Nimesulide, a preferential cyclooxygenase 2 inhibitor, suppresses peroxisome proliferator-activated receptor induction of cyclooxygenase 2 gene expression in human synovial fibroblasts: evidence for receptor antagonism.

Abstract

To characterize the inhibitory effects of therapeutic concentrations of the nonsteroidal antiinflammatory drug nimesulide (NIM) on peroxisome proliferator-activated receptor (PPAR)-induced cyclooxygenase 2 (COX-2) gene expression in human synovial fibroblasts (HSFs) from patients with osteoarthritis (OA) and to define the intracellular mechanisms mediating the response. PPARalpha and PPARgamma messenger RNA (mRNA) expression and protein synthesis in OA HSFs were measured by reverse transcription-polymerase chain reaction and electrophoretic mobility shift assay, respectively. Experiments investigating endogenous and overexpressed PPARalpha and PPARgamma activation of COX-2 mRNA and protein were conducted by incubating nontransfected and transfected cells with increasing concentrations of cognate ligands WY-14,643 (alpha agonist), ciglitasone (gamma agonist), and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) in the absence or presence of NIM and NS-398 (1 microM). COX-2 mRNA and protein were measured by Northern and Western blotting procedures, respectively. Receptor activation studies were evaluated by cotransfecting pSG5-Gal 4 DNA binding domain (DBD)-PPARalpha ligand binding domain (LBD) or pSG5-Gal 4 DBD-PPARgamma LBD chimeric constructs with a 5x Gal 4 enhancer site tk-tataa-luciferase reporter under ligand stimulation in the presence or absence of increasing concentrations of NIM. Gene transactivation analyses were conducted by treating cells overexpressing cytomegalovirus (CMV)-PPARalpha or CMV-PPARgamma expression constructs with either a PPAR response element (PPRE)-luciferase construct containing 3 DR1 acyl-coenzyme A (acyl-CoA) oxidase gene response elements or human COX-2 promoter constructs with WY-14,643, ciglitasone, and 15d-PGJ(2) in the presence or absence of increasing concentrations of NIM. Human synovial cells expressed functional PPAR isoforms, PPARalpha and PPARgamma. Neither receptor agonists nor antagonists modulated the intracellular protein levels of PPAR. PPARalpha and, especially, PPARgamma mediated the induction of COX-2 gene expression by receptor agonists. Stimulation of COX-2 mRNA expression and protein synthesis by 15d-PGJ(2) appeared to occur through a receptor-independent process. NIM inhibited PPAR agonist stimulation of COX-2 expression and synthesis in a dose-dependent manner in both nontransfected cells and cells overexpressing both receptor isoforms. NIM potently abrogated basal and ligand-stimulated PPRE(3X) DR1 acyl-CoA oxidase-driven luciferase activity and also human PPRE-containing COX-2 promoter activity. PPAR-mediated induction of COX-2 expression and synthesis in human OA synovial fibroblasts is inhibited by therapeutic concentrations of NIM through the functional antagonism of ligand-dependent receptor activation, with the resultant suppression of PPAR-dependent transactivation of target genes (e.g., COX-2).