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The Journal of Immunology, 2004, 172: 7677-7683.
Copyright © 2004 by The American Association of Immunologists

15-Deoxy-{Delta}12,14-Prostaglandin J2 Inhibits Glucocorticoid Binding and Signaling in Macrophages through a Peroxisome Proliferator-Activated Receptor {gamma}-Independent Process1

Adeline Cheron, Julie Peltier, Joëlle Perez, Agnès Bellocq, Bruno Fouqueray and Laurent Baud2

Institut National de la Santé et de la Recherche Médicale, Unité 489, Service d’Explorations Fonctionnelles Multidisciplinaires, AP-HP Hôpital Tenon, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
15-Deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) is involved in the control of inflammatory reaction. We tested the hypothesis that 15d-PGJ2 would exert this control in part by modulating the sensitivity of inflammatory cells to glucocorticoids. Human U937cells and mouse RAW 264.7 cells were exposed to 15d-PGJ2, and binding experiments were performed with [3H]dexamethasone as a glucocorticoid receptor (GR) ligand. 15d-PGJ2 caused a transient and concentration-dependent decrease in [3H]dexamethasone-specific binding to either cells through a decrease in the number of GR per cell without significant modification of the Kd value. These changes were related to functional alteration of the GR rather than to a decrease in GR protein. They did not require the engagement of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), because the response to 15d-PGJ2 was neither mimicked by the PPAR{gamma} agonist ciglitazone nor prevented by the PPAR{gamma} antagonist bisphenol A diglycidyl ether. 15d-PGJ2 altered GR possibly through the interaction of its cyclopentenone ring with GR cysteine residues because the cyclopentenone ring per se could mimic the effect of 15d-PGJ2, and modification of GR cysteine residues with methyl methanethiosulfonate suppressed the response to 15d-PGJ2. Finally, 15d-PGJ2-induced decreases in glucocorticoid binding to GR resulted in parallel decreases in the ability of GR to activate the transcription of a glucocorticoid-inducible reporter gene and to reduce the expression of monocyte chemoattractant protein-1. Together these data suggest that 15d-PGJ2 limits glucocorticoid binding and signaling in monocytes/macrophages through a PPAR{gamma}-independent and cyclopentenone-dependent mechanism. It provides a way in which 15d-PGJ2 would exert proinflammatory activities in addition to its known anti-inflammatory activities.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Arachidonate is converted through the cyclooxygenase (COX)3 pathway to PGD2 and by nonenzymatic dehydration of PGD2 to 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) during both the initial and the resolution phase of inflammatory process (1). Administration of COX inhibitors during the first phase inhibits inflammation, whereas their administration during the later phase exacerbates it (1). This observation raises the possibility that 15d-PGJ2 could exert sequentially pro- and anti-inflammatory activities.

The proinflammatory properties of 15d-PGJ2 remain poorly defined. In vitro studies have only shown that 15d-PGJ2 increases monocyte/macrophage expression of the neutrophil-selective chemokine IL-8 (2). By contrast, anti-inflammatory activities of 15d-PGJ2 have been well documented; they are mainly related to its ability to limit inflammatory cell accumulation and activation (3). 15d-PGJ2 inhibits endothelial cell expression of adhesion molecules (e.g., VCAM1 and ICAM1) and promotes leukocyte apoptosis (3). In addition, 15d-PGJ2 inhibits the transcription of genes coding for proinflammatory proteins, including IL-1{beta}, TNF-{alpha}, COX2, NO synthase-2, and matrix metalloproteinases (4, 5). This control is associated with the inactivation of transcription factors such as NF-{kappa}B, AP-1, and STATs (4). Two molecular mechanisms are potentially involved. First, 15d-PGJ2 binds to the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear receptor superfamily that decreases the availability of coactivators, and thereby prevents the activity of transcription factors (6). Alternatively, 15d-PGJ2 inhibits multiple steps in the NF-{kappa}B signaling pathway by PPAR{gamma}-independent mechanisms (7). These mechanisms involve direct interactions of a highly reactive electrophilic carbon atom in the cyclopentenone ring of 15d-PGJ2 with critical cysteine residues in target proteins (8).

Glucocorticoids play also a crucial role in the resolution of inflammation (9). They exert this role through intracellular glucocorticoid receptors (GR) that regulate gene transcription in two different ways (10). First, GR may form a homodimer that binds glucocorticoid-responsive elements (GREs) in the 5' upstream promoter or enhancer region of glucocorticoid-responsive genes to increase or repress the rate of their transcription. Alternatively, GR monomer may interact directly with other transcription factors, such as AP-1 and NF-{kappa}B, to suppress their efficacy. Thus, the availability of GR is a major factor limiting glucocorticoid action (11). During the inflammatory response, this availability is controlled by glucocorticoids themselves and by a great diversity of pro- and anti-inflammatory mediators, including IL-4, IL-10, IL-13, somatostatin, and TGF-{beta}1 (12).

Whether such a control is exerted by 15d-PGJ2 as well is not known. Therefore, the present study was undertaken to explore the effects of 15d-PGJ2 on glucocorticoid binding and signaling. Evidence was obtained that 15d-PGJ2 limits glucocorticoid binding and signaling in human and murine monocytes/macrophages through a PPAR{gamma}-independent mechanism. It provides a way in which 15d-PGJ2 could exert proinflammatory activities during the inflammatory process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

The promonocytic U937cell line was obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°C in a culture medium (RPMI 1640 with 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin) supplemented with 10% FCS under a 5% CO2-95% air atmosphere. These cells were differentiated to a mature macrophage-like phenotype, as previously described (13): 0.5 x 106 cells/ml culture medium supplemented with 2.5% FCS were pretreated for 24 h with 1.2% DMSO, washed, and resuspended in 0.5 ml/well culture medium (24-well plate). They were treated with PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) for 3 h at 37°C, washed, and then incubated with the indicated concentrations of 15d-PGJ2 (Biomol, Plymouth Meeting, PA). The mouse cell line RAW 264.7 was obtained from American Type Culture Collection. Cells were grown to confluence in DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin under a 5% CO2-95% air atmosphere (14).

Cell viability analysis

Cells were trypsinized, washed, and resuspended in 0.2% trypan blue in PBS. Nonviable cells taking up blue dye were counted using a hemocytometer.

GR assay

A whole cell binding assay was used to determine GR number and affinity in untreated and 15d-PGJ2-treated cells (14). PMA-differentiated U937cells or RAW 264.7 cells were incubated in culture medium supplemented with 2.5% FCS and containing [3H]dexamethasone (85.0 Ci/mmol; Amersham Pharmacia Biotech Europe, Saclay, France) with or without unlabeled dexamethasone (Sigma-Aldrich). For Scatchard analysis, the concentration of [3H]dexamethasone ranged from 0.5 to 64 nM; for one-point binding assays, 10 nM [3H]dexamethasone was used. Unlabeled dexamethasone was used at a concentration of 20 µM. After incubation at 37°C for 3 h, monolayers were washed six times with cold PBS, and cells were lysed in 1 N NaOH. Lysates were harvested and counted in a beta spectrometer.

Supplementary binding assays were performed using a cytosol fraction, as previously described (15, 16). RAW 264.7 cells were harvested by scraping into culture medium. The pellet was suspended in 1 ml of buffer (10 mM HEPES, 1 mM EDTA, and 20 mM sodium molybdate, pH 7.4) and ruptured with a plastic homogenizer. The homogenate was centrifuged for 1 h to 18,500 x g at 4°C, and the supernatant referred to as cytosol was frozen until further analysis. For binding assays, cytosol fraction (0.25 ml) was incubated for 4 h at 4°C with 10 nM [3H]dexamethasone together with or without unlabeled dexamethasone. Bound and unbound [3H]dexamethasone were separated by the charcoal-dextran technique (16).

Western blotting

Culture medium was removed, and RAW 264.7 cells were resuspended in 1.5 ml of PBS using a cell scraper. The cells were then pelleted by centrifugation (600 x g, 4°C, for 10 min), frozen at –80°C for 4 h, thawed, and resuspended in 100 µl of ice-cold protease inhibitory buffer (1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin in 50 mM Tris (pH 7.4), 100 mM NaCl, 2 mM EDTA, and 1% Nonidet P-40). The lysate was centrifuged (4,000 x g, 4°C, for 30 min). A portion of the supernatants was reserved for protein determination, and the protein concentration in supernatant samples was adjusted with the protease inhibitory buffer. These samples were boiled for 3 min in Laemmli loading buffer and subjected to electrophoresis on a 7.5% polyacrylamide-SDS gel. Proteins were electroeluted onto a nitrocellulose membrane (Immobilon-P; Millipore, Bedford, MA) that was blocked for 18 h at 4°C in 10% nonfat dry milk solution in PBS and 0.1% Tween. GR was detected using a polyclonal Ab (1/100 dilution; Affinity Bioreagents, Golden, CO) and a peroxidase-labeled anti-IgG secondary Ab (1/10,000 dilution). Thereafter, the membrane was developed with the ECL detection reagent (Amersham Pharmacia Biotech Europe).

Transient transfection and luciferase assay

U937 cells were transiently transfected with a glucocorticoid-inducible reporter plasmid containing a minimal thymidine kinase promoter with two consensus GRE upstream of the gene for luciferase ([GRE]2 TK-Luc, a gift from Dr. M.-G. Catelli, Centre National de la Recherche Scientifique, Paris, France), using the DEAE-dextran procedure (14). Thereafter, these cells were differentiated with PMA and exposed to 15d-PGJ2 and/or dexamethasone before luciferase activity was assayed as previously described (17).

ELISA for monocyte chemoattractant protein-1 (MCP-1)

RAW 264.7 cells were incubated with or without 2.5 µM 15d-PGJ2 for 2 h and then with increasing concentrations of dexamethasone (10–100 nM) for 1 h, before being challenged with LPS (10 ng/ml). The cell supernatants were harvested after 6 h, centrifuged to remove cell debris, and stored at –80°C until they were analyzed for MCP-1. Concentrations of MCP-1 were measured by ELISA (Quantikine mouse JE/MCP-1; R&D Systems, Minneapolis, MN). The minimum detectable concentration was 2 pg/ml.

PMA-differentiated U937cells were preincubated with or without 5 µM 15d-PGJ2 for 3 h. Thereafter, these cells were cultured for 6 h with dexamethasone (10–100 nM), and MCP-1 concentrations were measured in the supernatants by ELISA (Quantikine human MCP-1; R&D Systems).

Statistics

Results are presented as the mean ± SD. Comparisons between groups of values were made with Student’s t test. A difference between groups of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
15d-PGJ2 limits glucocorticoid-specific binding to monocytes/macrophages

To assess initially the sensitivity of monocytes/macrophages to 15d-PGJ2, RAW 264.7 cells and PMA-differentiated U937cells were exposed for 3 h to varying concentrations of 15d-PGJ2 before binding experiments were performed. The addition of 15d-PGJ2 caused a concentration-dependent decrease in [3H]dexamethasone-specific binding to either strain of cells (Fig. 1, A and B). The maximal effect was seen at 10 µM. These changes could not be related to differences in cell viability because cell exposure to 15d-PGJ2 for 3 h did not affect their capacity to exclude trypan blue (Fig. 1C). In PMA-differentiated U937cells, the above changes in [3H]dexamethasone-specific binding were detectable after 0.5 h, peaked after 3 h, and thereafter decreased progressively (Fig. 1D).



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  		FIGURE 1. Effect of 15d-PGJ2 on [3H]dexamethasone-specific binding. RAW 264.7 cells (A) and PMA-differentiated U937cells (B) were exposed to the indicated concentrations of 15d-PGJ2 for 3 h before the whole cell binding assay was performed with 10 nmol/L [3H]dexamethasone. C, RAW 264.7 cells were exposed to the indicated concentrations of 15d-PGJ2 for 3 h before the percentage of dead cells was assessed by measuring trypan blue uptake. D, PMA-differentiated U937cells were exposed to 2.5 µM 15d-PGJ2 for the indicated periods before the whole cell binding assay was performed with 10 nmol/L [3H]dexamethasone. Values are the mean ± SD of three to six determinations. *, p < 0.05 compared with the15d-PGJ2-untreated control.

 
To determine whether 15d-PGJ2 promoted a decrease in GR number or affinity, saturation binding and Scatchard analysis were performed (Fig. 2). Exposure of PMA-differentiated U937cells to 2.5 µM 15d-PGJ2 for 3 h yielded a 50% decrease in the average number of GR sites per cell (control cells, 106,366 ± 30,984; 15d-PGJ2-treated cells, 54,336 ± 4,340; p < 0.005; n = 3) without significant modification of the Kd value (control cells, 14.3 ± 7.7 nM; 15d-PGJ2-treated cells, 17.3 ± 7.6 nM).



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  		FIGURE 2. Effect of 15d-PGJ2 on the number and the affinity of [3H]dexamethasone binding sites in PMA-differentiated U937cells. A and B, Saturation binding (A) and Scatchard analysis (B) of [3H]dexamethasone binding data obtained from untreated cells ({circ}) and cells exposed to 2.5 µM 15d-PGJ2 for 3 h (•). One representative experiment of three is shown.

 
These results raised the question of whether 15d-PGJ2 might decrease the steady state levels of GR protein. Western blotting experiments were therefore performed. Exposure of RAW 264.7 cells (Fig. 3) or PMA-differentiated U937cells (data not shown) to 15d-PGJ2 for 3 h did not modify GR levels, indicating that 15d-PGJ2-induced changes in [3H]dexamethasone-specific binding were related to functional alteration of the GR rather than to adecrease in GR protein.



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  		FIGURE 3. Western blot analysis of the effect of 15d-PGJ2 on GR protein level. RAW 264.7 cells were exposed to the indicated concentrations of 15d-PGJ2 for 3 h. One representative experiment of four is shown.

 
15d-PGJ2 limits glucocorticoid-specific binding through a PPAR{gamma}- and membrane receptor-independent process

15d-PGJ2 binds to PPAR{gamma} and affects the inflammatory process through both PPAR{gamma}-dependent and -independent mechanisms. In addition, U937cells and RAW 264.7 cells express PPAR{gamma}, at high and low levels, respectively (4). Thus, we assessed the involvement of PPAR{gamma} in the response to 15d-PGJ2, by examining the effects of PPAR{gamma} antagonist and agonist. Exposure of RAW 264.7 cells or PMA-differentiated U937cells to the PPAR{gamma} antagonist bisphenol A diglycidyl ether (BADGE) (18, 19) augmented [3H]dexamethasone-specific binding, but did not interfere with the 15d-PGJ2 response (Fig. 4, A and B). Similarly, exposure of RAW 264.7 cells or PMA-differentiated U937cells to the potent PPAR{gamma} agonist ciglitazone did not affect [3H]dexamethasone-specific binding (Fig. 4, C and D), suggesting that PPAR{gamma} is not involved in the response to 15d-PGJ2.



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  		FIGURE 4. 15d-PGJ2 limits glucocorticoid-specific binding through a PPAR{gamma}-independent process. 1) The influence of BADGE, a PPAR{gamma} antagonist, on the response to 15d-PGJ2 was tested. RAW 264.7 cells (A) or PMA-differentiated U937cells (B) were preincubated for 10 min with or without 100 µM BADGE and incubated for 3 h with or without 5 µM 15d-PGJ2. Thereafter, the whole cell binding assay was performed with 10 nmol/L [3H]dexamethasone. Values are the mean ± SD of triplicate determinations. 2) Effect of ciglitazone, a PPAR{gamma} agonist, on glucocorticoid binding. RAW 264.7 cells (C) or PMA-differentiated U937cells (D) were exposed to the indicated concentrations of ciglitazone for 3 h. Thereafter, the whole cell binding assay was performed with 10 nmol/L [3H]dexamethasone. Values are the mean ± SD of triplicate determinations. *, p < 0.05 compared with the15d-PGJ2-untreated control.

 
At a lower concentration than that required for PPAR{gamma} activation, 15d-PGJ2 can act through binding to a cell membrane receptor (20). Thus, we also determined whether 15d-PGJ2 could affect [3H]dexamethasone-specific binding in the absence of cell membrane. Cytosol fraction was isolated from RAW 264.7 cells and used in a binding assay. After 1-h cytosol exposure to 5 µM 15d-PGJ2, [3H]dexamethasone-specific binding decreased from 170.6 ± 3.0 to 118.6 ± 14.5 fmol/mg protein (p < 0.05; n = 3). Together these results suggest that 15d-PGJ2 limited glucocorticoid-specific binding through a PPAR{gamma}- and membrane receptor-independent process.

15d-PGJ2 limits glucocorticoid-specific binding through the interaction of its cyclopentenone ring with GR

15d-PGJ2 is characterized by the presence of a cyclopentenone ring that contains an electrophilic carbon (7, 8). This carbon can react covalently by means of the Michael addition reaction with nucleophiles such as the free sulfhydryls of cysteine residues in proteins. The hormone binding domain of GR contains such cysteine residues, and their oxidation or chemical modification affects ligand binding (21). Thus, we first hypothesized that 15d-PGJ2 would inhibit glucocorticoid binding through a direct modification of the hormone binding domain of GR by the cyclopentenone ring system. Consistent with this hypothesis, cyclopentenone (2-cyclopenten-1-one), a compound that contains also an unsaturated carbonyl group, caused a concentration-dependent decrease in [3H]dexamethasone-specific binding to RAW 264.7 cells (Fig. 5A). Efficient concentrations were ~125–500 µM, as previously described in other cell models (7, 22). In contrast, cyclopentanone and cyclopentene, which contain a saturated carbonyl and a double bond without carbonyl, respectively, had no effect (data not shown). As a further confirmation of this hypothesis, methyl methanethiosulfonate (MMTS), a membrane-permeant, cysteine-specific, modifying reagent (16), decreased [3H]dexamethasone-specific binding to RAW 264.7 cells and completely prevented the response to 15d-PGJ2 (Fig. 5B).



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  		FIGURE 5. 15d-PGJ2 limits glucocorticoid-specific binding through the interaction of its cyclopentenone ring with GR. A, Effect of cyclopentenone on glucocorticoid binding. RAW 264.7 cells were exposed to the indicated concentrations of cyclopentenone for 3 h. Thereafter, the whole cell binding assay was performed with 10 nmol/L [3H]dexamethasone. B, Influence of MMTS, a cysteine-specific modifying reagent, on the response to 15d-PGJ2. RAW 264.7 cells were preincubated for 30 min with (•) or without ({circ}) 500 µM MMTS and exposed to the indicated concentrations of 15d-PGJ2 for 3 h. Thereafter, the whole cell binding assay was performed with 10 nmol/L [3H]dexamethasone. C, Influence of NAC, a scavenger of reactive oxygen species, on the response to 15d-PGJ2. RAW 264.7 cells were preincubated for 10 min with (•) or without ({circ}) 5 mM NAC and exposed to the indicated concentrations of 15d-PGJ2 for 3 h. Thereafter, the whole cell binding assay was performed with 10 nmol/L [3H]dexamethasone. Values are the mean ± SD of three or four determinations. *, p < 0.05 compared with the15d-PGJ2-untreated control.

 
Cyclopentenone PGs, including 15d-PGJ2, induce an intracellular oxidative stress that may underlie part of their biological effects (23). As oxidative stress limits the ligand binding activity of the GR (24), the question arises as to whether 15d-PGJ2 would inhibit glucocorticoid binding indirectly, through the oxidation of critical cysteine residues in the GR. However, exposure of RAW 264.7 cells to N-acetylcysteine (NAC), an antioxidant, did not affect the response to 15d-PGJ2 (Fig. 5C), indicating that such a mechanism was not required.

15d-PGJ2 limits glucocorticoid signaling and anti-inflammatory efficiency

We assessed whether 15d-PGJ2-induced changes in glucocorticoid binding were associated with changes in glucocorticoid signaling. Given that glucocorticoids regulate gene transcription in part via the binding of GR to GRE (10), we used a reporter gene under the control of a promoter including two copies of the GRE (Fig. 6). Exposure of PMA-differentiated U937cells transfected with [GRE]2 TK-Luc to dexamethasone for 4 h resulted in a 5-fold increase in luciferase activity. The addition of 15d-PGJ2 caused a concentration-dependent decrease in this response.



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  		FIGURE 6. Effect of 15d-PGJ2 on glucocorticoid signaling. U937cells were transiently transfected with the reporter plasmid [GRE]2 TK-Luc, differentiated with PMA, and exposed to the indicated concentrations of 15d-PGJ2 for 3 h. Thereafter, they were incubated with (•) or without ({circ}) 5 nM dexamethasone for 4 h before luciferase levels were measured. Values are the mean ± SD of triplicate determinations. *, p < 0.05 compared with the15d-PGJ2-untreated control.

 
We also evaluated the pathophysiologic relevance of the 15d-PGJ2-induced changes in glucocorticoid binding and signaling. As a marker of glucocorticoid efficacy, we examined the regulation of MCP-1 expression. Dexamethasone decreased in a concentration-dependent manner the expression of MCP-1 in both LPS-stimulated RAW 264.7 cells (Fig. 7A) and PMA-differentiated U937cells (Fig. 7B). The addition of 15d-PGJ2 significantly limited this response. Finally, consistent with the binding studies, 15d-PGJ2-induced changes in glucocorticoid anti-inflammatory efficiency were mimicked by cyclopentenone (Fig. 7C) and MMTS (Fig 7D).



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  		FIGURE 7. Effect of 15d-PGJ2 on glucocorticoid anti-inflammatory efficiency. A, RAW 264.7 cells were incubated with (•) or without ({circ}) 2.5 µM 15d-PGJ2 for 2 h and then with the indicated concentrations of dexamethasone for 1 h before being challenged with LPS (10 ng/ml). MCP-1 concentrations were measured in the supernatants by ELISA after 6 h. B, PMA-differentiated U937cells were preincubated with (•) or without ({circ}) 5 µM 15d-PGJ2 for 3 h. Thereafter, these cells were cultured for 6 h with the indicated concentrations of dexamethasone, and MCP-1 concentrations were measured in the supernatants by ELISA. C, RAW 264.7 cells were incubated with or without 250 µM cyclopentenone for 2 h and then with 50 nM dexamethasone for 1 h before being challenged with LPS (10 ng/ml). MCP-1 concentrations were measured in the supernatants by ELISA after 6 h. D, RAW 264.7 cells were incubated with or without 500 µM MMTS for 30 min and then with 50 nM dexamethasone for 1 h before being challenged with LPS (10 ng/ml). MCP-1 concentrations were measured in the supernatants by ELISA after 6 h. Values are the mean ± SD of values obtained in three or four independent experiments. *, p < 0.05 compared with the untreated control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that 15d-PGJ2 transiently inhibits glucocorticoid binding and signaling in monocytes/macrophages. The mechanism for this effect is likely to be through the interaction of the 15d-PGJ2 cyclopentenone ring with cysteine residues in constituents of the GR activation pathway.

The lowest 15d-PGJ2 concentration required to inhibit GR binding and signaling in monocytes/macrophages was ~100 nM. As the amounts of 15d-PGJ2 released in vitro into the medium of RAW 264.7 cells stimulated with LPS reach ~80 nM (25), the amounts of 15d-PGJ2 generated in vivo at the inflammatory site are presumably sufficient for it to play a role in modulating GR efficiency, although this may not be the case in all human tissues (26). Clearly, this role cannot be explained by a reduction in cell viability. Indeed, 15d-PGJ2 concentrations up to 5 µM did not affect RAW 264.7 cell viability, and the 15d-PGJ2 effect on GR binding was mostly reversible. These results are consistent with previous studies showing that 15d-PGJ2 induces an apoptotic response only at concentrations >5 µM (27).

After 15d-PGJ2 addition, decreases in GR binding were fast, being detectable after 30 min. This suggests that the response to 15d-PGJ2 is related to rapid processes such as GR degradation or GR post-translational modifications that impede ligand binding. An alternative would be that 15d-PGJ2 decreases GR gene transcription and, hence, GR expression. This latter possibility is supported by the observation that 15d-PGJ2 can significantly reduce COX2 gene transcription and COX2 expression after <3 and 4 h, respectively (28). However, this possibility is unlikely because GR protein amounts were not significantly modified by effective concentrations of 15d-PGJ2. In addition, decreased GR gene transcription would have no repercussions on GR protein availability after a <3-h period, because the GR turnover rate is slow (half-life of GR protein reaches 19 h) (29, 30).

Interestingly, 15d-PGJ2-induced modifications in GR binding were independent of PPAR{gamma} activation, as suggested by two observations. First, although U937cells express high levels and RAW 264.7 cells express negligible levels of PPAR{gamma} (4, 31), 15d-PGJ2 exhibited a similar effectiveness in the two cell lines. Second, the response to 15d-PGJ2 was neither mimicked by the specific 15d-PGJ2 agonist ciglitazone nor modified by the 15d-PGJ2 antagonist BADGE. In agreement with this observation, 15d-PGJ2 has been shown previously to control several functions of monocytes/macrophages through PPAR{gamma}-independent mechanisms (32).

15d-PGJ2 is characterized by the presence of a cyclopentenone ring with an electrophilic carbon that is able to form Michael adducts with nucleophiles such as free sulfhydryls in cysteine residues (7, 8). By this mechanism, 15d-PGJ2 has been shown to directly affect the activity of proteins, including I{kappa}B and NF-{kappa}B (7, 8). In our study several lines of evidence suggest that the response to 15d-PGJ2 depends on an interaction between the 15d-PGJ2 cyclopentenone ring and cysteine residues in constituents of the GR activation pathway. First, the response to 15d-PGJ2 was mimicked by 2-cyclopenten-1-one. That levels of 2-cyclopenten-1-one required to modify GR binding were 100-fold higher than levels of 15d-PGJ2 could be explained by the fact that 2-cyclopenten-1-one and 15d-PGJ2 contain one and two electrophilic carbons, respectively. Second, cell exposure to MMTS, a membrane-permeant, cysteine-specific, modifying reagent, decreased GR binding and completely suppressed the response to 15d-PGJ2. The five cysteine residues contained in the hormone binding domain of GR (21) are presumably the main target for the two electrophilic carbons of 15d-PGJ2. Consistent with this mechanism, modifications of cysteine residues in the hormone binding domain by oxidation, nitrosylation, or chemical alteration have been shown previously to limit the GR binding capacity (15, 24, 33). An alternative explanation would be that 15d-PGJ2 modifies cysteine residues in constituents of the GR activation pathway indirectly by promoting an oxidative stress. Indeed, 15d-PGJ2 may decrease antioxidant proteins such as glutathione and glutathione peroxidase, and, hence, increase the availability of reactive oxygen species that react with NO to form peroxinitrites (23, 26). Nevertheless, this indirect mechanism is unlikely, because NAC, an antioxidant, did not prevent the response to 15d-PGJ2.

In addition to its effect on the binding capacity of GR, 15d-PGJ2 promoted a decrease in the ability of GR to control the expression of MCP-1, a proinflammatory chemokine. This response is presumably due to a decrease in the transcriptional activity of GR, as suggested by experiments using [GRE]2 TK-Luc-transfected cells. Alternatively, this response may be caused by a decrease in the molecular interaction of GR monomer with other transcription factors, such as AP-1 and NF-{kappa}B. Thus, at the onset of the inflammatory process, when high levels of 15d-PGJ2 are produced (1), 15d-PGJ2 would participate in the development of inflammation by limiting both the binding and the anti-inflammatory efficiency of corticoids. The availability of corticoids depends partly on the activity of 11{beta}-hydroxysteroid dehydrogenase-1, a reductase that converts inactive cortisone to the active GR agonist cortisol (34). Interestingly, PPAR{gamma} ligands have been shown to down-regulate the expression of 11{beta}-hydroxysteroid dehydrogenase-1 (35). Thus, in vivo, 15d-PGJ2 would blunt glucocorticoid efficiency by two different ways, by limiting the availability of cortisol through a PPAR{gamma}-dependent mechanism and by impeding its binding to GR through a PPAR{gamma}-independent mechanism.

In summary, we have identified a novel molecular mechanism by which 15d-PGJ2 may exert proinflammatory effects in addition to its numerous anti-inflammatory effects. This finding highlights the necessity to precise the timing of either pro- or anti-inflammatory effects of 15d-PGJ2 throughout the inflammatory process before identifying 15d-PGJ2 as a potential target for therapeutic intervention.


   Acknowledgments
 
We thank M.-G. Catelli for the [GRE]2 TK-Luc construct, and N. Sabirhoussen for secretarial assistance.


   Footnotes
 
1 This work was supported by Institut National de la Santé et de la Recherche Médicale and Faculté de Médecine Saint-Antoine. Back

2 Address correspondence and reprint requests to Dr. Laurent Baud, Institut National de la Santé et de la Recherche Médicale, Unité 489, Hôpital Tenon, 4 rue de la Chine, 75020 Paris, France. E-mail address: laurent.baud{at}tnn.ap-hop-paris.fr Back

3 Abbreviations used in this paper: COX, cyclooxygenase; BADGE, bisphenol A diglycidyl ether; 15d-PGJ2, 15-deoxy-{Delta}12,14-PGJ2; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; MCP-1, monocyte chemoattractant protein-1; MMTS, methyl methanethiosulfonate; NAC, N-acetylcysteine; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; TK-Luc, thymidine-luciferase. Back

Received for publication October 23, 2003. Accepted for publication April 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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