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Endocrinology Vol. 147, No. 5 2490-2495
Copyright © 2006 by The Endocrine Society

Enhancement of Cortisol-Induced 11ß-Hydroxysteroid dehydrogenase Type 1 Expression by Interleukin 1ß in Cultured Human Chorionic Trophoblast Cells

Wenjiao Li1, Lu Gao1, Yan Wang, Tao Duan, Leslie Myatt and Kang Sun

School of Life Sciences, The First Maternal and Fetal Care Hospital, Fudan University (W.L., Y.W., T.D., K.S.), and Second Military Medical University (L.G.), Shanghai 200433, China; and Department of Obstetrics and Gynecology (L.M.), College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Dr. Kang Sun, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, People’s Republic of China. E-mail: sunkang2000{at}yahoo.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chorion is the most abundant site of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) expression within intrauterine tissues. It is important to study the regulation of 11ß-HSD1 expression in the chorion in terms of local cortisol production during pregnancy. Using real-time PCR and enzyme activity assay, we found that cortisol (1 µM) and IL-1ß (10 ng/ml) for 24 h significantly increased 11ß-HSD1 mRNA expression and reductase activity in cultured human chorionic trophoblasts. A further significant increase of 11ß-HSD1 mRNA expression and reductase activity was observed with cotreatment of cortisol and IL-1ß. To explore the mechanism of induction, 11ß-HSD1 promoter was cloned into pGL3 plasmid expressing a luciferase reporter gene. By transfecting the constructed vector into WISH cells, an amnion-derived cell line, we found that cortisol (1 µM) or IL-1ß (10 ng/ml) significantly increased reporter gene expression. Likewise, an additional increase in reporter gene expression was observed with cotreatment of cortisol and IL-ß. To explore the physiological significance of 11ß-HSD1 induction in the chorion, we studied the effect of cortisol on cytosolic phospholipase A2 and cyclooxygenase 2 expression. We found that treatment of chorionic trophoblast cells with cortisol (1 µM) induced both cytosolic phospholipase A2 and cyclooxygenase 2 mRNA expression. We conclude that cortisol up-regulates 11ß-HSD1 expression through induction of promoter activity, and the effect was enhanced by IL-1ß, suggesting that more biologically active glucocorticoids could be generated in the fetal membranes in the presence of infection, which may consequently feed forward in up-regulation of prostaglandin synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS ARE involved in both maturation of the fetal lung and parturition via stimulation of surfactant formation in the fetal lung and prostaglandin E2 (PGE2) synthesis in human intrauterine tissues, respectively (1, 2, 3). Therefore, defining the sources of glucocorticoids during pregnancy would have significant impact on the understanding of human fetal organ maturation as well as the mechanism of human parturition. Previous work by Sun and Challis has shown that fetal membranes express abundant 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts biologically inactive cortisone into active cortisol in humans (4). Alfaidy et al. (5) found that the expression of 11ß-HSD1 in the fetal membranes increased with gestational age, indicating increasing cortisol is produced with gestational age in the fetal membranes. Because the fetal membranes are in close contact with the amniotic fluid, cortisol derived from 11ß-HSD1 in the fetal membranes would diffuse easily into amniotic fluid where it can access the fetus through fetal swallowing (6). Thus, fetal membranes may have a role as an extraadrenal source of cortisol for the fetus during pregnancy (6, 7). Moreover, fetal membranes are the major site of prostaglandin synthesis during pregnancy (8, 9), the production of which was increased by glucocorticoids (10, 11, 12). Therefore, cortisol produced by 11ß-HSD1 in the fetal membranes may enhance prostaglandin production in a paracine or an autocrine way in the fetal membranes. Thus, the regulation of 11ß-HSD1 expression in the fetal membranes may impact human fetal organ maturation as well as parturition. Recent work by Sun and Myatt (13) showed that the up-regulation of 11ß-HSD1 expression by glucocorticoids was greatly enhanced in the presence of proinflammatory cytokines in the amnion cells. Because proinflammatory cytokines are a major stimulus to preterm labor in chorioamnionitis (14, 15), it is of prime importance to examine whether this phenomenon also holds true in the chorion, the most abundant site of 11ß-HSD1 expression within the intrauterine tissues. To address this issue, we studied the effect of cotreatment of cortisol and IL-1ß on 11ß-HSD1 mRNA expression in cultured human chorionic trophoblast cells. The mechanism of this effect was further explored in amnion-derived WISH cells transfected with vectors carrying the 11ß-HSD1 promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chorionic trophoblast and WISH cell preparations
Fetal membranes were collected at term from elective cesarean section patients not in labor under a protocol approved by Fudan University Institutional Review Board and with informed consent from patients. Chorionic trophoblasts were prepared using a modification of Kliman’s method, as described previously (16). Briefly, chorion was peeled off the amnion, and the residual blood was washed off chorionic tissue with cold normal saline. The minced chorionic tissue was digested with 0.125% (wt/vol) trypsin (Life Technologies, Inc., Grand Island, NY) and 0.1% (wt/vol) collagenase type 1 (Worthington, NJ) three times for 60 min. The chorionic cells were loaded onto a stepwise 5–75% (vol/vol) Percoll (Amersham Biosciences, Uppsala, Sweden) gradient composed of increments of 5% Percoll and centrifuged at 2500 x g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml (corresponding to 35 and 50% Percoll) were collected and plated at a density of 2 x 106 cells per well in six-well plates in DMEM culture medium (Sigma Chemical Co., St. Louis, MO) containing 10% (vol/vol) fetal calf serum (FCS) (Life Technologies) and antibiotic-antimycotic (Life Technologies). The cells were cultured at 37 C in 5% CO2 in air. Our previous work has demonstrated that cells prepared using the above method are predominantly chorion-derived trophoblast rather than decidual cells (17). Human amnion-derived WISH cells (American Type Culture Collection, Manassas, VA) were cultured in 24-well plates in DMEM supplemented with antibiotic-antimycotic and 10% FCS.

Cell treatment and RNA extraction
On the third day of chorionic trophoblast cell culture, cells were washed with PBS and changed to FCS-free DMEM. Cortisol (Sigma) with or without IL-1ß (Life Technologies) were added into the culture medium to achieve final concentrations of 0.001–1 µM for cortisol and 10 ng/ml for IL-1ß. In another set of experiments, the cells were incubated with cortisol (1 µM) in the presence or absence of glucocorticoid receptor antagonist RU486 (1 µM). Incubation with the above treatments was carried on for 24 h. After removal of the culture medium, cells were washed with PBS and then scraped off the plate in cell lysis buffer (supplied with RNeasy Kit; QIAGEN, Valencia, CA). Subsequent extraction and purification of total RNA from the cells was conducted using the RNeasy Kit (QIAGEN) according to the protocol provided by the company. The extracted RNA was then quantified spectrophotometrically at 260 nm, and integrity was assessed by agarose-formaldehyde gel electrophoresis.

11ß-HSD1 reductase activity assay
[3H]Cortisone was prepared by oxidizing [3H]cortisol (specific activity, 64.0 Ci/mmol) (Amersham Life Science, Little Chalfont, UK) with chromium trioxide and was purified by thin-layer chromatography (TLC) before it was used for 11ß-HSD1 reductase activity assay. 11ß-HSD1 reductase activity was assayed according to our previously published report (17). In brief, for the measurement of 11ß-HSD1 activity in cortisol and IL-1ß-treated chorion cells, cells were washed with PBS three times 24 h after the treatments, and the culture medium was changed to FCS-free medium. Each treatment was performed in duplicate or triplicate for each preparation of cells. 11ß-HSD1 reductase activity was measured using 1 µM cortisone (Sigma) containing 200,000 cpm [3H]cortisone with incubation for 12 h at 37 C. This incubation time was chosen from a preliminary study to maintain 11ß-HSD1 activity in a linear range. Our previous study has shown that the Michaelis-Menten constant (Km) value of 11ß-HSD1 reductase activity measured in cultured chorionic trophoblast cells is 1.4 µM (17). To measure conversion, a mixture of cortisol and cortisone (40 µg each) was added to the collected medium to allow visual localization of the steroids when subjected to TLC. Steroids in the media were extracted with ethyl acetate. The recovery rate of [3H]cortisol or [3H]cortisone was measured by adding [3H]cortisol or [3H]cortisone to culture medium and processing the samples in an identical fashion. The mean recovery rates of [3H]cortisol and [3H]cortisone were 86.7 and 88.3%, respectively. The extract was dried, reconstituted with ethyl acetate (100 µl), and applied to a TLC plate (Fisher Scientific, Pittsburgh, PA). Cortisol and cortisone were separated in the solvent system chloroform/ethanol (95:5, vol/vol), steroids were visualized under UV light, scraped off, and extracted with ethyl acetate. The solvent was dried, scintillation fluid added, and the radioactivity counted in a liquid scintillation counter. 11ß-HSD1 reductase activity was expressed as the percentage of cortisol formed from cortisone.

PCR and quantitative real-time PCR (QT-RT-PCR)
To measure 11ß-HSD1 mRNA level in response to cortisol and IL-1ß treatment, QT-RT-PCR analysis was carried out using an iCycler (Bio-Rad Laboratories Inc., Hercules, CA).

RNase-free DNase (Invitrogen, Carlsbad, CA) treatment of the extracted total RNA was performed before RT-PCR. DNase-treated RNA (1.0 µg) was reverse-transcribed with oligo(deoxythymidine)15 primer using the Superscript II kit (Life Technologies). Some RNA samples with no reverse transcriptase enzyme were used as control to further check the absence of genomic DNA contamination in the samples. Reverse-transcription product (cDNA) was diluted for subsequent PCR and QT-RT-PCR. Paired oligonucleotide primers for amplification of human 11ß-HSD1 were designed using Primer Designer (Scientific and Educational Software) against the sequence downloaded from GenBank with the accession no. NM005525. The primer sequences are as follows: forward, 5'-CTCAACCACATCACCAACAC-3'; reverse, 5'-TCTGATGGAGGAGAAGAACC-3'. To control sampling errors, PCR for the housekeeping gene ß-actin was routinely performed on each sample. The primer sequences for human ß-actin were as follows: forward, 5'-GGGAAATCGTGCGTGACATTAAG-3'; reverse, 5'-TGTGTTGGCGTACAGGTCTTTG-3'. To exclude any DNA interference, primers were designed spanning at least one intron.

QT-RT-PCR solution consisted of 2.0 µl diluted RT-PCR product, 0.2 µM of each of the paired primers, and 10 µl real-time PCR SYBR Green Master Mix (Toyobo, Osaka, Japan). QT-RT-PCR conditions were optimized according to preliminary experiments. The annealing temperature was set at 61 C, and amplification cycles were set at 45 cycles. The temperature range to detect the melting temperature of the PCR product was set from 60–95 C. The specificity of the primers was verified by examining the melting curve as well as subsequent sequencing of the QT-RT-PCR products. The cycles of QT-RT-PCR needed (cycle threshold) to reach the fluorescence threshold value were used to determine mRNA level. The absolute mRNA level in each sample was calculated according to a standard curve set up using serial dilutions of known amounts of specific templates against corresponding cycle threshold values. Then the ratio of the target gene over the reference gene in each sample was obtained to the normalized expression of the target gene.

Cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2) mRNA levels in response to cortisol treatment in cultured chorionic trophoblast cells were measured in the same way as 11ß-HSD1 with specific primers designed, respectively, for cPLA2 (forward, 5'-ATGGCCTTGGTGAGTGATTC-3'; reverse, 5'-TCAGGATCTGCTACAGCTGC-3') and COX2 (forward, 5'- TGTGCAACACTTGAGTGGCT-3'; reverse, 5'- ACTTTCTGTACTGCGGGTGG-3') against the sequences downloaded from GenBank (accession no. XM_051896 and NM_000963).

Cloning of 11ß-HSD1 promoter into pGL3 enhancer vector containing luciferase reporter gene
The cloned sequence of 1.178 kb in length upstream to the translation start site of the human 11ß-HSD1 gene (GenBank no. AL031316) was kindly provided by Dr. Kaiping Yang (University of Western Ontario, London, Ontario, Canada). The following paired oligonucleotide primers were designed against the cloned sequence: primer 1, 5'-GGGGTACCTTTTTCCCCGCTCTACTGATAACT-3'; primer 2, 5'-TTCTCGAGCCGACAGGGAGCTGGCCTGAAGACT-3'. Restriction sites (bold letters) for KpnI and XhoI were designed into the primers so that the amplified cDNA carried restriction sites at both ends. Amplification of the 1.178-kb 11ß-HSD1 gene promoter was carried out with PCR using the cloned sequence of 11ß-HSD1 gene promoter as template. PCR cycles were as follows: first denatured in 94 C for 4 min, followed by 30 cycles at 94 C for 55sec, 50 C for 55sec, and 72 C for 2 min, and concluded with one cycle at 72 C for 10 min. The PCR product and pGL3 enhancer vector containing enhanced firefly luciferase reporter gene (Promega, Madison, WI) were then cut with KpnI and XhoI to produce cohesive ends, which was then ligated. Subcloning of constructed plasmid was conducted, and the cloned sequence was verified by both examination of the insert size released by KpnI and XhoI digestion and sequence analysis of the insert.

Transfection of WISH cells with pGL3 enhancer vector and assay of the reporter gene activity
WISH cells were grown on 24-well plates in DMEM containing 10% FCS. At about 90% confluence, the cells were washed with PBS, and the medium was changed to antibiotic-free DMEM containing 10% FCS. Then the cells were cotransfected with 0.5 µg/well of the constructed pGL3 enhancer vector and 0.05 µg/well of internal control vector phRL expressing Renilla luciferase using lipofectamine 2000 (Invitrogen) in Opti-MEM (Life Technologies). Twelve hours after transfection, the culture medium was changed to serum-free medium, and the cells were treated with cortisol (1 µM) in the presence and absence of either RU486 (1 µM) or IL-1ß (10 ng/ml) for 24 h. Culture media were then discarded, and the cells were washed with PBS and lysed with lysis buffer (Promega). The cellular debris was pelleted by centrifugation (12,000 x g for 1 min at 4 C). The supernatant was used for luciferase activity assay using the Dual-Luciferase assay system (Promega) with a luminometer (Berthold Technologies, Lumat LB 9507). The ratio of 11ß-HSD1 promoter-driven firefly luciferase activity against Renilla luciferase activity was obtained to correct differential transfection efficiency in each well and to express the promoter activity.

Statistical analysis
All data are reported as mean ± SEM. Paired Student’s t test or two-way ANOVA followed by the Student-Newman-Keuls test was used to assess significant differences between groups. Significance was set at P < 0.05. The values for n refer to the number of experiments performed with different cell preparations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of cortisol and IL-1ß on 11ß-HSD1, cPLA2, and COX-2 mRNA expression and 11ß-HSD1 reductase activity in cultured chorionic trophoblast cells
The melting curve of QT-RT-PCR showed a single sharp peak of melting temperature value for both 11ß-HSD1 and ß-actin PCR products. Sequence analysis of 11ß-HSD1 and ß-actin PCR products showed full alignment with the corresponding sequences of human 11ß-HSD1 and ß-actin genes in GenBank (data not shown). Cortisol significantly increased 11ß-HSD1 mRNA expression at 0.1 and 1 µM (P < 0.05) but not at 0.001 and 0.01 µM in cultured chorionic trophoblast cells (Fig. 1AGo). RU486 (1 µM), a glucocorticoid receptor antagonist, significantly inhibited the induction of 11ß-HSD1 mRNA expression by cortisol (1 µM) (Fig. 1BGo). IL-1ß (10 ng/ml) treatment alone significantly increased 11ß-HSD1 mRNA expression in cultured chorionic trophoblast cells (Fig. 2AGo). However, a significantly enhanced 11ß-HSD1 mRNA expression was observed with the combination of cortisol (1 µM) and IL-1ß (10 ng/ml) compared with cortisol alone (P < 0.05) (Fig. 2AGo). Likewise, cortisol or IL-1ß treatment alone also increased 11ß-HSD1 reductase activity in cultured chorionic trophoblast cells (P < 0.01) (Fig. 2BGo). Again, a further enhanced 11ß-HSD1 reductase activity was also found with combined treatment of cortisol and IL-1ß as compared with either cortisol or IL-1ß alone (P < 0.01) (Fig. 2BGo). To explore the physiological significance of 11ß-HSD1 induction in the chorion, we studied the effect of cortisol on cPLA2 and COX-2 expression, the two key enzymes involved in prostaglandin synthesis. In response to cortisol treatment (1 µM for 24 h), both cPLA2 and COX2 mRNA levels were significantly increased in cultured chorionic trophoblast cells (P < 0.05) (Fig. 3Go).


Figure 1
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  		FIG. 1. A, Effect of cortisol (0.001–1 µM) on 11ß-HSD1 mRNA expression in cultured human chorionic trophoblast cells. *, P < 0.05 compared with control group; n = 4 separate experiments. B, Effect of glucocorticoid receptor antagonist RU486 (RU) (1 µM) on the induction of 11ß-HSD1 mRNA expression by cortisol (F) (1 µM) in cultured human chorionic trophoblast cells. *, P < 0.05 compared with control group; #, P < 0.05 compared with cortisol group; n = 3 separate experiments.

 

Figure 2
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  		FIG. 2. Effect of combined treatment with cortisol (F) (1 µM) and IL-1ß (10 ng/ml) on 11ß-HSD1 mRNA expression (n = 4 separate experiments) (A) and 11ß-HSD1 reductase activity (n = 5 separate experiments) (B) in cultured human chorionic trophoblast cells. *, P < 0.05; **, P < 0.01 compared with control group; #, P < 0.05; ##, P < 0.01 compared with cortisol group.

 

Figure 3
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  		FIG. 3. Effect of cortisol (1 µM) on cPLA2 (n = 5 separate experiments) (A) and COX-2 (n = 4 separate experiments) (B) mRNA expression in cultured human chorionic trophoblast cells. *, P < 0.05 compared with control group.

 
Effect of cortisol and IL-1ß on 11ß-HSD1 promoter-driven reporter gene expression in WISH cells
There was high basal 11ß-HSD1 promoter activity in WISH cells transfected with pGL3 vector carrying a 1.178-kb fragment of 11ß-HSD1 promoter (data not shown), which was reflected as reporter gene luciferase activity. Upon stimulation by cortisol (1 µM) or IL-ß (10ng/ml), luciferase activities, normalized by internal transfection efficiency control, were significantly increased (P < 0.05) (Fig. 4AGo). Consistent with the induction of 11ß-HSD1 mRNA expression, an enhanced 11ß-HSD1 promoter activity was also observed with combined treatment of cortisol and IL-1ß compared with cortisol alone (P < 0.05) (Fig. 4AGo). The stimulation of 11ß-HSD1 promoter activity by cortisol was also blocked by glucocorticoid receptor antagonist RU486 (1 µM; P < 0.05) (Fig. 4BGo).


Figure 4
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  		FIG. 4. A, Effect of combined treatment with cortisol (F) (1 µM) and IL-1ß (10 ng/ml) on 11ß-HSD1 promoter activity in amnion-derived WISH cells. *, P < 0.05, compared with control group; #, P < 0.05 compared with cortisol alone; n = 6 separate experiments. B, Effect of glucocorticoid receptor antagonist RU486 (RU) (1 µM) on the induction of 11ß-HSD1 promoter activity by cortisol (F) (1 µM) in amnion-derived WISH cells. *, P < 0.05 vs. control group; #, P < 0.05 compared with cortisol alone; n = 6 separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we present evidence that expression of 11ß-HSD1 in chorionic trophoblast cells is enhanced by cotreatment with cortisol and IL-1ß, which complements our earlier study showing enhancement of 11ß-HSD1 expression by combined glucocorticoids and proinflammatory cytokines in amniotic cells (13). This suggests a likely consistent regulatory pattern of 11ß-HSD1 expression by glucocorticoids and proinflammatory cytokines in both layers of human fetal membranes. Because chorion is the most abundant site of 11ß-HSD1 expression within intrauterine tissues (4), it may be more important in terms of local cortisol production during pregnancy.

Although maternal adrenal glands secrete increasing amount of glucocorticoids with gestational age (18), most of them are prevented from entering the fetus by the action of placental 11ß-HSD2, which converts cortisol into biologically inactive cortisone in human (19). Moreover dehydroepiandrosterone sulfate rather than glucocorticoids comprises the major products of human fetal adrenal glands (20). Therefore, cortisol derived from fetal membranes during human pregnancy could be crucial for fetal organ maturation. Early work by Murphy (6) showed that the cortisol to cortisone ratio of amniotic fluid is considerably higher than that of cord serum, indicating that the chorionic membrane plays a role as an important extraadrenal source of fetal cortisol in human amniotic fluid. Moreover, Carson et al. (21) demonstrated that isotope-labeled cortisol injected into amniotic fluid could be traced both in the fetal membranes and several fetal tissues such as lung, liver, and adrenal glands, further strongly supporting the proposed role of 11ß-HSD1 in the fetal membranes in fetal organ maturation. And the speculation is further strengthened by observations of relatively normal lung development seen in some anencephalic fetuses despite poor adrenal function (22) and of impaired fetal lung maturation in 11ß-HSD1 knockout mice (23). Because cortisol levels rise considerably both in the maternal plasma and amniotic fluids in late gestation and during labor (18, 24), the expression of 11ß-HSD1 in fetal membranes could be under sustained stimulation by glucocorticoids. Because 11ß-HSD1 manifests mainly a reductase activity in vivo regenerating cortisol from cortisone (25), stimulation of 11ß-HSD1 expression by cortisol may thus elicit a positive feedback on 11ß-HSD1 expression. As demonstrated in this study, the induction of 11ß-HSD1 expression required relatively high cortisol concentration (0.1 and 1 µM); thus, we speculate that this positive feedback would not be triggered unless cortisol levels reach a certain high level in vivo. It has been reported that micromolar range of cortisol concentrations could be achieved in maternal and fetal plasma as well as in amniotic fluid at late gestation and during labor (6, 18, 24). The effective doses of cortisol observed in this study fell in the physiological range of cortisol at late gestation and during labor.

It has been reported in the literature that glucocorticoids and proinflammatory cytokines increase 11ß-HSD1 expression in a number of cell types including adipocytes, smooth muscle cells, fibroblasts, and osteoblasts (26). Of interest, we found in this study that the positive feedback of glucocorticoids on induction of 11ß-HSD1 mRNA expression is further enhanced by the proinflammatory cytokine IL-1ß. It has been very well documented in the literature that infection is the leading cause of preterm delivery (27) with chorioamnionitis as the most common type of infection in preterm labor, especially in preterm rupture of membranes (14, 15, 28). Infection of membranes will then result in the activation of macrophages by lipopolysaccharides in these tissues. Consequently, activated macrophages will release proinflammatory cytokines such as IL-1ß and activate local stromal cells, which will further release proinflammatory cytokines (14, 15). Both IL-1ß and lipopolysaccharides have been shown to induce preterm labor by stimulating prostaglandin output in the intrauterine tissues (14, 29). In addition, glucocorticoids have also been shown to exert potent stimulation on prostaglandin production in amnion cells (10, 11). Here we found that cortisol stimulated cPLA2 and COX-2 mRNA expression, the two key enzymes involved in prostaglandin synthesis. Based upon these findings, we speculate that more prostaglandins and biologically active glucocorticoids will be locally available in the fetal membranes or in the amniotic fluid as a result of activation of 11ß-HSD1 expression in the presence of chorioamnionitis. This could, at least in part, be the mechanism underlying infection-induced preterm labor. On the other hand, accumulation of cortisol in amniotic fluid due to 11ß-HSD1 activation by cortisol and IL-ß may also make relatively more cortisol available for the fetal organ maturation in case of infection-induced preterm labor. Again, clinical and experimental observations show that fetal exposure to inflammation can induce lung maturation (30). Thus, the activation of 11ß-HSD1 expression by infection in the fetal membranes might have a dual role in terms of initiation of labor and fetal organ maturation.

It is well recognized that the major mechanism accounting for glucocorticoid’s antiinflammatory action is via suppression of the response of the immune system to proinflammatory cytokines. In the absence of glucocorticoids, proinflammatory cytokines cause phosphorylation of inhibitory-{kappa}B{alpha} (I{kappa}B{alpha}), which holds nuclear factor-{kappa}B (NF{kappa}B) in its inactive form (31). Phosphorylation of I{kappa}B{alpha} triggers the release of NF{kappa}B from the complex (31), and released NF{kappa}B then migrates into the nucleus, where it regulates gene expression related to inflammation (31). Glucocorticoids effectively suppress this immune cell activation by either inducing the expression of I{kappa}B{alpha} (31) or interfering directly the function of NF{kappa}B (31). However, we found the combination of glucocorticoids and proinflammatory cytokines further induced the expression of 11ß-HSD1 mRNA in both amnion and chorion, which is in obvious contrast to the opposing effects of glucocorticoids and proinflammatory cytokines at inflammatory sites in non-intrauterine tissues. This unique interaction of glucocorticoids and proinflammatory cytokines on 11ß-HSD1 expression in the fetal membranes may play a crucial role in both initiation of labor and fetal organ maturation.

Sequence analysis of human 11ß-HSD1 gene revealed several transcription factor binding sites in the promoter region, including putative glucocorticoid response elements (GRE), an estrogen-like response element, NF{kappa}B-binding sites, and consensus CAAT boxes (32). Previous studies have shown that estrogen regulates baboon 11ß-HSD1 gene expression via its promoter in JEG-3 cells (33) and that the transcription factor C/EBP (CAAT binding protein) is a potent activator of 11ß-HSD1 in hepatoma cells (34). We demonstrate for the first time that cortisol activates human 11ß-HSD1 promoter activity, which is further enhanced by IL-1ß. Both glucocorticoids and IL-1ß may affect gene expressions via up-regulating C/EBP expression in different cell types (34, 35, 36). Therefore, it is likely that the induction of 11ß-HSD1 expression by both cortisol and IL-ß is mediated through C/EBP. However, other regulatory pathways such as a NF{kappa}B-binding site for IL-1ß and GRE for glucocorticoids may be involved in the regulation of 11ß-HSD1 mRNA expression.

The effects of glucocorticoids are normally mediated through intracellular glucocorticoid receptor (GR). Using immunocytochemistry, Sun et al. (37) have demonstrated that nuclear GR was found in amnion epithelium, mesenchyme, and the chorion laeve. We demonstrated that the induction of 11ß-HSD1 expression by cortisol was blocked by cotreatment with the receptor antagonist RU486, which is capable of blocking both GR and progesterone receptor (38). Progesterone did not affect the expression of 11ß-HSD1 in the chorion trophoblast but has a potent inhibitory effect on 11ß-HSD2 expression in placenta trophoblast (39). We have also previously found that trilostane, a potent inhibitor of endogenous progesterone synthesis, did not affect the expression of 11ß-HSD1 in amnion fibroblasts. Therefore, inhibition by RU486 of cortisol-induced 11ß-HSD1 expression is very likely through GR rather than progesterone receptor. However, whether the induction of 11ß-HSD1 by glucocorticoids in the fetal membranes involves the interaction of GR and the putative GRE or other transcription factor binding elements of 11ß-HSD1 gene awaits further study.

In conclusion, these results suggest that more biologically active glucocorticoids can be generated from the fetal membranes in the presence of infection, especially when concurrent glucocorticoid treatment is given, which may consequently feed forward to up-regulate prostaglandin synthesis within fetal membranes.


   Footnotes
 
This work was supported by the National Natural Science Foundation of China (30470655 and 30570680) and Ministry of Education of China (20050246015).

All the authors have nothing to declare.

First Published Online February 9, 2006

1 W.L. and L.G. contributed equally to this work. Back

Abbreviations: COX-2, Cyclooxygenase 2; cPLA2, cytosolic phospholipase A2; FCS, fetal calf serum; GR, glucocorticoid receptor; GRE, glucocorticoid response elements; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; I{kappa}B{alpha}, inhibitory-{kappa}B{alpha}; NF{kappa}B, nuclear factor-{kappa}B; QT-RT-PCR, quantitative real-time PCR; TLC, thin-layer chromatography.

Received December 20, 2005.

Accepted for publication January 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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