Involvement of nuclear factor k B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes

Abstract

Objective . To evaluate involvement of the transcription factor nuclear factor k B (NF- k B) in the increased expression of cyclooxygenase-2 (COX-2) stimulated by interleukin-1Β (IL-1Β) in primary rheumatoid synoviocytes. Methods . We treated early-passage rheumatoid synoviocytes with IL-1Β and examined the time course of NF- k B translocation to the nucleus by Western blot analysis, as well as NF- k B binding to the COX-2 promoter/enhancer by electrophoretic mobility shift assay. We correlated the time course of NF- k B binding with expression of COX-2 messenger RNA (mRNA) and protein. Synoviocytes were then treated with either sense or antisense phosphorothioate-modified oligonucleotides derived from the transcription start site of the human NF- k B p65 RNA. We analyzed NF- k B binding to the COX-2 promoter and COX-2 protein levels after these treatments. Results . IL-1Β rapidly stimulated the translocation of the p65, p50, and c-rel NF- k B subunits from the cytoplasm to the nucleus. Electrophoretic mobility shift assay demonstrated binding to 2 NF- k B sites within the COX-2 promoter/enhancer, with a time course identical to that of nuclear localization of NF- k B. Supershift analysis revealed that binding activity was due primarily to the p65–p50 heterodimer and the p50 homodimer. With appropriate lag time after NF- k B binding, COX-2 mRNA and protein were increased. Pretreatment of RA synoviocytes with NF- k B p65 antisense oligonucleotides resulted in decreased binding to the COX-2 promoter and decreased COX-2 protein expression. Conclusion . These data demonstrate that signaling via the NF- k B pathway is involved in regulation of COX-2 expression induced by IL-1Β in RA synoviocytes.