Effect of signal inhibitors on MIF-induced RANKL expression in RA human synovial fibroblasts. RA synovial fibroblasts were pretreated with 20 μM LY294002, 10 μM SB203580, 1 μM SP600125, 10 μM PD98059, 50 μM AG490, 100 nM cyclosporin A, 10 μM parthenolide, or 10 μM of curcurmin, then cultured with 5 ng/mL of rhMIF for 72 hours. (a) After RA synovial fibroblasts were incubated with the signal inhibitors and rhMIF, mRNA was extracted and measured using real-time PCR. The data represent the mean and standard deviation of five separate experiments. (b) MIF activates the phosphorylation of Akt, p38 MAPK, STAT3, IκBα, and c-Jun in RA synovial fibroblasts. The activated forms of Akt, p38 MAPK, STAT3, IκBα, and c-Jun were detected by western blot analysis in RA synovial fibroblasts stimulated with rhMIF, while the amounts of total Akt, p38 MAPK, STAT3, IκBα, and c-Jun were unchanged. The data represent one of three independent experiments. *P < 0.05 and **P < 0.005. MIF, macrophage migration inhibitory factor; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor kappa-B ligand.