222. Molecular mechanisms involved in PGE2 release induced by the snake venom metalloproteinase BaP1 in synoviocytes

Abstract

Background: Snake venom metalloproteinases (SVMPs) and Matrix metalloproteinases (MMPs) share common domain organization and exhibit identical Zn-binding motif. Studies on SVMPs may thus provide insights into the functions of MMPs. Levels of these enzymes are increased in inflamed articular joints. Articular synovial fibroblasts (B type) are the main cells involved in production and release of inflammatory mediators during joint inflammatory processes, being PGE2 a major mediator. BaP1 is a 22.7 kDa SVMP from B. asper snake venom and contains only the metalloproteinase domain. This enzyme displays potent inflammatory activities both in vivo and in vitro experimental models. In this study we investigated the effects of this SVMP on isolated synoviocytes focusing on the release of prostaglandin E2 (PGE2) and the molecular mechanisms involved in this effect. Methods: B type synoviocytes isolated from rat knee joints synovial membranes (CEUIAB 576/09) were used. Levels of PGE2 were measured by enzyme immunoassay and protein expression of the PGE2 receptor EP4 was determined by Western blotting. Participation of both NF-kB and EP4 receptor in the release of PGE2 and COX-2 protein expression induced by BaP1 was evaluated in cells pretreated with the inhibitors SN50 (50 μg/mL) and AH23848 (30 μM), respectively. Results: BaP1 induced release of PGE2 from B type synoviocytes after 1, 3, 6 and 12 h, but not 30 min incubation, in comparison with control cells incubated with RPMI alone. BaP1 induced EP4 protein expression (52 and 65 kDa) by synoviocytes (30min – 6h). Moreover, inhibition of NF-kB or EP4 receptor significantly decreased BaP1-induced PGE2 release and COX-2 protein expression. Discussion: These data indicate the ability of BaP1 to induce biosynthesis of PGE2 and COX-2 expression in isolated B type synoviocytes. These effects are mediated by NF-kB pathway. Moreover, data demonstrated that EP4 receptor regulates BaP1-induced PGE2 production and COX-2 expression through a positive feedback loop, and strongly suggest that this subtype of PGE2 receptor contributes for amplification of BaP1-induced release of PGE2. Conclusion: BaP1 can directly stimulate synoviocytes for synthesis of PGE2 and expression of both COX-2 enzyme and EP4 receptor. These findings suggest novel mechanisms of action displayed by metalloproteinases in synoviocytes.