Figure 4

PIP-18 suppresses IL-stimulated p38 MAPK phosphorylation. (a) Rheumatoid arthritis (RA) synovial fibroblast (SF) cells were preincubated at 37°C for one hour with various inhibitors at optimal concentrations: phospholipase inhibitor from python (PIP)-18 (5 μM), LY315920 (5 μM), SB202190 (10 μM), PD98059 (1 μM) or SP600125 (5 μM), and stimulated with rhIL-1β (10 ng/ml) for 30 minutes before assaying for p38, Erk and JNK phosphorylation, using cell-based ELISA. For control of systematic variation, blank control wells (without cells) as well as experimental control wells (seeded cells without any treatment) were included. Phosphorylation index (Pi) was calculated as relative levels of the phosphorylated form of mitogen-activated protein kinase (MAPK)/total MAPK levels. Values are mean ± standard error of the mean (SEM) of three separate experiments presented as fold increase of Pi of experimentally treated cells relative to control cells without any treatment. (b) RA SF from separate experiments were pretreated with inhibitors as in (a), followed by stimulation with hrIL-1β (10 ng/ml) for 16 hours, and supernatants analyzed for secretory phospholipase A₂(sPLA₂) and matrix metalloproteinase (MMPs) as indicated. Values expressed as % IL-1β stimulation are mean ± SEM for four experiments for each condition. PIP-18 was more effective in suppressing MMP/sPLA₂ production (***P < 0.001 vs IL), while LY315920, p38 and Erk inhibitors were relatively less effective (*P < 0.05 vs IL). *P < 0.05, **P < 0.01 (one-way analysis of variance with Bonferroni's post hoc test); for pair-wise comparisons (IL without inhibitor versus IL with each of the inhibitor used in the study).