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Mechanisms of Hormone Action; |
CIHR Group in Development and Fetal Health,3 Department of Physiology and Obstetrics and Gynecology and Medicine, University of Toronto, Ontario, Canada M5S 1A8
Department of Obstetrics and Gynecology,4 Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan
ABSTRACT
Placental hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2) plays an important role in pregnancy maintenance and fetal maturation. In the event of intrauterine infection, lipoxygenase (LOX) metabolites are produced in the placenta and contribute to preterm labor and adverse fetal outcomes. On the other hand, LOX metabolites are involved in production of progesterone, which is required for pregnancy maintenance. In this study, we evaluated the interaction between the LOX pathway, progesterone, and HSD11B2. Specifically, we hypothesized that LOX metabolites would alter HSD11B2 and this effect would be mediated by progesterone. We cultured human term placental trophoblasts in the presence and absence of the LOX inhibitors Nordihydroguaiaretic acid (NDGA), AA861, and Baicalein; the LOX metabolites Leukotriene B4 and 12(S)-Hydroxyeicosatetraenoate (12-HETE); and progesterone and progesterone receptor antagonist RU486. By radiometric conversion assay, real-time quantitative PCR, Western blot analysis, and ELISA, we examined HSD11B2 enzyme activity, HSD11B2 mRNA and HSD11B2 protein expression, and progesterone output. LOX metabolites down-regulated HSD11B2 activity and HSD11B2 expression. LOX inhibitors up-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were attenuated by addition of LOX metabolites. Net progesterone output was increased by LOX metabolites and decreased by LOX inhibitors. Progesterone down-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were blocked by RU486. Furthermore, the suppressive effect of 12-HETE on HSD11B2 activity was also reversed by RU486. We conclude that HSD11B2 in human placental trophoblasts is decreased by progesterone and increased by inhibition of endogenous LOX metabolites, and that a component of the effect of LOX metabolites on HSD11B2 is mediated by their stimulation of endogenous progesterone output.
HSD11B2, human placental trophoblast, hydroxysteroid 11-beta dehydrogenase 2, lipoxygenase pathway, progesterone
In this study, we have examined the interactions between the lipoxygenase (LOX) products of arachidonic acid metabolism, progesterone, and hydroxysteriod 11-beta dehydrogenase 2 (HSD11B2) in the human placenta. In human pregnancy, placental HSD11B2 localizes to the syncytiotrophoblasts and exhibits an oxidase (cortisol to cortisone) activity under physiological conditions. HSD11B2 plays a pivotal role in restricting the passage of maternal cortisol to the fetus [1, 2]. Glucocorticoids play an important role in parturition, fetal growth, and maturation [3], although excessive amounts of glucocorticoids may result in intrauterine growth restriction [4–6]. Therefore, the HSD11B2 enzyme serves as a critical barrier, protecting the fetus from the high levels of maternal glucocorticoid that are present during pregnancy [7].
Increased prostaglandin production is a key step in the parturition process in the presence or absence of infection [8, 9]. It is proposed that cortisol from either maternal or fetal adrenal can up-regulate prostaglandin synthesis and down-regulate prostaglandin metabolism in placental and chorionic trophoblasts. Prostaglandins are formed from arachidonic acid by the activity of the two isoforms of the enzyme cyclooxygenase. However, there are at least two other major pathways of arachidonic acid metabolism that result in biologically active products, namely the epoxide pathway (cytochrome P450) and the LOX pathway [10], and the latter is classified into three main lines: 5-LOX, 12-LOX, and 15-LOX [11, 12]. The LOX pathway generates hydroperoxyeicosatetraenoic acid compounds that are metabolized to a range of eicosanoids, including leukotrienes [13]. LOX metabolites are produced in the placenta [14] and may stimulate myometrial activity [13]. Previous studies have shown that prostaglandins and leukotriene B4 (LTB4) inhibited activity of HSD11B2 in human choriocarcinoma JEG-3 cells, and that prostaglandins down-regulated expression of HSD11B2 in human chorionic and placental trophoblasts [15]. However, the biological roles of LOX metabolites in parturition and the regulation of placental cortisol metabolism are still unknown.
Progesterone, a major steroid of the human placenta required for pregnancy maintenance [16], down-regulates HSD11B2 activity and HSD11B2 mRNA expression in human term placental and chorionic trophoblasts [17]. It has been reported that the LOX pathway is involved in progesterone production in the ovary [18, 19]. However, there is little information about the involvement of the LOX pathway in the production of progesterone by the placenta and whether effects of LOX metabolites on HSD11B2 regulation might be mediated through effects on progesterone production.
Therefore, in this study we sought to investigate the effect of the metabolites of the LOX pathway on HSD11B2 regulation and endogenous progesterone production. We then evaluated the possible interaction between the LOX pathway and progesterone in the regulation of HSD11B2 in human placental trophoblasts.
Placental tissues were obtained from uncomplicated, normal term pregnancies after elective Cesarean section in the absence of labor. None of the patients received glucocorticoids or prostaglandin synthase inhibitor. Collection and processing of human placenta was approved by the local hospital ethical committees of Mount Sinai Hospital in Toronto and the University of Toronto. Informed patient consent was obtained in all cases.
Placental Trophoblast Cell Culture
Placental trophoblasts were isolated from term placental cotyledons and cultured by the modification of the technique described by Kliman et al. [20], as we published before [21]. Briefly, cotyledonary tissues were dissected aseptically from fetal membranes and blood vessels, rinsed in saline, and digested with 0.125% tripsin (Sigma, St. Louis, MO) and 0.02 % deoxyribonuclease-I (Sigma) in Dulbecco modified Eagle medium (DMEM, Sigma) three times for 30 min each. The dispersed placental cells were filtered through a 200-µm pore size nylon gauze, loaded onto a continuous Percoll (Sigma) gradient (5%–70% at step increments of 5%), and centrifuged at 2500 x g for 20 min to separate different cell types. Isolated cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected and plated in 24-well plates at a density of 106/cm2 (for radiometric conversion assay and measurement of progesterone) or in 60-mm dishes at a density of 107/cm2 (for cell collection and protein extraction) in DMEM containing 10% fetal bovine serum (Sigma) and 1% antibiotic antimycotic solution (Sigma). The cells were maintained for 72 h at 37°C in humidified 5% CO2 and 95% air before experimentation. Under these conditions, these cells aggregated to form a syncytium corresponding to syncytiotrophoblast.
After 72 h of culture, placental trophoblasts were washed two times with equilibrated Hanks balanced salt solution (Sigma) and cultured for 16 h in serum-free DMEM before treatment. After 16 h of incubation, the medium was changed and the cells were treated as follows: 1) for 24 h with medium alone; 2) for 24 h with the LOX inhibitors nordihydroguaiaretic acid (NDGA; 5-, 12-, and 15-LOX inhibitor; 10–7–10–5 M; Sigma), AA861 (5-LOX inhibitor, 10–7–10–5 M; Sigma), or Baicalein (12-LOX inhibitor, 10–6–10–4 M; Sigma); 3) for 12 h with the LOX metabolites LTB4 (5-LOX product, 10–10–10–6 M; Sigma) or 12(S)-Hydroxyeicosatetraenoic acid (12-HETE; 12-LOX product, 10–10–10–6 M; Sigma) after 12 h pre-incubation with medium alone; 4) for 12 h with LOX metabolites after 12 h pre-incubation with LOX inhibitors; 5) for 24 h with progesterone (10–6 M; Steraloids Inc., Wilson, NH) or progesterone receptor antagonist RU486 (10–5 M; Sigma) alone; 6) for 24 h with progesterone with NDGA; and 7) for 12 h with LOX metabolites after 12 h preincubation with RU486. Cell viability, assessed by trypan blue staining, was over 90% before and after all treatments.
RNA Isolation and cDNA Synthesis
At the end of the treatment, cells were harvested and RNA was isolated with the TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Potential genomic DNA contamination was removed with DNA-free DNAse Treatment and Removal Reagents (Ambion Inc., Austin, TX). The integrity of RNA samples (2 µl) was confirmed on agarose gel electrophoresis by the presence of two sharp bands representing 28S and 18S rRNA after staining with ethidium bromide. RNA was then quantified spectrophotometrically at 260 nm. Total mRNA (1 µg) was used for cDNA synthesis. The mRNA was reverse-transcribed with an oligo (dT) 12–18 primer Omniscript RT Kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer's instructions.
Quantitative Real-Time PCR Analysis
Real-time PCR was performed with the Platinum Quantitative PCR SuperMix Kit (Invitrogen, Burlington, ON, Canada). Quantitative real-time PCR mix consisted of 1 µl diluted cDNA, 0.5 µM of each paired primer, 12.5 µl of Platinum Mix from the kit (containing Platinum TaqDNA), 0.8 µl of SYBR green (1:1000 diluted; Invitrogen), and 9.7 µl of diethyl pyrocarbonate-treated H2O in 25 µl final volume. The primer sequences were as follows: HSD11B2 mRNA forward primer 5'-AGTAGTTGCTGATGCGGA and reverse primer 5'-CATGCAAGTGCTCGATGT, as published [15]. To control sampling errors, PCR for housekeeping gene 18S ribosomal RNA was performed on each sample. The primer sequences for 18S ribosomal RNA were as follows: forward primer 5'-TTGTTGGTTTTCGGAACTGAGGC and reverse primer 5'-GGCAAATGCTTTCGCTCTGGTC. For non-template-negative control we used complete DNA amplification mix where the target cDNA template was replaced with water. Quantitative PCR was performed using Rotor Gene (Montreal Biotech Inc., Dorval, QC, Canada) for 45 cycles (denaturation at 95°C for 10 min, specific annealing at 56°C for 30 sec, and extension at 72°C for 30 sec). To assess linearity and efficiency of PCR amplification, standard curves for HSD11B2 and 18S ribosomal RNA were generated by using serial dilutions of cDNA, prepared by reverse transcription of RNA, and isolated from untreated control cells. Standard curves for both sets of primers demonstrated a high linearity between the threshold cycle (Ct) and the log of the starting cDNA copy number, with a correlation coefficient (R value) greater than 0.992. Data were quantified using the comparative Ct quantification method. To control sampling errors, the ratio of cycle threshold for HSD11B2 and 18S ribosomal RNA was determined to quantify the relative HSD11B2 expression. Data from the samples that had an average amplification reaction efficiency exceeding the confidence interval for the average amplification efficiency of untreated control samples were excluded from the analysis. A melt curve analysis was carried out on the products of amplification reaction to ascertain the melting temperature of the product. Single major peaks for both products were observed. Experiments were performed with duplicates for each data point. The size of PCR product was assessed by agarose gel electrophoresis, the products were purified using QIAquick PCR Purification Kit (Qiagen), and the sequence was confirmed by Inn Biotech Inc. (Toronto, ON, Canada).
Protein Extraction and Western Blot Analysis
Cultured cells were scraped from the dishes and mixed with cold RIPA lysis buffer (100 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, and 50 mM Tris-HCl; pH 7.5) containing Mini EDTA-free protease inhibitors (Boehringer Mannheim Biochemicals, Mannheim, Germany) and 100 µM sodium orthovanadate (Sigma) for 30 min on ice. The cell lysates were vortexed vigorously and centrifuged at 12 000 rpm for 30 min at 4°C. The supernatant was collected, and the protein content was determined by the Bradford method using a protein assay kit (Bio-Rad Laboratories Inc., Mississauga, ON, Canada) with BSA as a standard.
Western blot analysis was performed as described previously [22]. Briefly, 40 µg of the protein extracts were subjected to a standard 10% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membrane (Bio-Rad). The nitrocellulose blots were blocked overnight at 4°C with 10% nonfat milk in Tris-buffered saline with 0.1% Tween-20 (TBST; Bio-Rad) and incubated with primary antibody for HSD11B2 protein (1:1000 dilution, with 10% nonfat milk solution in TBST) for 2 h at room temperature. The primary antibody was a polyclonal rabbit anti-human HSD11B2 antibody (Alpha Diagnostic International, San Antonio, TX). After five 5-min washes with TBST, the blots were incubated with a donkey anti-rabbit IgG coupled to horse radish peroxidase (Amersham Life Science, Baie d'Urfe, QC, Canada) at 1:4000 dilution for 1 h and then washed with TBST five times for 5 min each. Enhanced chemiluminescence detection reagents (Amersham Life Science) were added to the membranes for 1 min, and then the blots were exposed to X-OMAT blue film (Kodak, Rochester, NY) overnight. The intensities of the immunoreactive bands were measured by scanning (6200C scanner; Hewlett Packard Co., Mississauga, ON, Canada) and analyzed by using Scion Image software (version 4.0.2; Scion Co., Frederick, MD). Protein bands were digitalized, and the mean pixel density was analyzed to optical density units for each protein. To standardize for protein loading, the blots were stripped and reprobed with a monoclonal mouse anti-human β-actin antibody (Sigma A-5316; 1:10 000 dilution, with 5% nonfat milk solution in TBST). Each optical density value for HSD11B2 was then divided by the optical density value for β-actin to achieve a final relative optical density value for HSD11B2. Experiments were performed with triplicates for each data point.
Determination of HSD11B2 Enzyme Activity
At the end of treatment, the cells were washed two times with serum-free DMEM to remove the treatment compounds. The level of HSD11B2 activity in intact cells was determined by measuring the rate of cortisol to cortisone conversion, as described previously [22]. Briefly, the cells were incubated for 2 h at 37°C in serum-free DMEM containing 10 nM [3H] cortisol (specific activity, 259 x 1010 Bq/mmol; Perkin Elmer Life Sciences, Inc., Boston, MA) as substrate in a 1-ml total volume. At the end of incubation, the medium was collected, and a mixture of cortisol and cortisone (40 µg each) was added to allow subsequent localization of the steroids during purification by thin-layer chromatography (TLC). Steroids in the medium were extracted with ethyl acetate and applied to a TLC plate (Silica gel GF; Fisher Scientific, Pittsburg, PA). Cortisol and cortisone were separated in the solvent system chloroform/ethanol (95:5 vol/vol). The bands containing the labeled cortisol and cortisone were visualized under UV light, scraped off the plate, and extracted with ethyl acetate. The solvent was dried, scintillation fluid (Ready Safe; Beckman Coulter Inc., Fullerton, CA) was added, and the radioactivity was counted using a Tri-Carb 2100TR Liquid Scintillation Counter (Packard Instrument Co., Meridien, CT). The rate of cortisol to cortisone conversion was calculated, and blank values (defined as the amount of conversion in the absence of cells) were subtracted from the experimental values. Experiments were performed with duplicates for each data point.
Progesterone concentration in the medium was measured by specific ELISA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. At the end of the treatment, culture medium was collected and applied to the assay. The intra- and interassay coefficients of variation were 3.8% and 4.9%, respectively, and the minimum detectable limit of the assay was 7.8 pg/ml. Experiments were performed with duplicates for each data point.
All data are shown as mean ± SEM. Statistical comparisons were made by one-way ANOVA, followed by Student-Newman-Keuls test. Statistical significance was set at P < 0.05. Calculations were performed using Sigma Stat (Jandel Scientific Software, San Rafael, CA).
Effect of the LOX Pathway on HSD11B2 Enzyme Activity, HSD11B2 mRNA, and HSD11B2 Protein Expression
The enzyme activity assay revealed that HSD11B2 was significantly up-regulated with the 3 LOX inhibitors NDGA (10–6 and 10–5 M, P < 0.01 and P < 0.05), AA861 (10–7, 10–6, and 10–5 M; P < 0.01, P < 0.001, and P < 0.05), and Baicalein (10–5 and 10–4 M, both P < 0.01) in a dose-dependent manner (Fig. 1A). In contrast, HSD11B2 was significantly down-regulated with LTB4 (10–7 and 10–6 M, both P < 0.05) or 12-HETE (10–7 and 10–6 M, P < 0.05 and P < 0.01) (Fig. 1B). Furthermore, the effects of LOX inhibitors were significantly attenuated by LOX metabolites (NDGA plus LTB4, NDGA plus 12-HETE, AA861 plus LTB4, Baicalein plus 12-HETE; all P < 0.05; Fig. 1C).
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HSD11B2 mRNA was increased with NDGA (10–6 M, P < 0.01) and reduced with LTB4 (10–8 M, P < 0.05) or 12-HETE (10–8 M, P < 0.05). Furthermore, the effect of NDGA on HSD11B2 was attenuated by 12-HETE in a dose-dependent manner (10–8 and 10–7 M, both P < 0.05; Fig. 2A).
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Using Western blot analysis, we were unable to detect any significant differences in HSD11B2 protein in cells treated with LOX inhibitors/metabolites alone. However, the levels of HSD11B2 in cells treated with NDGA or AA861 were reduced by 12-HETE or LTB4 (NDGA plus LTB4, NDGA plus 12-HETE, AA861 plus LTB4; all P < 0.05; Fig. 2B).
Effect of LOX Pathway on Progesterone Output
Net progesterone output was significantly decreased by NDGA (10–6 and 10–5 M, P < 0.05 and P < 0.01) or Baicalein (10–5 and 10–4 M, both P < 0.05) in a dose-dependent manner (Fig. 3A). In contrast, both LTB4 (10–6 M) and 12-HETE (10–6 M) significantly increased progesterone output (both P < 0.05; Fig. 3B) and also reversed the inhibitory effect on progesterone output in cells treated with NDGA (both P < 0.05; Fig. 3C). Progesterone concentration in the "control" medium (cultured for 24 h with no treatment) was 33.58 ± 8.53 ng/ml.
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Interaction of LOX Pathway and Progesterone on HSD11B2 Enzyme Activity, HSD11B2 mRNA, and HSD11B2 Protein Expression
To examine whether the effect of LOX compounds on HSD11B2 was mediated through progesterone, we conducted a further series of studies. Exogenous progesterone significantly attenuated HSD11B2 activity (P < 0.001; Fig. 4A), HSD11B2 mRNA (P < 0.01; Fig. 4B), and HSD11B2 protein expression (P < 0.05; Fig. 4C), and these effects were significantly blocked by RU486 (all P < 0.05). The inhibitory effect of progesterone on HSD11B2 activity was also reversed by NDGA (P < 0.05; Fig. 4A). RU486 alone modestly increased HSD11B2 activity (P = 0.055; Fig. 4A), HSD11B2 expression (P < 0.05; Fig. 4B), and HSD11B2 expression (P < 0.05; Fig. 4C), and the effect on HSD11B2 activity, although not on HSD11B2 or HSD11B2 expression, was enhanced further with concurrent addition of NDGA (Fig. 4A). Furthermore, the suppressive effect of 12-HETE on HSD11B2 activity was also reversed by RU486 (P < 0.05; Fig. 4A).
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We have examined interactions between the LOX pathway of arachidonic acid metabolism and progesterone on the levels of HSD11B2 enzyme activity, HSD11B2 mRNA expression, and HSD11B2 protein expression in human placental trophoblasts. Our results point to interrelationships between these pathways that could be important in affecting HSD11B2 activity during normal pregnancy and at times of infection, and would therefore impact the efficacy with which the placenta serves as a barrier to the passage of cortisol from the maternal to the fetal compartment.
We found up-regulation of HSD11B2 activity and HSD11B2 expression in the presence of LOX inhibitors, implying a role for endogenous LOX products in inhibiting HSD11B2. In contrast, we found down-regulation of HSD11B2 activity and HSD11B2 expression with exogenous LOX metabolites LTB4 and 12-HETE, and these LOX metabolites also reduced the effects of LOX inhibitors on HSD11B2 activity, HSD11B2 expression, and HSD11B2 expression. Thus we have demonstrated, for the first time in primary cultures of human placental trophoblast, evidence for potential regulation of HSD11B2 by products of the LOX pathways. Because it is well established that progesterone reduces HSD11B2, we examined the interaction between the LOX pathway and progesterone output by the cells. We found that progesterone output was significantly decreased by inhibitors of the LOX pathway and was increased by LTB4 and 12-HETE. Thus, effects of LOX metabolites on HSD11B2 could be direct, as inferred from the first series of experiments, or indirect through stimulation of endogenous progesterone output.
We next confirmed that in this cell type, HSD11B2 activity, HSD11B2 expression, and HSD11B2 expression were suppressed by exogenous progesterone, and these effects were blocked by addition of the progesterone receptor antagonist RU486. Furthermore, RU486 itself up-regulated HSD11B2 expression and HSD11B2 expression, presumably by blocking the action of endogenous progesterone. Importantly, the suppressive effect of 12-HETE on HSD11B2 activity was reversed by RU486, implying a role for endogenously produced progesterone on HSD11B2. These studies do not preclude a direct effect of LOX metabolites on HSD11B2. However, they do suggest interaction between the LOX pathway and progesterone such that the inhibitory effect of LOX metabolites on HSD11B2 might be mediated, at least in part, through their stimulation of endogenous progesterone output, an effect that is blocked in the presence of RU486.
The present studies have been conducted with cultured primary trophoblasts rather than the JEG-3 choriocarcinoma-derived cell line. We have not attempted to delineate the full time course relationship of these interactions, but have shown clearly their dose dependency. We have interpreted our results as indicating that the amounts of RU486 used in these experiments are able to antagonize the actions of endogenously produced progesterone, which could be overcome at the higher concentrations of exogenous progesterone added in other experiments. Our studies on the output of progesterone in response to LTB4 and 12-HETE by the cells would not be inconsistent with this contention.
Previous studies have demonstrated that the metabolites of the 5- and 12-LOX pathways are major products of arachidonic acid metabolism in human intrauterine tissues [14, 23] and that the physiological levels of LOX metabolites in human umbilical plasma or amniotic fluid are within nanomolar concentrations [24, 25]. Because the effect of NDGA, a nonspecific inhibitor of the 5-, 12-, and 15-LOX pathways, was similar to that of the more specific LOX inhibitors, AA861 and Baicalein, we could not discriminate which specific LOX pathway was the more important in the regulation of HSD11B2. However, our results suggest that endogenous LOX metabolites, produced under pathological conditions such as infection, might have a key role in regulation of HSD11B2.
Previous studies concerning prostaglandins, produced locally within the decidua, fetal membranes, and placenta, have shown that these cyclooxygenase products play a key role in the initiation of human labor and also down-regulate HSD11B2 in human chorionic and placental trophoblasts [15]. HSD11B2 is expressed in the human placenta from early gestation and remains active throughout pregnancy and labor [26–28]. The present studies show that rising concentrations of LOX products from trophoblast cells, in addition to prostaglandins, may contribute to a decrease in HSD11B2 in late pregnancy. LOX metabolites are produced not only by the placenta and chorionic trophoblasts, but also by leukocytes and macrophages recruited into these tissues in response to infection, usually causing inflammation [29]. Recent clinical reports have associated fetal growth restriction with inflammation [30]. Placental HSD11B2 activity is significantly reduced in the growth-restricted fetus [31], and there is a correlation between placental HSD11B2 activity and birth weight [32–34]. Therefore, this additional source of LOX metabolites could potentially contribute to greater transfer of maternal glucocorticoids across the placenta to the fetus during infection and inflammation, with deleterious effects on fetal development and potentially restrictive effects on growth.
Net progesterone output was significantly decreased by NDGA and Baicalein, but increased by LTB4 and 12-HETE in a dose-dependent manner. Further, the inhibitory action of NDGA on progesterone output was reversed by both LTB4 and 12-HETE. Previous studies have revealed an interaction between the LOX pathway and progesterone production in the mammalian ovary [18, 19]. Our present results indicate that LOX metabolites, especially produced through the 12-LOX pathway, might play a role in the regulation of progesterone output by the human placenta. Previous studies have also suggested that both glucocorticoids and progesterone might stimulate LTB4 production by human fetal membranes at term [35]. Thus, there appears to be a positive feed-forward relationship between progesterone and LTB4 products in trophoblast cells, and these augment the effects of each other in suppressing HSD11B2. In vivo these controls could be exerted in either an autocrine or paracrine manner (Fig. 5).
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In summary, we have demonstrated that the LOX pathway is involved in the regulation of both HSD11B2 and progesterone output by human placental trophoblast cells. We suggest that the effect of LOX metabolites on HSD11B2 might be mediated, at least in part, through progesterone. This novel interaction enhances the inhibition of HSD11B2, with subsequent effects on the glucocorticoid environment of the placenta and fetus.
FOOTNOTES
1Supported by the Canadian Institute of Health Research operating grant (to J.R.G.C.). 
Correspondence: 2Kazuyo Sato, Department of Physiology, University of Toronto, Room 3344, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8. FAX: 416 978 4373; e-mail: kazusato{at}mail.tains.tohoku.ac.jp
Received: 5 August 2007.
First decision: 31 August 2007.
Accepted: 8 November 2007.
REFERENCES
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