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Differential mechanism of NF-kappaB 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-kappaB activation pathway in rheumatoid arthritis (RA) synovial cells.


METHODS
Quantitative polymerase chain reaction was performed to detect the tumor necrosis factor alpha (TNFalpha)-induced cytokine gene expression of interleukin-1beta (IL-1beta) 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-kappaB (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1beta. 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, IkappaBalpha, phospho-p38, phospho-ERK, and phospho-JNK.


RESULTS
Both DEX and CpdA efficiently inhibited IL-1beta gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNFalpha-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent IkappaBalpha degradation. CpdA also displayed profound effects on TNFalpha-induced MAPK activation. The effects of CpdA on TNFalpha-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNFalpha-induced IKK phosphorylation, IkappaBalpha degradation, p65 nuclear translocation, or MAPK activation in RA FLS.


CONCLUSION
DEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-kappaB activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (nongenomic), may explain the gene-inhibitory effects of CpdA in RA FLS.