Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation.
Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.
Roshak
A K
AK
SmithKline Beecham Pharmaceuticals, SmithKline Beecham Pharmaceuticals, Immunopharmacology, UW2532, King of Prussia, Pennsylvania 19406, USA.
Jackson
J R
JR
McGough
K
K
Chabot-Fletcher
M
M
Mochan
E
E
Marshall
L A
LA
D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D015152:Blotting, Northern; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology*; D016209:Interleukin-8 / Q000096:biosynthesis; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism* / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D013583:Synovial Membrane / Q000187:drug effects*; D051996:Transcription Factor RelA; D015854:Up-Regulation / Q000187:drug effects
D007375:Interleukin-1; D016209:Interleukin-8; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D051996:Transcription Factor RelA; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2; D015232:Dinoprostone