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.


CONCLUSIONS
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.
Gossye
Valerie
V
Ghent University, Ghent, Belgium.
Elewaut
Dirk
D
Bougarne
Nadia
N
Bracke
Debby
D
Van Calenbergh
Serge
S
Haegeman
Guy
G
De Bosscher
Karolien
K
D000085:Acetates; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D014439:Tyramine / Q000031:analogs & derivatives; D019892:eIF-2 Kinase / Q000378:metabolism
C104564:2-(4-acetoxyphenyl)-2-chloro-N-methylethylamine; D000085:Acetates; D005938:Glucocorticoids; D053583:Interleukin-1beta; D016328:NF-kappa B; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D019892:eIF-2 Kinase; D051550:I-kappa B Kinase; D020929:Mitogen-Activated Protein Kinase Kinases; D014439:Tyramine