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Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, Child Health Research Institute, and Lawson Health Research Institute, Departments of Obstetrics and Gynaecology and Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 4G5
Address all correspondence and requests for reprints to: Dr. K. Yang, Child Health Research Institute, Room A5-132, Victoria Research Laboratories, Westminster Campus, 800 Commissioners Road East, London, Ontario, Canada N6A 4G5. E-mail: kyang{at}uwo.ca.
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Abstract |
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Introduction |
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The placental enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) is thought to play a pivotal role in controlling fetal exposure to maternal glucocorticoid by catalyzing the unidirectional conversion of cortisol to its inactive metabolite, cortisone (7, 8, 9). Within the human placenta, 11ß-HSD2 is predominantly expressed in the syncytiotrophoblast layer (10, 11, 12), the site of fetal-maternal exchange, and its expression increases across gestation (13, 14). Moreover, placental 11ß-HSD2 activity and expression are reduced in pregnancies complicated by intrauterine growth restriction (IUGR) (13, 15), and IUGR is a characteristic feature of the syndrome of apparent mineralocorticoid excess, in which point mutations in the HSD11B2 gene render the enzyme inactive (16). Collectively, these observations underscore the critical role placental 11ß-HSD2 may play in human fetal development. However, our understanding of the regulation of 11ß-HSD2 in the human placenta is incomplete.
The present study was designed to determine whether 11ß-HSD2 in the human placenta is subject to auto-regulation by glucocorticoids for the following reasons. First, prenatal glucocorticoid administration is a common practice in high-risk obstetrics to accelerate fetal lung maturation and decrease perinatal morbidity and mortality (17). Second, maternal glucocorticoid treatment in sheep and rodents consistently results in fetal growth restriction, in contrast to the apparent lack of growth-reducing effect in humans (18). Third, glucocorticoids have been shown to exert species-specific effects on placental 11ß-HSD2, increasing its expression in baboons (19), while decreasing the expression in sheep (20, 21). It is conceivable that the glucocorticoid-induced down-regulation of placental 11ß-HSD2 in sheep would allow more maternal glucocorticoids (both endogenous and exogenous) to reach fetal circulation leading to fetal growth restriction. Thus, it is of particular importance to study the effects of glucocorticoids on placental 11ß-HSD2 in the human. In this report, we provide the first evidence that glucocorticoids increase 11ß-HSD2 activity and expression in cultured human trophoblasts by a dual mechanism involving an enhanced rate of HSD11B2 gene transcription and increased 11ß-HSD2 mRNA stability.
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Materials and Methods |
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[1,2,6,7-3H(N)]Cortisol (80 Ci/mmol) and was purchased from DuPont Canada Inc. (Markam, Canada). Nonradioactive cortisol and cortisone were obtained from Steraloids Inc. (Wilton, NH). Dexamethasone (Dex), (11ß,17ß)-11-[4-(dimethylamino)-phenyl]-17-hydroxy-17-(1-propynl)estra-4,9-dien-3-one (RU-486), cycloheximide (CHX), and 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DRB) were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON). Polyester-backed thin-layer chromatography (TLC) plates were obtained from Fisher Scientific Ltd. (Nepean, Canada). All solvents used were from VWR Canlab (Mississauga, Canada). Cell culture supplies were obtained from either Canada Life Technologies (Burlington, Canada) or Fisher Scientific. General molecular biology reagents were from Life Technologies, Inc. (Burlington, Canada) or Pharmacia Canada Inc. (Baie D’Urte, Quebec, Canada). Oligonucleotides were synthesized by a Pharmacia Gene Assembler and purified with NAP-50 columns (Pharmacia) according to the manufacturer’s instructions. The human 11ß-HSD2 cDNA (22) was labeled with [32P]dCTP (DuPont Canada; 3000 Ci/mmol) by random priming. Double-stranded DNA was prepared and sequenced by a standard automated sequencing protocol at the John P. Robarts Research Institute DNA Sequencing Facility (London, Canada).
Placental trophoblast cell culture
Human placentas were obtained from uncomplicated normal term pregnancies after elective caesarean section in accordance with the established guidelines of the ethics committee at St. Joseph’s Health Care and the University of Western Ontario. Placental trophoblast cells were isolated using a modification of the method of Kliman (23), as described (24). Briefly, villous tissues were dissected free from fetal membranes and blood vessels, and the residual blood was washed off with cold 0.9% NaCl. The tissue was then digested with 0.125% (wt/vol) trypsin (Sigma-Aldrich Canada Ltd., Oakville, Canada) and 0.02% (wt/vol) deoxyribonuclease-I (Sigma) in DMEM containing antibiotics (5 µg/ml penicillin/streptomycin and 10 µg/ml gentamycin; Canada Life Technologies) for 30 min at 37 C. After the first digestion, the supernatant containing dispersed placental cells was collected. The aforementioned digestion procedure was repeated two more times with the addition of fresh digestion media. The dispersed placental cells from each of the three digestions were filtered through a nylon mesh (Nitrex 40 IN WN MONO FIL, Sefar Canada, Inc., Scarborough, Canada) and loaded onto a 5–70% (vol/vol) Percoll (Sigma) gradient (5% step increments of 3 ml of Percoll), and sedimented at 2,500 x g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected and plated in 24-well plates (Nunc, Canada Life Technologies) or 35-mm dishes (Nunc, Canada Life Technologies) at a density between 2.5–5.0 x 105 cells/cm2 in medium 199 (M199; Canada Life Technologies) containing 10% (vol/vol) fetal bovine serum (Canada Life Technologies) and 5 µg/ml penicillin/streptomycin (Canada Life Technologies) and 10 µg/ml gentamycin (Canada Life Technologies). The cells were maintained at 37 C in humidified 5% CO2-95% air for the duration of the experiment. Treatment of trophoblast cells with various compounds was started immediately after plating, unless indicated otherwise. Each treatment was performed in triplicate, and a total of three to four independent experiments were carried out.
Assay of 11ß-HSD2 activity: radiometric conversion assay
11ß-HSD2 activity in intact cells was determined by a standard radiometric conversion assay, as previously described (24). Briefly, the cells were incubated for 1 h at 37C in serum-free medium containing approximately 50,000 cpm [3H]cortisol and 100 nM unlabeled cortisol. At the end of incubation, the medium was collected and a mixture of cortisol and cortisone (40 µg each) was added to aid in the visualization of steroids after TLC purification. The steroids in the media were extracted with ethyl acetate. The extracts were dried and the residues resuspended in ethyl acetate (50 µl). A fraction of the resuspension was spotted on a TLC plate, and the cortisol and cortisone were separated using a chloroform/methanol (9:1, vol/vol) solvent system. The separated bands containing the radioactively labeled cortisol and cortisone were identified by UV light illumination of the cold carriers. The solvent was dried, the bands were cut out and placed into scintillation vials, and scintillation fluid (Scintisafe Econo 1, Fisher Scientific) was added. The radioactivity was counted in a liquid scintillation counter. The rate of cortisol to cortisone conversion was calculated, the blank values (defined as the amount of conversion in the absence of cells) were subtracted, and the rate was expressed as picomoles cortisone formed per 106 cells per hour.
Assessment of 11ß-HSD2 mRNA and glucocorticoid receptor (GR) mRNA: real-time quantitative RT-PCR
To determine whether changes in 11ß-HSD2 activity after the different treatment regimens were associated with alterations in 11ß-HSD2 mRNA levels, the relative abundance of 11ß-HSD2 mRNA in trophoblast cells was assessed by two-step real-time quantitative RT-PCR. Given that GR is known to be subjected to auto-regulation by glucocorticoids in other organ systems both in vivo and in vitro (25, 26, 27), changes in the level of GR mRNA in cultured trophoblast cells after Dex treatment for various times were determined as described below.
Briefly, total RNA was extracted from cultured cells using RNeasy Mini Kit (QIAGEN Inc., Mississauga, Canada) coupled with on-column DNase digestion with the RNase-free DNase set (QIAGEN) according to the manufacturer’s instructions. Total RNA (0.5 µg) was reverse-transcribed in a total volume of 20 µl with the high-capacity cDNA archive kit (Applied Biosystems, Forest City, CA) following the manufacturer’s instructions. For every RT reaction set, one RNA sample was set up without reverse-transcriptase enzyme to provide a negative control. Gene transcript levels of 18S rRNA (housekeeping gene) and 11ß-HSD2 mRNA were quantified separately by TaqMan assays with the TaqMan universal PCR master mix (Applied Biosystems) and the universal thermal cycling parameters (2 min at 50 C and 10 min at 95 C, followed by 40 cycles of 15 sec at 95 C and 1 min at 60 C) on an ABI PRISM7900HT sequence detection system (Applied Biosystems). Levels of 18S rRNA were assessed with TaqMan rRNA control reagents (Applied Biosystems), and those of 11ß-HSD2 with custom-designed TaqMan assays. Primers (60 nM each) and TaqMan minor groove binder probe (200 nM) for human 11ß-HSD2 were designed using the Primer Express software (Applied Biosystems) (Table 1
), and their optimal concentrations were determined following guidelines developed for sequence detection systems by Applied Biosystems.
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), and the optimal concentrations were determined empirically. The SYBR Green I assay was performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a modified universal thermal cycling condition (2 min at 50 C and 10 min at 95 C, followed by 40 cycles of 10 sec each at 95 C, 60 C, and 72 C) with the standard disassociation/melting parameters (15 sec each at 95 C, 60 C, and 95 C) on the ABI PRISM7900HT sequence detection system (Applied Biosystems). The specificity of the SYBR Green I assay was verified by performing a melting curve analysis (single peak with a melting temperature of 83.2 C) and by subsequent sequencing of the PCR products. Levels of 18S rRNA, 11ß-HSD2 mRNA, and GR mRNA in each RNA sample were quantified by the relative standard curve method (Applied Biosystems). Briefly, standard curves for 18S rRNA, 11ß-HSD2, and GR were generated by performing a dilution series of the untreated control cDNA. For each RNA sample, the relative amounts of 18S rRNA, 11ß-HSD2 mRNA, and GR mRNA were obtained, and the ratio of 11ß-HSD2 mRNA to 18S rRNA and the ratio of GR mRNA to 18S rRNA were calculated. For each experiment, the amount of 11ß-HSD2 mRNA or GR mRNA at any given time point under various treatment conditions is expressed relative to the amount of transcript present in the untreated control.
Assessment of de novo protein synthesis involvement
Trophoblast cells were cultured as outlined above, and at the time of plating the translational inhibitor CHX (400 nM) was added. One hour later the cells were treated with Dex (100 nM), and at 24 h the cells were harvested for RNA isolation and real-time RT-PCR analysis.
Assessment of 11ß-HSD2 mRNA stability
The trophoblast cells were cultured as outlined above and treated with Dex (100 nM) for 18 h. Transcription was then stopped with DRB (75 µM), and cells were harvested at discrete times (0–12 h) thereafter for RNA isolation and real-time RT-PCR analysis.
Isolation of HSD11B2 gene and construction of the reporter construct
The human HSD11B2 gene was cloned, isolated, and characterized by a conventional cloning protocol, as previously described (28). Briefly, a human genomic DNA library (Stratagene, La Jolla, CA) was screened by plaque hybridization using the human 11ß-HSD2 cDNA as a probe. Positive plaques were isolated, and their inserts were subjected to restriction digestion followed by Southern blotting. A 7-kb restriction fragment containing the 5'-end of the gene was then subcloned into pBluescript KS. After extensive restriction mapping, a 4.5-kb 5'-flanking fragment of the HSD11B2 gene was constructed into the pGL3-Basic vector (Promega Corp., Madison, WI), which contains a luciferase reporter gene (pGL3-HSD11B2P + 4.5 kb). The NcoI site (i.e. the translational start codon in the human HSD11B2 gene) at the 3'-end of the construct was destroyed by S1 nuclease treatment before ligation and confirmed subsequently by DNA sequencing.
Transient transfection and reporter gene assay
Trophoblast cells were isolated as outlined above and plated at a density of 3 x 105 cells/cm2 in 24-well plates. One hour after plating, the cells were cotransfected with 0.8 µg/well of the pGL3-HSD11B2P + 4.5 kb and 0.2 µg/well of a cytomegalovirus promoter (pCMV) ß-galactosidase plasmid (Promega) or with 0.8 µg/well pGL3-Basic and 0.2 µg/well pCMV-ß-galactosidase (negative control). All transfections were carried out in serum-free medium 199 for 1 h with Transfast transfection reagent (Promega) at a ratio of 2:1 (transfection reagent to DNA) according to the manufacturer’s instructions. At the end of transfection, cells were fed with fresh medium containing 10% serum with or without 100 nM Dex. Luciferase and galactosidase activities were analyzed 48 h after transfection by the luciferase assay system (Promega) and the ß-galactosidase enzyme assay system (Promega), respectively. Luciferase activity was measured in a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany) and normalized against ß-galactosidase activity. Each transfection was performed in triplicate, and a total of three independent experiments were carried out.
Statistical analyses
Results are presented as mean ± SEM of three to four independent experiments (i.e. tissues from different patients), as indicated. Statistical analyses of 11ß-HSD2 activity and mRNA as well as GR mRNA data were performed using one-way ANOVA followed by Tukey’s post hoc test. HSD11B2 promoter activity data were analyzed by a standard t test. Significance was set at P < 0.05. Calculations were performed with SPSS software version 9.0 (Chicago, IL).
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Results |
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Treatment of trophoblasts with the synthetic glucocorticoid Dex (100 nM) increased 11ß-HSD2 activity approximately 1.5-fold after 12 h (P < 0.05, Fig. 1A) and 2-fold by 24 h (P < 0.05, Fig. 1A). The endogenous glucocorticoid cortisol also increased 11ß-HSD2 activity in a concentration-dependent manner but with a dose-response curve shifted to the right such that an overall 10 times increase in its concentration was required to elicit a similar response when compared with Dex (P < 0.05, Fig. 1B). Treatment of trophoblasts with the GR antagonist RU-486 (1 µM) blocked the glucocorticoid-induced increase in 11ß-HSD2 activity, indicating that the effect of glucocorticoids on 11ß-HSD2 is likely mediated by GR (Fig. 2A). In addition, Dex was ineffective in stimulating 11ß-HSD2 activity in trophoblast cells that had been cultured for 72 h (i.e. differentiated syncytiotrophoblasts; Fig. 2B), suggesting that the effect of Dex on 11ß-HSD2 may depend upon trophoblast differentiation.
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To determine whether the glucocorticoid-induced increase in 11ß-HSD2 activity was caused by enhanced expression of the enzyme, 11ß-HSD2 mRNA levels were assessed by real-time quantitative RT-PCR. Analogous to its effect on enzyme activity, Dex (100 nM) caused a time-dependent increase in the steady-state level of 11ß-HSD2 mRNA, with a significant increase by 12 h and a maximal increase after 24 h (P < 0.05, Fig. 3A). It is interesting to note that although there was a good correlation between changes in the level of 11ß-HSD2 mRNA and those of 11ß-HSD2 enzyme activity after Dex treatment, there was a modest difference between the two (
3-fold increase in mRNA vs. 2-fold increase in activity). To ascertain whether GR was subjected to auto-regulation by glucocorticoids in cultured trophoblast cells, the level of GR mRNA was also determined. Similar to its effects in other cell culture systems, Dex treatment (100 nM) resulted in a time-dependent decrease in the steady-state levels of GR mRNA with a 25% reduction at 6 h and a maximal 50% reduction by 12 h (Fig. 3B).
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Because the glucocorticoid-induced increase in 11ß-HSD2 expression may require the de novo synthesis of another protein that in turn would increase 11ß-HSD2 mRNA, we assessed mRNA levels after treating the trophoblasts with the protein synthesis inhibitor CHX. Addition of CHX (400 nM) did not block the stimulatory effect of Dex on 11ß-HSD2 mRNA, suggesting that de novo protein synthesis was not required (P < 0.05, Fig. 4). The efficacy of this concentration of CHX in blocking protein synthesis in these cells is demonstrated by the fact that even at higher concentrations (1–2 µM), CHX was unable to block the Dex-induced increase in 11ß-HSD2 mRNA, despite its profound pharmacological effects on basal 11ß-HSD2 mRNA (decreased by more than 90%; data not shown). Furthermore, a similar concentration of CHX was shown to be effective in abrogating the Dex-mediated decrease in fibronectin mRNA expression in cultured human trophoblasts (29).
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In theory, the steady-state levels of 11ß-HSD2 mRNA can be up-regulated by decreasing the rate at which 11ß-HSD2 mRNA is degraded and/or by increasing the rate of HSD11B2 gene transcription. To determine whether glucocorticoids enhance the stability of 11ß-HSD2 mRNA, trophoblast cells were treated with DRB (75 µM), an inhibitor of mRNA synthesis, in the presence or absence of Dex. Treatment with Dex prolonged the half-life of 11ß-HSD2 mRNA from 5.1 to 9.0 h (Fig. 5A), indicating that glucocorticoids enhance the stability of 11ß-HSD2 mRNA. Because the observed increase in 11ß-HSD2 mRNA stability cannot account completely for the enhanced mRNA levels, it remained possible that glucocorticoids may also increase HSD11B2 gene transcription.
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To determine whether glucocorticoids also increase the rate of HSD11B2 gene transcription, trophoblasts were transiently transfected with a luciferase construct containing the 4.5-kb 5'-flanking region of the human HSD11B2 gene. Treatment with Dex (100 nM) resulted in a significant increase in HSD11B2 promoter activity (P < 0.05; Fig. 5B).
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Discussion |
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Owing to the apparent species differences in the glucocorticoid mediated auto-regulation of placental 11ß-HSD2 and considering that prenatal glucocorticoid administration is a common practice in managing premature delivery, it is imperative to determine the effect of glucocorticoids on 11ß-HSD2 in the human placenta. In the present study, we demonstrate that glucocorticoids stimulate 11ß-HSD2 activity and mRNA expression in cultured human trophoblast cells during differentiation by enhancing 11ß-HSD2 mRNA stability and the rate of HSD11B2 gene transcription. Moreover, we present evidence that the glucocorticoid-induced increase in 11ß-HSD2 mRNA does not require de novo protein synthesis.
It is well established that isolated cytotrophoblasts in culture do not proliferate but undergo spontaneous differentiation into multinucleated syncytiotrophoblasts over a period of 48–72 h (23). This morphological differentiation is accompanied by acquisition of the ability to produce a range of hormones, factors, and enzymes that are the hallmarks of biochemical differentiation, some of which include human chorionic gonadotropin, progesterone, and syncytin (23). In a previous study, we reported that the expression of 11ß-HSD2 was induced during in vitro trophoblast differentiation (24). Because glucocorticoids are known to stimulate trophoblast differentiation (38), the effects of glucocorticoids on 11ß-HSD2 expression may be compounded by differentiation. Indeed, Dex was ineffective in stimulating 11ß-HSD2 activity in cultured trophoblasts after differentiation.
Given that GR is known to be down-regulated by glucocorticoids in other organ systems both in vitro and in vivo (25, 26, 27), we examined the effects of Dex on GR mRNA expression in cultured trophoblast cells during differentiation. Dex caused a time-dependent decrease in the level of GR mRNA, indicating that in human trophoblasts GR is subject to auto-regulation by glucocorticoids. Because we have shown in the present study that glucocorticoids also auto-regulate 11ß-HSD2, it is tempting to speculate that glucocorticoids regulate their own actions at multiple levels in the human placenta.
As a first step in delineating the molecular mechanism(s) by which glucocorticoids stimulate placental 11ß-HSD2 expression, we sought to determine whether glucocorticoid treatment alters the half-life of 11ß-HSD2 mRNA. Our results demonstrate that Dex prolongs the half-life of 11ß-HSD2 mRNA in cultured trophoblast cells, indicating that the glucocorticoid-induced increase in 11ß-HSD2 mRNA is mediated, at least in part, by enhancing mRNA stability. Our present findings are consistent with previous studies in which glucocorticoids have been shown to increase the expression of a number of genes by altering mRNA stability (39, 40, 41). Although the precise mechanism(s) by which glucocorticoids enhance 11ß-HSD2 mRNA stability remains to be explored, we speculate that activated GRs may interact with mRNA binding proteins (42) to stabilize 11ß-HSD2 mRNA. Alternatively, similar to their effects on human GH mRNA stability (43), glucocorticoids may increase the length of the 11ß-HSD2 mRNA polyadenosine tail. In addition, 11ß-HSD2 mRNA may possess a glucocorticoid-responsive stabilizing element, as has been demonstrated in the 3' noncoding region of the phosphoenolpyruvate carboxykinase mRNA (39). The precise mechanism of action of the glucocorticoid-responsive stabilizing element on the phosphoenolpyruvate carboxykinase mRNA has not yet been established, but it is postulated to regulate the polyadenosine tail, a 102-nucleotide stretch of alternating purines and pyrimidines, or an AUUUA motif (39).
To determine the effects of glucocorticoids on placental HSD11B2 gene transcription, we transfected trophoblast cells with a luciferase reporter gene construct driven by the HSD11B2 promoter. Our preliminary findings demonstrate that glucocorticoid treatment increases HSD11B2 promoter activity, suggesting that the glucocorticoid-induced increase in placental 11ß-HSD2 mRNA is mediated by a dual mechanism involving both an enhanced rate of HSD11B2 gene transcription and increased mRNA stability. It is well established that activated GRs modulate gene transcription by either directly binding to glucocorticoid-response elements (GREs) in target genes or indirectly through interactions with other transcription factors (44). Despite the apparent lack of a GRE consensus sequence within the 4.5-kb HSD11B2 promoter (TFSEARCH, version 1.3), it remains possible that activated GRs may interact with a GRE-like sequence in the HSD11B2 promoter to increase gene transcription. Alternately, glucocorticoids may increase the rate of HSD11B2 gene transcription indirectly through protein-protein interactions, with factors such as Sp1 and/or Sp3 that have been identified previously as the two critical transcription factors involved in regulating HSD11B2 promoter activity in JEG-3 cells (45, 46). It is noteworthy that a GR-mediated gene transcriptional effect that occurs independently of de novo protein synthesis operating through mechanisms other than traditional GREs has been reported previously (47). Obviously, additional studies will be required to elucidate the precise molecular mechanisms underlying the glucocorticoid-induced up-regulation of HSD11B2 gene transcription.
Placental 11ß-HSD2 expression increases with advancing gestation (13, 14), and this increase coincides with increased circulating glucocorticoid levels in pregnant women (5, 6). In the present study, we provide the first evidence that glucocorticoids increase the expression of 11ß-HSD2 in cultured human trophoblast cells. If this occurs in the human placenta in vivo, our findings would suggest that the elevated glucocorticoid levels in maternal circulation may be responsible for increasing the expression of placental 11ß-HSD2 across gestation.
In humans, single and multiple courses of antenatal glucocorticoid treatment do not appear to be associated with reduced birth weight, contrary to what is seen in several animal models, including mice, rats, and sheep (18). For instance, a single course of maternal glucocorticoid administration in sheep results in 15% reduction in birth weight, with severe growth restriction after multiple courses (48, 49). Although the reasons for the apparent species differences in fetal response to maternal glucocorticoid treatment are unknown, it is tempting to speculate that these differences may be explained partially by the opposite effects of glucocorticoids on placental 11ß-HSD2 in humans and sheep. It is conceivable that in humans the glucocorticoid-induced increase in placental 11ß-HSD2 could limit fetal exposure to maternally derived glucocorticoid via the placenta. Thus, the auto-regulation of placental 11ß-HSD2 by glucocorticoids would represent an important mechanism by which the fetus may be protected from detrimental exposure to elevated levels of maternal glucocorticoids.
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Acknowledgments |
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Footnotes |
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Abbreviations: CHX, Cycloheximide; Dex, dexamethasone; DRB, 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside; GR, glucocorticoid receptor; GRE, glucocorticoid-response element; 11ß-HSD2, 11ß-hydroxy- steroid dehydrogenase type 2; IUGR, intrauterine growth restriction.
Received January 22, 2004.
Accepted July 21, 2004.
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-adrenergic signaling. Am J Physiol Regul Integr Comp Physiol 281:R1966–R1974
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) 100 nM Dex for 18 h. The cells were then treated with DRB (75 µM) in the absence or presence of Dex (100 nM) (defined as time zero). At the indicated time points thereafter, total cellular RNA was isolated, and the steady-state level of 11ß-HSD2 mRNA was assessed by real-time quantitative RT-PCR, as described in Materials and Methods. Each data point is expressed as a percentage of control and represents the ratio of 11ß-HSD2 mRNA to 18S rRNA. Data are expressed as the mean ± SEM (n = 3; regression lines: control R2 = 0.9986; Dex R2 = 0.9204). B, Effects of Dex on HSD11B2 promoter activity. The isolated trophoblasts were transfected with a luciferase reporter gene construct driven by the HSD11B2 promoter, as described in Materials and Methods. One hour after transfection, the cells were treated with or without Dex (100 nM) for 48 h. At the end of treatment, luciferase activity was determined, and data are expressed as the mean ± SEM (n = 3; *, P < 0.05 vs. control).