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The Journal of Immunology, 2003, 171: 3245-3252.
Copyright © 2003 by The American Association of Immunologists

Glucocorticoid Receptor Nitration Leads to Enhanced Anti-Inflammatory Effects of Novel Steroid Ligands 1

Mark J. Paul-Clark2,*, Fiorentina Roviezzo2,{dagger}, Roderick J. Flower*, Giuseppe Cirino{dagger}, Piero Del Soldato{ddagger}, Ian M. Adcock§ and Mauro Perretti3,*

* The William Harvey Research Institute, Queen Mary School of Medicine and Dentistry, London, United Kingdom; {dagger} Department of Pharmacology, University of Naples, Naples, Italy; {ddagger} NicOx, Nice, France; and § Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has recently emerged that posttranslational modification of proteins via nitration of tyrosine residues can alter their function. In this study, we describe that specific nitration of the glucocorticoid receptor (GR) by NCX-1015, a novel NO-donating prednisolone derivative (prednisolone 21-[4'-(nitrooxymethyl)benzoate), results in an enhancement of GR-mediated events. Incubation of PBMC and U937 cells with 1–10 µM NCX-1015 caused faster activation of GR as assessed by augmented 1) binding to [3H]dexamethasone, 2) dissociation from heat shock protein 90, and 3) nuclear translocation. PBMCs treated with NCX-1015 contained GR that had undergone tyrosine nitration. The chemistry facilitating the increase in steroid binding capacity observed with NCX-1015 is specific, because changing the position of the NO-donating group or ubiquitous nitration by addition of an NO donor was unable to mimic this event. In vivo treatment with NCX-1015 provoked GR nitration and faster heat shock protein 90 dissociation as assessed in peritoneal cells. Accordingly, NCX-1015, but not prednisolone or other derivatives, produced a rapid inhibition of the early neutrophil recruitment and mediator generation in a model of peritonitis. In conclusion, we report here for the first time that posttranslational modification of GR by this novel nitrosteroid is associated with its enhanced anti-inflammatory activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid (GC) 4 therapy is widely used to control human disorders particularly inflammatory diseases, however, long-term use is often associated with unwanted side effects (1, 2). We have recently described a different approach to the development of innovative GCs that takes advantage of the anti-inflammatory actions of NO. The novel NO-releasing derivative of prednisolone, termed NCX-1015 or nitroprednisolone, displayed a more potent anti-inflammatory profile with reduced side effects in experimental models of acute and chronic inflammation compared with its parent compound (3, 4, 5).

The functionality of the GC receptor (GR) is tightly regulated by several receptor-associated proteins (RAP), including heat shock protein (hsp)90, hsp70, and the immunophilins FK-binding protein 51 and cyclosporine A binding protein 40 (6, 7). Although the number of RAP putatively associated with GR is ever increasing (8), experiments of reconstitution of the GR complex have shown that hsp90 and hsp70 are the only RAP essential for unveiling the high affinity receptor-binding cleft, with other RAP determining GR stability and transport into the nucleus (9, 10, 11). Thus, one of the first events following GR binding to its ligands (synthetic or natural GCs) is hsp90 dissociation and exposure of the nuclear localization sequence (12, 13). The GR-ligand complex then dimerizes and translocates into the nucleus, where it modulates the transcription of the genes containing the GC response element within their promoter region. The GR-ligand complex can also affect gene expression indirectly by interfering with the function of cognate transcription factors (7). For example, activated GR can bind directly to NF-{kappa}B blocking the downstream induction of several genes (14, 15). Thus, GR protein-protein interaction is fundamental in controlling receptor localization and function.

GR is a phosphoprotein containing numerous potential phosphorylation sites (16). Evidence obtained during the past 10 years suggests that altered GR phosphorylation status can affect GR-ligand binding, hsp90 interactions, receptor subcellular localization, nuclear-cytoplasmic shuttling, and transactivation potential (17). Recent results demonstrate tyrosine nitration as a novel biochemical mechanism able to modulate protein function (18). In the present study, we sought to substantiate the pharmacological profile of NCX-1015 and the related new family of nitrosteroids with the molecular and cellular analyses of its effects following interaction with GR. We report a novel modulation of selected biochemical and cellular events associated with GR activation, and provide evidence that these phenomena have in vivo relevance, thus they may contribute to the overall more potent anti-inflammatory profile of this new GC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthesis of prednisolone derivatives NCX-1006, NCX-1015, and NCX-1016

Fig. 1 reports the chemical structures of prednisolone, dexamethasone, and the nitrosteroids used in this study. Prednisolone 21-[4'-(nitrooxymethyl)benzoate (NCX-1015), prednisolone 21-[4'-(oxymethyl)benzoate (NCX-1016), and prednisolone 21-[4'-(nitrooxy)butyrate (NCX-1006) were synthesized at NicOx Research Institute (Milan, Italy). The synthesis of NCX-1015 and NCX-1016 (Fig. 1, B and C) has already been described in detail (4). Batches of NCX-1006 were prepared following a two-step synthesis procedure with an overall yield of ~70%. A solution of prednisolone (33.3 mmol in tetrahydrofurane) was added to 49.9 mmol of 4-bromobutiryl chloride and triethylamine. The reaction was stirred for 4 h at room temperature and the solvent was evaporated under vacuum. After treatment of the residue with ethyl acetate and water, and removal of the insoluble material, the intermediate (prednisolone 21-(4'-bromobutyrrate)) was obtained by dehydration with sodium sulfate and concentrated under reduced pressure; the product was further purified by silica gel chromatography (31.19 mmol, yield 65%). This intermediate was then treated with silver nitrite (43.66 mmol), acetonitrile (100 ml), and tetrahydrofurane (200 ml) in the dark for 18 h at 80°C. The precipitate was filtered off, the solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography, and crystallized from tetrahydrofurane. The final product, prednisolone 21-[4'-(nitrooxy)butyrate] was obtained as a white powder, with a m.w. of 491.6 (Fig. 1C). The structure of NCX-1006 (Fig. 1D) was confirmed by nuclear magnetic resonance (with 1H and 13C) and infrared analyses. This preparation was tested for endotoxin and found to contain <1 endotoxin unit per milligram as determined by the Limulus assay (BioWhittaker, Walkersville, MS).



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  		FIGURE 1. Chemical structures of prednisolone derivatives. A, Chemical structure of prednisolone and dexamethasone (added for reference purposes). B–D, Different esterase-sensitive chemical moieties were added to position 21 of prednisolone to yield (B) NCX-1015 or prednisolone 21-[(4'-nitro-oxymethyl)benzoate], (C) NCX-1016 or prednisolone 21-[(4'-hydroxymethyl)benzoate], and (D) NCX-1006 or prednisolone 21-[(4'-nitro-oxy)butyrate]. All compounds were synthesized at NicOx Research Institute as described in Materials and Methods.

 
Whole cell GR binding assay

Human PBMC (70% lymphocytes, 30% monocytes), isolated as described (19), and the immortalized monomyelocytic cell line U937, cultured as reported (20), were used. The binding assay described by Molijn et al. (21) was used, with minor modifications. Briefly, cells (106 in 1 ml) were incubated with [3H]dexamethasone (50 nM; specific activity 89 Ci/mmol; Amersham International Biotech, Buckinghamshire, U.K.) with or without other GCs, for 1 h at 37°C. After five washings with 0.01 M ice-cold PBS, cell-bound [3H]dexamethasone was quantified by liquid scintillation and the specific concentration was calculated by subtracting the nonspecific binding (determined with a 1000-fold excess cold dexamethasone). Scatchard plot analysis was performed to determine the dissociation constant Kd and the maximal number of binding sites (Bmax) values using a concentration range of 1.57–50 nM [3H]dexamethasone.

Prednisolone or NCX-1016 (10 µM in both cases) were also incubated with the cells in the presence of the following NO donors: 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-traizine (NOC-18, 5 µM; Alexis, Nottingham, U.K.) and sodium nitroprusside (SNP; 10 µM; Sigma-Aldrich, Dorset, U.K.). NCX-1015 (3–10 µM) was added to cells also in combination with the following chemicals: the soluble guanylate cyclase inhibitor [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ, 5 µM; Alexis) (22); an activator of soluble guanylate cyclase 3-(5'-hydroxymethyl-2'-furyl)-1-bezylindazole (YC-1, 50 µM; Calbiochem, Nottingham, U.K.) (23); a cell-permeable inhibitor of protein G kinase KT35823 (5 µM; Calbiochem); sodium azide (0.2% w/v; Sigma-Aldrich, Dorset, U.K.); the energy metabolism inhibitors 2-deoxy-glucose and DNP (4.5 mM; Sigma-Aldrich) (24); the protein synthesis inhibitor cycloheximide (10 mM; Sigma-Aldrich) (25); the hsp90 re-association inhibitor geldanamycin (10 mM) (26); and the nuclear transport inhibitor okadaic acid (50 µM) (27). Finally, NCX-1015 was also coadded with the GR antagonist RU486 (mifepristone; 10 µM) (28).

GR immunoprecipitation (IP) experiments

U937 cells or PBMCs (106 cells/ml) were incubated with test compounds for either 0.5 or 2 h at 37°C in 5% CO2 atmosphere. Cell extracts in 0.01 M Tris-HCl (pH 8) supplemented with 0.15M NaCl and a protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany). Lysates (100 µg of protein) were diluted with 100 µl of mild IP buffer (as above plus 0.5% Nonidet P-40) and incubated on ice for 30 min. Extracts were precleared with 20 µl of protein A/G with agarose (50:50 mix; Santa Cruz Biotechnology, San Diego, CA) and 5 µl of normal mouse IgG (500 µg/ml) for 1 h at 4°C. After microcentrifugation, 20 µl of protein A/G with agarose conjugated with 5 µg of anti-human-GR (clone 8E9; Serotec, Oxford, U.K.) or anti-nitrotyrosine (clone 1A6; Upstate Biotechnology, Lake Placid, NY) and incubated overnight (4°C) with constant rotation. Immune complexes were resolved by gel electrophoresis on 10% SDS-polyacrylamide gels; after transfer onto nitrocellulose membranes, hsp90, hsp70, and hsp40 proteins were detected using specific polyclonal rabbit Abs (1/2,000, 1/20,000, and 1/3,000, respectively; Stressgen Biotechnologies, Victoria, British Columbia, Canada). In experiments for nitrotyrosine, Western blot analysis was carried-out using the anti-human GR Ab (1/1000) or the rabbit anti-GR polyclonal IgG (1/1000; Santa Cruz Biotechnology). In the latter case, doublets were observed. In all cases, the signal was amplified with a HRP-linked goat anti-rabbit secondary Ab (1/2000; DAKO, Cambridgeshire, U.K.) and visualized using BioMax MR-1 film (Kodak, Rochester, NY) after incubation with Luminol (ECL; Amersham International Biotech).

Anti-human Abs reacted with murine GR and heat shock proteins and were used in IP experiments with mouse peritoneal cell (>=80% macrophages), collected after treatment with NCX-1015 or prednisolone (13.8 µmol/kg i.p. or s.c.; -30 min or -2 h).

Immunocytochemistry

U937 cells (2 x 106) were cultured as above in 12-well plates in the presence of dexamethasone, prednisolone, NCX-1016, NCX-1015, or vehicle control (RPMI 1640 containing 0.1% DMSO) for either 0.5 or 2 h. Aliquots (105 cells) were removed, cytospun (200 x g for 4 min) onto glass slides, air-dried, then fixed in ice-cold acetone-methanol (50:50 v/v) at -20°C for 10 min. Cells were stained for GR (rabbit anti-GR 1/50; Santa Cruz Biotechnology) using a previously described protocol for intracellular fluorescence staining (29). Stained cells were observed under an oil immersion objective lens by confocal microscopy with a Leica confocal microscope (Buckinghamshire, U.K.), equipped with a 488- and 514-nm dual band argon laser (Leica), and images collected using TCSNT software (Leica).

Flow cytometric analysis of GR and CD163 expression

PBMC (106 cells) were incubated in a humidified environment containing 5% CO2 for either 2 or 24 h at 37°C, for GR and CD163 determination, respectively. GR was detected with clone 8E9 mAb (25 µg/ml; Serotec, Oxford, U.K.), whereas CD163 expression was quantified with a mouse anti-human CD163 (clone Mac2-48, 20 µg/ml; provided by Dr. N. J. Goulding, William Harvey Research Institute, London U.K.). Analysis was performed in a BD Biosciences FACScan (Abingdon, U.K.), using CellQuest software (BD Biosciences). Monocyte and lymphocyte populations were identified for their forward and side scatter properties.

Model of acute inflammation

Animal work was approved by Home Office U.K. (Ministry of Interior, London, U.K.) (project license; PPL 70/4804). Zymosan peritonitis was induced as previously reported (3). Differential cell counts were performed under light microscopy (Olympus B061; London, U.K.), whereas the content of PGE2 in selected cell-free lavage fluids was determined with a specific enzyme immunoassay (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Concentrations of the CXC chemokine KC were determined with a specific ELISA (R&D Systems, Abingdon, U.K.).

Data handling and statistical analysis

In vitro experiments were conducted in triplicate and repeated at least three times with different cell preparations (GR binding assay; immunoprecipitations; GR nuclear translocation). In vivo, data are reported as mean ± SEM of n animals per group. For IP experiments, Western blots were analyzed by densitometrical analysis completed using SCION image (National Institutes of Health, Bethesda, MD). Differences among the experimental groups were determined by one-way ANOVA followed by the Dunnett’s test taking a p value < 0.05 as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Higher GR binding following cell incubation with NCX-1015

PBMC displayed an avid binding to [3H]dexamethasone (Kd = 19.4 ± 7.2 nM; Bmax = 25.19 ± 1.42 pM, mean ± SE from four experiments). Competition assays using prednisolone produced a concentration-dependent reduction of the amount of tracer specifically bound to GR (Fig. 2A). A similar effect was measured with all nitrosteroids, with the exception being NCX-1015 at concentrations between 1 and 10 µM, which produced a marked increase in radioactive tracer bound (Fig. 2A; higher concentrations could not be used due to solubility problems). The same profile was obtained in U937 cells (Fig. 2B). Neither the denitrated derivative NCX-1016 nor NCX-1006, which is linked to NO via an aliphatic spacer, potentiated binding (Fig. 2C). In 20 distinct preparations, NCX-1015 (10 µM) augmented [3H]dexamethasone binding by 7.3- ± 0.5-fold in PBMCs (p < 0.05). In this experimental condition, a marked increase in Bmax (864 pM) and a concomitant decrease in binding affinity (approximate Kd of 275 nM) was measured by Scatchard plot analysis. Importantly, total GR levels in PBMC and U937 cells were not modified by cell incubation with 10 µM NCX-1015 or prednisolone. Values of median fluorescence intensity were as follows: 510 ± 40, 560 ± 30, and 480 ± 35 U were measured in monocytes treated with vehicle, prednisolone, or NCX-1015, respectively (n = 3; not significant). Similar GR values across treatments were also obtained in the lymphocyte population (GR immunoreactivity gave values around the 300 mark; data not shown).



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  		FIGURE 2. Cumulative displacement curves of synthetic GC on 3[H]dexamethasone (Dex) binding to GR in PBMCs and U937 cells. A, Concentration-dependent (0.01–10 µM) displacement by prednisolone and NCX-1015 of [3H]Dex (10 nM) binding to PBMCs (1 x 106 cells; binding assay performed for 1 h at 37°C). B, As in A, using the U937 monocytic cell line (1 x 106 cells per treatment). C, Effect of NCX-1016 (prednisolone 21-[(4'-hydroxymethyl)benzoate]) and NCX-1006 (prednisolone 21-[(4'-nitroxy)butyrate]) in PBMCs: compare with the concentration-response curve produced by NCX-1015 and shown in A. Data are expressed as mean ± SEM of n >= 3 experiments performed in triplicate; *, p < 0.05 vs vehicle control.

 
The potentiation of GR binding assay by NCX-1015 required an intact metabolism of the cells, as it was absent when the assay was conducted at 4°C (not shown), or if the metabolic machinery was blocked (effect of sodium azide or 2-deoxy-glucose and DNP; Table I). The GR antagonist RU486 abrogated NCX-1015 increase of GR binding (Table I). The effect of NO in this experimental condition was determined in a more detailed manner. Two distinct NO donors (NOC-18 and SNP) did not modify the GR binding when added to PBMC alone or together with 10 µM prednisolone or NCX-1016 (Table I). In addition, drugs interfering with guanylate cyclase activation and function were inactive. The guanylate cyclase inhibitor ODQ, or an inhibitor (KT5823) and an activator (YC-1) of cGMP-dependent kinases, was tested (Table I).


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  		Table I. Modulation of GR binding by NCX-1015 and other compoundsa

 
Posttranslational modifications of GR

U937 cell incubation with 10 µM NCX-1015 resulted in faster dissociation of hsp90 from GR in comparison to an equimolar dose of prednisolone. Fig. 3A illustrates this set of data, with a representative blot obtained at 0.5 h incubation, and the densitometric analysis from three separate experiments. In two experiments we determined the effect of 3 µM NCX-1015: this concentration produced intermediate potentiation in the GR binding assay (Fig. 3A), and similarly, dissociated hsp90 from GR (at the 30 min time point) less effectively than 10 µM NCX-1015 (data not shown). In addition, incubation of U937 cells with 10 µM NCX-1015 resulted in a retardation of hsp70/GR association as seen at 2 h (Fig. 3B). At this time point, NCX-1015 and prednisolone produced a clear distinct profile of association/dissociation for these two proteins. Similar findings were obtained with freshly purified PBMCs (Fig. 3, C and D).



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  		FIGURE 3. Effect of GR interaction with prednisolone or NCX-1015 on RAP as detected by IP in PBMCs and U937 cells. PBMCs or U937 cells (1 x 106 cells/ml) were treated with 10 µM NCX-1015 or prednisolone (Pred), or with vehicle (RPMI 1640 containing 2% FCS, 1% glutamine, 1% penicilline-streptomycin solution and 0.1% DMSO), at 37°C in a humidified environment for either 0.5 or 2 h. Protein extracts (100 µg) of the subsequent lysates were immunoprecipitated with an anti-human GR mAb (clone 8E9). Western blot analysis was used to determine the presence of hsp90 (A and C) and hsp70 (B and D). Each panel shows representative blots (with duplicate determinations) and histograms reporting the densitometric analysis and expressed as percent of the value of the vehicle treatment (data are mean ± SEM from at least three distinct experiments). *, p < 0.05 vs corresponding prednisolone group.

 
The specificity of the NCX-1015 effects was confirmed with experiments of receptor nitration. Fig. 4A demonstrates that PBMC treatment with 10 µM NCX-1015, as early as 0.5 h, provokes nitrotyrosine formation within the GR-ligand complex, with the extent of nitration being concentration-dependent (1–10 µM; not shown). In comparison, IP of vehicle-treated cells with an anti-nitrotyrosine Ab failed to detect GR (Fig. 4A). Coaddition of the GR antagonist RU486 prevented the nitrating effect of NCX-1015 (Fig. 4B), making an important parallel with the results produced in the binding assay (Table I).



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  		FIGURE 4. Effect of NCX-1015 on GR nitration and activation. A, Left panel, PBMC (1 x 106 cells/ml) were incubated with 10 µM NCX-1015 or with vehicle (RPMI 1640 containing 2% FCS, 1% glutamine, 1% penicilline-streptomycin solution and 0.1% DMSO), for 0.5 h at 37°C. Right panel, Addition of RU486 (100 µM) prevents the nitrating effect produced by 10 µM NCX-1015. In all cases, soluble protein extracts (100 µg) were immunoprecipitated with an anti-nitrotyrosine mAb (clone 1A6), and subject to Western blot analysis to determine the presence of human GR. Arrow highlights GR nitration found selectively after cell incubation with NCX-1015. B, GR-dependent gene induction as detected with the sensitive cell surface receptor CD163, after treatment with prednisolone, NCX-1015, NCX-1016, and NCX-1006 (1–10 µM in all cases), was analyzed by FACS and expressed as median fluorescent intensity (MFI) units. *, p < 0.05 vs Pred.

 
NCX-1015 effect on GR translocation into the nucleus and gene activation

In resting U937 cells, the large majority of GR was found in the cytoplasm (91 ± 5%; Fig. 5B). Short cell incubation with 10 µM prednisolone or NCX-1016 increased nuclear GR localization to 28 ± 4% and 35 ± 6%, respectively (Fig. 5, D and E). However, GR nuclear translocation was further augmented by 10 µM NCX-1015 to 89 ± 11% of positive cells (Fig. 5F). This effect was similar to that produced with 1 µM dexamethasone (94 ± 9% of positive cells; Fig. 5C).



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  		FIGURE 5. Effect of NCX-1015 on GR nuclear localization. U937 cells (1 x 106 cells/ml) were incubated with either mouse IgG control (negative control) (A), vehicle (B), 1 µM dexamethasone (positive control) (C), 10 µM prednisolone (D), 10 µM NCX-1016 (E), or 10 µM NCX-1015 (F) in a humidified environment for 0.5 h. Cell aliquots (105) were permeabilized and incubated with an anti-human GR mAb (clone 8E9). Following second Ab labeling (FITC), and nuclear counterstaining with 4',6'-diamidino-2-phenylindole, cells were visualized and analyzed by confocal microscopy. Insets in B and F show confocal images without 4',6'-diamidino-2-phenylindole nuclear stain.

 
Overnight PBMC incubation with prednisolone, NCX-1016 or NCX-1006 resulted in a concentration-dependent increase in CD163 levels on the monocyte surface; however, cells incubated with NCX-1015 had significantly higher CD163 levels, at both 1 and 10 µM compound concentration (Fig. 4B).

Rapid activation of GR in vivo is associated with increased anti-inflammatory activity

Next, we sought to provide in vivo relevance to the results obtained with isolated cells. Intraperitoneal injection of an anti-inflammatory dose of NCX-1015 (3), provoked rapid dissociation of hsp90, and a retarded reassociation of hsp70, when compared with prednisolone (Fig. 6, A and B), indicating longer lasting GR activation. Importantly, systemic treatment of mice with NCX-1015 (either i.p. or s.c.) resulted in detectable GR nitration, as seen at the 30 min time point (Fig. 6C).



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  		FIGURE 6. Ex vivo analyses of GR activation in murine macrophages. A and B, Mice were treated i.p. with vehicle (100 µl of peanut oil), prednisolone, or NCX-1015 at the dose of 13.8 µmol/kg 30 min or 2 h before peritoneal lavage and cell collections. Protein extracts (100 µg) of the subsequent lysates were immunoprecipitated with an anti-human GR mAb (clone 8E9) shown to recognize the murine Ag. Western blot analysis was used to determine the presence of hsp90 (A) and hsp70 (B). Each panel shows representative blots (with duplicate determinations) and histograms reporting the densitometric analysis and expressed as percent of the value of the vehicle treatment (data are mean ± SEM from at least four distinct animals). *, p < 0.05 vs corresponding prednisolone group. C, Vehicle or NCX-1015 (13.8 µmol/kg i.p. or s.c.) were given to animals 30 min before peritoneal cell collection. Soluble protein extracts (100 µg) were immunoprecipitated with an anti-nitrotyrosine mAb (clone 1A6), and were subject to Western blot analysis (polyclonal anti-GR rabbit IgG) to determine the presence of mouse GR. Samples are pooled from three mice per treatment. Similar data were obtained in three distinct experiments.

 
In the zymosan peritonitis model of inflammation evaluated at the 2 h time point, NCX-1015, given at time 0 concomitantly with zymosan, reduced neutrophil influx by >50%. In contrast, prednisolone, NCX-1016, or NCX-1006, under the same conditions were inactive (Fig. 7). Similar findings were obtained when the steroids were given according to a prophylactic protocol, i.e., 2 h after zymosan injection (Fig. 7). As expected (3), pretreatment (-1 h) of mice with each steroid produced a significant antimigratory effect, with NCX-1015 being more potent. The results on cell influx were mirrored by the inhibitory actions of these steroids upon the release of the CXC chemokine KC in the lavage fluids (Table II) and PGE2 (not shown).



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  		FIGURE 7. Analysis of the anti-inflammatory effects of different GCs in the zymosan peritonitis model. Mice were treated i.p. with vehicle (100 µl of peanut oil) or the dose of 13.8 µmol/kg prednisolone, NCX-1016, NCX-1006, or NCX-1015 at the reported times respective to the injection of zymosan (1 mg at time 0). Peritoneal cavities were washed either 2 or 4 h later, and polymorphonuclear cell (PMN) accumulation was determined under light microscopy. Data are reported as percent of inhibition vs vehicle control group, and are mean ± SEM of n = 8 animals per group. PMN influx in the vehicle groups was 4.6 ± 0.3 and 13.6 ± 0.9 x 106 PMN per mouse, at 2 and 4 h postzymosan, respectively. *, p < 0.05 vs respective vehicle control as calculated on original values.

 

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  		Table II. Inhibition of mouse CXC chemokine KC (CXC ligand 1) release in inflammatory exudates by NCX-1015 and other steroidsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have characterized a novel mechanism of GR activation by an NO-releasing derivative of prednisolone, NCX-1015, and provided an explanation for its enhanced anti-inflammatory activity. Using this new steroid compound to produce NO-mediated events within the microenvironment of bound GR, we could demonstrate higher degrees of ligand-mediated GR activation and GR-dependent cellular responses. The impact of these intracellular events on GC control of experimental inflammation could also be assessed.

All four prednisolone derivatives examined (the native compound, NCX-1015, NCX-1016, and NCX-1006) were able to displace [3H]dexamethasone from GR in both PBMC and U937 cells. However, at higher concentrations NCX-1015 augmented the extent of GR binding to the tracer. This effect was concentration-dependent, and not mimicked by the hydroxy analog of NCX-1015, NCX-1016. The enhanced GR binding capacity measured after NCX-1015 incubation was not due to higher expression of GR protein. In addition, the need for a defined chemistry was clear because another NO-releasing prednisolone derivative, NCX-1006 equipped with a spacer different from that of NCX-1015 (see Fig. 1), was unable to potentiate steroid binding. Similarly, NCX-1015-like derivatives with the nitrosoester in position orto or meta of the aromatic ring did not alter GR binding profile (M.J.P.-C., unpublished data).

In an aqueous environment NCX-1015 is stable, whereas it slowly releases NO species in biological fluids due to the presence of endogenous esterases (3). Though the enzymatic degradation of NCX-1015 occurs with a slow rate, the principle is similar to that adopted for other anti-inflammatory compounds containing this linker (30). For instance, in human platelet-rich plasma, NCX-1015 releases NO with a peak at ~45–60 min postincubation. A similar rate of release is also seen following i.p. injection in mice (3). For all these reasons, we then tested the effect of exogenously added NO in this assay. In contrast to NCX-1015, the combined incubation of NOC-18 or SNP (equimolar to 10 µM NCX-1015) with either prednisolone or NCX-1016 did not alter GR binding characteristics of PBMC. The fact that the canonical NO-guanylcyclase pathway (31) was not involved in this fine modulation of GR binding was also demonstrated by the lack of effect of selective inhibitors of cGMP synthesis and functions. Thus, the new compound NCX-1015 can modify GR functions after binding to the receptor as indicated by the displacement produced up to 1 µM, and the inhibitory action of GR antagonist RU486. Also, these effects are unlikely to be mediated by a diffuse release of NO species in the microenvironment to alter the GR-binding phenotype, whereas they require an intact cellular energy metabolism. In addition, performing the binding assay on cytosolic protein extracts NCX-1015 failed to elicit any augmentation of tracer bound to the receptor (M.J.P.-C., unpublished data).

Recent studies suggest that GR can undergo posttranslational modulation. In fact, an enhanced phosphorylation status of GR can affect its ligand binding profile (32), interaction with specific RAP ligand (16, 33) and nuclear-cytoplasmic shuttling (27, 34, 35). Moreover, recent data from Cidlowski and colleague (36) showed that decreased phosphorylation in mouse GR reduces transactivation of a 2x GC response element promoter and alters the localization of the unbound receptor without affecting that of the ligand-bound GR responsiveness. Interestingly, protein nitration has more recently become a focus of interest where, like phosphorylation, it can augment or down-regulate the functions of a given protein. Examples are the inducible NO synthase, for which nitration inhibits enzymatic activity, (37) and an isoform of protein kinase C, in which the net effect is increased translocation and activation (18). Along these lines, IL-10 nitration has recently been reported to augment protein activity (38). NCX-1015 induced rapid tyrosine nitration of GR in a concentration-dependent fashion, consistent with the effects on GR functions discussed above. The specificity of GR nitration was controlled in two different manners: first, NCX-1015-induced GR nitration was abolished by the GR antagonist RU486; second, the nonspecific tyrosine nitrating agent tetranitromethane (39) though nitrating GR, did not modify the binding assay (M.J.P.-C., unpublished data). Thus, we propose a model in which this new steroid derivative binds to GR before causing nitration at a specific tyrosine residue.

It is unclear which tyrosine residue(s) are the target of NCX-1015 and this is the subject of ongoing work. However, within the ligand-binding domain of GR (residues 521–777) (40) 3 of 12 tyrosine residues act as potential phosphorylation, and these sites might be subject to nitration (Ref. 41 ; NetPhos 2.0, http://www.cbs.dtu.dk/servises/netphos/). Therefore, because the possible targets sites are small, a high degree of stoichiometry may be required to target these tyrosine residues, as suggested by the lack of effect with NCX-1006 and the NO-donors. Interestingly, 1 of 12 tyrosine residues, Tyr548, is contained within the amino acid sequence (residues 547–551) that is crucial to stabilize the GR dimer (40). Another plausible candidate is Tyr735 because it is exposed to the ligand and is essential in binding recognition (40). Thus, GR activation may be determined by a combination of rapid posttranslational modifications including specific nitration events: this is consistent with the results obtained in the IP experiments. NCX-1015, but not prednisolone or NCX-1016, produced faster dissociation of hsp90, GR nuclear translocation, and retarded reassociation of hsp70, all events pointing to faster kinetics of GR activation (6, 7). NCX-1015-mediated potentiation of GR binding further affects classical GR-dependent downstream events including nuclear translocation and CD163 expression (8, 42).

Interestingly, addition of millimolar concentrations of NO donors to mouse L929 fibroblastic cells has shown to reduce GR binding and activity (43). However, NO is often referred to as a double-edged sword, with opposite effects in relation to the concentrations used (44, 45). The concentrations of NCX-1015 (3–10 µM) used here to promote GR binding and activation are likely to be reached in a pathophysiological environment: clinically relevant doses of prednisolone, 20 and 60 mg give plasma concentrations of 0.5 and 1.5 µM, respectively (46, 47). Higher concentrations are reached following intra-articular administration of this steroid, a route often used for GC-mediated clinical management of rheumatoid arthritis (48).

Finally, we analyzed this process in an in vivo context. Mouse GR in peritoneal cells reacted to an anti-inflammatory dose of NCX-1015 and prednisolone similarly to the human receptor in PBMC, as demonstrated by the altered kinetics of interaction with hsp90 and hsp70. These events were coherently associated with GR nitration, as seen 30 min postinjection with NCX-1015. More importantly, both i.p. and s.c. administration of this nitrosteroid led to nitration of peritoneal macrophage GR. As NCX-1015 dissociates slowly, i.e., it produces NO species with slow kinetics (3), it is likely that a good proportion of the intact compound binds to GR and then nitrates it. Our previous analysis of the zymosan peritonitis indicated that both prednisolone and NCX-1015, administered 1 h before zymosan, inhibited selected markers of inflammation. It is noteworthy that in this experimental model, NCX-1015 displayed anti-inflammatory efficacy with an apparent ID50 of ~1 µmol/kg (whereas prednisolone required a 10 times higher dose; Ref. 3), that is similar to the degree of inhibition attained with annexin 1-derived peptides (~1 µmol/kg) and lipoxin analogues (~0.5 µmol/kg) in models of zymosan-induced inflammation (49).

In the present study, though, we selected "stringent" experimental conditions, in which compounds were allowed to act for short 2-h time frames. NCX-1015 was the only steroid able to significantly inhibit neutrophil influx when administered at time 0 (i.e., together with the inflammogen) or even 2 h after the inflammogen (i.e., according to a therapeutic protocol). Relevantly, prednisolone, NCX-1016, and NCX-1006 were not active in these conditions, whereas they displayed the expected inhibitory property if administered prophylactically on the more established inflammatory response (4 h time point). These in vivo data of peritonitis fit well with the rapid GR activation (measured as hsp90 dissociation) produced by NCX-1015 in peritoneal cells compared with prednisolone.

In conclusion, we propose a molecular mechanism for the enhanced therapeutic profile of NCX-1015, namely modulation of GR binding and function. This effect relies upon a stringent structural requirement of the NO-steroid/receptor interaction, and it is the results of posttranslation modifications of GR. These findings may clearly impact on the development of potent new GC for use in clinical therapy.


   Footnotes
 
1 This work was supported by a project grant from NicOx (Nice, France). M.P. is a Senior Research Fellow of the Arthritis Research Campaign U.K., whereas R.J.F. is a Principal Research Fellow of the Wellcome Trust U.K. Back

2 M.J.P.-C. and F.R. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Mauro Perretti, Department of Biochemical Pharmacology, The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, U.K. E-mail address: M.Perretti{at}qmul.ac.uk Back

4 Abbreviations used in this paper: GC, glucocorticoid; GR, GC receptor; RAP, receptor-associated protein; hsp, heat shock protein; Bmax, maximal number of binding sites; SNP, sodium nitroprusside; IP, immunoprecipitation. Back

Received for publication March 31, 2003. Accepted for publication July 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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