Involvement of nuclear factor kB in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes
Version of Record online: 12 DEC 2005
Copyright © 1997 American College of Rheumatology
Arthritis & Rheumatism
Volume 40, Issue 2, pages 226–236, February 1997
How to Cite
Crofford, L. J., Tan, B., McCarthy, C. J. and Hla, T. (1997), Involvement of nuclear factor kB in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis & Rheumatism, 40: 226–236. doi: 10.1002/art.1780400207
- Issue online: 12 DEC 2005
- Version of Record online: 12 DEC 2005
- Manuscript Accepted: 21 AUG 1996
- Manuscript Received: 7 JUN 1996
- NIH. Grant Number: P60-AR20557
- NIH. Grant Numbers: HL-49094, DK-45659
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.