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Abstract

Objective. To evaluate involvement of the transcription factor nuclear factor kB (NF-kB) 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-kB translocation to the nucleus by Western blot analysis, as well as NF-kB binding to the COX-2 promoter/enhancer by electrophoretic mobility shift assay. We correlated the time course of NF-kB 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-kB p65 RNA. We analyzed NF-kB 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-kB subunits from the cytoplasm to the nucleus. Electrophoretic mobility shift assay demonstrated binding to 2 NF-kB sites within the COX-2 promoter/enhancer, with a time course identical to that of nuclear localization of NF-kB. Supershift analysis revealed that binding activity was due primarily to the p65–p50 heterodimer and the p50 homodimer. With appropriate lag time after NF-kB binding, COX-2 mRNA and protein were increased. Pretreatment of RA synoviocytes with NF-kB 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-kB pathway is involved in regulation of COX-2 expression induced by IL-1β in RA synoviocytes.