Differential effects of RU486 reveal distinct mechanisms for glucocorticoid repression of prostaglandin E 2 release Joanna E. Chivers 1 , Lisa M. Cambridge 1 , Matthew C. Catley 1 , Judith C. Mak 1 , Louise E. Donnelly 1 , Peter J. Barnes 1 and Robert Newton 2 1 Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London, Faculty of Medicine, London, UK; 2 Department of Biological Sciences, University of Warwick, Coventry, UK In A549 pulmonary cells, the dexamethasone- and budeso- nide-dependent repression of interleukin-1b-induced pros- taglandin E 2 release was mimicked by the s teroid antagonist, RU486. Conversely, whereas dexamethasone and budeso- nide were highly effective inhibitors of interleukin-1b- induced cyclooxygenase (COX)/prostaglandin E synthase (PGES) activity and COX-2 expression, RU486 (< 1 l M ) was a poor inhibitor, but was able to efficiently antagonize the effects of dexamethasone and budesonide. In addition, both dexamethasone and RU486 repressed [ 3 H]arachido- nate release, which is consistent with an effect a t the level of phospholipase A 2 activity. By contrast, glucocorticoid response element-dependent transcription was unaffected by RU486 but induced by dexamethasone and budesonide, whilst dexamethasone- and budesonide-dependent repres- sion of nuclear factor-jB-dependent transcription was maximally 30–40% and RU486 (< 1 l M ) was without significant effect. Thus, two pharmacologically distinct mechanisms of glucocorticoid-dependent repression of prostaglandin E 2 release are revealed. First, glucocorticoid- dependent repression of arachidonic acid is mimicked by RU486 and, second, repression of COX/PGES is antagon- ized by RU486. Finally, whilst all compounds induced glucocorticoid receptor translocation, no role for glucocor- ticoid response element-dependent transcription is suppor- ted in these inhibitory processes and only a limited role for glucocorticoid-dependent inhibition of nuclear factor-jBin the repression of COX-2 is indicated. Keywords: corticosteroid; cyclooxygenase; epithelial cell; glucocorticoid receptor; prostaglandin E 2 . Synthetic glucocorticoids are potent repressors of inflam- mation and are a first-line therapy for i nflammatory diseases [1]. However, their clinical usage is limited by immunosup- pression as well as by metabolic effects, including increased gluconeogenesis, i ncreased blood glucose, amino and fatty acid mobilization, and loss of bone [2]. In addition, endogenous glucocorticoids p articipate in feedback inhibi- tion of the hypothalamo-pituitary-adrenal axis, and long- term high-dose synthetic glucocorticoid usage may cause hypothalamo-pituitary-adrenal insufficiency and glucocor- ticoid dependency. Glucocorticoids are believed to act primarily via the glucocorticoid receptor (GR), which is maintained as an inactive cytoplasmic c omplex with heat shock proteins (hsp) and immunophilins [3]. Following ligand binding and complex dissociation, the GR translocates to the nucleus where it binds glucocorticoid response elemen ts (GREs), as a dimer, to promote the transcription of responsive genes [2]. However, the GR may also act a s a monomer to inhibit key inflammatory transcription factors, such as nuclear factor-jB(NF-jB) and activator protein-1, by direct interaction, competition for cofactors or by modifying the chromatin structure to prevent the expression of inflamma- tory genes [1,2]. Inflammatory prostaglandins, produced by the arachi- donic acid cascade, play a pathophysiological role in edema, bronchoconstriction, fever and hyperalgesia [4]. Arachidonic acid, released from cell membranes by phospholipase A 2 (PLA 2 ), is converted to prostaglandin H 2 (PGH 2 ) by c yclooxygenase enzymes ( COX), and further modified by specific isomerases and reductases to produce b iologically relevant prostaglandins, including prostaglandin E 2 (PGE 2 ), which is the major prostaglan- din product of both airway epithelial and A549 cells [5]. In inflammation, the inducible COX, COX-2, is normally up-regulated a nd accounts for the elevated levels of prostaglandins [4]. Conversely, COX-2 expression is highly sensitive to glucocorticoid inhibition, suggesting that inhibition of COX-2 is critical in t he repression of prostaglandins by glucocorticoids. As cytokine-induced COX-2 and PGE 2 release are highly NF-jB-dependent in A549 cells [6], and treatment with dexamethasone pro- foundly represses PGE 2 release a nd COX-2 expression [7], Correspondence t o R. Newton, Department of Biological Sciences, University of Warwick, Coven try CV4 7AL, UK. Fax: +44 2476 523701; Tel.: +44 2476 574187; E-mail: robert.newton@imperial.ac.uk Abbreviations: C OX, cyclooxygenase; CRE, cyclic A MP response element; DAPI , 4¢,6¢-diamidino-2-phenylinole dihydrochloric hydrate; EGF, ep idermal growth f actor; GR, glucocorticoid receptor; GRE, glucocorticoid response e lement; hsp, heat shock pr otein; IL,interleukin;NF-jB, nuclear factor-jB; PGE 2 , p rostaglandin E 2 ; PGES, prostaglandin E synthase; PLA 2 , phosph olipase A 2 ; SFM, serum-free media. (Received 13 January 2 004, revised 1 6 A ugust 2 004, accepted 23 August 2004) Eur. J. Biochem. 271, 4042–4052 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04342.x we have used this system to further explore the mecha- nisms of glucocorticoid action. Materials and methods Cell culture A549 cells were cultured to confluence, as described previously [7]. Following overnight incubation in serum- free media (SFM), d rugs (dexamethasone, budesonide, ionomycin, R U486) were a dded 1 h before stimulation with interleukin-1b (IL-1b) (R & D Systems, Oxon, UK). Dexamethasone and budesonide (both Sigma, Poole, UK) were dissolved in Hank’s balanced salt solution (Sigma). Ionomycin and RU486 (both Sigma) were dissolved in ethanol. Final concentrations of ethanol were less than 0.1% (v/v). PGE 2 release, COX/prostaglandin E synthase (PGES) activity and COX-2 expression PGE 2 released into the medium was measured using a commercially available PGE 2 antibody (Sigma) [5,8]. For the assay of combined COX/PGES activity, cells were rinsed with SFM prior to incubation at 37 °Cfor10minin SFM supplemented with 3 0 l M arachidonic acid, and released PGE 2 was taken as a index of COX/PGES a ctivity [5,8]. Northern and Western blot analyses were performed as described previously [7]. Reporter cell lines and luciferase assay A549 cells containing the N F-jB-dependent r eporter, 6jBtkluc, have been described previously [9]. The 1·GRE- dependent and 2·GRE-dependent reporters, pGL3.neo TATA.GRE and pGL3.neo.TATA.2GRE, respectively, were based on the parent vector pGL3.neo.TATA, which contains a modified minimal b-globin promoter, as pre- viously described [10]. This was digested at the SmaI site, upstream of t he minimal promoter, and double- stranded oligonucleotides (sense strand: 5¢-GC TGTACAG GATGTTCTAG-3¢ and 5¢-GCTGTACAGGATGTTC TAGGCTGTACAGGATGTTCTAG-3¢), containing one or two copies of a consensus GRE site (underlined) [11], were inserted to produce pGL3.neo.TATA.GRE and pGL3.neo.TATA.2GRE, respectively. A 2·GRE(mut ) reporter was generated as described above, but using a mutated 2·GRE oligonucleotide (sense strand 5¢-GC TcaACAGGATcaTCTAGGCTcaACAGGATcaT CTAG-3¢) (mutated bases in lower case). The cyclic AMP response element (CRE)-dependent reporter, which con- tains six CRE s ites, was as previously described [ 12]. A549 cells, stably harboring the luciferase reporters, were gener- ated as previously described [9]. Prior to experiments, confluent plates of reporter cells were incubated overnight in serum-free, G-418-free, media. Cells were subsequently harvestedin1· reporter lysis buffer (200 ll) (Promega) 6 h after treatment for luciferase activity assay (Promega). As each well is confluent and a ll the cells contain the reporter construct, we find reporter activity to be highly reproducible, and normalization to a second reporter is unnecessary [9]. [ 3 H]Arachidonic acid release As previously described [8], cells were incubated overnight in 0.5 mL of S FM supplemented with 0.125 lCi [5,6,8,9,11,12,14,15- 3 H]arachidonic acid (Amersham Phar- macia). Cells were washed twice prior to treatment with dexamethasone or RU486. After 1 h, supernatants were changed to fresh SFM containing 2 mgÆmL )1 fatty acid-free BSA (Sigma) plus drugs prior to stimulation. Supernatants were collected and cells washed prior to harvesting in 1% (w/v ) S DS. Relea se of [ 3 H]arachidonic acid, or its metabolites, was expressed as a percentage of the total incorporated. Ligand binding At 80% c onfluence, A549 cells cultured in T175 flasks w ere transferred to SFM and harvested the following day in cell dissociation solution (C-5789; Sigma). Cells (1.5–4 · 10 6 cells per mL) were incubated overnight at 4 °C with increasing concentrations of [ 3 H]dexamethasone, in the p re sence of 10 l M dexamethasone, to determine nonspecific binding. F ree radioligand was removed by t he rapid filtration of cells through glass-fibre filters (GF/B) presoaked i n NaCl/P i (PBS), 0.1% ( v/v) polyethylenimine, using a cell harvester [M-24R Brandel, SEMAT Technical (UK) Ltd, St. Albans, Hertfordshire, UK]. Filters were combined with Filtron-X scintillant (National D iagnostics, Atlanta, GA, USA) and radioactivity was measured using a beta counter (2200CA Tri-carb Liquid Scintillation Ana- lyser; Canberra Packard, Berks., UK). K d and B max. values were determined using saturation binding isotherms and Scatchard analysis, [Bound]/[Free] vs. [Bound], where the x-intercept ¼ B max. and the gradient ¼ ) 1/K d (Fig. 1 A) ( PRISM 3; GraphPad, San Diego, CA, USA). Relative binding affinity was assessed by incubating cells with an increasing concentration of unlabelled steroid and 4 n M [ 3 H]dexamethasone overnight at 4°C. Bound and free radioligand were separated as d escribed above. Specific binding was c alculated by s ubtraction of nonspecific from total binding, and Cheng–Prusoff analysis was performed to determine the K i value: K i ¼ IC 50 /{1 + ([Free Count]/K d )}, where IC 50 is the concentration that results i n 50% inhibi- tion (Table 1) [13]. Immunocytochemistry Cells grown on coverslips were transferred at 70% conflu- ence to SFM f or 24 h. After incubation with steroid for the indicated times, cells were washed with NaCl/P i (PBS) and fixed with 4% (w/v) paraformaldehyde before successive incubations in 0.5% (v/v) Nonidet P-40 and 100 m M glycine. Coverslips were blocked in NaCl/P i (PBS) contain- ing 0 .1% ( v/v) Tween- 20, 0.1% (w/v) BSA and 10% (v/v) human serum prior to incubation for 1 h in 5 lgÆmL )1 rabbit anti-human GR (PA1–511A; Affinity Bioreagents Inc., Golden, CO, USA) or rabbit isotype con trol (Dako, Glostrup, Denmark). After washing with NaCl/P i (PBS) containing 0.1% (v/v) Tween and incubation with biotin- ylated anti-rabbit immunoglobulins (Dako) for 1 h, cells were incubated with fluorescein isothiocyanate (FITC)- linked s treptavidin (Dako) for 1 h. Nuclei were then stained Ó FEBS 2004 Glucocorticoid repression: differential mechanisms (Eur. J. Biochem. 271) 4043 with 1 l M 4¢,6¢-diamidino-2-phenylinole dihydrochloric hydrate (DAPI) (Sigma) and coverslips were mounted on glass slides using Citifluor mounting fluid (Citifluor Ltd, London, UK), prior to analysis using a Leica TCS 4D confocal microscope (Leica Microsystems, M ilton Keynes, UK) equipped with argon, krypton, and ultraviolet lasers. Confocal images w ere acquired at ·40 magnification using TCS NT software (Leica Microsystems). Statistical analysis Statistical a nalysis was perf ormed u sing analysis of variance ( ANOVA ) w ith a Dunn’s post-test, unless specifically stated otherwise in the figure legends. Significance was taken at P-values of < 0.05 (*), < 0.01 (**) and < 0 .001 (***). Results Repression of PGE 2 release, COX/PGES activity and COX-2 expression As reported previously [7,14], untreated A549 cells released low levels of PGE 2 (1.2 ± 0.2 ngÆmL )1 ) and showed low levels of combined COX/PGES activity (3.1 ± 0 .6 ng Æ mL )1 Æmi n )1 ), which were both increased upon stimulation with IL-1b (1 ngÆmL )1 ) (22.6 ± 3.7 ngÆmL )1 and 0 50 100 -7-6 -5 -10 -9 -8 -7 -6 -5 NS IL-1 Log [Bud] (M)Log [RU486] (M) 0 50 100 -7-6 -5 -10 -9 -8 -7 -6 -5 NS IL-1 Log [Bud] (M)Log [RU486] (M) PGE 2 release (% IL-1 ) 0 50 100 -10 -9 -8 -7 -6 -5 Log [Steroid] (M) NS IL-1 0 50 100 0 50 100 Log [RU486] (M) -10 -9 -8 -7 -6 -5 NS IL-1 IL+Dex Log [RU486] (M) -10 -9 -8 -7 -6 -5 NS IL-1 IL+Dex 0 50 100 0 50 100 -7-6 -5 -10 -9 -8 -7 -6 -5 NS IL-1 Log [Dex] (M)Log [RU486] (M) -7-6 -5 -10 -9 -8 -7 -6 -5 NS IL-1 Log [Dex] (M)Log [RU486] (M) PGE 2 release (% IL-1 ) COX/PGES activity (% IL-1 ) -11 COX/PGES activity (% IL-1 ) 0 50 100 -10 -9 -8 -7 -6 -5 Log [Steroid] (M) NS IL-1 -11 IL+Bud IL+Bud ABC D Fig. 1. The e ffect of dexamethasone, budesonide and R U486 on inte rleukin-1b (IL-1 b)-dependent prostaglandin E 2 (PGE 2 ) release and cyclooxygenase (COX)/prostaglandin E synthase (PGES) activity. (A) A549 cells were cultured with various concentrations of dexamethasone (j), budesonide (h) or RU486 (.) for 1 h prior to stimulation with IL-1b (1 ngÆmL )1 ) or no stimulation (NS). (B) Cells were treated with dexamethasone (Dex) (0.1 l M )(j) or b udesonide (0.1 l M )(h), in th e presence of increasing c oncen trations of RU486, for 1 h prior to stimulation w ith IL-1b (1 ngÆmL )1 ) or no stimulation (NS). (C and D ) Cells were treated w ith various c onc entrations of d examet hasone (C) or budesonide (D) in the abse nce (j)or presence of RU486 at 0 .1 l M (h), 1.0 l M (d)or10.0l M (s) fo r 1 h prior to stimulation with IL-1b (1 n gÆmL )1 ) or no stimulation (NS). In a ll cases, PGE 2 release ( upper panels of A, B an d C and t he left panel o f D) and COX/ PGES activity (lower panels of A, B and C a nd the right p anel of D) were analy zed a fter 24 h. Data ( A a nd B, n ¼ 5–7; C, n ¼ 4; D, n ¼ 4) are e xpressed a s a percentage of the response to IL-1b an d p lotted as mean ± SEM. T he following levels of significanc e w ere e stablished , e xpresse d a s P-values of < 0.05 (* ), < 0 .01 (**) and < 0.001 (***). (A) (upper p anel) Budesonide at 10 )8 (**), 10 )7 M (***) a nd 10 )6 M (***); dexamethaso ne a t 1 0 )8 M (**), 10 )7 M (***), 10 )6 M (***) and 10 )5 M (***); and RU486 at 10 )6 (*) and 10 )5 (***). (A) (lower panel) Budesonide and dexamethasone at 10 )7 (***), 10 )6 (***) and de xamethasone at 10 )5 M (***). (B) (lower panel) Bud esonide + RU486 at 10 )6 M and 10 )5 M (both ***); and dexamethasone + R U486 a t 10 )6 M and 1 0 )5 M (both ***). 4044 J. E. Chivers et al. (Eur. J. Biochem. 271) Ó FEBS 2004 32.8 ± 2.0 ng ÆmL )1 Æmin )1 , respectively). In each case the IL-1b-induced release of PGE 2 and combined COX/PGES activity were repressed in a concentration-dependent man- ner to near-basal levels by dexamethasone [50% effective concentration (EC 50 ) values of 1.9 n M and 3.2 n M , respect- ively) and budesonide (EC 50 values of 2.6 n M and 7.8 n M , respectively) (Fig. 1A, upper and lower panels). Similarly, RU486 produced a concentration-dependent repression of IL-1b-induced PGE 2 release (EC 50 ¼ 33.1 n M ) (Fig. 1A, upper panel), yet was considerably less effective against combined COX/PGES activity, with concentrations of less than 1 l M being without significant effect (EC 50 ¼ 5 l M ) (Fig. 1 A, lower panel). This effect was even more apparent when RU486 was used to antagonize the responses to dexamethasone and budesonide. Thus, whereas the glucocorticoid-depend ent inhibition of IL-1b-induced PGE 2 release was not antag- onized (Fig. 1B, upper p anel), the i nhibition of COX/PGES activity was effectively antagonized by RU486 (Fig. 1B, lower panel). The abilities of dexamethasone and budeso- nide to inhibit both PGE 2 release and COX/PGES activity were further t ested in t he presence of various concentrations Table 1. K i and functional properties of steroid ligands in A549 cells. Cheng–Prusoff analy sis wa s pe rformed using 50% inhibitory concentration (IC 50 ) v alues and glu cocortic oid receptor (GR) number gen erated by s aturation and c ompetition-bindin g studies (see Fig. 6). Data (n ¼ 3–5) are presented as m e an ± SEM. S ee the text for a full description of o utpu t measurements. COX, c ycloo xygenase; EC 50 , 50% effective concentratio n; GRE, glucocorticoid response element; NF-jB, n uclear f actor-jB; PGE2, p rostaglandin E 2 ; PGES, prostaglandin E syn thase. Steroid ligands Radioligand binding EC 50 (n M ) for steroid effect on various functional outputs K i (n M ) PGE 2 release COX/PGES activity GRE (23GRE) NF-jB (6jBtk) Dexamethasone 4.9 ± 1.3 1.9 ± 1.4 3.2 ± 1.3 54.5 ± 23.6 3.2 ± 2.2 Budesonide 1.2 ± 0.4 2.6 ± 1.6 7.8 ± 1.9 65.3 ± 29.7 6.6 ± 3.5 RU486 0.5 ± 0.2 33.1 ± 2.4 4995 ± 23 – 1434 ± 837 A B C D Fig. 2. Effect of d examethasone and RU48 6 on cyclooxygenase-2 ( COX-2) expression. (A) Cells were either not stimulated ( NS) or pretreated w ith various concentrations of dexam ethasone (Dex) or RU 486 for 1 h prior to stimulation with interleukin-1b (IL -1b)(1ngÆmL )1 ). Cells were harvested a t 6 h for RNA, and N orthern blot (NB) a nalysis was performed f or COX-2 and glyc eraldehyde-3-phosphate dehydrogenase (GAPDH). Cells harvested at 24 h were subject to Western blot (WB) analy sis for COX-2. (B) Cells were pretreat ed for 1 h with dexamethasone (0.1 l M )(Dex) in the presence of various concentrations of RU486. Cells were harvested as described in (A) f or Northern and Western blot analyses. In each case, blots representative of t hree or m ore su ch experimen ts a re shown. (C) Following densitometric analysis, data (n ¼ 4–6) (upper panels, Western blots; lo wer p an els, N orthern blots) from th e e xperiments in (A) were expressed as a percentage of IL -1b, treated and plotted as m ean ± SEM. (j), De xametha sone; (.), RU486. (D ) D ata ( n ¼ 4–5) f rom the experiments i n ( B) were plotted a s d escribed in (C). Ó FEBS 2004 Glucocorticoid repression: differential mechanisms (Eur. J. Biochem. 271) 4045 of RU486 (Fig. 1C,D). As shown by the rightwards shift and the reduced apparent efficacy o f the inhibition curves described for both dexamethasone and budesonide, the glucocorticoid-dependent repression of COX/PGES activity was clearly antagonized by increasing the concentration of RU486. However, in marked contrast, RU486 primarily resulted in an increased overall inhibition of the response curves described for dexamethasone and budesonide on PGE 2 release, as shown by the progressive flattening of the respective lines (Fig. 1C,D). These data are t herefore indicative of a primary inhibitory effect of RU486 on PGE 2 release, but not on combined COX/PGES activity. Analysis of COX-2 mRNA and protein expression, which is responsible for the inflammatory release of PGE 2 from A549 cells [5,15], often revealed basal l evels of expression, as reported previously [16]. However, in each case, and as previously shown, COX-2 expression was dramatically in creased by treatment with IL-1b [7,14]. Consistent with the combined COX/PGES data, the analysis of COX-2 mRNA and protein expression revealed a concentration-dependent inhibition of COX-2 expression by dexamethasone, whereas RU486 showed little effect except at h igh doses (Fig. 2A,B). Consistent with Fig. 1B, 0.1 l M dexamethasone a lmost t otally repressed both mRNA and protein expression of COX-2, and this effect was efficiently antagonized by RU486 (Fig. 2B). Effect of dexamethasone and RU486 on arachidonic acid release To investigate the possibility of an effect of steroids upstream of COX-2, ce lls were loaded with [ 3 H]arachidonic acid prior to stimulation in the presence of dexamethasone or RU486. As I L-1b alone is a poor activator of arachidonic acid release [8], cells were also treated with ionomycin or with IL-1b + ionomycin, w hich provides a Ca 2+ stimulus, causing translocation and membrane association of cyto- solic (c)PLA 2 to markedly enhance cPLA 2 activity [8 ,17,18]. IL-1b, ionomycin and IL-1b + ionomycin increased [ 3 H]arachidonic acid release by 1.6-fold, 3.2-fold and 7.2- fold, respectively (Fig. 3A). In each case, dexamethasone produced repressions of 50, 61 and 68%, whilst RU486 resulted in rep ressions of 58, 5 3 and 63%, respectively. To further characterize this inhibition, cells were treated with various concentrations of either dexamethasone or RU486 prior t o s timulation with IL-1b + i onomycin. I n each case, a concentration-dependent inhibition of [ 3 H]arachidonic acid release (EC 50 ¼ 1 8.7 ± 10.6 and 26.2 ± 11.6 n M , respectively) was observed, thereby confirming the inde- pendent inhibitory effect of RU486 acting at the level of arachidonic acid release (Fig. 3B). Transactivation and transrepression by glucocorticoids and RU486 The effect of dexamethasone and R U486 was a nalyzed on GRE-dependent and N F-jB-dependent transcription. From the 1·GRE r eporter, pGL3.neo.TATA.GRE, GRE-dependent transcription was increased by  4.5-fold (EC 50 ¼ 46 .7 ± 17.7) by dexamethasone and fivefold (EC 50 ¼ 53.5 ± 20.8 n M , r espectively) by budesonide (Fig. 4 A). Similarly the 2·GRE-driven reporter, pGL3.neo.TATA.2GRE, gave r ise t o o ver a 15-fold (EC 50 ¼ 54.5) induction by dexamethasone and a 20-fold induction (EC 50 ¼ 65.3 n M ) by budesonide (Fig. 4B). No response was observed with reporters containing either mutated GRE elements (pGL3.neo.TATA.2GREmut) or no GRE s ites (pGL3.neo.TATA) ( data not shown), w hich confirms the specificity of these reporter systems for the presence of GRE sites. In e ach case, RU486 showe d little or no ability to activate GRE-dependent transcription (Fig. 4 A,B), but demonstrated a profound ability to antag- onize both 1·GRE and 2·GRE reporter activity induced by 0.1 l M of either dexamethasone or bu desonide (Fig. 4C,D). Analysis of IL-1b-induced NF-jB-dependent transcrip- tion revealed a modest 30–40% inhibition (EC 50 ¼ 3 H arachidonic acid release (% of total incorporated) NS IL-1 Iono IL+Iono Dex Ru486 0 5 10 * * * ** ** ** *** *** ** ** ** * 0 50 100 3 H arachidonic acid release (% IL-1 + ionomycin) -10 -9 -8 -7 -6 -5 Log [Steroid] (M) NS IL+Iono A B Fig. 3. Inhibition of arachidonic acid release by dexamethasone and RU486. ( A) Following loading with [ 3 H]arachidonic acid, c ells were either not treated or pretreated with dexameth asone (1 l M )(Dex)or RU486 (1 l M ) for 1 h. Cells were then either not stimulated (NS) or stimulated wi th interleukin-1b (IL-1b)(1ngÆmL )1 ), ion om ycin (3 l M ) (Iono) or both together (IL + Iono), and the supe rnantants and cells were harvested after 1 h for liqui d s cintillation counting. Data (n ¼ 4 or 5) are shown as arachidonate release expressed as a percentage of the total incorporated ± SEM. Significance was assessed using the Student’s t-test. *P <0.05,**P < 0 .01. (B) Cells were treated as in (A) except that various concentrations of either dexamethasone (j)or RU486 (.) were added p rior t o t he IL-1b (1 ngÆmL )1 )+ionomycin (3 l M ) stimulus. After harvesting, 1 h fo llowing stimulation, arachi- donate re lease as a fraction of the total incorporated was expressed as a percentage of the IL-1b + ionomyc in stimulus and plotted as mean ± SEM. Significance was assessed using analysis of va riance ( ANOVA ) with a Dunn’s post-test. **P < 0.01, ***P <0.001. 4046 J. E. Chivers et al. (Eur. J. Biochem. 271) Ó FEBS 2004 3.2 ± 1.3 a nd 7.8 ± 1 .9 n M ) by d examethasone and budesonide, respectively, and just over a 50% inhibition by 10 l M RU486 (Fig. 5A). RU486 was without effect at 0.1 l M and required to be present at concentrations of  100-fold higher than either dexamethasone or budesonide to achieve similar levels (30–40%) of inhibition. It is worth noting that the inhibition of NF-jB by RU486 correlates very closely with the effects o bserved on COX activity and COX-2 expression (Figs 1 and 2 ). In ad dition, the ability of RU486 to antagonize the repressive effects of 0.1 l M dexamethasone or budesonide was examined. In each case, a concentration-dependent antagonism was observed up to a maximum of 0.1 l M RU486 (Fig. 5B). Above this concentration, increasing levels of inhibition were observed owing to the r epressive effect of RU486 acting alone (data not shown and see Fig. 5 A). The expression of COX-2 m ay also depen d on activatin g transcription factors (ATFs) and activator protein-1 (AP-1)- like factors acting at a CRE site located in the proximal region of the COX-2 promoter [19–21]. Consistent with this, we have previously found that a CRE-driven reporter construct was unresponsive to cAMP in A549 cells, but responded t o IL-1b [10]. This was not believed to reflect a general problem with this reporter, as strong cAMP-indu- cibility has be en d emonstrated in other experimental systems [12]. Consistent with these earlier fin dings, IL-1b was s hown to induce r eporter activity twofold (Fig. 5C). In each c ase, both dexamethasone (0.1 l M ) and RU486 (10 l M )were found to produce marked repressive effects (Fig. 5C). Binding affinity of steroid ligands and effect on GR translocation Saturation binding studies using [ 3 H]dexamethasone dem- onstrated one-site binding in A549 cells and revealed 16 500 ± 2700 GR/cell w ith an affinity of 1.36 ± 0.10 n M , which is consistent with other reports, including primary epithelial cells, indicating an affinity in the low n M range (Fig. 6A) [22–24]. Competitive binding studies were performed to examine the relative GR-binding affinity of these steroid ligands, and the following rank order of affinity was observed : RU486 > budesonide > dexameth- asone (Fig. 6B). The appropriate K i values are given in Table 1. 0 1 2 3 4 5 6 -11 -10 -9 -8 -7 -6 -5 0 5 10 15 20 -11 -10 -9 -8 -7 -6 -5 NS NS Log [Steroid] (M)Log [Steroid] (M) Fold activation Fold activation 0 20 40 60 80 100 120 -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 -10 -9 -8 -7 -6 -5 Log [RU486] (M)Log [RU486] (M) Luciferase activity (% Dex) Luciferase activity (% Dex) NS Dex Bud RU486 NS Dex Bud RU486 *** *** *** *** *** *** 1 GRE 1 GRE 2 GRE 2 GRE AB DC Fig. 4. Effect o f d examethasone, budesonide and RU486 on glucocorticoid response element (GRE)-dependent tr anscription. (A) 1·GRE o r ( B) 2·GRE A549 reporter cells were either not stimulated (NS) or treated with various concentrations of dexamethasone (j), budesonide (h)or RU486 (.). After 6 h, cells were harvested f or luciferase assay. Data ( n ¼ 6–10), expressed as f old induction, are plotted as m eans ± SEM. (C) 1·GRE and (D) 2·GRE A549 reporter cells were activated by dexamethasone (0.1 l M )(j) or budesonide (0.1 l M )(h) in the presence of various concentrations of RU486. Cells were harvested as described above, and luciferase activity, expressed as a percentage of the activity induced by dexamethasone (0.1 l M ), was plotted a s mean ± SEM. The effect of no stimulation (NS), or of stimulation with dexamethasone (0.1 l M )(Dex), budesonide (0.1 l M ) ( Bud) or RU486 (10 l M ) a lone, is also shown. All data are n ¼ 6–10. In (A) and (B), the indicated levels of significance apply to both budesonide and dexamethasone. In addition, the followi ng levels of significance were established, expressed as P-values of < 0.05 (*), < 0.01 (**) and < 0.001 (*** ). (B) B udesonide at 10 )8 M (**) and dexamethasone at 10 )8 M (*). (C) B udeson ide + RU486 at 1 0 )7 M (**), 10 )6 M (**) and 10 )5 M (***); dexamethasone + RU486 at 10 )7 M (**), 10 )6 M and 10 )5 M (***). (D) Budesonide + RU486 at 10 )6 M (**), and 10 )5 M (**); dexamethas one + RU486 a t 10 )7 M (*), 10 )6 M (**) and 10 )5 M (**). Ó FEBS 2004 Glucocorticoid repression: differential mechanisms (Eur. J. Biochem. 271) 4047 Nuclear translocation of GR by dexamethasone and RU486 Dexamethasone induced a rapid (within 15 min) transloca- tion of GR from the c ytoplasm to the nuclear compartment, with complete translocation observed by 1 h ( Fig. 7A ). Similarly, and as e xpected, nuclear translocation of GR was also induced by budesonide (Fig. 7B). In addition, RU486 was also efficient at inducing GR nuclear translocation, indicating that binding of the antagonist can result in dissociation of the cytoplasmic hsp–GR complex (Fig. 7B). Analysis of an isotype-control a ntibody revealed no s igni- ficant i mmunoreactivity, suggesting that the observed signal was GR-specific (Fig. 7C). Discussion In the above studies, dexamethasone and budesonide produced a near-total inhibition of both PGE 2 and COX/ PGES activity, and acted with similar efficacies (Table 1) and potencies. However, whilst the steroid receptor antag- onist, RU486, showed reversal of both C OX-2 expression and COX/PGES a ctivity, which is c onsistent with a GR-dependent mechanism, RU486 was incapable of ant- agonizing the repression of IL-1b-induced PGE 2 release produced by either dexamethasone or budesonide. I n fact, RU486 resulted in the progressive repression of PGE 2 release at increasing concentrations. Analysis of RU486 alone on IL-1b-induced PGE 2 release revealed a concentra- tion-dependent inhibition of PGE 2 release, yet s howed little or no effect on COX/PGES activity or COX-2 expression until RU486 concentrations of 1 l M were reached. This clear discrepancy stro ngly suggests that RU486 may exert an inhibitory effect upstream o f COX-2, possibly at the level of PLA 2 and arachidonic acid release. This proposal was confirmed by the analysis of [ 3 H]arachidonate release, which revealed concentration- dependent inhib ition by both dexamethasone and RU486. Interestingly, the EC 50 values for repression of PGE 2 release, and the repression of arachidonic acid release by RU486 (33.1 and 26.2 n M , respectively), correlate closely and therefore support the suggestion of a mechanistically distinct action for RU486 at the level of arachidonic acid release. We therefore conclude that these data docu ment the existence o f at least two f unctionally distinct processes for the inhibition of inflammatory PGE 2 release by ste roid s. In the fi rst mechanism, glucoco rticoids, such as dexameth- asone or budesonide, inhibit the expression of COX-2, and this response is antagonized efficiently by RU486. This contrasts w ith a second, and p harmacologically distinct mechanism, which occurs at the level of arachidonic acid release, in which the actions of glucocorticoids are mimicked by RU486. Previous reports have also documented the inhibition of arachidonic acid release in A549 cells by dexamethasone [25]. H owever, t hese authors did not report any inhibition by RU486 (10 n M ) alone [26], and showed a 50% antag- onism of the dexamethasone-dependent repression when using RU486 at 10 l M [25]. In an attempt to reconcile the apparent differences between the r esults of these reports and those of the present study, it is noticeable that different mechanisms of stimulation w ere u sed in each of the studies, and this alone could account for any differen ces. Further- more, inspection of our current data on the repression of both PGE 2 release and arachidonic acid release, suggests that the effects of 10 n M RU486 could be at the margins of experimentally discernable r epression (see Figs 1A and 3B). We also note that Croxtall et al. did not seemingly test higher concentrations of RU486 acting alone for an inhibitory effect o n epidermal g rowth factor (EGF)-stimu- lated arachidonic acid release [25]. This therefore leaves open the possibility that the incomplete antagonism of RU486 observed on dexamethasone-dependent repression of EGF-stimulated arachidonic acid release is, in fact, Luciferase activity (% IL-1 ) Dex RU486 80 70 60 -10 -9 -8 -7 Dex Bud IL-1 Log [Steroid] (M) NS IL-1 -10 -9 -8 -7 -6 -5 0 100 50 Luciferase activity (% of IL-1 ) IL-1 NS Fold activation 0 1 2 3 * ** NF- B NF- B CRE 100 90 Log [RU486] (M) A B C Fig. 5. Transrepression by dexamethasone, budesonide and RU486. (A) 6jBtk reporter cells were either not treated or were treated with various concentrations of dexamethasone (j), budesonide (h)orRU486(.) for 1 h, prior to stimulation with IL-1b (1 ngÆmL )1 ) or no stimulation (NS). After 6 h, cells were harvested for analysis in the luciferase a ssay. Data ( n ¼ 8), expressed a s percentage of the r esponse to IL-1 b stimulation, are plotted as mean ± SEM. Significance was established, expressed as P-values of < 0.05 (*), < 0.01 (**) and < 0.001 (***), for: budesonide at 10 )8 M (*), 10 )7 M (**) and 10 )6 M (**); dexamethasone at 10 )7 M (**), 10 )6 M (***) and 10 )5 M (***); and RU486 at 10 )5 M (**). (B) 6jBtk reporter cells, were treated with dexamethasone (0.1 l M )(j) or budesonide (0.1 l M )(h) in the presence of various concentrations of RU486. Luciferase assay data (n ¼ 7–9), expressed as a percentage of the response to IL-1b, are plotted as mean ± SEM. The effect of IL-1b + dexamethas one (0.1 l M )(Dex)andIL-1b + bud esonide (0.1 l M ) ( Bud) alone are sh own. ( C) C RE re porter c ells w ere e ither n ot t reated o r were treated for 1 h with dexamethasone (0.1 l M )orRU486(10l M ) prior to no stimulation (NS) or s timu lation with IL-1b (1 ngÆmL )1 ), as in dicated. Cells were harvested after 6 h for analysis in the luciferase assay, as described above. Data (n ¼ 6), expressed as fold activation, are plotted as mean ± SEM. *P < 0.05, **P <0.01. 4048 J. E. Chivers et al. (Eur. J. Biochem. 271) Ó FEBS 2004 attributable to a partial agonistic effect of RU486 acting alone [25]. It is well established that glucocorticoids can repress the transcription of inflammatory genes via transcription factors such as NF-jB [1,2]. However, whilst s ome degree (30–40% inhibition) of glucocorticoid-dependent inhibition of NF-jB-dependent transcription was observed in response to both dexamethasone and budes- onide, this effect is clearly insufficient to account for the near-complete repression of COX-2 expression or PGE 2 release observed with each of these compounds. As PGE 2 release and COX-2 expression in A549 cells is highly NF-jB-dependent, and this level of inhibition of NF-jB-dependent transcription correlates very well with our previous observation that the IL-1b-induced COX-2 transcription rate was inhibited by  40 % by dexameth- asone, we are compelled to suggest that additional mechanisms of glucocorticoid-dependent repression of COX-2 must also e xist [6,7]. Similarly, whilst G RE- dependent transcription was robustly increased following dexamethasone and budesonide treatment, this mechan- ism is unlikely t o account for the repression of COX-2 o r COX/PGES activity, as the EC 50 for this effect is greater than 10-fold more than that required for the inhibition of PG E 2 release o r COX/PGES a ctivity (Tab le 1 ). Interestingly, this shift in the concentration–response curve for transactivation effects at GREs (EC 50 values of 54.5 and 65.3 n M for dexamethasone and budesonide, respectively) when compared with transrepression, for example of NF-jB(EC 50 values of 3.2 and 7.8 n M for dexamethasone and budesonide, respectively), has been previously reported , although t he exact mechanistic explanation is currently lacking [27]. Therefore, in respect of COX-2, these data suggest that other, non-NF-jB- mediated and probably non-GRE-mediated, mechanisms of dexamethas one-dependent inhibition must be in operation to account for the full repression of COX-2 and COX/PGES activities in these cells. By contrast, the inhibition of NF-jB-dependent tran- scription by high c oncentrations of RU486 correlated v ery closely, in terms of both apparent efficacy a nd potency, with the inhibition of COX/PGES activity, thereby providing further strength to the argument that additional mecha- nisms, other than the inhibition of NF-jB, account for the inhibition by dexamethasone. However, the basis of this inhibition by RU486 is currently unclear to us because these levels of steroid are vastly in excess of that necessary to saturate GR, as suggested by our own, and previously reported [24,28], ligand-binding studies (Fig. 6). It is pos- sible that at these high concentrations RU486 is acting in a GR-independent manner. Notwithstanding the inhibition at high doses, it is clear that at concentrations of 1 or 0.1 l M , RU486 sho ws a limited or no effect on NF-jB-dependent transcription, yet is effective at inhibiting both PGE 2 and arachidonic acid release, suggesting that the inhibition of NF-jB plays no role in this response. Previous studies have suggested that, relative to dexamethasone, RU486 is a poor inducer of glucocorti- coid-dependent transcription [29–35]. Similarly, in the present study, RU486-induced GRE-dependent transcrip- tion from either a 1·GRE or a 2·GRE reporter was virtually absent, and this is consistent with data from primary human bronchial epithelial cells [24]. These data therefore raise the po ssibility that R U486 inhibits arachidonic acid release via a mechanism that is independent of transcription. Indeed, the rapid dexa- methasone-dependent repression of EGF-induced release of arachidonic acid was previously shown to be actino- mycin D insensitive and therefore independent of tran- scription [25]. In this respect, RU486 has previously been shown to mimic other nongenomic glucocorticoid responses, including the down-regulation of GR itself [36,37]. Certainly, our data indicate that RU486, can, like dexamethasone and budesonide, bind to and induce the nuclear translocation of GR. We therefore speculate that binding of ligand, including antagonists such as RU486, to GR, and complex dissociation, may be sufficient for the inhibition of arachidonic acid release and that this represents a mechanistically distinct event from the inhibition of inflammatory gene express ion . In this context i t is notable that various nongenomic actions of steroid hormones have been id entified [38,39], which raises the possibility o f ligand-dependent nongenomic anti-inflammatory functions for GR or for GR-associated -11 -10 -9 -8 -7 -6 -5 0 25 50 75 100 Log [Steroid] (M) Specific binding (%) 0 10 20 30 0 2.5 5.0 7.5 10 Log [Dex] (nM) Specific Binding x 10 3 (dpm) Specific Binding x10 2 (dpm) 0 5 0 7 5 1 0 0 0 1 2 3 4 Bound/Free x 10 3 2 5 A B Fig. 6. Analysis of glucocorticoid receptor (GR) number and relative affinity o f ligands. (A) A typical saturation–binding isotherm, showing specific GR binding using 2.4 · 10 6 cells and resulting Scatchard analysis (inset), wh ere the ratio o f free t o bound radioligand is p lotted against log [steroid ] to give a straight line with a gradient equal to )1/K d and and an x intercept that equals B max . (B) Competition binding curves showing relative affinity in A549 cells, where dexa- methasone ( j), bu desonide ( h), RU24858 ( d)orRU486(.)compete with 4 n M [ 3 H]dexamethasone to b ind the GR . D ata are pre sented a s mean ± SEM for n ¼ 3–5 observations. Ó FEBS 2004 Glucocorticoid repression: differential mechanisms (Eur. J. Biochem. 271) 4049 proteins present in the GR–hsp complex. Finally, we should point out that a number of effects of glucocor- ticoids, which are independent of the classical GR, are also reported to occur a nd these could help to e xplain our results [39]. Thus, the mineralocorticoid receptor may mediate glucocorticoid responsiveness in the brains of GR knockout mice [40]. In addition, a pharmacologically distinct pool of membrane-localized glucocorticoid recep- tors have been identified by various authors [39]. For example, a membrane glucocorticoid receptor has been biochemically identified in amphibians [41]. However, it is currently unclear whether this represents a version of the classical GR [42] or P-glycoprotein/multiple drug resistance gene, a member of the ATP-binding cassette (ABC) transporters [43,44], or some other receptor [45]. In this context, P-glycoprotein is of interest as it actively exports certain steroids, and blocking its function has been shown to promote glucocorticoid actions [46,47]. In conclusion, we present data w hich further confirm t hat the inhibition of NF-jB-dependent transcription cannot account for all the repressive effects of glucocorticoids on inflammatory genes such as COX-2. Furthermore, we present e vidence t hat glucocorticoids and RU486 also inhibit the r elease of arachidonic acid via a process that does not involve either inhibition of NF-jB or the activation of GRE-mediated transcription and which is mechanistically distinct from the inhibition of COX-2. 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Differential effects of RU486 reveal distinct mechanisms for glucocorticoid repression of prostaglandin E 2 release Joanna E. Chivers 1 ,. distinct mechanisms of glucocorticoid- dependent repression of prostaglandin E 2 release are revealed. First, glucocorticoid- dependent repression of arachidonic