Differential Mechanism of NF-κB Inhibition by Two Glucocorticoid Receptor Modulators in Rheumatoid Arthritis Synovial Fibroblasts

Objective. To investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor (GR)-activating compounds, dexamethasone (DEX) and Compound A (CpdA), interfere with the NF-kappa B activation pathway in rheumatoid arthritis (RA) synovial cells. Methods. Quantitative polymerase chain reaction was performed to detect the tumor necrosis factor alpha (TNF alpha)-induced cytokine gene expression of interieukin-1 beta (IL-1 beta) and to investigate the effects of DEX and CpdA in RA fibroblast-like synoviocytes (FLS) transfected with small interfering RNA (siRNA) against GR (siGR) compared with nontransfected cells. Immunofluorescence analysis was used to detect the subcellular distribution of NF-kappa B (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1 beta. Signaling pathways were studied via Western blotting of siGR-transfected cells, compared with nontransfected and nontargeting siRNA-transfected control cells, to detect the regulation of phospho-IKK, I kappa B alpha, phospho-p38, phospho-ERK, and phospho-JNK. Results. Both DEX and CpdA efficiently inhibited IL-1 beta gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNF alpha-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent I kappa B alpha degradation. CpdA also displayed profound effects on TNF alpha-induced MAPK activation. The effects of CpdA on TNF alpha-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNF alpha-induced IKK phosphorylation, I kappa B alpha degradation, p65 nuclear translocation, or MAPK activation in RA FLS. Conclusion. DEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-kappa B activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (non-genomic), may explain the gene-inhibitory effects of CpdA in RA FLS.