Interleukin 1 (IL-1) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A 2 (PLA 2 ) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFB consensus site. Immunoblot analysis identified NFB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFB consensus motif. An NFB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE 2 production (IC 50 ؍ ϳ2 M), indicating a role of NFB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1-stimulated PGE 2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA 2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFB 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 B motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA 2 gene induction and support the IL-1 activation and participation of distinct NFB protein dimers in RSF prostanoid and IL-8 formation.Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation and hyperproliferation of the synovial lining (1). Enhanced levels of the cytokine, interleukin (IL)-1, 1 perpetuate the disease process through up-regulation of a multitude of factors leading to eicosanoid formation, matrix degradation, bone resorption, and proliferation in the joint (2-6). We and others have demonstrated that human rheumatoid synovial fibroblast (RSF) prostaglandin (PG) E 2 accumulation in response to IL-1 is a direct result of the coordinate up-regulation of 85-kDa phospholipase A 2 (PLA 2 ) and the induction of COX II (6 -8). Indeed, we reported that depletion of IL-1-induced 85-kDa PLA 2 to basal levels by antisense severely compromised the ability of RSF to make PGE 2 . However, the mechanism(s) by which IL-1 regulates 85-kDa PLA 2 and COX II gene induction in this system have not been elucidated.IL-1 is a potent activator of nuclear factor B (NFB) (1, 9, 10) in other cell systems, and this transcription factor in turn regulates a wide variety of inflammatory and immunoregulatory genes (10 -16). 5Ј-flanking regulatory regions for both the human 85-kDa PLA 2 and COX II genes have recently been isolated (17, 18), and sequence analysis has identified a number of possible transcription factor consensus binding motifs, including NFB. The putative NFB motif in the 85-kDa PLA 2 promoter is located at Ϫ1099 base pairs (17), whereas the NFB consensus site in the human COX II promoter is located at Ϫ233 base pairs (18).NFB is a dimeric DNA binding protein comprised of members of the NFB/Rel/dorsal family of protei...
Rheumatoid arthritis and periodontitis are inflammatory diseases modulated by proinflammatory cytokines [e.g. interleukin (IL-1) 1 and tumour necrosis factor alpha], which activate local fibroblasts to do the following: (1) proliferate, (2) induce gene expression and (3) produce destructive metalloproteinases. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor (composed of HIF-1alpha and HIF-1beta/aryl hydrocarbon receptor nuclear transporter) that is modulated by hypoxia. HIF-1 binds to and induces several genes containing an HIF-1 consensus-binding site, including vascular endothelial growth factor and several glycolytic enzymes. Through differential screening of a human synovial fibroblast cDNA library, we identified HIF-1alpha as a clone up-regulated by IL-1. The mRNA for HIF-1alpha subunit was increased 3-4-fold by Northern blot analysis after cells had been incubated for 3 h in the presence of IL-1. In addition, IL-1 increased the binding of the heterodimer HIF-1 to the HIF consensus sequence. These results suggest that HIF-1 might have a role in inflammation, possibly in attempting to re-establish homoeostasis.
Interleukin-1 (IL-1) treatment of synovial cells from rheumatoid arthritis and osteoarthritis patients resulted in a dose-dependent secretion of phospholipase A2 (PLA2). IL-1 also stimulated prostaglandin E2 and plasminogen activator synthesis, in parallel with PLA2 activation; all 3 were detectable within 6 hours of IL-1 treatment and peaked by 24 hours. Synovial cell PLA2 required calcium (5 mM) and a neutral pH (7.5) for maximal activity and appears similar to the PLA2 in synovial fluid, which has been described previously. We conclude that PLA2 can be induced by IL-1, and its secretion may contribute significantly to the inflammatory actions of IL-1.
IL-4 inhibits IL-1 induction of both collagenase and stromelysin, as well as PGE2 production, in human synovial fibroblasts. The inhibition occurs at least in part at the level of transcription, does not affect binding of transcription factor AP-1, and appears to involve a mechanism that is independent of the ability of IL-4 to inhibit production of PGE2.
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