Enhancement of phosphorylation and transcriptional activity of the glucocorticoid receptor in human synovial fibroblasts by nimesulide, a preferential cyclooxygenase 2 inhibitor
Abstract
Objective
To examine the effect of 2 nonsteroidal antiinflammatory drugs (NSAIDs), nimesulide (NIM), a preferential cyclooxygenase 2 (COX-2) inhibitor, and naproxen (NAP), on the functional parameters and transcriptional activity of the glucocorticoid receptor (GR) system in cultured human synovial fibroblasts (HSF).
Methods
HSF were incubated with NIM (0.3, 3, and 30 μg/ml), NAP (15, 30, and 90 μg/ml), and dexamethasone (DEX; 0.01, 0.1, and 1 μM) on a time- and dose-dependent basis. The numbers of GR binding sites per cell were determined by radioligand receptor assay. Total cellular, cytoplasmic, or nuclear GR protein was measured by Western analysis using a specific anti-human GR antibody. Phosphorylation of GR was determined by specific immunoprecipitation of protein extracts from 32P-orthophosphate–labeled HSF. Mitogen-activated protein kinase p44/42 (MAPK) phosphorylation was followed by Western analysis using a specific anti-phosphoMAPK antibody. Levels of activated nuclear GR capable of binding specifically to a 32P-labeled oligonucleotide harboring the glucocorticoid/hormone response element (GRE) were evaluated by gel electrophoretic mobility shift analysis. The effects of NIM and DEX on transcriptional activation of the mouse mammary tumor virus (MMTV) promoter was determined by transfecting HSF with MMTV-luciferase (reporter gene) constructs.
Results
NIM had no effect on the number of GR binding sites, in contrast to NAP and DEX. NIM and NAP did not influence cellular GR protein levels or nucleocytoplasmic shuttling, although DEX lowered GR messenger RNA and protein levels after 48 hours. NIM, but not NAP, markedly increased MAPK phosphorylation (suggesting an increase in MAPK cascade activity), GR phosphorylation, GR binding to GRE, and transcriptional activation of MMTV promoter through the GRE site in the promoter.
Conclusion
This study is the first to report that the antiinflammatory effects of NIM, an NSAID, may be partly related to its activation of the GR system.
