs and P4 may thus act through independent receptors or may compete for binding at the GR. Furthermore, characterization of the structure and promoter activity of the mouse 15-PGDH gene, which shares 87% homology with the human gene, has shown the presence of several GREs, but no progesterone response elements (39), suggesting that regulation of this gene by both GCs and P4 may be via competition at the level of the GR or the GRE. Karalis et al. (40) suggested that antagonistic GC and P4 regulation of CRH output by placental trophoblast cells occurs via competition for binding to the GR. It is possible that a similar antagonism of the effects of P4 through competition by GCs occurs in placenta and chorion in relation to PGDH activity and mRNA expression.

The following series of experiments was designed to explore the mechanisms of GC and P4 antagonism of PGDH activity and mRNA expression in human fetal membranes and placenta. As PR have been shown to be either absent or present at extremely low levels at term in chorion and placenta, we hypothesized that P4 may be acting at least in part at the GR/GRE to maintain PGDH activity and expression throughout gestation. As F has a higher affinity than P4 for GR, we hypothesized that as the concentration of F rises at term, F would competitively displace P4 at the GR to down-regulate PGDH activity and expression. To examine this possibility we localized GR and PR in human fetal membranes and placenta and in cultured human chorion and placental trophoblast cells. We then treated chorion and placental cells with GCs and/or progestins in the presence or absence of a GR antagonist and measured corresponding levels of PGDH activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection, protein extraction, and Western blot hybridization

Duplicate samples of placenta, amnion, and chorio-decidua were obtained from uncomplicated, normal term pregnancies after elective cesarean section at Mt. Sinai Hospital (Toronto, Canada). The collection of tissues for these studies was approved according to the bioethical requirements of Mt. Sinai Hospital and the University of Toronto human experimentation and ethics committees. Rat liver and ovary were processed as positive controls for GR and PR, respectively. Protein concentrations were determined by the Bradford assay (41).

Protein samples (50 and 200 µg each) were solubilized in Laemmli sample buffer (10% sodium dodecyl sulfate, 0.5 M Tris, glycerol, and 0.2% bromophenol blue; Bio-Rad Laboratories, Inc., Richmond, CA), heated at 55 C for 15 min, separated by PAGE (100 V for 2 h) as previously described (42) on an 8% bis-acrylamide gel/4% stacking gel [1.5 M Tris (pH 8.8), 10% sodium dodecyl sulfate, 30% bis-acrylamide, and 10% ammonium persulfate], and then electrophoretically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.), run at 110 V for 2 h at 4 C. Blots containing immobilized proteins were blocked overnight at 4 C in 5% skim milk powder in PBS-Tris (PBS-T) with constant agitation. Primary antibodies (Abs; polyclonal rabbit antihuman GR Ab, 1:100; monoclonal mouse antihuman PR Ab, 1:100; both from Affinity BioReagents, Inc., Golden, CO) were diluted in blocking solution and incubated with the blots for 1 h at room temperature. Blots were rinsed five times for 5 min each time in PBS-T. Rabbit (1:1000; goat antirabbit IgG; Amersham Pharmacia Biotech, Little Chalfont, UK) or mouse (1:3000; sheep antimouse IgG; Amersham Pharmacia Biotech) secondary antiserum conjugated with horseradish peroxidase was also diluted in blocking solution and incubated with membranes for 1 h at room temperature, followed by six 5-min washes in PBS-T. Detection of proteins on Western blots was achieved using the Amersham ECL Detection System (Amersham Pharmacia Biotech). Blots were exposed to x-ray film (X-OMAT Blue XB-1, Eastman Kodak Co., Rochester, NY), and the relative intensity of protein signals was quantified by computerized image analysis (MCID, Imaging Research, Inc., St. Catherines, Canada; laser scanner from Molecular Dynamics, Inc., Sunnyvale, CA; ImageQuant software, PD Biosciences, Mountain View, CA). To ensure specificity, the primary Ab was preabsorbed with peptide/Ab (1:1, wt/vol; GR and PR peptide from Affinity BioReagents, Inc.). Before use, the preabsorbed Ab was centrifuged at 178,000 x g for 30 min at 4 C, and the supernatant fraction was substituted for the primary Ab in the overnight incubation.

Chorion and placental tissue culture

Human chorio-decidual tissue and placentas were obtained from uncomplicated normal term pregnancies after elective cesarean section or spontaneous vaginal delivery (n = 12 patients) at Mt. Sinai Hospital. Trophoblast cells from chorio-decidual tissue and placental cotyledons were isolated and cultured by a modification of the technique described by Kliman et al. (43) and as previously reported (10, 44, 45).

Steroid and steroid receptor antagonist treatment of cultured cells

Trophoblast cells were grown for 3 d, then incubated for 24 h in serum-free fresh medium containing F (1 µM), cortisone (1 µM), dexamethasone (DEX; 1 µM), P4 (1 µM), medroxyprogesterone acetate (MPA; 1 µM; all from Sigma-Aldrich Corp., St. Louis, MO), ß-methasone (1 µM; Celestone Soluspan, Schering-Plough Pty Ltd.), trilostane (synthesized at Schering AG, Berlin, Germany; gift from Dr. M. Novy, Oregon Health Sciences Center, Portland, OR; 1 µM), 21-hydroxy-6,19-oxidopregn-4-ene-3,20-dione (21OH-6OP; GR antagonist, 1 µM; Steraloids, Inc., Wilton, NH), or combinations of these compounds. Control cultures were maintained without additives or in the presence of 21OH-6OP alone. After 24 h the medium was replaced with fresh medium containing PGF2{alpha} (100 ng/ml; 282 nM) without steroids for 4 h. Media were then collected and stored at –80 C for later assessment of PGDH activity by RIA of the concentration of 13,14-dihydro-15-keto PGF2{alpha} (PGFM), the stable metabolite of PGF2{alpha}, in the culture medium.

Immunohistochemistry

Samples of term human placenta and fetal membranes were washed twice a day for 3 d in PBS (0.01 M, pH 7.4) after initial fixation in 4% paraformaldehyde/0.2% glutaraldehyde and stored in 70% ethanol at 4 C. Fetal membranes were rolled, and the placenta was trimmed before embedding it in paraffin wax. The paraffin blocks were then sectioned at 5 µm.

The purity of the cultured cell preparation was assessed at the end of each experiment by immunohistochemistry (46). After 3 d of culture cells were washed twice with PBS (0.01 M, pH 7.4) and then fixed in 4% paraformaldehyde/0.2% glutaraldehyde and dehydrated in ethanol through a series of washes (two at 50%, two at 70%, and two at 90%). Chamber slides were stored in 90% ethanol at 4 C until ready to stain.

Sections and cultured cells were stained for immunoreactive (IR-) GR and IR-PR by the avidin-biotin procedure (Vector ABC kit, Vector Laboratories, Inc., Burlingame, CA) as described previously (10). GR were localized with a polyclonal rabbit antihuman Ab (ABR, PA1-511) at a dilution of 1:500, and PR were localized with a monoclonal mouse antihuman Ab (Affinity BioReagents, Inc., MA1-410) at a dilution of 1:100 for sectioned tissues and 1:50 for cultured cells. All antibodies were diluted in Ab dilution buffer (1 g BSA and 0.02 g sodium azide in 100 ml 0.01 M PBS, pH 7.4). For negative controls the primary Ab was either substituted with Ab dilution buffer or nonimmune rabbit serum (1:2000 dilution) or preabsorbed with 1:1 (wt/vol) of peptide/Ab (GR and PR peptide from ABR). Before use, the preabsorbed Ab was centrifuged at 178,000 x g for 30 min at 4 C, and the supernatant fraction was substituted for the primary Ab in the overnight incubation.

PGFM RIA

The activity of PGDH was assessed by measuring the PGFM content in duplicate aliquots (10 and 50 µl) of culture medium by a modification of the RIA technique described by PerSeptive Biosystems, Inc. (Framingham, MA). PGFM antiserum (100 µl; raised in rabbits; PerSeptive Biosystems, Inc.), diluted 1:10, and 100 µl [3H]PGFM (10,000–15,000 cpm of 13,14-dihydro-15-keto-[5,6,8,11,12,14-N-3H]PGF2{alpha}; Amersham Pharmacia Biotech) was added to each tube. Volumes were adjusted with BGG-phosphate buffer [10 mM PO4, 0.85% (wt/vol) NaCl, 0.02% (wt/vol) KCl, 0.1% (wt/vol) bovine {gamma}-globulin (Sigma-Aldrich Corp.), and 0.1% (wt/vol) NaN3, in deionized water, pH 7.0] to a total volume of 0.6 ml in 12 x 75-mm borosilicate tubes (Maple Leaf brand, Sigma-Aldrich Corp.). Tubes were vortexed and incubated overnight at 4 C. The interassay coefficient of variation was 10.8 ± 2.4% (±SEM; n = 12).

RNA extraction and Northern blotting analysis

Cells in petri dishes were mechanically dispersed by scraping with a rubber policeman for 1 min in the presence of TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) and then incubating them for 5 min at room temperature to permit complete dissociation of nucleoprotein complexes. Total RNA was extracted from tissues by a method based on that described by Chomczynski and Sacchi (47). Trial extractions demonstrated that 2 ml TRIzol reagent were sufficient to obtain total RNA of suitable purity (OD260/280nm = 1.6–1.8) from cultured chorion and placental trophoblast cells plated in petri dishes. Samples were then stored at -80 C in 75% ethanol.

Northern blot hybridization was carried out using an 800-bp fragment of the PGDH cDNA sequence as a probe (7, 48). Blots were exposed to Kodak X-AR film with an intensifying screen for 5–7 d. After autoradiographic exposure, the blots were stripped and reprobed with a cDNA for mouse 18S ribosomal RNA as an internal standard. Relative ODs (ROD) were determined by computerized image analysis (MCID, Imaging Research, Inc., St. Catherines, Canada). The values for ROD were determined after different exposure times to ensure that the values obtained were within the linear range of the autoradiographic film and densitometer. Results are expressed as the ratio of the RODs of the PGDH mRNA/18S ribosomal RNA hybridization signals.

Statistical analysis

Results are expressed as the mean ± SEM for the number of observations (patients) studied. The effects of treatment on concentrations of PGFM in the culture medium were examined by one-way ANOVA corrected for repeated measures when appropriate. Differences between treatments were examined by Student-Newman-Keuls multiple range tests, when the data were not distributed normally. ROD determinations were analyzed by t test; statistical significance was set at P < 0.05. Calculations were performed on SigmaStat (Jandel Scientific, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell characterization

After 72 h of culture, chorion trophoblast cells either remained as single cells or formed clumps, whereas placental trophoblast cells formed syncytial aggregates, suggested to correspond with placental syncytiotrophoblast (49). Both chorion and placental trophoblast cell cultures were predominantly cytokeratin positive (chorion, 85–95%; placenta, >90%) and predominantly vimentin negative, as described previously (10), suggesting the presence of mainly trophoblast cells with a few fibroblast or decidual cells. Both chorion and placental trophoblast cell cultures were positive for IR-PGDH (10). By trypan blue exclusion staining the percent viability of cultured cells before and after treatment was determined to be greater than 95%.

Distribution of IR-GR and IR-PR in human fetal membranes and placenta by Western blot hybridization and immunohistochemical analysis

GR, shown predominantly as a 97-kDa band by Western blot, was present in all samples of human amnion, chorio-decidua, and placenta (Fig. 1Go). PR, shown as three bands (94, 120, and 140 kDa), was also present in all samples of human amnion, chorio-decidua, and placenta. Preabsorption with corresponding peptides indicated the specificity of bands.



View larger version (54K):
[in this window]
[in a new window]
  		Figure 1. Distribution of IR-GR and IR-PR in human amnion, chorio-decidua, and placenta by Western blot hybridization (n = 2). GR is present as three bands of 120, 97, and 55 kDa. Preabsorption (right column) eliminates all of the bands. PR is present as three bands; one of 74 kDa, a doublet/triplet band of 120 kDa, and one band of approximately 140 kDa. Panels in the right column represent preabsorption blots for each of the steroid receptors. Both steroid receptors were present in all tissues examined.

 
GR were present in both intact fetal membranes (amnion, chorion, and decidua; Fig. 2Go, A and B) and chorion trophoblast cells (Fig. 2Go, C and D). GR was heterogeneously distributed throughout the amnion and chorion and, to a lesser extent, within decidual cells. Both nuclear and cytoplasmic GR stainings were seen in cultured chorion trophoblast cells. IR-PR was localized predominantly within the decidua, with relatively little staining in the amnion and chorion (Fig. 2Go, I and J). Perinuclear PR staining was seen in cultured chorion trophoblast cells (Fig. 2Go, K and L).



View larger version (132K):
[in this window]
[in a new window]
  		Figure 2. Immunohistochemical staining for the GR (A–H), and PR (I–P) in human fetal membranes, placenta, and cultured chorion and placental trophoblast cells 72 h after culture. Brown indicates positive staining. A, B, I, and J, Intact sections of fetal membranes; C, D, K, and L, cultured chorion trophoblast cells; E, F, M, and N, intact sections of placenta; G, H, O, and P, are cultured placental trophoblast cells; B, D, F, and H, negative controls for GR; J, L, N, and P, negative controls for PR. All panels are magnified x400.

 
In placenta, strong GR staining was present throughout the villous tissue (Fig. 2Go, E–H), whereas some PR staining was found in placental syncytiotrophoblast (Fig. 2Go, M and N). However, quantitative comparisons between the two staining patterns could not be made. Cultured placental trophoblast cells were positive for both GR and PR (Fig. 2Go, G, H, O, and P).

Effect of 21OH-6OP (a GR antagonist) on GC regulation of PGDH activity and mRNA expression in cultured chorion and placental trophoblast cells

In chorion trophoblast cells, F (1 µM), cortisone (1 µM), DEX (1 µM), and ß-methasone (1 µM) significantly (P < 0.05) decreased PGFM output (mean basal value, 15.1 ± 1.7 ng/ml; n = 8 each; Fig. 3Go) and PGDH mRNA expression [by 56% (n = 8), 32% (n = 8), 64% (n = 12), and 79% (n = 8), respectively; Fig. 4Go, A and B]. Coincubation with 21OH-6OP (1 µM) significantly reversed F, cortisone, DEX, and ß-methasone inhibition of PGFM output (n = 4 each; Fig. 3Go). Coincubation with 21OH-6OP also reversed F, cortisone, and DEX inhibition of PGDH mRNA expression, although ß-methasone inhibition of PGDH mRNA expression remained significantly below basal (n = 4 each; Fig. 4Go, A and B). This less than full restoration may reflect the more marked inhibitory effect of ß-methasone alone than in combination with the other GC and the use of a single equimolar dose of competing 21OH-6OP.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Effect of 21OH-6OP (a GR antagonist; 1 µM; n = 4) on cortisol, cortisone, DEX, and ß-methasone (1 µM each; n = 8) regulation of PGDH activity in cultured term human chorion trophoblast cells. All values are the mean ± SEM. *, P < 0.05 vs. basal.

 


View larger version (36K):
[in this window]
[in a new window]
  		Figure 4. A, Effect of 21OH-6OP (a GR antagonist; 1 µM; n = 4) on cortisol (n = 8), cortisone (n = 8), DEX (n = 12), and ß-methasone (n = 8; 1 µM each) regulation of PGDH mRNA levels determined by Northern blotting analysis in cultured term human chorion trophoblast cells. Control, n = 8. All values are the mean ± SEM. *, P < 0.05 vs. basal. B, Representative Northern blots of PGDH mRNA levels in cultured term human chorion trophoblast cells after treatment with cortisol, cortisone, DEX, and ß-methasone in the absence or presence of the GR (21OH-6OP). PGDH mRNA is shown as two bands of 3.4 and 2.0 kb. Changes in the larger 3.4-kb band were measured and analyzed. 18S ribosomal RNA is shown as an internal standard to correct for variations in gel loading and transfer.

 
In placental trophoblast cells, F (1 µM), DEX (1 µM), and ß-methasone (1 µM) again significantly (P < 0.05) decreased PGFM output (mean basal value, 10.2 ± 0.7 ng/ml; n = 8 each; Fig. 5Go) and PGDH mRNA expression (Fig. 6Go, A and B). However, cortisone (1 µM) had no effect on PGFM output by placental trophoblast cells (Fig. 5Go), in contrast to its effects on chorion trophoblast cells (Fig. 3Go). Coincubation with 21OH-6OP (1 µM) significantly reversed F, DEX, and ß-methasone inhibition of PGFM output (n = 4 each; Fig. 5Go) and PGDH mRNA expression (n = 4 each; Fig. 6Go, A and B), and did not alter the lack of effect seen with cortisone (n = 4). Treatment of cells with 21OH-6OP (1 µM; n = 4) alone did not affect PGFM formation or PGDH mRNA levels in either chorion or placenta.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Effect of 21OH-6OP (a GR antagonist; 1 µM; n = 4) on cortisol, cortisone, DEX, and ß-methasone (1 µM each; n = 8) regulation of PGDH activity in cultured term human placental trophoblast cells. All values are the mean ± SEM. *, P < 0.05 vs. basal.

 


View larger version (36K):
[in this window]
[in a new window]
  		Figure 6. A, Effect of 21OH-6OP (a GR antagonist; 1 µM; n = 4) on cortisol (n = 8), cortisone (n = 8), DEX (n = 12), and ß-methasone (n = 8; 1 µM each) regulation of PGDH mRNA levels determined by Northern blotting analysis in cultured term human placental trophoblast cells. Control, n = 8. All values are the mean ± SEM. *, P < 0.05 vs. basal. B, Representative Northern blots of PGDH mRNA levels in cultured term human placental trophoblast cells after treatment with cortisol, cortisone, DEX, and ß-methasone in the absence or presence of the GR (21OH-6OP) antagonist. PGDH mRNA is shown as two bands of 3.4 and 2.0 kb. Changes in the larger 3.4-kb band were measured and analyzed. 18S ribosomal RNA is shown as an internal standard to correct for variations in gel loading and transfer.

 
Effect of 21OH-6OP (a GR antagonist) on P4 and MPA regulation of PGDH activity in cultured chorion and placental trophoblast cells

Treatment of cells with 21OH-6OP (1 µM) in the presence or absence of P4 (1 µM) did not alter PGFM formation in either chorion (Fig. 7Go) or placental (Fig. 8Go) trophoblast cells. Trilostane (1 µM) significantly inhibited PGFM output in chorion (mean basal value, 15.0 ± 0.7 ng/ml; n = 4; P < 0.05; Fig. 7Go) and placenta (mean basal value, 10.7 ± 1.1 ng/ml; n = 4; P < 0.05; Fig. 8Go). Coincubation of cells with trilostane (1 µM) plus P4 (1 µM) reestablished basal PGFM output in chorion and placenta, and the addition of 21OH-6OP (1 µM) to cells pretreated with trilostane (1 µM) blocked P4 (1 µM) stimulation of PGFM output in both cell types. Incubation of cells with MPA (1 µM; stable progestin analog) significantly stimulated PGFM output in chorion trophoblast cells by 53 ± 17.9% above basal (Fig. 7Go) and by 59 ± 21.1% above basal in placental trophoblast cells (Fig. 8Go), and coincubation of cells with 21OH-6OP plus MPA blocked MPA stimulation of PGFM output.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 7. Effect of 21OH-6OP (a GR antagonist) on P4 and MPA regulation of PGDH activity in cultured term human chorion trophoblast cells. All values are the mean ± SEM (n = 4). *, P < 0.05 vs. basal.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 8. Effect of 21OH-6OP (a GR antagonist) on P4 and MPA regulation of PGDH activity in cultured term human placental trophoblast cells. All values are the mean ± SEM (n = 4). *, P < 0.05 vs. basal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have localized GR and PR by Western blotting analysis to human amnion, chorion, and placenta and by immunohistochemistry to cultured chorion and placental trophoblast cells. F and the synthetic GCs, DEX and ß-methasone, significantly decreased PGDH mRNA levels and activity in chorion and placenta, suggesting that the effect of GC on PGDH may be at the transcriptional level. Coincubation of cells with GCs and the GR antagonist (21OH-6OP) significantly reversed GC inhibition of PGDH mRNA expression and activity. Similarly, the GR antagonist blocked the stimulation of PGDH seen in response to added P4 in the presence of trilostane and the response to MPA. These results raise the possibility that the effects of P4 on PGDH may be mediated in part through the GR.

Our earlier experiments, however, using a PR antagonist could be interpreted as evidence for a PR-mediated effect in cultured chorion and placental trophoblast cells. There was no effect of RU486, both an anti-GC and an antiprogestin, on the dose-dependent inhibition of PGDH activity seen in response to F (10). However, RU486 alone inhibited PGDH activity, and the addition of P4, MPA, or R5020 to these cells restored PGDH activity (10). Thus, it appeared that RU486 was acting predominantly as an antiprogestin (and/or a GC agonist) in this culture system. PGDH activity was also reduced by treatment with a more specific P4 antagonist, onapristone, or a 3ß-HSD inhibitor (trilostane), but was restored by the addition of P4 (10). Treatment of cells with MPA or R5020, two stable progestins, resulted in an increase in PGDH activity; onapristone treatment decreased basal PGDH activity, but this was reversed by exogenous P4, again suggesting effects mediated via PR.

Three protein isoforms of PR have been reported, PR-A, PR-B, and PR-C. All three receptors are produced from a single gene by transcription at distinct promoters (50, 51, 52). A recent study has shown by RT-PCR that only the A form of PR is present in human placenta during late gestation (27), consistent with P4 up-regulation of PGDH via the GR, as PR-A is thought to have predominantly repressive effects. Attia et al. (53) suggested that the presence of the inhibitory PR-A isoform and the absence of the stimulatory isoform, PR-B, may explain P4 resistance in human endometriotic tissue. In contrast, PR-A was found to be a stronger trans-activator than PR-B for the expression of IGF-binding protein-1 in human endometrial stromal cells (54). Recently, in myometrial smooth muscle cells, both PR-A and PR-B caused a ligand-dependent activation of PGDH (55); however, given the tissue-specific regulatory nature of these PR isoforms, it is uncertain whether both isoforms have the same effect on PGDH in chorion and placenta. We have not yet determined which specific PR isoforms are expressed in cultured chorion and placental trophoblast cells or whether there is a shift in isoform expression at the onset of labor.

P4 action via GR was recently shown to be critically important for regression of the rat corpus luteum near the end of pregnancy (56, 57). P4, acting through GR, was able to enhance its own levels by down-regulating the expression of 20{alpha}-HSD, an enzyme that catabolizes P4 and reduces P4 secretion by the corpus luteum. Karalis et al. (17) also suggested that regulation of CRH in human placenta by P4 is mediated via GR. A number of other groups have also demonstrated physiological actions of P4 via GR in the absence (58) or presence (59, 60, 61, 62, 63, 64, 65, 66) of PR.

We have shown that GCs (including DEX, ß-methasone, F, and cortisone in chorion), acting via the GR, inhibit PGDH mRNA expression and activity in chorion and placental trophoblast cells. The GR antagonist (21OH-6OP) we used in these experiments has been shown to be highly specific for the GR in the rat (67). The necessity of examining a range of treatments within cells prepared from tissue collected from a single patient precluded our generating simultaneously full dose-response curves. In the present experiments we used 21OH-6OP at a single high concentration. It remains possible that in man 21OH-6OP at the dose we have used could block the effects of P4 through interaction at the PR. In control cultures, 21OH-6OP alone had no significant effect on PGDH mRNA levels or activity, in contrast with drugs that block P4 synthesis, such as trilostane, or P4 action, such as onapristone or RU486, which reduce endogenous P4 availability or activity and decrease PGDH mRNA expression. 21OH-6OP antagonized the up-regulation of PGDH by MPA or added P4 in the presence of trilostane, consistent with either the effect of P4 being exerted in part through a receptor other than PR (probably GR) or a weak antiprogestin effect of 21OH-6OP at equimolar doses. Erman et al. (68) have also shown that DEX significantly decreases PGDH activity, whereas other groups have reported that DEX either stimulates (69, 70) or has no effect (71) on PGDH activity in various cell types. A recent study by Tong and Tai (72) has shown almost complete inhibition of PGDH protein expression and activity with DEX in human promonocytic cells.

GC-GR can interact with the GRE or negative GRE (nGRE) directly to mediate changes in gene transcription. However, activated GR appears to down-regulate gene expression primarily by modulating either the binding or activation of transcription factors, such as CCAAT/enhancer-binding protein (73, 74), activating protein-1 (AP-1) (73, 75), nuclear factor-{kappa}B (NF-{kappa}B) (76, 77), cAMP response element-binding protein (CREB) (78), and some signal transducer and activator of transcription (STAT) proteins, such as STAT3, STAT5, and STAT6 (79, 80). Furthermore, while GRE binding involves a GR homodimer, interaction with the transcription factors AP-1 and NF-{kappa}B involves only a single GR monomer. Thus, inhibition of AP-1- and NF-{kappa}B-dependent genes by GR is transcriptional and rapid and does not require protein synthesis (81).

Of particular interest is the finding that GR modulates CREB action. CREB binds to the cAMP response element (CRE) in a given promoter. cAMP and Ets-1 are two of the many transcription factors that complex with CREB to bind to the CRE and induce transcription (78, 82). Greenland et al. (55), in transfection experiments, found that P4 stimulated PGDH promoter activity was enhanced in the presence of cAMP. The F-GR interaction with CREB may thus block or reduce P4 up-regulation of PGDH by inhibiting cAMP action. Greenland et al. (55) also found that Ets family members, Ets-1, Ets-2, and polyomavirus enhancer activator 3, potently stimulated the transcriptional activity of the PGDH promoter. GR has also been shown to interact with an integrator molecule termed CREB-binding protein (CBP) (83, 84). A number of transcription factors, including CREB, c-Fos, c-Jun, and Ets-1, have been shown to interact with CBP to mediate genomic effects (82, 84). GC negative regulation of PGDH may involve a cluster of these transcription factors that combine into a large complex via CBP and subsequently bind at the Ets/AP-1/CREB element in the PGDH promoter. Thus, it remains possible that the effect of P4/MPA to increase PGDH transcription could also be indirect (e.g. via Ets), given the absence of demonstrated P4 response element within the PGDH promoter region, and that GC can block this action via protein-protein interactions between activated GR and the stimulatory transcription factor involved.

GCs appear to regulate gene expression in a highly tissue-specific manner. In the hypothalamus, GCs inhibit CRH gene transcription via GR (85, 86); in contrast, CRH mRNA levels are increased by GCs in cultured human placental trophoblast cells (87) and are unaffected by GCs at several extrahypothalamic central nervous system sites (88, 89) even though GR are present (90). Similarly, GCs down-regulate PGHS-2 expression in amnion WISH cells (91) and in most other cell types by interference with the NF-{kappa}B signaling system (92); in contrast they up-regulate PGHS-2 expression in human breast adenocarcinoma cells (93), and in human fetal membranes (94, 95, 96), presumably via GR. Given the highly tissue-, cell-, and time-specific nature of transcription factor expression, it is not surprising that GCs have such variable effects on expression of the same gene in different tissues and perhaps even in the same tissue at different times. Furthermore, given that the PGDH promoter has five AP-1, two CRE, four Ets and a human transcription factor Sp1 gene/AP2 site (39), but no nGRE, it is likely that GR interaction with AP-1 and CREB proteins represents the mechanism by which GCs overcome P4 action at the GRE to down-regulate local PGDH expression and activity at term.

11ß-HSD1 in the chorion and 11ß-HSD2 in the placenta may play a role in mediating interactive effects of P4 and GCs. We have shown previously that 11ß-HSD isozymes, by altering the local concentrations of F in chorion and placental trophoblast cells, can modulate PGDH activity and expression (44). Cortisone, a biologically inactive GC, significantly decreased PGDH mRNA expression and activity in chorion, but not placenta. Cortisone inhibition of PGDH mRNA expression and activity in chorion was abolished in the presence of the GR antagonist, in agreement with a GC effect mediated via GR.

Interestingly, changes in PGDH activity at term in the rat placenta correlate with regional differences in 11ß-HSD found in two morphologically and functionally distinct placental zones (basal and labyrinth) (97, 98, 99). PGDH activity decreases over the last 4 d of rat pregnancy in the labyrinth zone, where decreased 11ß-HSD2 and increased 11ß-HSD1 activity were demonstrated. In contrast, PGDH activity increases over the same time period in the basal zone, where 11ß-HSD2 activity was reported to increase. Thus, locally generated GC levels by 11ß-HSD isozymes in these two placental regions in the rat appear to regulate local PG concentrations through effects on PGDH activity. A similar correlation between 11ß-HSD2 and PGDH in human placenta was demonstrated in preeclamptic patients (100). A 3-fold decrease in 11ß-HSD2 mRNA levels was found in preeclamptic patients, a decrease significantly correlated with a 2-fold reduction in PGDH mRNA levels, suggesting that a diminished metabolism of placental F leads to reduced PGDH mRNA expression by an autocrine/paracrine mechanism.

Alfaidy et al. (101) demonstrated the ability of PGs to increase 11ß-HSD1 activity in chorion and decrease 11ß-HSD2 activity in placenta. An increase in PGs at term could increase local concentrations of F in these tissues, thereby shifting the balance in favor of GC over P4 occupancy of GR, resulting in the inhibition of PGDH activity and expression. PGF2{alpha} administration to pregnant rats has recently been shown to decrease 3ß-HSD and increase 20{alpha}-HSD activity in vivo (57). This would suggest that local P4 concentrations, in addition to local F concentrations, are also regulated by PGs. This feedforward loop would serve to further decrease local P4 levels at term, allowing F to act at the GR to down-regulate PGDH and further increase local PGs levels.

In conclusion, the results from this study suggest that F inhibition and P4 maintenance of PGDH activity may be mediated by competition for downstream effector systems in human chorion and placenta. These results are consistent with the hypothesis that in vivo PGDH activity may reflect opposing effects of F and P4 exerted at least in part via GR. The increase in PG levels in fetal membranes, at term or preterm, could stimulate myometrial contractility and cervical dilatation or act locally to alter 11ß-HSD1 or matrix metalloproteinase activity. The increase in PG output from the placenta may also be involved in mediating changes in uteroplacental blood flow.


   Acknowledgments
 
We thank Cristine Botsford and Dr. S. J. Lye (Mt. Sinai Hospital, Toronto, Canada) for assistance in collecting tissues.


   Footnotes
 
This work was supported by the Canadian Institutes for Health Research.

Abbreviations: Ab, Antibody; AP-1, activating protein-1; CBP, CREB binding protein; CRE, cAMP response element; CREB, cAMP response element-binding protein; DEX, dexamethasone; F, cortisol; GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HSD, hydroxysteroid dehydrogenase; IR-, immunoreactive; MPA, medroxyprogesterone acetate; NF-{kappa}B, nuclear factor-{kappa}B; 21OH-6OP, 21-hydroxy-6,19-oxidopregn-4-ene-3,20-dione; P4, progesterone; PBS-T, PBS-Tris; PG, prostaglandin; PGDH, prostaglandin dehydrogenase; PGFM, 13,14-dihydro-15-keto PGF2{alpha}; PR, progesterone receptor; ROD, relative OD.

Received October 31, 2002.

Accepted March 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Challis JRG, Lye SJ 1994 Parturition. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; vol 2:985–1031
  2. Wiqvist N, Lindblom B, Wikland M, Wilhelmsson L 1983 Prostaglandins and uterine contactility. Acta Obstet Gynecol Scand 113(Suppl):23–29
  3. Bennett PR, Elder MG, Myatt L 1987 The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility. Prostaglandins 33:837–844[CrossRef][Medline]
  4. Ellwood DA, Mitchell MD, Anderson ABM, Turnbull AC 1980 The in vitro production of prostanoids by the human cervix during pregnancy: preliminary observations. Br J Obstet Gynaecol 87:210–214[Medline]
  5. Calder AA, Greer IA 1992 Prostaglandins and the cervix. Baillieres Clin Obstet Gynaecol 6:771–786[CrossRef][Medline]
  6. Keirse MJNC, Turnbull AC 1975 Metabolism of prostaglandins within the pregnant uterus. Br J Obstet Gynaecol 82:887–893[Medline]
  7. van Meir CA, Matthews SG, Keirse MJNC, Ramirez MM, Bocking A, Challis JRG 1997 15-Hydroxyprostaglandin dehydrogenase (PGDH): implications in preterm labor with and without ascending infection. J Clin Endocrinol Metab 82:969–976[Abstract/Free Full Text]
  8. Sangha RK, Walton JC, Ensor CM, Tai HH, Challis JRG 1994 Immunohistochemical localization, messenger ribonucleic acid abundance, and activity of 15-hydroxyprostaglandin dehydrogenase in placenta and fetal membranes during term and preterm labor. J Clin Endocrinol Metab 78:982–989[Abstract]
  9. van Meir CA, Ramirez MM, Matthews SG, Calder AA, Keirse MJNC, Challis JRG 1997 Chorionic prostaglandin catabolism is decreased in the lower uterine segment with term labour. Placenta 18:109–114[Medline]
  10. Patel FA, Clifton VL, Chwalisz K, Challis JRG 1999 Steroid regulation of prostaglandin dehydrogenase activity and expression in human term placenta and chorio-decidua in relation to labor. J Clin Endocrinol Metab 84:291–299[Abstract/Free Full Text]
  11. Patel FA, Gibb W, Challis JRG 1999 Cortisol and progesterone regulation of prostaglandin dehydrogenase (PGDH) mRNA in human fetal membranes and placenta at term [Abstract 429]. 46th Annual Meeting of the Society for Gynecologic Investigation, Atlanta, GA, 1999
  12. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
  13. Yamamoto KR 1985 Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet 19:209–252[CrossRef][Medline]
  14. Amero SA, Kretsinger RH, Moncrief ND, Yamamoto KR, Pearson WR 1992 The origin of nuclear receptor proteins: a single precursor distinct from other transcription factors. Mol Endocrinol 6:3–7[Free Full Text]
  15. Nordeen SK, Suh BJ, Kuehnel B, Hutchison III CA 1990 Structural determinants of a glucocorticoid receptor recognition element. Mol Endocrinol 4:1866–1873[Abstract/Free Full Text]
  16. Pratt WB 1993 The role of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J Biol Chem 268:21455–21458[Free Full Text]
  17. Karalis K, Goodwin G, Majzoub JA 1996 Cortisol blockade of progesterone: a possible molecular mechanism involved in the initiation of human labor. Nat Med 2:556–560[CrossRef][Medline]
  18. Padayachi T, Pegoraro RJ, Hofmeyr J, Joubert SM, Norman RJ 1987 Decreased concentrations and affinities of oestrogen and progesterone receptors of intrauterine tissue in human pregnancy. J Steroid Biochem 26:473–479[CrossRef][Medline]
  19. Padayachi T, Pegoraro RJ, Holloway G, Joubert SM, Norman RJ 1989 Changes in the binding characteristics of oestrogen and progesterone in uterine and intrauterine tissue with the progression of human pregnancy. J Steroid Biochem 33:535–540[CrossRef][Medline]
  20. Padayachi T, Pegoraro RJ, Rom L, Joubert SM 1990 Enzyme immunoassay of oestrogen and progesterone receptors in uterine and intrauterine tissue during human pregnancy and labour. J Steriod Biochem Mol Biol 37:509–511[CrossRef][Medline]
  21. Khan-Dawood FS, Dawood MY 1984 Estrogen and progesterone receptor and hormone levels in human myometrium and placenta in term pregnancy. Am J Obstet Gynecol 150:501–505[Medline]
  22. McCormick PD, Razel AJ, Spelsberg TC, Coulam CB 1981 Absence of high-affinity binding of progesterone (R5020) in human placenta and fetal membranes. Placenta Suppl 3:123–132[Medline]
  23. Rezapour M, Backstrom T, Lindblom B, Ulmsten U 1997 Sex steroid receptors and human parturition. Obstet Gynecol 89:918–924[CrossRef][Medline]
  24. Wu WX, Brooks J, Millar MR, Ledger WL, Glasier AF, McNeilly AS 1993 Immunolocalization of oestrogen and progesterone receptors in the human decidua in relation to prolactin production. Hum Reprod 8:1129–1135[Abstract/Free Full Text]
  25. Chibbar R, Wong S, Miller FD, Mitchell BF 1995 Estrogen stimulates oxytocin gene expression in human chorio-decidua. J Clin Endocrinol Metab 80:567–572[Abstract]
  26. Rivera J, Cano A 1989 Oestrogen and progesterone receptors in human term placenta. Measurement by binding assays and immunological methods. Placenta 10:579–588[Medline]
  27. Shanker YG, Rao AJ 1999 Progesterone receptor expression in the human placenta. Mol Hum Reprod 5:481–486[Abstract/Free Full Text]
  28. Cudeville C, Mondon F, Robert B, Rebourcet R, Mignot TM, Benassayag C, Ferre F 2000 Evidence for progesterone receptors in the human fetoplacental vascular tree. Biol Reprod 62:759–765[Abstract/Free Full Text]
  29. Rossmanith WG, Wolfahrt S, Ecker A, Eberhardt E 1997 The demonstration of progesterone, but not of estrogen, receptors in the developing human placenta. Horm Metab Res 29:604–610[Medline]
  30. Giannopoulos G, Jackson K, Tulchinsky D 1983 Specific glucocorticoid binding in human uterine tissues, placenta and fetal membranes. J Steroid Biochem 19:1375–1378[CrossRef][Medline]
  31. Weisbart M, Huntley FM 1997 The presence of cortisol receptors in the human amnion. J Steroid Biochem Mol Biol 63:339–344[CrossRef][Medline]
  32. Whittle WL, Gibb W, Challis JRG 2000 The characterization of human amnion epithelial and mesenchymal cells: the cellular expression, activity and glucocorticoid regulation of prostaglandin output. Placenta 21:394–401[CrossRef][Medline]
  33. Sun M, Ramirez M, Challis JRG, Gibb W 1996 Immunohistochemical localization of the glucocorticoid receptor in human fetal membranes and decidua at term and preterm delivery. J Endocrinol 149:243–248[Abstract/Free Full Text]
  34. Selman PJ, Wolfswinkel J, Mol JA 1996 Binding specificity of medroxyprogesterone acetate and proligestone for the progesterone and glucocorticoid receptor in the dog. Steroids 61:133–137[CrossRef][Medline]
  35. Beato M, Truss M, Chavez S 1996 Control of transcription by steroid hormones. Ann NY Acad Sci 784:93–123[Medline]
  36. Ojasoo T, Dore JC, Gilbert J, Raynaud JP 1988 Binding of steroids to the progestin and glucocorticoid receptors analyzed by correspondence analysis. J Med Chem 31:1160–1169[CrossRef][Medline]
  37. Philibert CG et al. 1991 From RU 38486 towards dissociated anti-glucocorticoid and antiprogesterone. Front Horm Res 19:1–17
  38. Ogle TF, Beyer BK 1982 Steroid-binding specificity of the progesterone receptor from rat placenta. J Steroid Biochem 16:147–150[CrossRef][Medline]
  39. Matsuo M, Ensor CM, Tai HH 1997 Characterisation of the genomic structure and promotor of the mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene. Biochem Biophys Res Commun 235:582–586[CrossRef][Medline]
  40. Karalis K, Majzoub JA 1995 Regulation of placental corticotropin-releasing hormone by steroids. Possible implication in labor initiation. Ann NY Acad Sci 551–555
  41. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  42. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  43. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss III JF 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:1567–1582[Abstract/Free Full Text]
  44. Patel FA, Sun K, Challis JRG 1999 Local modulation by 11ß-hydroxysteroid dehydrogenase of glucocorticoid effects on the activity of 15-hydroxyprostaglandin dehydrogenase in human chorion and placental trophoblast cells. J Clin Endocrinol Metab 84:395–400[Abstract/Free Full Text]
  45. Sun K, Yang K, Challis JRG 1997 Differential regulation of 11ß-hydroxysteroid dehydrogenase type 1 and 2 by nitric oxide in cultured human placental trophoblast and chorionic cell preparation. Endocrinology 138:4912–4920[Abstract/Free Full Text]
  46. Cheung PYC, Walton JC, Tai HH, Riley SC, Challis JRG 1990 Immunocytochemical distribution and localization of 15-hydroxyprostaglandin dehydrogenase in human fetal membranes, decidua, and placenta. Am J Obstet Gynecol 163:1445–1449[Medline]
  47. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  48. Ensor CM, Yang JY, Okita RT, Tai HH 1990 Cloning and sequence analysis of the cDNA for human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. J Biol Chem 265:14888–14891[Abstract/Free Full Text]
  49. Kliman HJ, Feinman MA, Strauss III JF 1987 Differentiation of human cytotrophoblast into syncytiotrophoblast in culture. Trophoblast Res 2:407–421
  50. Wei LL, Hawkins P, Baker C, Norris B, Sheridan PL, Quinn PG 1996 An amino-terminal truncated progesterone receptor isoform, PRc, enhances progestin-induced transcriptional activity. Mol Endocrinol 10:1379–1387[Abstract/Free Full Text]
  51. Conneely OM, Kettelberger DM, Tsai MJ, Schrader WT, O’Malley BW 1989 The chicken progesterone receptor A and B isoforms are products of an alternate translation initiation event. J Biol Chem 264:14062–14064[Abstract/Free Full Text]
  52. Kastner P, Bocquel MT, Turcotte B, Garnier JM, Horwitz KB, Chambon P, Gronemeyer H 1990 Transient expression of human and chicken progesterone receptors does not support alternative translational initiation from a single mRNA as the mechanism generating two receptor isoforms. J Biol Chem 265:12163–12167[Abstract/Free Full Text]
  53. Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE 2000 Progesterone receptor isoform A but not B is expressed in endometriosis. J Clin Endocrinol Metab 85:2897–2902[Abstract/Free Full Text]
  54. Gao J, Mazella J, Tang M, Tseng L 2000 Ligand-activated progesterone receptor isoform hPR-A is a stronger transactivator than hPR-B for the expression of IGFBP-1 (insulin-like growth factor binding protein-1) in human endometrial stromal cells. Mol Endocrinol 14:1954–1961[Abstract/Free Full Text]
  55. Greenland KJ, Jantke I, Jenatschke S, Bracken KE, Vinson C, Gellersen B 2000 The human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene promoter is controlled by Ets and activating protein-1 transcription factors and progesterone. Endocrinology 141:581–597[Abstract/Free Full Text]
  56. Sugino N, Telleria CM, Gibori G 1997 Progesterone inhibits 20{alpha}-hydroxysteroid dehydrogenase expression in rat corpus luteum through the glucocorticoid receptor. Endocrinology 138:4497–4500[Abstract/Free Full Text]
  57. Telleria CM, Stocco CO, Stati AO, Deis RP 1999 Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy. Steroids 64:760–766[CrossRef][Medline]
  58. Shyamala G, McBlain WA 1979 Distinction between progestin- and glucocorticoid-binding sites in mammary glands. Apparent lack of cytoplasmic progesterone receptors in lactating mammary glands. Biochem J 178:345–352[Medline]
  59. Xu XF, Hoebeke J, Bjorntorp P 1990 Progestin binds to the glucocorticoid receptor and mediates antiglucocorticoid effect in rat adipose precursor cells. J Steroid Biochem 36:465–471[CrossRef][Medline]
  60. Rousseau GG, Baxter JD, Higgins SJ, Tomkins GM 1973 Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells. J Mol Biol 79:539–554[CrossRef][Medline]
  61. Suthers MB, Pressley LA, Funder JW 1976 Glucocorticoid receptors: evidence for a second, non-glucocorticoid binding site. Endocrinology 99:260–269[Abstract/Free Full Text]
  62. Jones TR, Bell PA 1980 Glucocorticoid-receptor interactions. Studies of the negative co-operativity induced by steroid interactions with a secondary, hydrophobic, binding site. Biochem J 188:237–245[Medline]
  63. Svec F, Rudis M 1981 Progestin-induced enhancement of dexamethasone dissociation from glucocorticoid hormone receptors. Arch Biochem Biophys 212:417–423[CrossRef][Medline]
  64. Naylor PH, Gilani SS, Milholland RJ, Ip M, Rosen F 1981 In vivo antiglucocorticoids: comparison between in vivo activity and in vitro competition of progestins for the glucocorticoid receptor. J Steroid Biochem 14:1303–1309[CrossRef][Medline]
  65. Jahn GA, Houdebine LM, Djiane J 1987 Antiprogesterone and antiglucocorticoid actions of RU 486 on rabbit mammary explant cultures. Evidence for a persistent inhibitory action of residual progesterone upon the mammary tissue. J Steroid Biochem 28:371–377[CrossRef][Medline]
  66. Nordeen SK, Kuhnel B, Lawler-Heavner J, Barber DA, Edwards DP 1989 A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line. Mol Endocrinol 3:1270–1278[Abstract/Free Full Text]
  67. Vicent GP, Monteserin MC, Veleiro AS, Burton G, Lantos CP, Galigniana MD 1997 21-Hydroxy-6,19-oxidoprogesterone: a novel synthetic steroid with specific antiglucocorticoid properties in the rat. Mol Pharmacol 52:749–753[Abstract/Free Full Text]
  68. Erman A, Pitcock JA, Liston T, Brown P, Baer PG, Nasjletti A 1987 Biphasic effect of dexamethasone on urinary prostaglandins in rats: relation to alterations in renal medulla triglycerides and prostaglandin metabolism. Endocrinology 121:1853–1861[Abstract/Free Full Text]
  69. Xun CQ, Ensor CM, Tai HH 1991 Regulation of synthesis and activity of NAD+-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells. Biochem Biophys Res Commun 177:1258–1265[CrossRef][Medline]
  70. Moore PK, Hoult JRS 1980 Anti-inflammatory steroids reduce tissue prostaglandin synthetase activity and enhance prostaglandin breakdown. Nature 288:269–270[CrossRef][Medline]
  71. Brennand JE, Leask R, Kelly RW, Greer IA, Calder AA 1995 Changes in prostaglandin synthesis and metabolism associated with labour, and the influence of dexamethasone, RU486 and progesterone. Eur J Endocrinol 133:527–533[Abstract/Free Full Text]
  72. Tong M, Tai HH 2000 Dexamethasone inhibits the induction of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase by phorbol ester in human promonocytic U937 cells. Biochim Biophys Acta 1497:61–68[Medline]
  73. Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189–1204[CrossRef][Medline]
  74. Northrop JP, Crabtree GR, Mattila PS 1992 Negative regulation of interleukin 2 transcription by the glucocorticoid receptor. J Exp Med 175:1235–1245[Abstract/Free Full Text]
  75. Pfahl M 1993 Nuclear receptor/AP-1 interaction. Endocr Rev 14:651–658[Abstract/Free Full Text]
  76. McKay LI, Cidlowski JA 1998 Cross-talk between nuclear factor-{kappa}B and the steroid hormone receptors: mechanisms of mutual antagonism. Mol Endocrinol 12:45–56[Abstract/Free Full Text]
  77. Caldenhoven E, Liden J, Wissink S, van de Stolpe A, Raaijmakers J, Koenderman L, Okret S, Gustafsson JA, van der Saag PT 1995 Negative cross-talk between RelA and the glucocorticosteroid receptor: a possible mechanism for the anti-inflammatory action of glucocorticoids. Mol Endocrinol 9:401–412[Abstract/Free Full Text]
  78. Imai E, Miner JN, Mitchell JA, Yamamoto KR, Granner DK 1993 Glucocorticoid receptor-cAMP response element-binding protein interaction and the response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids. J Biol Chem 268:5353–5356[Abstract/Free Full Text]
  79. Zhang Z, Jones S, Hagood JS, Fuentes NL, Fuller GM 1997 STAT3 acts as a co-activator of glucocorticoid receptor signaling. J Biol Chem 272:30607–30610[Abstract/Free Full Text]
  80. Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional interactions between Stat5 and the glucocorticoid receptor. Nature 383:726–728[CrossRef][Medline]
  81. Saatcioglu F, Claret FX, Karin M 1994 Negative transcriptional regulation by nuclear receptors. Semin Cancer Biol 5:347–359[Medline]
  82. Yang C, Shapiro LH, Rivera M, Kumar A, Brindle PK 1998 A role for CREB binding protein and p300 transcriptional coactivators in Ets-1 transactivation functions. Mol Cell Biol 18:2218–2229[Abstract/Free Full Text]
  83. Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Tsai MJ, O’Malley BW 1997 Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141–164
  84. Janknecht R, Hunter T 1996 Transcription. A growing coactivator network. Nature 383:22–23[CrossRef][Medline]
  85. Herman JP, Schafer MK, Thompson RC, Watson SJ 1992 Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Mol Endocrinol 6:1061–1069[Abstract/Free Full Text]
  86. Agnati LF, Fuxe K, Yu ZY, Harfstrand A, Okret S, Wikstrom AC, Goldstein M, Zoli M, Vale W, Gustafsson JA 1985 Morphometrical analysis of the distribution of corticotrophin releasing factor, glucocorticoid receptor and phenylethanolamine-N-methyltransferase immunoreactive structures in the paraventricular hypothalamic nucleus of the rat. Neurosci Lett 54:147–152[Medline]
  87. Robinson BG, Emanuel RL, Frim DM, Majzoub JA 1988 Glucocorticoid stimulates expression of corticotropin-releasing hormone gene in human placenta. Proc Natl Acad Sci USA 85:5244–5248[Abstract/Free Full Text]
  88. Imaki T, Nahan JL, Rivier C, Sawchenko PE, Vale W 1991 Differential regulation of corticotropin-releasing factor mRNA in rat brain regions by glucocorticoids and stress. J Neurosci 11:585–599[Abstract]
  89. Frim DM, Robinson BG, Pasieka KB, Majzoub JA 1990 Differential regulation of corticotropin-releasing hormone mRNA in rat brain. Am J Physiol 258:E686–E692
  90. Alexis MN, Kitraki E, Spanou K, Stylianopoulou F, Sekeris CE 1990 Ontogeny of the glucocorticoid receptor in the rat brain. Adv Exp Med Biol 265:269–276[Medline]
  91. Wang Z, Tai HH 1999 Cyclic AMP response element mediates dexamethasone induced suppression of prostaglandin H synthase-2 gene expression in human amnion derived WISH cells. Prostaglandins Leukotrienes Essent Fatty Acids 60:243–248[CrossRef][Medline]
  92. McKay LI, Cidlowski JA 1999 Molecular control of immune/inflammatory responses: interactions between nuclear factor-{kappa}B and steroid receptor-signalling pathways. Endocr Rev 20:435–459[Abstract/Free Full Text]
  93. Kniss DA 1999 Cyclooxygenases in reproductive medicine and biology. J Soc Gynecol Invest 6:285–292[Medline]
  94. Economopoulos P, Sun M, Purgina B, Gibb W 1996 Glucocorticoids stimulate prostaglandin H synthase type-2 (PGHS-2) in the fibroblast cells in human amnion cultures. Mol Cell Endocrinol 117:141–147[CrossRef][Medline]
  95. Zakar T, Hirst JJ, Mijovic JE, Olson DM 1995 Glucocorticoids stimulate the expression of prostaglandin endoperoxide H synthase-2 in amnion cells. Endocrinology 136:1610–1619[Abstract]
  96. Blumenstein M, Hansen WR, Deval D, Mitchell MD 2000 Differential regulation in human amnion epithelial and fibroblast cells of prostaglandin E2 production and prostaglandin H synthase-2 mRNA expression by dexamethasone but not tumour necrosis factor-{alpha}. Placenta 21:210–217[CrossRef][Medline]
  97. Waddell BJ, Benediktsson R, Brown RW, Seckl JR 1998 Tissue-specific messenger ribonucleic acid expression of 11ß-hydroxysteroid dehydrogenase types 1 and 2 and the glucocorticoid receptor within rat placenta suggests exquisite local control of glucocorticoid action. Endocrinology 139:1517–1523[Abstract/Free Full Text]
  98. Burton PJ, Dharmarajan AM, Hisheh S, Waddell BJ 1996 Induction of myometrial 11ß-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid and protein expression late in rat pregnancy. Endocrinology 137:5700–5706[Abstract]
  99. Nagai K, Nabekura H, Mibe M, Ohshige T, Mori N 1991 Prostaglandin dehydrogenase activity in placenta and in maternal lung, kidney, and gastric mucosa during rat pregnancy. Mol Cell Endocrinol 80:153–163[CrossRef][Medline]
  100. Schoof E, Girstl M, Frobenius W, Kirschbaum M, Dorr HG, Rascher W, Dotsch J 2001 Decreased gene expression of 11ß-hydroxysteroid dehydrogenase type 2 and 15-hydroxyprostaglandin dehydrogenase in human placenta of patients with preeclampsia. J Clin Endocrinol Metab 86:1313–1317[Abstract/Free Full Text]
  101. Alfaidy N, Xiong ZG, Myatt L, Lye SJ, MacDonald JF, Challis JRG 2001 Prostaglandin F2{alpha} potentiates cortisol production by stimulating 11ß-hydroxysteroid dehydrogenase 1: a novel feedback loop that may contribute to human labor. J Clin Endocrinol Metab 86:5585–5592[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
P. J. Mark, S. Augustus, J. L. Lewis, D. P. Hewitt, and B. J. Waddell
Changes in the Placental Glucocorticoid Barrier During Rat Pregnancy: Impact on Placental Corticosterone Levels and Regulation by Progesterone
Biol Reprod, June 1, 2009; 80(6): 1209 - 1215.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
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]

Home page
 J EndocrinolHome page
R. Kancheva, M. Hill, D. Cibula, H. Vcelakova, L. Kancheva, J. Vrbikova, T. Fait, A. Parizek, and L. Starka
Relationships of circulating pregnanolone isomers and their polar conjugates to the status of sex, menstrual cycle, and pregnancy
J. Endocrinol., October 1, 2007; 195(1): 67 - 78.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M.-J. Lee, Z. Wang, H. Yee, Y. Ma, N. Swenson, L. Yang, S. S. Kadner, R. N. Baergen, S. K. Logan, M. J. Garabedian, et al.
Expression and Regulation of Glucocorticoid Receptor in Human Placental Villous Fibroblasts
Endocrinology, November 1, 2005; 146(11): 4619 - 4626.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
D. M. Duffy, B. L. Dozier, and C. L. Seachord
Prostaglandin Dehydrogenase and Prostaglandin Levels in Periovulatory Follicles: Implications for Control of Primate Ovulation by Prostaglandin E2
J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1021 - 1027.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
D. J. Toufexis, C. Davis, A. Hammond, and M. Davis
Progesterone Attenuates Corticotropin-Releasing Factor-Enhanced But Not Fear-Potentiated Startle via the Activity of Its Neuroactive Metabolite, Allopregnanolone
J. Neurosci., November 10, 2004; 24(45): 10280 - 10287.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, F. A.
Right arrow Articles by Challis, J. R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, F. A.
Right arrow Articles by Challis, J. R. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals