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Endocrinology Vol. 147, No. 12 5865-5872
Copyright © 2006 by The Endocrine Society

Role for Prostaglandins in the Regulation of Type 1 11ß-Hydroxysteroid Dehydrogenase in Human Granulosa-Lutein Cells

Kim C. Jonas, Christina Chandras, D. Robert E. Abayasekara and Anthony E. Michael

Department of Biochemistry and Molecular Biology (K.C.J., C.C., A.E.M.), Royal Free and University College Medical School, University College London, London WC1E 6BT, United Kingdom; Department of Veterinary Basic Science (K.C.J., C.C., D.R.E.A.), Royal Veterinary College, London NW1 0TU, United Kingdom; and Centre for Developmental and Endocrine Signalling (A.E.M.), Academic Section of Obstetrics & Gynaecology, Division of Clinical Developmental Sciences, St. George’s, University of London, London SW17 0RE, United Kingdom

Address all correspondence and requests for reprints to: Dr. Kim C. Jonas, Department of Veterinary Basic Science, Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom. E-mail: kjonas{at}rvc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
11ß-Hydroxysteroid dehydrogenase (11ßHSD) enzymes regulate glucocorticoid availability in target tissues. 11ßHSD1 is the predominant isoenzyme expressed and active in human granulosa-lutein (hGL) cells. This study investigated the effects of pharmacological inhibitors of prostaglandin (PG) synthesis on 11ßHSD1 activities and expression in hGL cells. The consequences for 11ßHSD1 of increasing exposure of hGL cells to PGs, either by treatment with exogenous PGs or by challenging cells with IL-1ß, were also assessed. Suppression of basal PG synthesis using four different inhibitors of PG H synthase enzymes [indomethacin, niflumic acid, meclofenamic acid (MA) and N-(2-cyclohexyloxy-4-nitorophenyl) methane sulfonamide (NS-398)] each resulted in significant decreases in both cortisol oxidation and cortisone reduction. Both activities of 11ßHSD1 were suppressed by up to 64 ± 6% (P < 0.05). Over 4 and 24 h, neither MA nor NS-398 affected the expression of 11ßHSD1 protein, suggesting enzyme regulation by PGs at the posttranslational level. When cells were cotreated for 4 h with PGHS inhibitors plus 30 nM PGD2, PGF2{alpha}, or PGE2, each PG overcame the suppression of cortisol oxidation by indomethacin or MA. Treatment of hGL cells with IL-1ß increased the concentrations of both PGE2 and PGF2{alpha}, accompanied by a 70 ± 25% increase in net cortisol oxidation. All three responses to IL-1ß were abolished when cells were cotreated with MA. These findings suggest a role for PGs in the posttranslational regulation of 11ßHSD1 activities in hGL cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WITHIN GLUCOCORTICOID target tissues, cortisol is interconverted with its inactive keto-steroid metabolite cortisone, by the 11ß-hydroxysteroid dehydrogenase (11ßHSD) enzymes. To date, two 11ßHSD enzymes have been cloned. Type 1 11ßHSD (11ßHSD1) is an ubiquitously expressed enzyme with a relatively low affinity for cortisol (Km>1 µM) (1, 2, 3). In vivo, the bidirectional reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent 11ßHSD1 enzyme acts predominantly as a reductase to reactivate cortisol from cortisone (4). However, recent studies have established that the net direction of 11ßHSD1 activity in a given cell is dependent on the redox state of NADPH within the lumen of the smooth endoplasmic reticulum. In most tissues, hexose-6-phosphate dehydrogenase maintains a high concentration of NADPH that favors the reductase activity of 11ßHSD1 (5, 6, 7, 8, 9). In contrast, in rat testis Leydig cells and in human ovarian granulosa-lutein cells, 11ßHSD1 acts predominantly as a dehydrogenase enzyme catalyzing the inactivation of glucocorticoids (10, 11, 12, 13). This has been attributed to the preferential usage of NADPH for steroid biosynthesis in steroidogenic cells such that NADPH is oxidized to NADP+ that can support the oxidative activity of 11ßHSD1 (14, 15).

The 11ßHSD2 enzyme is a high affinity, nicotinamide adenine dinucleotide-dependent dehydrogenase that exclusively inactivates cortisol to cortisone (16, 17, 18, 19). 11ßHSD2 is highly expressed in tissues involved in aldosterone-dependent water reabsorption, where it acts to prevent illicit occupation of the mineralocorticoid receptors by cortisol (18, 19).

Initial studies detected 11ßHSD activities in human granulosa-lutein (hGL) cells obtained from women undergoing in vitro fertilization and embryo transfer (20, 21, 22). Subsequent studies revealed that 11ßHSD1 is expressed in the oocyte (23, 24, 25) and ovarian surface epithelial cells (26, 27) as well as in luteinizing granulosa cells (11, 24, 28, 29). Moreover, the expression of mRNA transcripts for the two cloned 11ßHSD enzymes are differentially expressed in granulosa cells throughout the ovarian cycle (28, 29, 30). 11ßHSD2 mRNA is the predominant (if not exclusive) transcript expressed in granulosa cells during the follicular phase, switching to predominant expression of 11ßHSD1 mRNA at the time of ovulation in luteinizing granulosa cells and the corpus luteum. In hGL cells isolated from women undergoing controlled ovarian hyperstimulation for in vitro fertilization and allowed to lutenize in serum supplemented culture media, only 11ßHSD1 mRNA and protein are expressed with no detectable expression of 11ßHSD2 at either mRNA or protein levels (11, 24, 29). Hence, in this cell model, cortisol-cortisone interconversion can be attributed to the 11ßHSD1 enzyme.

There are many established endocrine and paracrine regulators of ovarian function including the prostaglandins (PGs). These inflammatory mediators are synthesized from arachidonic acid by the sequential cyclooxygenase and endoperoxide synthase activities of PG H synthase (PGHS) enzymes. In fetal membranes at the time of parturition, PG F2{alpha} (PGF2{alpha}) can increase regeneration of cortisol from cortisone by 11ßHSD1, creating the potential for a positive loop between local PG and cortisol synthesis (31, 32). Moreover, in studies of human ovarian surface epithelial cells, Hillier and colleagues (26, 27, 33, 34) have shown that LH and IL-1 can both induce increases in expression of 11ßHSD1 at 48 h of treatment. This rise in 11ßHSD1 expression in response to IL-1ß is preceded by increased expression of PGHS-2 at 12 h of treatment (34, 35), raising the possibility that the increased synthesis of proinflammatory PGs by PGHS-2 may actually mediate up-regulation of 11ßHSD1 expression and activity in response to LH and IL-1ß.

As granulosa cells commence functional luteinization in the periovulatory follicle, the predominant isoform of 11ßHSD expressed switches from 11ßHSD2 to 11ßHSD1 (reviewed in Ref. 14). Differentiation of follicular granulosa cells is associated with an increase in the expression of PGHS enzymes and PG receptors (36, 37). The resultant increases in PG synthesis and action are essential for the inflammatory event of ovulation, where PGs act to up-regulate matrix metalloproteinases and affect the vascularization of the follicle in preparation for follicular rupture, oocyte expulsion and subsequent follicular luteinization (36, 37). It has been hypothesized that the expression of 11ßHSD2 in the preovulatory follicle is required to inactivate cortisol and hence favor the proinflammatory environment required for ovulation. After ovulation, the isoform of 11ßHSD expressed switches from 11ßHSD2 to 11ßHSD1, providing an antiinflammatory environment through cortisol regeneration which acts to rapidly dampen the effects of the inflammatory mediators of ovulation e.g. PGs (26). If PGs were to coordinate the ovarian switch in 11ßHSD enzyme activities at ovulation, this would give rise to a paracrine feedback loop that would enable the PGs to control the local inactivation/regeneration of cortisol, ensuring that the proinflammatory actions of PGs required for ovulation are limited in both space and time.

Although a link between PG and cortisol metabolism has been observed in other reproductive tissues, the role of PGs in the regulation of 11ßHSD1 activities and expression in human ovarian cells remains unclear. Hence, the aim of the present study was to establish the importance of locally synthesized PGs in regulating the activities and/or expression of 11ßHSD1 in hGL cells. This aim was addressed by a combined approach of 1) inhibiting local PG synthesis using structurally dissimilar inhibitors of PGHS enzymes, and 2) increasing PG concentrations either by treating cells with exogenous PGs or challenging cells with the proinflammatory cytokine IL-1ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and culture
Cells were isolated as previously described by Webley et al. (38) from follicular aspirates of women undergoing controlled ovarian hyperstimulation for assisted conception at the Lister Private Hospital (Chelsea, London, UK). Follicular aspirates were collected with informed patient consent in accordance with the Declaration of Helsinki and as approved by the local ethics committee.

After isolation on 60% (vol/vol) Percoll, hGL cells were cultured for 48 h in 1:1 DMEM:Ham’s F12 medium supplemented with 10% (vol/vol) fetal calf serum (Life Technologies, Strathclyde, UK), 2 mM L-glutamine (Life Technologies), penicillin (87,000 IU/liter) (Sigma-Aldrich, Poole, UK), and streptomycin (87 mg/liter) (Sigma-Aldrich) in an atmosphere of 5% (vol/vol) CO2 in air. Cells were cultured in 1-ml volumes at a density of 5 x 104 viable cells/well in 24-well plates for measurement of 11ßHSD activities, and at a density of 1 x 105 viable cells/well for measurement of PG secretion. For immunoblotting, cells were plated in 2-ml volumes at a density of 1 x 106 viable cells/well in six-well culture plates. For all experiments, cell viability was assessed by exclusion of 0.4% (vol/vol) trypan blue dye.

Effect of PGHS inhibitors on PG synthesis
To confirm that the inhibitors of PGHS enzymes suppressed local PG synthesis, cells were transferred to serum-free 1:1 DMEM:Ham’s F12 medium and then treated for either 4 or 24 h with indomethacin (Sigma Aldrich), niflumic acid (NA) (Sigma-Aldrich), meclofenamic acid (MA) (Sigma-Aldrich) or N-(2-cyclohexyloxy-4-nitorophenyl) methane sulfonamide (NS-398) (Calbiochem, Nottingham, UK), each at final concentrations of 0, 0.01, 0.1, 1, 10, and 100 µM. Each compound was prepared at a stock concentration of 100 mM in either ethanol (indomethachin, NA, and MA) or dimethylsulfoxide (NS-398) and in all wells, the final concentration of organic solvent was maintained at 0.1% (vol/vol).

After the 4- or 24-h treatment period, culture plates were frozen at –20 C. Samples were subsequently thawed and PGE2 and PGF2{alpha} concentrations measured using RIAs previously described by Poyser (39) and by Kelly et al. (40), respectively. These assays were validated for use in hGL cell-conditioned medium in our laboratory by Fowkes et al. (41).

Effects of PGHS inhibitors on 11ßHSD1 activities and expression
To assess the effects of indomethacin, NA, MA and NS-398 on 11ßHSD1 activities, cells were preincubated for 48 h in serum-supplemented 1:1 DMEM:Ham’s F12 medium. After this, the cells were transferred to serum-free 1:1 DMEM:Ham’s F12 medium containing 0 µM to 100 µM indomethacin, NA, MA, or NS-398 for 4 or 24 h. 11ßHSD1 activities were assessed by radiometric conversion assays as previously described by Michael et al. (42). In brief, cells were incubated with either 100 nM [1,2,6,7-3H]-cortisol (Amersham Biosciences,Amersham, Buckinghamshire, UK), or 100 nM [1,2(n)-3H]-cortisone (Amersham Biosciences) for the last 4 h of the treatment period to assess net 11ß-dehydrogenase (11ß-DH) and net 11-ketosteroid reductase (11-KSR) activities, respectively. To terminate the reaction, steroids were extracted into 2 volumes of chloroform and evaporated to dryness under nitrogen gas at 45 C. The steroid residues were resuspended in 20 µl ethyl acetate containing 1 mM cortisol (Sigma-Aldrich) and 1 mM cortisone (Sigma-Aldrich) and resolved by thin-layer chromatography (TLC) on silica 60 TLC plates (Merck, Nottingham, UK) developed in an atmosphere of 92:8 chloroform:95% (vol/vol) ethanol. [3H]-cortisol and [3H]-cortisone were quantified using a Bioscan 200 TLC radiochromatogramme scanner (Lablogic, Sheffield, UK), and 11ßHSD1 activities were calculated as either picomoles cortisol oxidized to cortisone, or picomoles of cortisone reduced to cortisol, over 4 h (42).

Effects of PGHS inhibitors on expression of 11ßHSD1 protein were assessed using methods previously reported by Thurston et al. (13). Cells were preincubated for 48 h in serum-supplemented medium before being washed into prewarmed, serum-free 1:1 DMEM:Ham’s F12 medium. Cells were then treated for either 4 or 24 h with MA or NS-398 each at a concentration of 100 µM. This single concentration of MA/NS-398 was selected based on the maximal suppression of PGE2 and PGF2{alpha} synthesis (see Fig. 1Go).


Figure 1
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  		FIG. 1. Concentration-dependent effects of treatment with (A) MA for 4 h, (B) MA for 24 h, (C) NS-398 for 4 h, and (D) NS-398 for 24 h on production of PGE2 (open circles) and PGF2{alpha} (closed triangles) by hGL cells treated on d 3 of culture. Each data point represents the mean ± SEM for three to five independent experiments, expressed as a percentage of control at 0 µM MA/NS-398. (The 100% control values correspond to 0.202 ± 0.072 ng PGE2 and 0.097 ± 0.393 ng PGF2{alpha} per 100,000 cells for 4 h, and to 1.970 ± 0.335 ng PGE2 and 0.650 ± 0.047 ng PGF2{alpha} per 100,000 cells for 24 h, respectively.) Data were analyzed using one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison test for each PG; *, P < 0.05, and **, P < 0.01 vs. the control concentration for the respective PG measured in the absence of MA/NS-398.

 
After treatment, spent medium was aspirated from the six-well plate and cells were rinsed with ice-cold PBS (pH 7.5; Life Technologies) containing 200 µM sodium orthovanadate (Sigma-Aldrich). The PBS was immediately aspirated, and cells were lysed in a protein lysis buffer containing Tris-HCl [63.5 mM (pH 6.8), Sigma-Aldrich), 10% (vol/vol) glycerol (Sigma-Aldrich), 2% (wt/vol) SDS (Sigma-Aldrich) and protease inhibitor cocktail set I (Calbiochem, c/o Merck Biosciences). Cells were freeze-thawed and cell monolayers were scraped into 1.5-ml Eppendorf tubes (VWR International, Leicestershire, UK). The samples were heated for 5 min at 100 C and centrifuged at 3000 x g for 1 min to precipitate cell debris. The protein content of the cell lysates was determined using the bicinchoninic acid method as per the manufacturers’ instructions (Pierce Chemical Co., Chester, UK). Before gel electrophoresis, 5% (vol/vol) ß-mercaptoethanol (VWR International, UK) and 0.02% (wt/vol) bromophenol blue were added to the lysates.

Proteins from whole cell lysates (30 µg/lane) were separated using 10% (vol/vol) SDS-PAGE and transferred to an Immobilon polyvinylidene difluoride membrane using semi-dry Western blotting equipment (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK). Prestained molecular weight markers were run in parallel lanes (Bio-Rad Laboratories, Inc.). To prevent nonspecific antibody binding, membranes were incubated for 2 h at room temperature in 10% (wt/vol) BSA in Tris-buffered saline-Tween (TBST) [50 mM Tris, 150 mM NaCl, 0.02% (wt/vol) Tween 20 (pH 7.4); Sigma-Aldrich]. Membranes were incubated overnight at 4 C with the primary antibody raised against 11ßHSD1 at a dilution of 1/100 in 10% (wt/vol) BSA in TBST. Polyclonal 11ßHSD1 primary antibodies were commercially raised in sheep against the human 11ßHSD1 protein sequence (amino acids 19–33) (The Binding Site, Birmingham, UK). After overnight incubation with the primary antibody, the membranes were washed with TBST (6 x 10 min) before being incubated for 1 h with donkey antisheep peroxidase-conjugated IgG (Sigma-Aldrich) at an antibody dilution of 1/10,000 in 0.2% (wt/vol) TBST. After secondary antibody binding, membranes were washed in TBST (6 x 10 min), and immunoreactive proteins were visualized using the enhanced chemiluminescence (ECL) detection method, according to manufacturers’ instructions (Amersham Biosciences). Excess ECL reagent was removed and membranes were exposed to x-ray hyperfilm for 1–5 min.

To account for minor discrepancies in protein loading or transfer, the signal for each 11ßHSD1 protein band was standardized relative to the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) within the same lane. To quantitate GAPDH levels, after assessment of 11ßHSD1 expression, each membrane was stripped in 100 ml of stripping buffer [62.5 mM Tris-HCl, 2% (wt/vol) SDS, 100 mM ß-mercaptoethanol] at 50 C for 20 min in a water-bath (adapted from the manufacturer’s guidelines for the membrane-stripping procedure) (Amersham Biosciences). After incubation, the blot was removed and the stripping buffer discarded. The blot was transferred to wash in 150 ml TBST at maximum agitation on a Rotamax 120 (Hoeffer, c/o Fisher Scientific, Leicestershire, UK) for 15 min. This wash step was repeated four times in preparation for reprobing of the membrane for GAPDH using the same methodology as for 11ßHSD1 with the following exceptions. The primary antibody (1 in 1000 dilution in 10% (wt/vol) BSA in TBST) was a monoclonal GAPDH antibody raised in mouse against the human GAPDH peptide sequence (AbCam, Cambridge, UK) and the secondary antibody was a rabbit antimouse peroxidise conjugated IgG [1 in 5000 dilution in 10% (wt/vol) BSA in TBST, Sigma-Aldrich]. Levels of both 11ßHSD1 and GAPDH protein expression were assessed by densitometric analyses, performed using a Gel Doc 1000 system and Molecular Analyst software (Bio-Rad Laboratories Inc.).

Effects of exogenous PGs of 11ßHSD1 activities
After a 48 h preculture in serum-supplemented 1:1 DMEM:Ham’s F12 medium, cells were transferred to serum-free medium. Cells were then treated for 4 h with either 100 µM indomethacin or 100 µM MA, each in the presence and absence of 30 nM PGD2, 30 nM PGF2{alpha} or 30 nM PGE2. During the 4-h treatment period, the net 11ß-dehydrogenase activities of 11ßHSD1 were assessed using 100 nM [3H]-cortisol as substrate in the radiometric conversion assay.

In a subsequent experiment, the concentration-dependent effects of PGE2 on net cortisol oxidation were investigated in more detail. After a 48 h preculture in serum-supplemented medium, cells were transferred to serum-free medium and treated for 4 h with 0, 10, 100, and 1000 nM PGE2, during which time the net 11ß-dehydrogenase activities were assessed as described above. Previous studies have reported that at concentrations above 100 nM, PGE2 can act on hGL cells to stimulate progesterone synthesis (38, 43, 44). In view of our published data, which implicate progesterone as a paracrine inhibitor of 11ßHSD1 in hGL cells (13), we assessed the concentration-dependent effects of PGE2 on 11ßHSD1 activities in the presence and absence of 0.1 mM aminoglutethimide (AG): a concentration of AG that we have shown to suppress progesterone synthesis in hGL cells (13, 41).

Effects of IL-1ß on PG production and 11ßHSD1 activities
To assess the effects of IL-1ß on PG synthesis, hGL cells were preincubated for 48 h in serum-supplemented 1:1 DMEM:Ham’s F12 medium. After this, the cells were transferred to serum-free 1:1 DMEM:Ham’s F12 medium containing 0.1 mM AG and challenged with IL-1ß (10 ng/ml) in the presence and absence of 100 µM MA for 24 h. At the end of the experiment, culture plates were frozen at –20 C. Samples were subsequently thawed and PGE2 and PGF2{alpha} concentrations measured by RIA.

To assess effects on cortisol metabolism, cells were challenged (in serum-free medium containing 0.1 mM AG) for 24 h with IL-1ß (10 ng/ml) in the presence and absence of 100 µM MA. The net oxidation of 100 nM [3H]-cortisol by 11ßHSD1 was assessed over the final 4 h of each experiment using the radiometric conversion assay.

Statistical analyses
All experimental data are presented as the mean ± SEM for up to five independent replicates of each experimental design, where each experimental replicate was performed using cells from individual patients and each experimental condition was repeated in triplicate within an experiment. Due to differences in absolute levels of PG synthesis and enzyme activities between patients, the results for each experiment were standardized and presented as a percentage of control in the absence of treatments. However, all statistical analyses were performed using absolute data, rather than the internally referenced data.

When assessing the effects of indomethacin, NA, MA, and NS-389 on PG synthesis and 11ßHSD activities in hGL cells, a one-way ANOVA with repeated measures was performed, followed by Dunnett’s multiple comparison as the post hoc test. To assess the effects of single concentrations of MA and NS-398 on 11ßHSD1 protein expression in hGL cells, a paired t test was used. The effects of exogenous PGs, the concentration-dependent effects of PGE2 and all cellular responses to IL-1ß were each assessed by one-way ANOVA with repeated measures and the Dunnett’s multiple comparison. All statistical evaluations were performed using GraphPad Prism 3.02 software (San Diego, CA). Significance was assessed in all experiments as a probability value of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PGHS inhibitors on basal PG production
Each of the four tested PGHS inhibitors suppressed basal PGE2 and PGF2{alpha} concentrations at both 4 and 24 h of treatment. Maximum inhibition of PG production over 4 h was observed with 100 µM MA, which inhibited PGE2 and PGF2{alpha} production by 64.5 ± 17.2% (P < 0.01) and 60.1 ± 11.3% (P < 0.05), respectively (Fig. 1AGo). Over 24 h, 100 µM MA maximally inhibited both PGE2 and PGF2{alpha} production by approximately 50% (Fig. 1BGo). The concentrations of both PGE2 and PGF2{alpha} were similarly suppressed over both 4 and 24 h with NS-398 (Fig. 1Go, C and D), indomethacin and NA (data not shown).

Effect of PGHS inhibitors on 11ßHSD1 activities in hGL cells
Inhibition of PG synthesis by treatment of hGL cells for either 4 or 24 h with MA was associated with concentration-dependent decreases in both the 11ß-DH and 11-KSR activities of 11ßHSD1. Reduction of cortisone was suppressed by up to 50% at MA concentrations as low as 0.1 µM, whereas cortisol oxidation was decreased by up to 30% at 100 µM MA (Fig. 2Go, A and B).


Figure 2
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  		FIG. 2. Concentration-dependent effects of treatment with (A) MA for 4 h, (B) MA for 24 h, (C) NS-398 for 4 h, (D) NS-398 for 24 h on net 11ß-DH activities (open circles) and net 11-KSR activities (closed triangles) in hGL cells treated on d 3 of culture. Each data point represents the mean ± SEM for three to five independent experiments, expressed as a percentage of control at 0 µM MA/NS-398. (The 100% control values correspond to 13.4 ± 2.9 pmol cortisol oxidized and 3.5 ± 0.4 pmol cortisone reduced per 50,000 cells for 4 h in cells treated for 4 h, and to 8.1 ± 0.4 pmol cortisol oxidized and 4.2 ± 0.9 pmol cortisone reduced per 50,000 cells for 4 h in cells treated for 24 h). Data were analyzed using one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison test for each enzyme activity; *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs. the respective enzyme activity measured in the absence of MA/NS-398.

 
Both acute and chronic treatment of hGL cells with NS-398 inhibited cortisol-cortisone interconversion in a concentration-dependent manner (Fig. 2Go, C and D). 11ß-DH activity was significantly inhibited with 100 µM NS-398 by up to 40% at both time points (P < 0.05), whereas 11-KSR activity was suppressed by up to 50% at the same concentration of NS-398 (P < 0.01). Indomethacin and NA exerted similar effects in suppressing both the reduction of cortisone and the oxidation of cortisol in a concentration-dependent manner over both 4 and 24 h of treatment (data not shown).

Effects of MA and NS-398 on 11ßHSD1 protein expression
Treatment with 100 µM MA or 100 µM NS-398 had no significant effect on 11ßHSD1 protein expression even after 24 h (P > 0.05; Fig. 3Go).


Figure 3
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  		FIG. 3. Representative Western blots and mean ± SEM relative optical densities from three independent experiments for the expression of 11ßHSD1 protein in hGL cells treated for either 4 h (open bars) or 24 h (filled bars) with (A) 100 µM MA, or (B) 100 µM NS-398. The signal intensity for 11ßHSD1 protein expression was standardized within each lane relative to GAPDH.

 
Effects of exogenous PGs on 11ßHSD1 activities
In agreement with our previous published data (11, 13) the experiments depicted in Fig. 2Go confirmed that under these experimental conditions, the level of 11ß-DH activity was approximately 2- to 4-fold higher than the level of 11-KSR activity (8–10 pmol cortisol oxidized/4 h vs. 3–4 pmol cortisone reduced/4 h). Having confirmed this to be the case, in subsequent experiments we only assessed effects on the net 11ß-DH activity of 11ßHSD1.

As observed in the first series of experiments, treatment of hGL cells for 4 h with either 100 µM indomethacin or 100 µM MA decreased the net oxidation of cortisol to cortisone by 58 ± 6% (P < 0.05) and 64 ± 6% (P < 0.05), respectively (Fig. 4Go). However, when cells were cotreated with 30 nM PGD2, 30 nM PGF2{alpha} or 30 nM PGE2, the enzyme activities did not differ significantly from that measured in the absence of indomethacin or MA (Fig. 4Go).


Figure 4
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  		FIG. 4. Effects on net cortisol oxidation in hGL cells treated for 4 h on d 3 of culture with (A) 100 µM indomethacin (Indo), or (B) 100 µM MA, each in the presence and absence of 30 nM PGD2, 30 nM PGF2{alpha}, and 30 nM PGE2. Each data point represents the mean ± SEM for three independent experiments, expressed as a percentage of control in the absence of indomethacin or MA (open bars). (The 100% control value corresponds to 4.0 ± 0.1 pmol cortisol oxidized per 50,000 cells for 4 h). Data were analyzed using one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison test for each PGHS inhibitor; *, P < 0.05 vs. the rate of cortisol oxidation measured in the absence of indomethacin/MA.

 
When cells were treated for 4 h with 100 µM MA and cotreated with increasing concentrations of PGE2 in the absence of 0.1 mM AG, 10 nM PGE2, and 100 nM PGE2 increased cortisol metabolism by just over 40% relative to control cells incubated with MA alone (P < 0.05), but 1000 nM PGE2 had no significant effect on 11ßHSD activity (Fig. 5Go). When cells were cotreated with 0.1 mM AG, all enzyme activities were increased by at least 60% relative to enzyme activities measured in the absence of AG, and the stimulation of cortisol oxidation by PGE2 increased progressively at 10, 100, and 1000 nM PGE2 (Fig. 5Go).


Figure 5
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  		FIG. 5. Effects on net cortisol oxidation in hGL cells treated for 4 h on d 3 of culture with 0–1000 nM PGE2 in the absence (open bars) and presence (filled bars) of 0.1 M AG. Each data point represents the mean ± SEM for three independent experiments expressed as a percentage of control in the absence of PGE2 and AG. (The 100% control value corresponds to 6.9 ± 0.3 pmol cortisol oxidized per 50,000 cells for 4 h). Data were analyzed using one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison; *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs. the rate of cortisol oxidation measured at 0 nM PGE2 in the absence or presence of AG, as appropriate.

 
Effects of IL-1ß on PG synthesis and 11ßHSD1 activities
Having confirmed that progesterone was a potential confounding factor when cells were stimulated with PGE2, all experiments with IL-1ß were performed in the presence of 0.1 mM AG such that inhibition of cortisol metabolism by progesterone was not a consideration. Under these circumstances, IL-1ß increased PGE2 and PGF2{alpha} production and cotreatment with MA abolished both of the PG responses (Fig. 6Go). Furthermore, IL-1ß significantly increased 11ß-DH activity (P < 0.05; Fig. 7Go) and cotreatment with MA abolished this stimulation of cortisol oxidation.


Figure 6
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  		FIG. 6. Effects of treatment of hGL cells on d 3 of culture for 24 h with 10ng/ml IL-1ß on (A) PGE2 production, and (B) PGF2{alpha} production, each measured in the absence (open bars) or presence (filled bars) of 100 µM MA. Each data point represents the mean ± SEM for five independent experiments expressed as a percentage of control in the absence of IL-1ß and MA. (The 100% control values correspond to 0.103 ± 0.009 ng PGE2 and 0.160 ± 0.054 ng PGF2{alpha} per 100,000 cells.24 h). Data were analyzed using one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison; *, P < 0.05 vs. the control concentration for the respective PG measured in the absence of IL-1ß and MA.

 

Figure 7
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  		FIG. 7. Effects on net cortisol oxidation in hGL cells treated for 24 h on d 3 of culture with 10 ng/ml IL-1ß in the absence (open bars) or presence (filled bars) of 100 µM MA; all treatments were conducted in the presence of 0.1 mM AG. Each data point represents the mean ± SEM for five independent experiments expressed as a percentage of control in the absence of IL-1ß and MA. (The 100% control value corresponds to 9.7 ± 2.8 pmol cortisol oxidized per 50,000 cells for 4 h). Data were analyzed using one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison; *, P < 0.05 vs. the rate of cortisol oxidation measured in the absence of IL-1ß and MA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has investigated the importance of locally produced PGs in regulating the expression and activity of 11ßHSD1 in hGL cells. The suppression of locally produced PGs using four structurally dissimilar pharmacological inhibitors of PGHS enzymes decreased both the oxidative and reductive activities of 11ßHSD1. The suppressive effects of indomethacin and MA on cortisol oxidation could each be reversed by cotreatment with exogenous PGD2, PGF2{alpha}, or PGE2. Moreover, PGE2 increased 11ßHSD1 activity in a concentration-dependent manner and cytokine-stimulated increases in 11ßHSD1 activity were abolished by cotreatment with MA.

The half-life of the PGHS enzymes is approximately 10 min and PGs are rapidly inactivated by oxidation of the 15-hydroxyl group by 15-hydroxyPG dehydrogenase (PGDH). In this study, the four structurally distinct pharmacological inhibitors of PGHS were all able to suppress PG production within 4 h. Although the PGHS inhibitors would almost certainly have achieved significant inhibition of PG synthesis in 1 h or less, acute experiments were conducted over 4 h because this is the shortest duration over which we were able to make reliable measurements of cortisol metabolism by 11ßHSD1 in the hGL cells. At 24 h, each of the tested PGHS inhibitors suppressed PG synthesis by approximately 50%. Given that each test compound was only partially successful in inhibiting PG synthesis, we were unable to create a cellular environment devoid of PGs. Hence, it seems likely that, even in the presence of the highest concentrations of indomethacin, MA, NA, and NS-398, residual PGs will still have exerted a positive effect on 11ßHSD1 activities in the hGL cells.

Each of the PGHS inhibitors suppressed PG synthesis at different concentrations, which was reflected in differential potencies for suppressing the bidirectional activities of 11ßHSD1 in the hGL cells. The differences in the IC50s of each compound for inhibiting PGHS activities (45, 46) almost certainly relates to differences in their chemical structures (Fig. 8Go). Differences in the structures of the PGHS inhibitors used in this study may also have affected their rate of uptake into cells, their solubilities within the cytosol and/or the rate at which they were transported back out of the cells. However, even though each PGHS inhibitor is structurally distinct, all four compounds significantly inhibited PG production and exerted very similar suppressive actions on the bidirectional activities of 11ßHSD1 in hGL cells.


Figure 8
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  		FIG. 8. Chemical structures of the PGHS inhibitors (adapted from Cayman Chemicals, Ann Arbor, MI).

 
The suppression of local PG synthesis with structurally distinct PGHS inhibitors had differential effects on cortisol oxidation and cortisone reduction; in general, the PGHS inhibitors had a greater effect on the reduction of cortisone than on cortisol oxidation. However, this apparent finding may be an artifact arising from the differences in the absolute levels of 11ß-DH vs. 11-KSR activities. Control rates of cortisol oxidation were more than double the control rate of cortisone reduction (8–10 pmol cortisol oxidized/5 x 104 cells.4 h vs. 3–4 pmol cortisone reduced/5 x 104 cells for 4 h). Hence, a fixed decrease in the absolute amount of substrate conversion to product would represent a greater proportion of the low 11-KSR activity compared with the higher 11ß-DH activity.

The concentrations of the tested inhibitors of PGHS required to suppress 11ßHSD1 activities corresponded with the concentrations at which these compounds significantly inhibited PG synthesis. These data could be interpreted as indicating a positive autocrine/paracrine role for PGs in stimulating 11ßHSD1 activities in hGL cells. The simplest alternative explanation of these findings would be that each of the tested compounds exerted a direct inhibitory action on the 11ßHSD1 enzyme, independent of their effects on PG synthesis. However, the ability of exogenous PGD2, PGF2{alpha}, and PGE2 to overcome the suppression of 11ßHSD1 activities by inhibitors of PGHS suggests that the suppressive effects of these compounds on cortisol oxidation are probably due to the decrease in PG concentrations. The ability of PGE2 to increase cortisol oxidation in a concentration-dependent manner adds further support to a model in which locally synthesized PGs elevate 11ßHSD1 activity in hGL cells. Such a model parallels that proposed by Alfaidy et al. (31, 32), whereby PGF2{alpha} induces 11ßHSD1 activity in the fetal membranes at parturition.

In studying the effects of PGE2 on 11ßHSD1 activity, it proved necessary to control for the confounding effects of PGE2 on progesterone production. Our previous reports have shown that suppression of local progesterone synthesis using AG can increase cortisol metabolism in hGL cells, implicating progesterone as an autocrine/paracrine inhibitor of 11ßHSD1 (13). In the absence of AG, the highest tested concentration of PGE2 (1000 nM) would have stimulated progesterone synthesis. The fact that 1000 nM PGE2 could only stimulate 11ßHSD1 activity in the presence of AG suggests that under physiological circumstances, simultaneous stimulation of progesterone synthesis by PGE2 may limit the effect of the PG on 11ßHSD1 activity with progesterone acting as an autocrine/paracrine inhibitor to oppose enzyme stimulation by PGE2. Hence, in the presence of progesterone, PGE2 would only be expected to stimulate cortisol metabolism when present at sub-luteotrophic concentrations.

The addition of PGs to the outside of the cells is likely to activate preferentially the cell surface, G protein-coupled PG receptors. In contrast, endogenous production of PGs by intracellular PGHS enzymes is more likely to activate nuclear receptors, such as peroxisome proliferator-activated receptor-ß and -{delta}. In the present study, PGD2, PGE2, and PGF2{alpha} could restore cortisol metabolism to control levels when endogenous PG synthesis had been inhibited by MA or indomethacin, even though these three PGs activate very different transmembrane signaling cascades. In general, PGD2 and PGE2 activate the G{alpha}s-adenylyl cyclase-cAMP-protein kinase A pathway, whereas PGF2{alpha} activates the G{alpha}q/11-DAG/IP3-Ca2+-protein kinase C signaling cascade. Based on available evidence, it would be premature to speculate as to the exact signaling mechanisms by which extracellular PGs, or indeed intracellular PGs, regulate cortisol metabolism.

It is certainly true that the effects of exogenous PGs added outside the hGL cells may not accurately reflect the actions of endogenous PGs generated within the cells by PGHS enzymes. Therefore, the alternative approach of elevating PG synthesis by challenging cells with a proinflammatory cytokine was used. IL-1ß has been reported to increase PG synthesis in a variety of cells by up-regulating PGHS-2 (35). In hGL cells, IL-1ß stimulated parallel increases in the concentrations of both measured PGs (PGE2 and PGF2{alpha}) and also increased the net oxidation of cortisol by 11ßHSD1. (Although the stimulation of PGF2{alpha} by IL-1ß failed to achieve statistical significance, the large variance in absolute levels of PG synthesis reduced the power of the statistical tests for this experiment to just 30% with only three experimental replicates). Cotreatment of cells with MA abolished the effects of IL-1ß on both PG production and cortisol oxidation, consistent with the hypothesis that the elevation of intracellular PGs in response to IL-1ß mediates the stimulation of 11ßHSD1 activity by this proinflammatory cytokine in hGL cells.

In experimental conditions where progesterone synthesis was suppressed using AG to inhibit the activity of the rate-determining enzyme, cytochrome P450 cholesterol side-chain cleavage, it was notable that MA did not significantly inhibit either PGE2 accumulation or cortisol oxidation by 11ßHSD1. Although we have no explanation for this at present, this finding would suggest that steroid output from hGL cells influences the ability of MA to suppress basal PG synthesis and, in turn, 11ßHSD1 activities. However, even in the presence of AG, IL-1ß could stimulate parallel increases in the synthesis of both PGE2 and PGF2{alpha} and in 11ßHSD1 activity, all of which could be effectively blocked by cotreatment with MA.

On balance, the most likely explanation of the findings reported herein is that the effects of the PGHS inhibitors and IL-1ß on 11ßHSD1 activities reflect changes in PG synthesis. The fact that suppression of enzyme activities by MA and NS-398 were not accompanied by changes in 11ßHSD1 protein expression implicates PGs in the posttranslational stimulation of 11ßHSD1 activities in hGL cells. Our findings suggest that in the ovulatory follicle, increased PG synthesis and action may increase 11ßHSD1 activity that would mediate local regeneration of the antiinflammatory steroid cortisol, hence limiting further synthesis of PGs in a novel paracrine feedback loop.


   Acknowledgments
 
We thank the staff at the Lister Private Hospital assisted conception unit (Chelsea, London, UK) for providing the hGL cells used for this study, and Mrs. Sarah Winyard (St. George’s University of London) for her assistance in the final production of this manuscript.


   Footnotes
 
This work was supported by Medical Research Council Ph.D. studentship ref: G69/1756 (awarded in support of K.C.J.).

Disclosure statement: All authors have nothing to declare.

First Published Online September 7, 2006

Abbreviations: AG, Aminoglutethimide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hGL, human granulosa-lutein; 11ß-DH, 11ß-dehydrogenase; 11ßHSD, 11ß-hydroxysteroid dehydrogenase; 11ßHSD1, 11ßHSD1 type 1; 11-KSR, 11-ketosteroid reductase; MA, meclofenamic acid; NA, niflumic acid; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NS-398, N-(2-cyclohexyloxy-4-nitorophenyl) methane sulfonamide; PG, prostaglandin; PGF2{alpha}, PG F2{alpha}; PGHS, PG H synthase; TLC, thin-layer chromatography.

Received May 31, 2006.

Accepted for publication August 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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