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Endocrinology, doi:10.1210/en.2003-0780
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Endocrinology Vol. 144, No. 12 5568-5577
Copyright © 2003 by The Endocrine Society

Enhancement of Glucocorticoid-Induced 11ß-Hydroxysteroid Dehydrogenase Type 1 Expression by Proinflammatory Cytokines in Cultured Human Amnion Fibroblasts

Kang Sun and Leslie Myatt

Department of Obstetrics and Gynecology (L.M.), University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267; and Department of Physiology (K.S.), Second Military Medical University, Shanghai 200433, China

Address all correspondence and requests for reprints to: Dr. Kang Sun, Department of Physiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. E-mail: sunkang2000{at}yahoo.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids and proinflammatory cytokines may be involved in parturition by stimulation of prostaglandin production in the fetal membranes. The actions of glucocorticoids on the fetal membranes are amplified by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts biologically inactive cortisone into active cortisol. Whether glucocorticoids and proinflammatory cytokines regulate the expression of 11ß-HSD1 in the major prostaglandin-producing tissue, amnion, thus further increasing prostaglandin production, is not known. In this study, we found that term amnion fibroblasts had higher 11ß-HSD1 mRNA and activity per cell than amnion epithelial cells. Both isoforms of glucocorticoid receptor ({alpha} and ß) were expressed in amnion fibroblasts and epithelial cells. Quantitative real-time PCR showed that dexamethasone (0.01–1 µM) dose-dependently induced 11ß-HSD1 mRNA expression only in amnion fibroblasts but not in amnion epithelial cells. The induction of 11ß-HSD1 mRNA expression by dexamethasone was blocked by glucocorticoid receptor antagonist RU486. Although only a modest increase or no change in 11ß-HSD1 mRNA expression and activity was observed with IL-1ß (10 ng/ml) or TNF{alpha} (10 ng/ml) treatment, respectively, in amnion fibroblasts, combination of dexamethasone with either IL-1ß or TNF{alpha} significantly enhanced the induction of 11ß-HSD1 mRNA expression and activity, as compared with dexamethasone treatment alone. With prior induction of 11ß-HSD1 expression by dexamethasone, cortisone caused more prostaglandin E2 production in the amnion fibroblast. This study suggests that glucocorticoids can positively induce 11ß-HSD1 expression in amnion fibroblasts, an effect further strengthened by proinflammatory cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS AND PROINFLAMMATORY cytokines may play important roles in normal term and preterm labor (1, 2). In the sheep at term, glucocorticoids derived from the fetus trigger parturition by stimulating P-450 17{alpha} hydroxylase as well as prostaglandin (PG)H synthase type II (PGHS-2) or cyclooxygenase 2 expression in intrauterine tissues (1). This leads to progesterone withdrawal and increased estrogen and PG production before labor in the sheep, which ultimately leads to labor. Glucocorticoids are also among a variety of hormones involved in human parturition. Despite a lack of P-450 17{alpha} hydroxylase in human placenta, glucocorticoids nevertheless stimulate the production of PGs in intrauterine tissues (3). In addition, glucocorticoids paradoxically induce the expression of CRH in human placenta (4). The concentration of CRH in maternal circulation correlates to the timing of labor (5).

The actions of glucocorticoids on intrauterine tissues are regulated by 11ß-hydroxysteroid dehydrogenase (11ß-HSD), of which at least two isoforms have been recognized so far (6). Previous work by Sun and Challis (7) has shown that 11ß-HSD1 is mainly localized to human fetal membranes converting biologically inactive cortisone into active cortisol, whereas the expression of 11ß-HSD2 predominates in human placenta converting cortisol into cortisone (8). Placental 11ß-HSD2 provides the fetus with an effective barrier against maternal glucocorticoids during pregnancy (9). However, the function of 11ß-HSD1 in the fetal membranes is not very well understood. Sequence analysis of the 11ß-HSD1 gene revealed a putative glucocorticoid response element (GRE) in the promoter (10). Recent work by Sun et al. (11) showed that glucocorticoids up-regulated 11ß-HSD1 expression in the chorion, thus potentiating PG output by glucocorticoids. Whether this phenomenon also holds true in the major PG-producing tissue, amnion, is not known.

Proinflammatory cytokines such as IL-1ß and TNF{alpha} are key factors in infection-induced preterm labor (2). They stimulate PG synthesis in the fetal membranes as well as increase the production of estrogen and CRH in the placenta (12, 13). It has been reported that IL-1ß and TNF{alpha} induced 11ß-HSD1 expression in adipose tissue, ovary, and osteoblast (14, 15, 16); however, it is not known whether these cytokines modulate the expression of 11ß-HSD1 in the fetal membranes. The actions of glucocorticoids and proinflammatory cytokines usually oppose each other at sites of inflammation (17, 18), but both glucocorticoids and proinflammatory cytokines stimulate PG production in the fetal membranes (19, 20, 21). Therefore, we determined the interaction of glucocorticoids and proinflammatory cytokines in the regulation of 11ß-HSD1 expression in the fetal membranes.

PG synthesis increases in amnion tissue at term (22), and glucocorticoids were reported to stimulate PG production by human amnion cells in vitro (23). However, this effect appears to be restricted to stimulation of PGHS-2 and PGE2 production from amnion fibroblasts but not amnion epithelial cells (21, 24, 25). Thus, we performed studies in separate cultures of human amnion epithelial cells and fibroblasts to investigate the regulation of 11ß-HSD1 expression by glucocorticoids and proinflammatory cytokines in these distinct cell types.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Amnion epithelial and fibroblast cell preparations
Fetal membranes were collected at term from elective cesarean section patients not in labor under a protocol approved by the University of Cincinnati Institutional Review Board. Amnion was peeled off the chorion and washed three times in cold PBS (pH 7.5). For amnion epithelial cell preparation, amnion tissue was digested with 0.125% trypsin (Sigma, St. Louis, MO) and 0.02% DNase (Sigma) twice for 30 min at 37 C. The digestion media were collected, and the remaining amnion tissue was washed vigorously with PBS three times to wash residual epithelial cells off the amnion tissue. The wash solution was then combined with the previous trypsin digestion media. For the preparation of amnion fibroblasts, the remaining amnion tissue was further digested with 0.1% collagenase (Roche, Indianapolis, IN) at 37 C for 1 h. The digestion medium was then collected. Both trypsin (epithelium) and collagenase (fibroblast) digestion media were centrifuged at 2300 rpm for 15 min. Cell pellets were collected and re suspended in DMEM without phenol red (Sigma). Re suspended cells were loaded onto pre-prepared discontinuous Percoll (Sigma) gradients (5, 20, 40, and 60%), respectively, and the gradients were centrifuged at 2500 rpm for 20 min. A single band of cells around 20% Percoll concentration was collected and diluted with DMEM containing 10% fetal calf serum (FCS, Atlas, Fort Collins, CO) and antibiotic-antimycotic (Life Technologies, Inc., Grand Island, NY) to a density of 106 cells/ml. Cells (3 x 106) were plated in each well of a six-well plate. Cell culture was maintained at 37 C with a water saturated atmosphere of 5% CO2 in air.

Immunocytochemical staining for vimentin, cytokeratin, and 11ß-HSD1
To identify the cell types that were obtained after trypsin and collagenase digestion, immunocytochemical staining for cytokeratin (epithelial cell marker) and vimentin (mesenchymal cell marker) was carried out on cells cultured for 3 d on chamber slides using the avidin biotin peroxidase method (Vector ABC, Vector Laboratories, Burlingame, CA), as described previously (26). The cells were fixed with 4% paraformaldehyde. Before applying primary antibodies, endogenous peroxidase activity was quenched in 0.3% H2O2, and then the cells were incubated with normal blocking serum. The monoclonal vimentin antibody (Sigma) at 1:3000 dilution and cytokeratin antibodies (Sigma) at 1:1000 dilution were applied respectively as primary antibodies. To investigate whether 11ß-HSD1 protein was expressed in amnion fibroblasts and epithelial cells, a polyclonal primary antibody raised in goat was used at a dilution of 1:1000 (Santa Cruz Laboratories, Santa Cruz, CA). After incubation with the primary antibodies, appropriate secondary antibodies were then applied. After the cells were washed, cells were incubated with Vectastain ABC reagent. The color reactions were developed using either 3-amino-9-ethyl carbazole (red color) for cytokeratin and vimentin or diaminobenzidine tetrahydrochloride (brown color) for 11ß-HSD1. Cells were counterstained with Carazzi’s hematoxylin. To test the specificity of immunocytochemical staining, cells were also stained with preimmune serum or PBS instead of primary antibodies and then the same procedures as described above were followed.

Cell treatments and RNA extraction
On the third day of culture, amnion fibroblasts and epithelial cells were washed with PBS and culture medium was changed to FCS-free DMEM. Steroid hormones and cytokines were added into the culture medium to achieve final concentrations of 0.01–1.0 µM for dexamethasone (Sigma), 1.0 µM for cortisol (Sigma) and cortisone (Sigma), and 10 ng/ml for IL-1ß (Biosource, Camarillo, CA) and TNF{alpha} (Biosource). Dexamethasone with or without RU486 (1 µM, Sigma), IL-1ß (10 ng/ml), or TNF{alpha} (10 ng/ml) was also used to treat the cells. 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 into cell lysis buffer (supplied with RNeasy kit, QIAGEN, Valencia, CA). Subsequent extraction and purification of total RNA from the cells for analysis with PCR was conducted using RNeasy kit (QIAGEN) according to the protocol provided by the company. The extracted RNA was then quantified spectrophotometrically at 260 nm. The integrity of the extracted RNA was assessed by agarose-formaldehyde gel electrophoresis.

To evaluate the prior induction of 11ß-HSD1 expression by dexamethasone on PGE2 production upon cortisone treatment, amnion fibroblasts were treated with dexamethasone (1 µM) for 24 h and washed with PBS, and the cells were then treated with or without cortisone (1 µM) for another 24 h. Culture media were collected for PGE2 RIA.

11ß-HSD1 activity assay
To measure 11ß-HSD1 activity in cultured amnion fibroblasts and epithelial cells, cells were washed with PBS and culture medium was changed to FCS-free medium on the third day of culture. Cortisone was then added into the culture media to achieve a final concentration of 1 µM. Incubation was carried out with cortisone for 2, 4, 12, and 24 h. At these time points, the media were collected for cortisol RIA. For the measurement of 11ß-HSD1 activity in glucocorticoid and proinflammatory cytokine-treated amnion cells, cells were washed with PBS 24 h after the treatments and the culture medium was changed to FCS-free medium. Cortisone was then added into the culture media to achieve a final concentration of 1 µM. Incubation was carried out with cortisone for 12 h. This incubation time was chosen from a preliminary study to maintain 11ß-HSD1 activity in a linear range. Media were then collected for cortisol RIA. There was 0.5% cross-reaction of the cortisol antibody with cortisone in RIA. 11ß-HSD1 activity was evaluated according to cortisol levels measured in the sample.

11ß-HSD1 activity kinetic analysis
Kinetic analysis of 11ß-HSD1 reductase activity was performed using a fixed number of cells (3 x 106 cells) and reaction time (4 h) for both amnion epithelial and fibroblast cells but with varying amounts of cortisone substrate (0.5, 1, 2, 5, 10 µM). The conditions were chosen so that the initial velocity was linear with reaction time. Incubation media were collected for cortisol RIA. In all cases, the conversion in the blank well (no cells) was subtracted from that in the experimental wells before analysis. Michaelis constant (Km) values were calculated from Lineweaver-Burk plot [1/vi vs. 1/(s)] using the slope and y intercept.

Relative PCR and quantitative real-time PCR (QT-RT-PCR)
To measure 11ß-HSD1 mRNA levels, glucocorticoid receptor (GR){alpha} and GRß mRNA levels in the fibroblasts, and epithelial cells, relative PCR was performed on RNA extracted from the cultured fibroblasts and epithelial cells. To measure 11ß-HSD1 mRNA levels in response to glucocorticoid and proinflammatory cytokine treatments, QT-RT-PCR analysis was carried out using Smart Cycler (Cepheid, Sunnyvale, CA).

RNase-free DNase (Invitrogen, Carlsbad, CA) treatment of the extracted total RNA was performed before PCR. DNase-treated RNA (1.0 µg) was reverse transcribed with oligo(dT)12–18 primer using Superscript II kit (Invitrogen). Some RNA samples with no reverse transcriptase enzyme were used as controls to further check the absence of genomic DNA contamination in the samples. Reverse-transcription products (cDNA) were diluted for subsequent relative PCR and QT-RT-PCR. Relative PCR conditions were optimized according to preliminary experiments to achieve linear relationship between initial RNA concentration and PCR product. Paired oligonucleotide primers for amplification of human 11ß-HSD1 were designed using Primer Designer (Scientific and Educational Software, Durham, NC) against the sequence downloaded from GenBank. The primer sequences were as follows: forward 5'-GGAGCAGCCTCAGCACACTA3-' and reverse 5'-GGCAAGGCAGCTACAGTCAG3-'. Annealing temperature and PCR amplification cycles were set at 61 C and 35 cycles, respectively. GR{alpha} and GRß cDNAs were amplified using specific antisense primers that shared the same sense primer with sequences as follows: 5'-GGCAATACCAGGTTTCAGGAACTTACA-3' (GR{alpha}/ß forward), 5'-ATTTCACCATCTACTCTCCCATCACTG-3' (GR{alpha} reverse), and 5'-ATTATCCAGCACTTCATAGACACAAAT-3' (GRß reverse) (27). Annealing temperature was set at 60 C for both GR{alpha} and GRß. Thirty-six and 40 cycles were performed for GR{alpha} and GRß, respectively. 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' and reverse 5'-TGTGTTGGCGTACAGGTCTTTG-3'. All the PCR products were analyzed by electrophoresis in 1% agarose gel and the signals for all PCR product bands were measured with densitometer, and the ratio to ß-actin was calculated.

QT-RT-PCR solution consisted of 2.0 µl diluted cDNA product, 0.2 µM of each paired primer, 2.0 mM Mg2+, 100 µM deoxynucleotide triphosphates, 2 U Taq DNA polymerase, and 1x PCR buffer. SYBR green (BMA, Rockland, ME) was used as detection dye. QT-RT-PCR conditions were optimized according to a preliminary experiment. 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 C to 95 C. The same paired oligonucleotide primers for relative PCR amplification of human 11ß-HSD1 and ß-actin were used for QT-RT-PCR. The cycle thresholds of QT-RT-PCR reaching the fluorescence threshold value were used to determine mRNA level. To control sampling errors, the ratio of cycle threshold for 11ß-HSD1 and house keeping gene, ß-actin, was obtained to quantify the relative 11ß-HSD1 mRNA expression level. The specificity of the primers was verified by examining the melting curve as well as subsequent sequencing of the QT-RT-PCR products.

For sequencing, PCR products was cloned using TOPO cloning kit (Invitrogen). The mixture of PCR product and pCR4-TOPO vector was transformed into TOPO10 Escherichia coli cells and grown on a selective plate containing ampicillin overnight at 37 C. Positive colonies were picked and further cultured in Luria-Bertani medium containing ampicillin overnight at 37 C. Plasmid DNA was extracted from the harvested bacteria with QIAprep miniprep kit (QIAGEN) and was sent for sequencing at the DNA Core of the University of Cincinnati.

Statistical analysis
All data are reported as mean ± SEM. Student’s t test or one-way ANOVA followed by the Student-Newman-Keuls test was used 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 cell preparations from different patients.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of cultured human amnion fibroblasts and epithelial cells
Morphological examination of cells isolated with trypsin showed round-shaped epithelial cells that began to divide and form clusters around 24 h after plating. Immunocytochemical staining of the cells with the epithelial cell marker, cytokeratin, showed that more than 99% of the cells were positive for cytokeratin, suggesting an epithelial origin. Morphological examination of cells dispersed using collagenase showed that these cells were irregular in shape at the time of plating and took on a spindle appearance and began branching after overnight incubation. Immunocytochemical staining of these cells with the mesenchymal cell marker, vimentin, showed that more than 90% of the cells were positive, indicating the mesenchymal origin (Fig. 1Go). To examine the specificity of the staining, a mixed cell preparation was also made. Immunocytochemical staining for vimentin showed that only spindle-shaped fibroblasts but not round epithelial cells were stained positive (data not shown). In addition, cells stained with normal serum or PBS instead of specific primary antibody showed no obvious staining (data not shown).



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  		FIG. 1. Cultured amnion fibroblast (A and C) and epithelial cells (B and D) stained for mesenchymal cell marker vimentin (A and D) and epithelial cell marker cytokeratin (B and C). Mixed amnion fibroblast and epithelial cell culture stained for 11ß-HSD1 (E) and negative control (F). Red color indicates positive staining for vimentin and cytokeratin. Brown color indicates positive staining for 11ß-HSD1. Magnification, x100 (C and D), x200 (A, B, E, and F).

 
11ß-HSD1 expression and activity in cultured amnion fibroblasts and epithelial cells
Immunocytochemical staining showed that both epithelial cells and fibroblasts were positive for 11ß-HSD1, and the staining was localized in the cytoplasm of both cell types (Fig. 1EGo). No obvious staining was found in cells stained with preimmune serum instead of specific antibody (Fig. 1FGo). Relative PCR showed that 11ß-HSD1 mRNA was expressed in both cell types (Fig. 2AGo), but amnion fibroblasts expressed a significantly higher level of 11ß-HSD1 mRNA than amnion epithelial cells (Fig. 2BGo). 11ß-HSD1 activity assay showed that the conversion from cortisone to cortisol increased progressively with time both in amnion fibroblast and epithelial cell culture. Under the same conditions of cell density, substrate concentration, and incubation time, conversion of cortisone to cortisol in the amnion fibroblasts was significantly higher than in the amnion epithelial cells at 4, 12, and 24 h (Fig. 3Go). Kinetic analysis of 11ß-HSD1 activity showed the Km values for cortisone were 2.08 and 1.98 µM, respectively, for epithelial and fibroblast cells (Fig. 4Go), which compare favorably with previously published values for 11ß-HSD1 (6).



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  		FIG. 2. Expression of 11ß-HSD1 mRNA in amnion fibroblast and epithelial cells. Top, Agarose gel electrophoresis of the PCR products. Bottom, Relative mRNA expression (mean ± SEM). **, P < 0.01 vs. epithelial cell, n = 4 separate experiments.

 


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  		FIG. 3. Time course of 11ß-HSD1 activity in cultured amnion fibroblast and epithelial cells incubated with cortisone (1 µM). *, P < 0.05; **, P < 0.01 vs. epithelial cells at respective time points; mean ± SEM, n = 4 separate experiments.

 


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  		FIG. 4. Lineweaver-Burk plots for determination of kinetic parameters. Data from representative experiment for conversion of cortisone to cortisol in amnion epithelial cells (A) and fibroblast cells (B). Km values are average of three experiments.

 
Expression of GR{alpha} and GRß mRNA in cultured amnion fibroblast and epithelial cells
A relatively high level of GR{alpha} mRNA was expressed in both amnion fibroblast and epithelial cells, but there was no significant difference in expression levels between these two cell types (Fig. 5Go). A low level of GRß mRNA was detected in both amnion fibroblasts and epithelial cells. In some of the amnion fibroblast and epithelial cells, the expression level of GRß mRNA was too low to be detected (Fig. 5Go).



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  		FIG. 5. Expression of GR{alpha} and GRß mRNA in amnion fibroblast and epithelial cells. Top, Agarose gel electrophoresis of the PCR products. Bottom, Relative mRNA expression (mean ± SEM). n = 6 separate experiments.

 
Effects of glucocorticoids on the expression of 11ß-HSD1 mRNA in cultured amnion fibroblast and epithelial 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 100% alignment with the corresponding sequences of human 11ß-HSD1 and ß-actin genes in the gene bank (data not shown). Dexamethasone (0.01–1 µM) significantly increased 11ß-HSD1 mRNA expression in a dose-dependent manner only in amnion fibroblasts but not in amnion epithelial cells, whereas expression of ß-actin mRNA was not affected by dexamethasone treatment (Fig. 6Go). Cortisol (1 µM) similarly induced 11ß-HSD1 mRNA expression in amnion fibroblasts, whereas cortisone (1 µM) did not stimulate 11ß-HSD1 mRNA expression in amnion fibroblasts (Fig. 7Go). RU486 (1 µM), a GR and progesterone receptor (PR) antagonist, could significantly inhibit the induction of 11ß-HSD1 mRNA expression by dexamethasone (1 µM) (Fig. 7Go). RU486 treatment alone also caused a significant increase in the expression of 11ß-HSD1 mRNA in amnion fibroblasts but not to the same extent as dexamethasone (Fig. 7Go).



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  		FIG. 6. Effect of dexamethasone (dex) on 11ß-HSD1 mRNA expression in cultured amnion fibroblast and epithelial cells. Top, Representative fluorescence curves of QT-RT-PCR for 11ß-HSD1 and ß-actin mRNA expression in the corresponding samples. Bottom, Relative mRNA expression (mean ± SEM). *, P < 0.05; **, P < 0.01 vs. control; #, P < 0.05 vs. dex 0.01 µM; @, P < 0.05 vs. dex 0.1 µM (n = 4–5 separate experiments).

 


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  		FIG. 7. Effect of different glucocorticoids (1 µM) and RU486 (1 µM) on 11ß-HSD1 mRNA expression in cultured amnion fibroblasts. Dex, Dexamethasone, mean ± SEM. **, P < 0.01 vs. control; ##, P < 0.01 vs. dex; @, P < 0.01 vs. dex (n = 5–8 separate experiments).

 
Effects of combined dexamethasone and proinflammatory cytokine treatment on the expression and activity of 11ß-HSD1 in cultured amnion fibroblasts
Both IL-1ß (10 ng/ml) and TNF{alpha} (10 ng/ml) treatments alone modestly stimulated 11ß-HSD1 mRNA expression in amnion fibroblasts, but a statistically significant increase was observed only with IL-1ß treatment, not with TNF{alpha} (Fig. 8Go). However, a dramatic significant increase of 11ß-HSD1 mRNA expression was observed with both combined IL-1ß (10 ng/ml) and dexamethasone (0.1 µM) as well as with combined TNF{alpha} (10 ng/ml) and dexamethasone (0.1 µM) treatments, as compared with dexamethasone (0.1 µM) treatment alone. Likewise, although no significant changes of 11ß-HSD1 activity were observed with IL-1ß (10 ng/ml) or TNF{alpha} (10 ng/ml) treatment alone in amnion fibroblasts, the combination of IL-1ß (10 ng/ml) or TNF{alpha} (10 ng/ml) with dexamethasone (0.1 µM) caused an even more significant increase of 11ß-HSD1 activity in amnion fibroblasts, as compared with the dexamethasone (0.1 µM) treatment alone (Fig. 8Go).



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  		FIG. 8. Effect of IL-1ß (10 ng/ml) or TNF{alpha} (10 ng/ml) either alone or in combination with dexamethasone (dex, 0.1 µM) on 11ß-HSD1 mRNA expression and activity in cultured amnion fibroblasts after 24 h culture. Top, Representative fluorescence curves of QT-RT-PCR for 11ß-HSD1 mRNA expression. Middle, Histogram of the mean data of mRNA expression. Bottom, 11ß-HSD1 activity measured after incubation with cortisone (1 µM) for 12 h. *, P < 0.05; **, P < 0.01 vs. control (ctr); #, P < 0.05 vs. dex; mean ± SEM, n = 4–5 separate experiments.

 
Prior induction of 11ß-HSD1 by dexamethasone potentiates the effect of cortisone on PGE2 production in cultured amnion fibroblasts
To evaluate the prior induction of 11ß-HSD1 expression by dexamethasone on PGE2 production on cortisone treatment, amnion fibroblasts were pretreated with dexamethasone (1 µM) for 24 h and then washed. Subsequent treatment with or without cortisone (1 µM) was conducted for another 24 h. Culture media were collected for PGE2 measurement. PGE2 output from the cultured amnion fibroblasts was significantly increased by dexamethasone treatment (1 µM), the effect still being seen 24 h after removal of dexamethasone without subsequent cortisone treatment (Fig. 9Go). Subsequent addition of cortisone (1 µM) after removal of dexamethasone caused a further increase of PGE2 output but not in the cells without prior dexamethasone treatment (Fig. 9Go).



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  		FIG. 9. Effect of cortisone (1 µM) on PGE2 output in cultured amnion fibroblasts pretreated with or without dexamethasone (dex, 1 µM). Amnion fibroblasts were pretreated with dex (1 µM) for 24 h and then washed. Subsequent treatment with or without cortisone (1 µM) was conducted for another 24 h. Culture media were collected for PGE2 measurement. *, P < 0.05 vs. dex pretreatment without subsequent cortisone treatment; ##, P < 0.01 vs. respective control (ctr); mean ± SEM, n = 3 separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Early work using homogenized tissues demonstrated that term chorionic and placental tissues had fairly high 11ß-HSD reductase activity converting cortisone to cortisol, whereas amnion and myometrial tissues had negligible 11ß-HSD reductase activity (28). This 11ß-HSD reductase activity is now regarded as the predominant reaction catalyzed by 11ß-HSD 1 in intact cells (6). However, using immunocytochemical staining and Northern blotting methods, Sun and Challis (7) found that both 11ß-HSD 1 protein and mRNA were present in human amnion and chorion tissues. In the amnion, subepithelial mesenchymal cells with fibroblast appearance and discrete populations of epithelial cells were stained positive for immunoreactive 11ß-HSD 1 (7). In this study, separate amnion epithelial cell and fibroblast cultures were examined to determine 11ß-HSD1 expression in each cell type and regulation by glucocorticoids. By using these relatively pure amnion epithelial cell and fibroblast preparations, we found both cell types expressed 11ß-HSD 1 protein and mRNA as determined by immunocytochemistry and PCR, respectively, although amnion fibroblasts express higher 11ß-HSD 1 activity and mRNA level than amnion epithelial cells. The present study also showed that the Km values for cortisone in amnion epithelial and fibroblast cells compare favorably with previously published values for 11ß-HSD 1 (6), which provides further evidence for the expression of 11ß-HSD 1 in amnion epithelial and fibroblast cells. However, we cannot be certain whether the discrete rather than homogeneous 11ß-HSD 1 distribution in amnion epithelial cells could account for the low level of 11ß-HSD 1 expression in this cell type. Neither can we be certain whether this also accounts for the failure of induction of 11ß-HSD 1 expression by glucocorticoids in this cell type as demonstrated in this study.

Sippell et al. (29) showed that cortisol and corticosteroid levels rose by 12- and 3-fold, respectively, in the amniotic fluid between wk 14–16 and wk 36–38, whereas levels of the biologically inactive cortisone rose by 2-fold in the amniotic fluid between wk 14–16 and wk 31–35 and then remained constant until term. Because fetal membranes are nourished directly by amniotic fluid, cortisone in the amniotic fluid could provide a ready substrate source for 11ß-HSD 1 in the fetal membranes. Tanswell et al. (30) reported that human amniotic membrane (amnion plus chorion) contained increasing 11ß-HSD activity during pregnancy, thereby potentially contributing to the rising cortisol concentrations of amniotic fluid. Recent work by Alfaidy et al. (31) has also shown that 11ß-HSD 1 expression in amnion tissue increased with gestational age and was further increased in labor. However, how 11ß-HSD1 expression is regulated by gestational age or labor in the fetal membranes is poorly understood.

Sequence analysis of the human 11ß-HSD1 gene revealed a putative GRE sequence in the promoter region (10), suggesting the expression of 11ß-HSD1 is likely under the control of glucocorticoids. Although human placenta and fetal membranes do not produce de novo glucocorticoids per se, the maternal adrenal gland secrets a large amount of glucocorticoids during pregnancy. Glucocorticoid levels in maternal blood increase with gestational age and further increase in labor (32, 33). In addition, the fetal adrenal glands also start to secrete glucocorticoids in the last week of pregnancy (34, 35). These surges of glucocorticoids not only induce fetal organ maturation in preparation for a successful transition from intra- to extrauterine life but also have also been suggested to be integral to the cascade of events leading to the onset of parturition (1, 32, 35). Rajan et al. (36) showed that glucocorticoid induced the expression of 11ß-HSD1 in the rat hippocampus. Recent work by Sun et al. (11) showed that glucocorticoid up-regulated 11ß-HSD1 expression in the chorion. The present study further demonstrated that glucocorticoids could also positively feedback on the induction of 11ß-HSD1 expression in amnion fibroblasts. Thus, glucocorticoids are capable of inducing 11ß-HSD1 expression in both the amnion and chorion, both of which are crucial tissues for prostaglandin production during labor.

Effects of glucocorticoids are normally mediated through intracellular GRs. Two isoforms of GR (GR{alpha} and GRß) have been identified, which originate from the same gene by alternative splicing of the GR primary transcript (37). GR{alpha} is the predominant isoform that possesses steroid binding activity. Upon binding glucocorticoids, GR{alpha} translocates from cytoplasm to nucleus in which it acts as a transcription factor to regulate target gene expressions. Due to a lack of a steroid binding domain in the carboxyl terminus, GRß does not bind glucocorticoids (37). Studies (38) showed that GRß could inhibit the gene transactivating effect of GR{alpha} by forming impaired heterodimers with GR{alpha}. However, there were also studies challenging this concept (39). Most of the previous studies analyzing GR expression did not distinguish between GR{alpha} and GRß isoforms. By using immunocytochemistry, Sun et al. (40) have demonstrated that nuclear GR was found in amnion epithelium, mesenchyme, and the chorion leave. In this study, we found that both GR{alpha} and GRß mRNAs were expressed in amnion epithelial and fibroblast cells. These findings provide a molecular basis for the actions of glucocorticoids in the fetal membranes. We also found no significant differences in GR{alpha} mRNA expression between these two cell types, suggesting the failure of induction of 11ß-HSD1 expression by dexamethasone in amnion epithelial cells was at least not due to absence or low levels of GR{alpha} expression. We also demonstrated in this study that the induction of 11ß-HSD1 expression by dexamethasone was blocked by cotreatment with RU486. Although it has been very well recognized that RU486 is capable of blocking both GR and PR (41), progesterone did not affect the expression of 11ß-HSD1 in chorion trophoblasts but has a potent inhibitory effect on 11ß-HSD2 expression in placenta trophoblasts (42). It has also been reported that term human amnion has no detectable PR (43, 44). Because there is a putative GRE present in the promoter region of the 11ß-HSD1 gene (10), we propose that the blocking effect of RU486 on dexamethasone’s induction of 11ß-HSD1 expression was very likely through GR rather than PR. With regard to the induction of 11ß-HSD1 expression by RU486 treatment alone, we assume this effect is possibly due to the partial GR agonist effect of RU486. It has been recently reported that RU486-induced glucocorticoid receptor agonism is controlled by the receptor N terminus (45).

Of interest, we found in this study that the positive feedback of glucocorticoids on the induction of 11ß-HSD1 was further enhanced by cotreatment with proinflammatory cytokines. Infection is the leading cause of preterm delivery (1). Chorioamnionitis is the most common type of infection in preterm labor, especially in preterm rupture of membranes (46). Infection of membranes results in the activation of macrophages in these tissues. The activated macrophages then release proinflammatory cytokines such as IL-1ß and TNF{alpha} and activate local stromal cells that further release proinflammatory cytokines (46). Both IL-1ß and TNF{alpha} induce preterm labor by stimulating prostaglandin output in the intrauterine tissues (47). Studies have also demonstrated that IL-1ß and TNF{alpha} induced both 11ß-HSD1 mRNA and activity in human adipose tissue, osteoblast, and ovarian surface epithelial cells (14, 15, 16). The present study demonstrated that only IL-1ß but not TNF{alpha} treatments significantly up-regulated 11ß-HSD1 mRNA expression, and both IL-1ß and TNF{alpha} treatments had no effects on 11ß-HSD1 activity in amnion fibroblasts at the doses examined. However, combination of dexamethasone with either IL-1ß or TNF{alpha} further significantly induced 11ß-HSD1 mRNA and activity in amnion fibroblasts, as compared with dexamethasone treatment alone. Based on this finding, we propose that more biologically active glucocorticoids would be available locally in the fetal membranes or the amniotic fluid as a result of activation of 11ß-HSD1 activity and mRNA expression in the presence of chorioamnionitis.

It is well recognized that glucocorticoids effectively suppress immune cell activation induced by proinflammatory cytokines by two main mechanisms (48). Glucocorticoids inactivate the function of the proinflammatory cytokine mediator nuclear factor-{kappa}B (NF-{kappa}B) by inducing the expression of inhibitory {kappa}B{alpha} (48). This additional inhibitory {kappa}B{alpha} holds NF-kB in its inactive form in the cytoplasm (48). Additionally, potential binding of activated nuclear GR complexes to nuclear NF-kB may prevent the latter from binding to appropriate DNA response elements and contribute to steroid-mediated immunosuppression (48). However, in this study we found the combination of glucocorticoids and proinflammatory cytokines further induced the expression of 11ß-HSD1 mRNA in amnion fibroblasts, which is in obvious contrast to the opposing effects of glucocorticoids and proinflammatory cytokines at inflammation in nonintrauterine tissues. The detailed mechanisms of this paradoxical finding in amnion fibroblasts need further investigation. Nevertheless, understanding the paradoxical interaction of glucocorticoids and proinflammatory cytokines in the fetal membranes could provide important clues in understanding the mechanism of normal and preterm labor.

Parturition is a feed-forward process involving several positive feedback loops in terms of oxytocin and PG release (1). In this study, we found another possible feed-forward mechanism with regard to glucocorticoid and PG formation in the fetal membranes. Glucocorticoids induce the enzyme 11ß-HSD1, which catalyzes the formation of biologically active glucocorticoids in the fetal membranes. As a result, there would be more and more biologically active glucocorticoids formed in the fetal membranes and amniotic fluid before labor. The increasing level of biologically active glucocorticoids would not only participate in the induction of labor but also promote fetal organ maturation through fetal swallowing (49). Glucocorticoids have been shown to exert potent stimulation of PG output by both inducing the expression of PGHS-2 in the fetal membranes and inhibiting the expression of prostaglandin dehydrogenase in the chorion (3). Thus, under the influence of glucocorticoids, more PGs would be formed and less would be degraded by prostaglandin dehydrogenase. We also found in this study that dexamethasone potently stimulates PGE2 output in the amnion fibroblast, and cortisone caused further enhancement of PGE2 output with prior induction of 11ß-HSD1 expression by dexamethasone, suggesting that more cortisone was converted into cortisol with prior induction of 11ß-HSD1 expression, and cortisol in turn further promoted the production of PGE2 in amnion fibroblasts. Recent work by Alfaidy et al. (50) showed that PGs could stimulate 11ß-HSD1 activity in the chorion. Therefore, the increase of 11ß-HSD1 activity by dexamethasone treatment in amnion fibroblasts could be mediated in part by its stimulation on PG production. However, there could also be other mechanisms involved in the induction of 11ß-HSD1 expression and activity by glucocorticoids in amnion fibroblasts, such as a possible direct interaction with the putative GRE in the promoter region of the 11ß-HSD1 gene.

Taking all these results together, we propose that a positive feedback loop involving glucocorticoids, proinflammatory cytokines, PGs, and 11ß-HSD1 was formed locally in the fetal membranes at term or preterm labor. This positive feedback loop would produce abundant biologically active glucocorticoids and PGs in fetal membranes or amniotic fluid.


   Footnotes
 
This work was supported by NIH RO1 Grant HD31514 and the National Basic Research Program of China (G1999054000).

Abbreviations: FCS, Fetal calf serum; GR, glucocorticoid receptor; GRE, glucocorticoid response element; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; Km, Michaelis constant; NF-{kappa}B, nuclear factor-{kappa}B; PG, prostaglandin; PGHS, PG H synthase type II; PR, progesterone receptor; QT-RT-PCR, quantitative real-time PCR.

Received June 24, 2003.

Accepted for publication August 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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