Biological therapies such as tumor necrosis factor-α inhibitors have advanced the treatment of rheumatoid arthritis, but one-third of patients do not respond to such therapy. Furthermore, these inhibitors are now usually administered in combination with conventional disease-modifying antirheumatic drugs, suggesting they have not achieved their early promise. This study investigates a novel therapeutic target, proteinase-activated receptor (PAR)-2, in joint inflammation. Intra-articular carrageenan/kaolin (C/K) injection in mice resulted in joint swelling that was associated with synovial PAR₂ up-regulation. Inhibiting receptor up-regulation using small interfering RNA technology, as confirmed by immunoblotting, substantially reduced the inflammatory response in the joint. Serine proteinase-induced joint swelling was mediated primarily via PAR₂ activation, since the response to exogenous application of trypsin and tryptase was absent in PAR₂ knockout mice. Furthermore, serine proteinase inhibitors were effective anti-inflammatory agents in this model. Disrupting proteolytic activation of PAR₂ using antiserum (B5) directed to the receptor cleavage/activation site also attenuated C/K-induced inflammation, as did the similarly targeted PAR₂ monoclonal antibody SAM-11. Finally, we report the activity of a novel small molecule PAR₂ antagonist, N¹-3-methylbutyryl-N⁴-6-aminohexanoyl-piperazine (ENMD-1068), that dose dependently attenuated joint inflammation. Our findings represent a major advance in collectively identifying PAR₂ as a novel target for the future treatment of arthritis.

Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages.

Background and purpose: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)‐induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action.Experimental approaches: Cardamonin was isolated from the Greek plantA. absinthiumL. Its effects were assessed on LPS‐induced nitrite release and iNOS and COX‐2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFκB pathway, at the level of IκBαdegradation and phosphorylation of NFκB. Also its effects on NFκB and GAS/GAF‐DNA binding were assessed by EMSA.Key results: Cardamonin concentration‐dependently inhibited both NO release and iNOS expression but had no effect on COX‐2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IκBαor phosphorylation of NFκB. However, it inhibited NFκB DNA‐binding in both LPS‐stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNγ‐stimulated iNOS induction and GAS/GAF‐DNA binding.Conclusions and Implications: These results show that the inhibitory effect of cardamonin on LPS‐induced iNOS induction is not mediated via effects on the initial activation of the NFκB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX‐2 expression, its exact mechanism(s) of action has yet to be identified.British Journal of Pharmacology(2006)149, 188–198. doi:10.1038/sj.bjp.0706856

In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory κB kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NFκB-DNA binding and the activation of the upstream kinases IKKα and -β. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKCα, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKCα or PKCζ had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKKα activity but abolished that of IKKβ, whereas DN-PKCα but not DN-PKCζ substantially reduced trypsin-stimulated Flag-IKKβ activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.