Herpesvirus entry mediator (HVEM),

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...

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A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.