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Regular Article |
a Department of Physiology, Second Military Medical University, Shanghai 200433, People's Republic of China
b Departments of Physiology and Obstetrics and Gynecology, University of Western Ontario, London, Ontario, Canada N6C 2V5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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glucocorticoid receptor, parturition, pregnancy, steroid hormones, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and corticotropin-releasing hormone (CRH) from the fetal membranes and placenta [4–6]. These hormones are involved in the process of labor [7, 8]. Our previous work revealed differential distributions of 11ß-HSD1 and 11ß-HSD2 in the fetal membranes and placenta [9]. 11ß-HSD2 is localized to the syncytiotrophoblast of the placenta [10], whereas 11ß-HSD2 provides a functional barrier protecting the fetus from maternal glucocorticoids [11, 12]. 11ß-HSD1 was localized predominantly to the chorionic trophoblast [9], which is involved in the downregulation of prostaglandin dehydrogenase H (PGDH) by cortisone in the chorion [4]. A sequence resembling glucocorticoid response element (GRE) has been identified in the promoter region of the human 11ß-HSD1 gene [13]. Glucocorticoids induced the expression of 11ß-HSD1 in the hippocampus in vitro [14], but controversial results were obtained in the hepatocyte [15]. However, it is not known whether glucocorticoids modulate 11ß-HSD1 expression, thereby modulating the effect of cortisone on the synthesis of prostaglandin in the fetal membranes. In this study, we examined whether 11ß-HSD1 and glucocorticoid receptor (GR) are coexpressed in the chorionic trophoblast, which may provide the intracrine basis for the regulation of 11ß-HSD1 expression by glucocorticoids. Furthermore, the regulation of prostaglandin synthesis by cortisone was investigated on the basis of prior induction of 11ß-HSD1 expression by glucocorticoid in the cultured human chorionic trophoblast. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was carried out with the approval of the Human Ethics Committee of the Second Military Medical University. Twenty-two human placentae were collected from women who chose to undergo cesarean sections in the absence of term labor. The chorion was peeled off from the amnion and brought to the culture laboratory in ice-cold normal saline within 20 min after delivery.
Chorionic Trophoblast Culture
Chorionic trophoblasts were prepared using a previously described modification of the method of Kliman [16]. The residual blood was washed off chorionic tissue with cold normal saline. The minced chorionic tissue was digested with 0.125% (w/v) trypsin (Sigma, St. Louis, MO) and 0.2% (w/v) collagenase (Roche, Indianapolis, IN) three times for 60 min. The chorionic cells were loaded onto a 5–75% (v/v) Percoll (Pharmacia, Piscataway, NJ) gradient at step increments of 5% Percoll and centrifuged at 37°C and 2500 x g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml or 35% and 50% Percoll were collected and plated at a density of 4 x 106 cells/well in six-well plates in Dulbecco modified Eagle medium containing 10% (v/v) fetal calf serum (FCS). The cells were cultured for 3 days at 37°C in 5% CO2 before use. In previous studies, cells prepared using the above method were predominantly chorion-derived trophoblasts rather than decidual cells [16].
Double Immunocytochemical Staining
Double staining was carried out as described previously [17]. The cultured cells were plated on the poly-L-lysine-coated glass coverslips (10 x 10 mm). After 3 days in culture, the cells were fixed in 4% (w/v) paraformaldehyde for 10 min. Rabbit polyclonal 11ß-HSD1 antibody [9] and mouse monoclonal GR antibody (BUGR2; courtesy of Dr. B. Gametchu, Medical College of Wisconsin, Milwaukee, WI) were used as primary antibodies. The primary 11ß-HSD1 antibody was raised against a synthetic peptide corresponding to amino acids 278–289 from the deduced sequence of ovine 11ß-HSD1 and was used at a dilution of 1:400. Western blot analysis using this antibody revealed a 34-kDa immunoreactive protein in homogenates from Chinese hamster ovary cells transfected with 11ß-HSD1 cDNA [9]. The antibody was purified by affinity chromatography before use. Biotinylated goat anti-rabbit and goat anti-mouse IgG (Boster Biotech, Wuhan, China) was used as secondary antibodies for 11ß-HSD1 antibody and GR antibody, respectively. Immunoreactivity for GR was visualized by a streptavidin-biotin-alkaline phosphatase (SABC-POD; Boster Biotech) method with a blue-stained 5-bromocresyl-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) reaction. Immunoreactivity for 11ß-HSD1 was visualized using the streptavidin-biotin-peroxidase (SABC-AP; Boster Biotech) method with a red 3-amino-9-ethyl carbazole (AEC) reaction. Between these two immunocytochemical staining procedures, blocking solution (Boster Biotech) was used to rinse the cover slip to block the cross-reaction between the two immunostains. For a control, normal rabbit or mouse serum was used instead of the 11ß-HSD1 or GR primary antibodies, respectively, in double stainings.
Radiometric Conversion Assay of 11ß-HSD1 Activity in Chorionic Trophoblasts
For the determination of the effects of dexamethasone on 11ß-HSD1 activity, experiments were carried out on cultured chorion trophoblasts collected from six placentae. After 3 days in culture, culture medium was changed to FCS-free culture medium. The cells were treated with dexamethasone (0.1 µM) in the absence and presence of RU486 (1 µM) and progesterone (1 µM) for 24 h. The doses of the reagents and duration of treatments were chosen based on the results of preliminary dose- and time-dependent experiments. All the reagents were dissolved in ethanol as stock solutions and diluted 100 times with culture medium at the time of treatment. For a control, the cells were treated with vehicle used to make up the drugs. All the treatments were carried out in duplicate wells. After 24 h, the cells were washed with culture medium and incubated with the 11ß-HSD1 reductase substrate cortisone (1 µM, containing 200 000 cpm 3H-corticosterone) for 6 h to achieve linear conversion of cortisone to cortisol. 3H-cortisone was prepared by oxidation of 3H-cortisol (specific activity of 64 Ci/mmol; Amersham Life Science, Piscataway, NJ) with chromium trioxide [18] and was purified by thin layer chromatography (TLC). After incubation with the substrate, culture medium was collected for subsequent analysis of 11ß-HSD1 activity.
To determine the conversion of cortisone to cortisol, a mixture of cortisol and cortisone (40 µg) was added to the collected medium to allow localization of the steroids when subjected to TLC. Steroids in the medium were extracted with ethyl acetate. The extract from the medium was dried, reconstituted with ethyl acetate (100 µl), and applied to the TLC plate. Cortisol and cortisone in the samples were separated on the TLC plate in the solvent system chloroform/ethanol (95/5 v/v). Steroids were visualized under ultraviolet light, scraped off, and extracted with ethyl acetate. The solvent was dried, scintillation fluid was added, and the radioactivity was counted with a liquid scintillation counter (Wallac, Boston, MA). The 11ß-HSD1 reductase activity was expressed as the percentage of cortisol formed from total cortisone and was normalized per 106 cells [16].
Determination of 11ß-HSD1 mRNA Expression in Chorionic Trophoblasts
Six placentae were used for this experiment. Chorionic trophoblasts were cultured as above for 3 days, then the cells were treated with dexamethasone (0.1 and 1 µM) and/or RU486 (1 µM) or vehicle for 24 h. All treatments were carried out in duplicate wells. After 24 h, the cells were washed with ice-cold PBS. Total RNA was extracted with trizol from the treated chorionic trophoblasts and was quantified spectrophotometrically at 260 nm. Northern analysis was carried out using 30 µg RNA and specific human 11ß-HSD1 cDNA [19] labeled with 32P-dCTP. A cDNA for 18S rRNA was used as an internal standard to determine the relative amounts of RNA loaded into each well and the transfer efficiency.
The autoradiographs were scanned using a densitometer to determine the relative optical densities of 11ß-HSD1 and 18S rRNA hybridization signals. For each RNA sample, the signals for the transcripts were measured within the linear range of the densitometer, and the ratio of 11ß-HSD1 mRNA signals to 18S rRNA signals was calculated.
Induction of PGE2 Synthesis by Cortisone in Chorionic Trophoblasts Treated with Dexamethasone
After 3 days in culture, chorionic trophoblasts from six placentae were treated with dexamethasone (0.1 and 1 µM) and/or RU486 (1 µM) or vehicle for 24 h as above. All treatments were carried out in duplicate wells. The cells were then washed three times with culture medium and incubated with cortisone (1 µM) for another 24 h to achieve maximal conversion of cortisone to biologically active cortisol. After 24 h of incubation with cortisone, the medium was collected for RIA of PGE2 levels (RIA kit; Biotinge Tech, Beijing, China), and the trophoblasts were collected for the extraction of protein and subsequent analysis of prostaglandin H synthetase 2 (PGHS-2) levels with Western blot. The protein content in the samples was determined by a protein assay (Bio-Rad, Hercules, CA). Sample proteins (50 µg) were electrophoresed on 10% SDS-polyacrylamide gels and then transferred electrophoretically to a nitrocellulose membrane. The membrane was blocked in Tris-buffered saline containing 15% powdered skim milk and 0.1% Tween-20. Immunoblots were carried out with PGHS-2 primary antibody of rabbit origin (Cayman Chemical, Ann Arbor, MI) at a dilution of 1:1000. The internal control was ß-actin primary monoclonal antibody of mouse origin (Sigma) at a dilution of 1:10 000. Secondary antibodies against rabbit and mouse IgG (Sino-American Biotech, Beijing, China) were used against PGHS-2 primary antibody and ß-actin primary monoclonal antibody, respectively. Both of the secondary antibodies were conjugated to peroxidase and were used at a dilution of 1:1000. The enhanced chemiluminescence light-based detection system was used to detect the peroxidase activity. The light-emitting bands were detected with x-ray film. The exposed film was scanned using a densitometer to determine the optical density of each band, and the ratio of PGHS-2 signals to ß-actin signals was calculated.
Statistical Analysis
All data are reported as mean ± SEM. One-way ANOVA followed by the Student-Newman-Keuls test were used (SPSS, Chicago, IL) to assess significant differences between absolute values. Significance was set at P < 0.05. The values for n refer to the number of experiments performed with different chorionic trophoblast preparations from different patients.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After 3 days in culture, some of the chorionic trophoblasts clustered together and formed multinucleated syncytiotrophoblast cells, and some of the trophoblasts remained as single cells. Staining for 11ß-HSD1 revealed a cytoplasmic distribution in the chorionic trophoblasts. No staining for 11ß-HSD1 was found in the nucleus of the cell. Staining for GR was found in both the cytoplasm and the nucleus of the cell. Dual staining showed coexpression of 11ß-HSD1 and GR in the cytoplasm of chorionic trophoblasts (Fig. 1). For control of antibody specificity, dual staining using 11ß-HSD1 antiserum and normal mouse serum instead of GR antibody revealed just red 11ß-HSD1 staining, whereas dual staining using GR antibody and normal rabbit serum instead of 11ß-HSD1 antiserum revealed just blue GR staining (Fig. 1).
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Induction of 11ß-HSD1 Activity by Dexamethasone in Chorionic Trophoblasts
Radiometric conversion assay showed that the conversion of cortisone to cortisol by chorionic trophoblasts was in the linear range after 18–20 h of incubation with cortisone under our experimental conditions. After 20 h of incubation with cortisone, there was still an increase in the conversion, but it was no longer linear with the incubation time. The conversion rate was 85–950% by 24 h.
About 13% of cortisone was converted to cortisol by 11ß-HSD1 in the chorionic trophoblasts after 6 h of incubation with the substrate in the vehicle control group. Prior treatment of the cells with dexamethasone (0.1 µM) for 24 h increased the subsequent conversion of cortisone to cortisol by 2-fold (P < 0.01 vs. control) after 6 h of incubation with the substrate, and this increase was blocked by prior treatment with GR antagonist RU486 (1 µM) (P < 0.05 vs. dexamethasone, Fig. 2). Treatment of the cells with progesterone (1 µM) did not affect the conversion of cortisone to cortisol compared with the vehicle control.
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Induction of 11ß-HSD1 mRNA Expression by Dexamethasone in Chorionic Trophoblasts
The 1.5-kilobase 11ß-HSD1 mRNA was detected using the Northern blot hybridization method. The expression of 11ß-HSD1 mRNA was significantly increased by treating the chorionic trophoblasts with dexamethasone (0.1 and 1 µM) for 24 h in comparison with vehicle control (P < 0.05 or P < 0.01), and the induction by dexamethasone was blocked by cotreatment of the cells with GR antagonist RU486 (1 µM) (P < 0.05 vs. dexamethasone, Fig. 3). Although the induction of 11ß-HSD1 mRNA by 1 µM dexamethasone appeared to be greater than that by 0.1 µM dexamethasone, the difference was not significant (Fig. 3).
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Induction of PGE2 Secretion and PGHS-2 Expression by Cortisone in Chorionic Trophoblasts Treated with Dexamethasone
RIA revealed that the average PGE2 level induced by cortisone for 24 h was 572 ± 87 pg/ml in the culture medium of chorionic trophoblasts previously treated with vehicle. The secretion of PGE2 induced by cortisone for 24 h was significantly increased in the chorionic trophoblasts treated with dexamethasone (0.1 and 1 µM) compared with those treated with vehicle (P < 0.05 or P < 0.01, Fig. 4). This increase was abolished by prior combined treatment of the chorionic trophoblasts with dexamethasone and RU486 (1 µM) (P < 0.05 vs. dexamethasone, Fig. 4).
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Western blot revealed a PGHS-2 protein band around the position of molecular mass 72 kDa. The PGHS-2 level induced by cortisone (1 µM) for 24 h was significantly increased in the chorionic trophoblasts treated with dexamethasone (0.1 and 1 µM) compared with those treated with vehicle (P < 0.05). This increase was again abolished by prior combined treatment of the chorionic trophoblasts with dexamethasone and RU486 (1 µM) (P < 0.05 vs. dexamethasone, Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Glucocorticoids play a key role in parturition in nonprimate animals [20, 21]. In late gestation, with the maturation of the fetal hypothalamus-pituitary-adrenal axis, the fetal adrenal cortex begins to produce glucocorticoids. Glucocorticoids reach the placenta and fetal membranes and induce the expression of P450c17-hydroxylase in the placenta and production of PGF2 and PGE2 in the fetal membranes [21]. P450c17-Hydroxylase catalyzes the conversion of progesterone to estrogen [22]. Both estrogen and prostaglandins are important hormones involved in parturition [7, 22]. Although the human placenta does not express P450c17a-hydroxylase [22], human placenta and fetal membranes produce a large amount of CRH [23], which is correlated closely with the timing of labor [8, 24]. Glucocorticoids induce the production of CRH by the placenta and the production of prostaglandins by the fetal membranes in humans [4–6]. Therefore, glucocorticoids also play an important role in human parturition.
In the fetal membranes, the actions of glucocorticoids are amplified by the actions of 11ß-HSD1. Our previous work demonstrated abundant 11ß-HSD1 in the chorionic trophoblasts, where 11ß-HSD1 converts biologically inert cortisone to active cortisol thereby increasing the local levels of biologically active glucocorticoids [9, 16]. In nonpregnant women, the major source of cortisone is the kidney, where cortisone is formed from cortisol by the actions of 11ß-HSD2 [25]. In pregnant women, the placenta becomes another major source of cortisone in addition to the kidney. 11ß-HSD2 in the placenta provides a functional barrier to the passage of maternal cortisol to the fetal circulation by converting maternal cortisol to cortisone [12, 26, 27]. Our previous work showed that cortisone produced by the placenta has access to both maternal and fetal circulation [11]. In contrast to cortisol, cortisone is largely unbound to plasma proteins. Therefore, during pregnancy the increased level of cortisone in the maternal blood is an adequate source of substrate for 11ß-HSD1 in the fetal membranes [3].
Because cortisone and cortisol are the substrate and product, respectively, for 11ß-HSD1, use of cortisone or cortisol in the study of 11ß-HSD1 expression would complicate the situation. However, the synthetic glucocorticoid dexamethasone is a poor substrate for 11ß-HSD1 but is more potent than the endogenous glucocorticoids [28]. In addition, synthetic glucocorticoids are widely used in obstetrics to promote lung maturation in preterm infants. The underlying hazards of clinical use of dexamethasone during preterm delivery are not well understood, which prompted us to choose dexamethasone to study the regulation of 11ß-HSD1 gene expression in the chorionic trophoblast. The doses of dexamethasone used were based on our preliminary experiments and on the glucocorticoid levels in the maternal plasma and amniotic fluid at term [29].
The coexpression of 11ß-HSD1 and GR in the chorionic trophoblast provides the molecular basis of an intracrine regulation of 11ß-HSD1 by glucocorticoids through GR within the same cell. Sequence analysis of the 11ß-HSD1 gene revealed that there is a GRE-like motif present in the promoter region of the human 11ß-HSD1 gene [13]. However, whether glucocorticoids regulate chorionic 11ß-HSD1 expression remains unclear. To control the conversion rate within the linear range, we incubated the chorionic trophoblasts with cortisone for 6 h to study the effect of dexamethasone on 11ß-HSD1 activity. Dexamethasone upregulated both 11ß-HSD1 mRNA and activity levels in the chorionic trophoblasts, but this upregulation was blocked by RU486. These findings strongly suggest that glucocorticoid modulates the expression of 11ß-HSD1 through a GR-dependent intracrine mechanism in the chorion. Although RU486 also blocks progesterone receptor, the induction of 11ß-HSD1 levels by dexamethasone in the chorionic trophoblast as observed in this study is unlikely the result of a progesterone receptor-mediated effect. As demonstrated previously and in this study, progesterone has no effect on the expression or activity of 11ß-HSD1 in the chorionic trophoblast but has a potent inhibitory effect on 11ß-HSD2 expression in the placental trophoblast [30]. These findings suggest that dexamethasone stimulates the expression of 11ß-HSD1 by an intracrine mechanism involving GR within the same chorionic trophoblast. Whether this effect involves the interaction of GR and the putative GRE of 11ß-HSD1 gene awaits further study.
Cortisol increases the production of prostaglandins in the fetal membranes by either upregulating PGHS-2 levels or downregulating PGDH levels [4, 31]. The induction of 11ß-HSD1 expression in the chorion by glucocorticoids produced more biologically active cortisol from inert cortisone, which in turn would further amplify the effects of glucocorticoids on the production of PGE2 or PGF2 from the fetal membranes. This proposed mechanism is consistent with the fact that both secretion of PGE2 and expression of PGHS-2 induced by cortisone were higher in the chorionic trophoblasts that had been treated with dexamethasone than in those that had been treated with vehicle. Our previous work also demonstrated that cortisone is almost as effective as cortisol in downregulating the activity of PGDH in the human chorion [4]. This effect is reversed by addition of carbenoxolone [4], an 11ß-HSD inhibitor, which provides further evidence for an intracrine role of 11ß-HSD1 in the prostaglandin-releasing effects of glucocorticoids in the fetal membranes.
Prior treatment of chorionic trophoblasts with dexamethasone induced the expression of 11ß-HSD1, which then converted more cortisone to biologically active cortisol in the subsequent incubation with cortisone for 24 h. The higher local level of cortisol in turn exerted a positive feedback effect on the expression of 11ß-HSD1 and upregulated the production of prostaglandin. This cascade of events initiated by glucocorticoids may play an important role in the positive feed-forward mechanisms of labor.
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FOOTNOTES |
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2 Correspondence: Kang Sun, Department of Physiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R. China. FAX: 86 21 25072297; sunkang2000{at}yahoo.com
Received: 27 March 2002.
First decision: 16 April 2002.
Accepted: 4 June 2002.
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and -ß immunoreactivity in hippocampal cell cultures with special attention to GABAergic neurons. J Neurosci Res 2001 65:396-402[CrossRef][Medline]This article has been cited by other articles:
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  S. Sai, C. L. Esteves, V. Kelly, Z. Michailidou, K. Anderson, A. P. Coll, Y. Nakagawa, T. Ohzeki, J. R. Seckl, and K. E. Chapman Glucocorticoid Regulation of the Promoter of 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Is Indirect and Requires CCAAT/Enhancer-Binding Protein-{beta} Mol. Endocrinol., September 1, 2008; 22(9): 2049 - 2060. [Abstract] [Full Text] [PDF]
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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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 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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