Manipulation of Distinct NFκB Proteins Alters Interleukin-1β-induced Human Rheumatoid Synovial Fibroblast Prostaglandin E2 Formation

• DOI: 10.1074/jbc.271.49.31496
• Publication Date: 2002-07-26T14:51:35Z
• Authors: Amy K. Roshak; Jeffrey R. Jackson; Kevin McGough; Marie Chabot-Flet...; Eugene Mochan; Lisa A. Marshall
• Keywords: 0301 basic medicine; 0303 health sciences; Interleukin-8; Synovial Membrane; NF-kappa B; Transcription Factor RelA; Membrane Proteins; Fibroblasts; In Vitro Techniques; Oligonucleotides, Antisense; Blotting, Northern; Dinoprostone; Phospholipases A; Up-Regulation; Arthritis, Rheumatoid; Isoenzymes; Phospholipases A2; 03 medical and health sciences; Cyclooxygenase 2; Prostaglandin-Endoperoxide Synthases; Humans; Interleukin-1

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.