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 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.


CONCLUSIONS
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.
Crofford
L J
LJ
University of Michigan, Ann Arbor, USA.
Tan
B
B
McCarthy
C J
CJ
Hla
T
T
D001172:Arthritis, Rheumatoid / Q000473:pathology*; D015153:Blotting, Western; D004586:Electrophoresis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007375:Interleukin-1 / Q000502:physiology*; D016328:NF-kappa B / Q000494:pharmacology / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D013583:Synovial Membrane / Q000276:immunology / Q000473:pathology*; D013997:Time Factors; D051996:Transcription Factor RelA
D007375:Interleukin-1; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D011451:Prostaglandin-Endoperoxide Synthases