Involvement of mitogen-activated protein kinase and phosphoinositide 3-kinase-Akt pathways in chemerin-induced fibroblast-like synoviocyte activation. (A) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) were pretreated for 30 minutes with PD98059 (mitogen-activated protein kinase kinase inhibitor, 10 μM), SB203580 (p38 mitogen-activated protein kinase inhibitor, 10 μM), LY294002 (phosphoinositide 3-kinase inhibitor, 10 μM), or polymyxin B (50 μg/ml) before stimulation with or without 50 nM chemerin for 48 hours. IL-6 production in the supernatant was measured by ELISA. Data are presented as means ± SEM of one of four independent experiments analyzed in quadruplicate. LY = LY294002; PD = PD98059; polyB = polymyxin B; SB = SB203580. *P < 0.05 relative to treatment with 50 nM chemerin; ns, not significant. (B) and (C) Effects of PD98059, SB203580 and LY294002 on FLS motility were assessed by scrape motility assay. Representative two-dimensional photographs of FLS migratory process into the scraped cell-free area (0 and 24 hours) are shown in (B). The number of migrating cells in the scraped area was counted. Data represent the fold increase in the number of migrating cells, shown in (C). Data are presented as means ± SEM of one of four independent experiments analyzed in triplicate. *P < 0.05 relative to samples not treated with inhibitor; ns, not significant.