INVOLVEMENT OF NUCLEAR FACTOR KAPPA-B IN THE REGULATION OF CYCLOOXYGENASE-2 EXPRESSION BY INTERLEUKIN-1 IN RHEUMATOID SYNOVIOCYTES

Objective. To evaluate involvement of the transcription factor nuclear factor kappa B (NF-kappa B) in the increased expression of cyclooxyge nase-2 (COX-2) stimulated by interleukin-1 beta (IL-1 beta) in primary rheumatoid synoviocytes. Methods. We treated early-passage rheumatoid synoviocytes with IL-1 beta and examined the time course of NF-kappa B translocation to the nucleus by Western blot analysis, as well as NF -kappa B binding to the COX-2 promoter/enhancer by electrophoretic mob ility shift assay. We correlated the time course of NF-kappa B binding with expression of COX-2 messenger RNA (mRNA) and protein. Synoviocyt es were then treated with either sense or antisense phosphorothioate-m odified oligonucleotides derived from the transcription start site of the human NF-kappa B p65 RNA. We analyzed NF-kappa B binding to the CO X-2 promoter and COX-2 protein levels after these treatments. Results. IL-1 beta rapidly stimulated the translocation of the p65, p50, and c -rel NF-kappa B subunits from the cytoplasm to the nucleus. Electropho retic mobility shift assay demonstrated binding to 2 NF-kappa B sites within the COX-2 promoter/enhancer, with a time course identical to th at of nuclear localization of NF-kappa B. Supershift analysis revealed that binding activity was due primarily to the p65-p50 heterodimer an d the p50 homodimer. With appropriate lag time after NF-kappa B bindin g, COX-2 mRNA and protein were increased. Pretreatment of RA synoviocy tes with NF-kappa B p65 antisense oligonucleotides resulted in decreas ed binding to the COX-2 promoter and decreased COX-2 protein expressio n, Conclusion. These data demonstrate that signaling via the NF-kappa B pathway is involved in regulation of COX-2 expression induced by IL- 1 beta in RA synoviocytes.