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

Crofford LJ, Tan B, McCarthy CJ, Hla T
Arthritis Rheum. 1997 40 (2): 226-36

PMID: 9041934 · DOI:10.1002/art.1780400207

OBJECTIVE - To evaluate involvement of the transcription factor nuclear factor kappa B (NF-kappa B) in the increased expression of cyclooxygenase-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 mobility shift assay. We correlated the time course of NF-kappa 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-kappa B p65 RNA. We analyzed NF-kappa B binding to the COX-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. Electrophoretic mobility shift assay demonstrated binding to 2 NF-kappa B sites within the COX-2 promoter/enhancer, with a time course identical to that of nuclear localization of NF-kappa 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-kappa B binding, COX-2 mRNA and protein were increased. Pretreatment of RA synoviocytes with NF-kappa 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-kappa B pathway is involved in regulation of COX-2 expression induced by IL-1 beta in RA synoviocytes.

MeSH Terms (14)

Arthritis, Rheumatoid Blotting, Western Electrophoresis Gene Expression Regulation, Enzymologic Humans Interleukin-1 NF-kappa B Oligonucleotides, Antisense Promoter Regions, Genetic Prostaglandin-Endoperoxide Synthases RNA, Messenger Synovial Membrane Time Factors Transcription Factor RelA

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