Effect of nimesulide on glucocorticoid receptor activity in human ...

1 downloads 0 Views 210KB Size Report
Université de Montréal and Unité de recherche en arthrose, Centre de recherche ,. Centre Hospitalier de l'Université de Montréal (CHUM), Campus Notre-Dame ...
Rheumatology 1999;38(suppl. 1):11–13

Effect of nimesulide on glucocorticoid receptor activity in human synovial fibroblasts J.-P. Pelletier, J. A. Di Battista, M. Zhang, J. Fernandes, N. Alaaeddine and J. Martel-Pelletier Université de Montréal and Unité de recherche en arthrose, Centre de recherche , Centre Hospitalier de l’Université de Montréal (CHUM), Campus Notre-Dame, Montréal, Québec, Canada Abstract Fibroblasts from human synovial membranes were cultured with nimesulide, naproxen or dexamethasone. Nimesulide, but not naproxen, showed effects on the glucocorticoid system that may contribute importantly to its anti-inflammatory activity. Nimesulide at therapeutically relevant concentrations induced the intracellular phosphorylation and activation of glucocorticoid receptors, and activated their binding to the target genes. Naproxen or dexamethasone markedly reduced the number of glucocorticoid receptor binding sites, in contrast to nimesulide, which had no significant effect. KEY WORDS: Glucocorticoid receptor, Synovial fibroblast, Nimesulide.

more detailed study on the response of the GR system in human synovial fibroblasts (HSF) to NIM. Comparative experiments were conducted with the NSAID NAP, and the anti-inflammatory steroid/GR activator dexamethasone (DEX). We chose this human fibroblast model because synovial lining cells are known to play an important role in the pathophysiology of arthritic joint destruction [7–9].

Nimesulide (NIM; 4-nitro-2-phenoxymethanesulphonanilide) is a preferential cyclooxygenase-2 (COX-2) inhibitor [1–3]. Recent data suggest that NIM has many other effects in addition to the well-described inhibition of prostaglandin synthesis in various cell types. Using human osteoarthritic synovial fibroblasts in culture, we showed that therapeutic concentrations of NIM or naproxen (NAP) in vitro could reduce the synthesis of urokinase (uPA) and IL-6 while increasing the production of plasminogen activator inhibitor-1 (PAI-1) [4]. Furthermore, NIM could suppress matrix metalloprotease synthesis by cartilage explants in vitro [5]. Taken together, these results suggest that the drug can inhibit cartilage catabolism through mechanisms not associated with the inhibition of COX-2 activity and eicosanoid synthesis. Studies have shown a reduction in glucocorticoid receptor (GR) binding sites in human synoviocytes/ chondrocytes by non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and NAP, both nonselective COX-1/COX-2 inhibitors. These reductions could be counteracted by co-incubating with prostaglandin E2 (PGE2), PGE1 or the PGE1 analogue misoprostol [6], suggesting a relationship between COX activity and GR binding sites. However, we could not define the contribution of each COX isoform to GR binding because indomethacin and NAP inhibit both COX-1 and COX-2. In view of recent results that NIM can affect several metabolic pathways, we performed a

Materials and methods Specimens of synovial membranes were obtained at necropsy from donors free of arthritic disease. HSF were released by sequential enzymatic digestion, and cultured until confluence in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics at 37°C in a humidified atmosphere of 5% CO2 in air [6]. The cells were incubated in fresh serum-free medium for 24 h before the experiment; only these primary or first-passage cells were used in the experiments. Radioligand binding assays were conducted as described previously [10, 11] after using the test compounds (0.3, 3, 30 µg/ml NIM; 30 µg/ml NAP; 0.01, 0.1, 1 µMDEX). Receptor-bound radioactivity was measured as reported previously [10]. Cytosolic and nuclear protein extracts were prepared as described below. Cytosolic extract from control and treated cells (100 µg in buffer: 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 µg/ml each of aprotinin, leupeptin and pepstatin, 1% NP-40, 1 mM sodium orthovanadate and 1 mM NaF) was subjected to sodium dodecylsulphate–polyacrylamide gel electro-

Correspondence to: J.-P. Pelletier, Unité de recherche en arthrose, Centre de recherche, Centre Hospitalier de l’Université de Montréal (CHUM), Campus Notre-Dame, 1560 rue Sherbrooke est, Montréal (Québec) H2L 4M1, Canada.

11

© 1999 British Society for Rheumatology

12

J.-P. Pelletier et al.

FIG. 1. Nimesulide induced the intracellular phosphorylation and activation of the glucocorticoid receptors and activated their binding to the target genes.

phoresis (SDS–PAGE) under reducing conditions, followed by Western analysis as previously described [10, 11]. The antibodies used were a rabbit polyclonal anti-human GR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and monoclonal antibodies to phosphop44/42 MAPK and to total MAPK (both from New England Biolabs, Beverly, MA, USA). For nuclear extracts, cells were first lysed in ice-cold hypotonic lysis buffer containing 10 mM HEPES–KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM PefablocTM, 10 µg/ml each of aprotinin, leupeptin and pepstatin, 1 mM sodium orthovanadate, 1 mM NaF and 1% Nonidet P-40. The nuclear extracts were recovered by centrifugation and used for Western analysis according to Miller et al. [12]. For [32P]orthophosphate labelling, HSF (3–5 × 106 cells/well) previously stimulated with DEX (1 h), NIM or NAP, were pre-incubated in phosphate- and phenol-red-free DMEM containing 0.5% FCS and 100 µCi/ml of [32P]orthophosphate for 48 h, and incubated for 4 h.

Nuclear extracts were incubated for 16 h with the anti-human GR antibody. The immune complexes were precipitated with Protein A–agarose slurry, eluted with hot SDS–PAGE buffer, and subjected to gel electrophoresis and autoradiography. The sequence of the oligonucleotide primers for the polymerase chain reactions (PCR) was for the GR primers 5′-AGCAGTGTGCTTGCTCAGGAGAGGG-3′, which corresponds to position 46–70 bp of the N-terminal sequence, and 5′-GAGAGGCTTGCAGTCCTCATTCGAG-3′ (anti-sense) from position 720–744 bp. Total RNA was extracted with the Trizol reagent, and 2 µg was reverse-transcribed and then subjected to PCR as previously described [13]. Nuclear extracts were prepared from control and treated cells as in the previous section. Double-stranded oligonucleotides containing consensus and mutant glucocorticoid response element (GRE) sequences (Santa Cruz Biotechnology) were end-labelled with [32P]ATP using T4 polynucleotide kinase. Binding reactions were

Efficacy and safety: a balanced link for nimesulide

conducted with 30 µg of nuclear extract and 32P-labelled oligonucleotide probe. Binding complexes were resolved by non-denaturing polyacrylamide gel electrophoresis and prepared for autoradiography. The effect of NIM and DEX on transcriptional activation of the mouse mammary tumour virus (MMTV) promoter was determined by transfecting HSF with MMTV-Luciferase (LUC, reporter gene) constructs. Luciferase values, expressed as light units, were normalized to the level of β-galactosidase activity.

Results NIM had no effect on the number of GR binding sites, in contrast to NAP and DEX, which caused marked reductions (75 and 85% respectively). NIM or NAP did not influence cellular GR protein levels or nucleocytoplasmic shuttling, although DEX lowered GR mRNA and protein levels. NIM, but not NAP, markedly induced MAPK phosphorylation (suggesting an increase in MAPK cascade activity), GR phosphorylation, GR binding to GRE and transcriptional activation of the MMTV promoter through the GRE site in the promoter (5.5-fold, 30 µg/ml).

Discussion Clinical studies indicate that NIM has several favourable characteristics as an anti-inflammatory drug [14]. New molecular targets have been identified that help to explain some of the therapeutic effects of this drug. The present results indicate modulation of the GR system by NIM, so that some of the anti-inflammatory effects of NIM in vivo might be due to phosphorylation and activation of the GR with the resultant changes in the expression of glucocorticoid target genes (Fig. 1). To our knowledge, ours is the first report of this type of drug acting on the GR system. The effects of NIM, in terms of GR binding, phosphorylation and DNA binding, contrast markedly with those of NAP. This is probably not unexpected as the two NSAIDs differ markedly in their chemical composition, relative specificities for the COX isoenzymes, mechanisms of action and pharmacokinetics [15].

Acknowledgements The authors would like to thank Ms S. Fiori, C. Byrne and F. Roy-Huppen for their expert secretarial assistance, Dr Kay Kiansa for the cell culture work, and Dr J. Drouin for providing the MMTV-LUC plasmid. Supported in part by the Medical Research Council of Canada and Helsinn Healthcare SA, Lugano-Pazzallo, Switzerland.

References 1. Tavares IA, Bishai PM, Bennett A. Activity of nimesulide

13

on constitutive and inducible cyclooxygenases. Arzneimittelforschung 1995;45:1093–5. 2. Vago T, Bevilacqua M, Norbiato G. Effect of nimesulide action time dependence on selectivity towards prostaglandin G/H synthase/cyclooxygenase activity. Arzneimittelforschung 1995;45:1096–8. 3. Famaey JP. In vitro and in vivo pharmacological evidence of selective cyclooxygenase-2 inhibition by nimesulide: an overview. Inflamm Res 1997;46:437–46. 4. Pelletier JP, Mineau F, Fernandes JC, Kiansa K, Ranger P, Martel-Pelletier J. Two NSAIDs, nimesulide and naproxen, can reduce the synthesis of urokinase and IL-6 while increasing PAI-1, in human OA synovial fibroblasts. Clin Exp Rheumatol 1997;15:393–8. 5. Pelletier JP, Martel-Pelletier J. Effects of nimesulide and naproxen on the degradation and metalloprotease synthesis of human osteoarthritic cartilage. Drugs 1993;46 (suppl. 1):34–9. 6. Pelletier JP, Di Battista JA, Ranger P, Martel-Pelletier J. The reduced expression of glucocorticoid receptors in synovial cells induced by NSAIDs can be reversed by prostaglandin E1 analog. J Rheumatol 1994;21:1748–52. 7. Pelletier JP, Roughley PJ, Di Battista JA, McCollum R, Martel-Pelletier J. Are cytokines involved in osteoarthritic pathophysiology? Semin Arthritis Rheum 1991;20:12–5. 8. Arend WP, Dayer JM. Cytokines and cytokine inhibitors or antagonists in rheumatoid arthritis [review]. Arthritis Rheum 1990;33:305–15. 9. Firestein GS, Paine M, Littman BH. Gene expression (collagenase, tissue inhibitor of metalloproteinases, complement, and HLA-DR) in rheumatoid arthritis and osteoarthritis synovium: quantitative analysis and effect of intra-articular corticosteroids. Arthritis Rheum 1991; 34:1094–105. 10. Di Battista JA, Mengkun Zhang, Martel-Pelletier J, Fernandes J, Alaaeddine N, Pelletier JP. Nimesulide, a preferential cyclooxygenase-2 (COX-2) inhibitor, enhances phosphorylation and transcriptional activity of the glucocorticoid receptor (GR) in human synovial fibroblasts. Arthritis Rheum 1999; in press. 11. Di Battista JA, Martel-Pelletier J, Wosu LO, Sandor T, Antakly T, Pelletier JP. Glucocorticoid receptor mediated inhibition of interleukin-1 stimulated neutral metalloprotease synthesis in normal human chondrocytes. J Clin Endocrinol Metab 1991;72:316–26. 12. Miller C, Zhang M, Zhao J, Pelletier JP, Martel-Pelletier J, Di Battista JA. Transcriptional induction of cyclooxygenase-2 gene by okadaic acid inhibition of phosphatase activity in human chondrocytes: co-stimulation of AP-1 and CRE nuclear binding proteins. J Cell Biochem 1998;69: 392–413. 13. DiBattista JA, Martel-Pelletier J, Antakly T, Tardif G, Cloutier JM, Pelletier JP. Reduced expression of glucocorticoid receptor levels in human osteoarthritic chondrocytes. Role in the suppression of metalloprotease synthesis. J Clin Endocrinol Metab 1993;76:1128–34. 14. Carr DP, Henn R, Green JR, Bottcher I. Comparison of the systemic inhibition of thromboxane synthesis, antiinflammatory activity and gastro-intestinal toxicity of non-steroidal anti-inflammatory drugs in the rat. Agents Actions 1986;19:374–5. 15. Ouellet M, Percival MD. Effect of inhibitor timedependency on selectivity towards cyclooxygenase isoforms. Biochem J 1995;306:247–51.