Regulatory effect of tacrolimus on RANKL and OPG expression in FLS under IL-6/sIL-6R stimulation. (A) After pretreatment of cultured FLS with IL-6/sIL-6R (100 ng of both), incubation with tacrolimus at several concentrations led to a consistently marked reduction of RANKL at the mRNA (*P <0.001 at 10, 100, 1,000 nM of tacrolimus) and protein levels (‡P <0.05 at 100 nM and †P <0.01 1,000 nM of tacrolimus). In contrast, tacrolimus at dosages of 100 and 1,000 nM significantly increased levels of OPG mRNA (*P <0.001 for each dosage). Expression of OPG protein was consistently observed (†P <0.01 of 100 and 1,000 nM). (B) In western blot analysis for RANKL expression after tacrolimus treatment, tacrolimus was found to inhibit RANKL expression in FLS stimulated by IL-6/sIL-6R (100 ng of both). In contrast, OPG expression was increased following tacrolimus treatment. (C) Tacrolimus was shown to inhibit the expression of RANKL in FLS after stimulation with IL-6/sIL-6R in the immunofluorescence assay. (D) The inhibitory effect of tacrolimus (1 µM) on RANKL expression was more prominent than that of other anti-inflammatory drugs such as methotrexate (1 µg) and dexamethasone (1 µg). OPG mRNA expression was increased by tacrolimus and methotrexate but not by dexamethasone. (*P <0.001, †P <0.01, ‡P <0.05 versus IL-6/sIL-6R-treated FLS). Data are determined in three independent experiments. FLS, fibroblast-like synociocytes; IL-6, interleukin-6; OPG, osteoprotegerin; RANKL, receptor activator of NF-κB ligand; sIL-6R, soluble interleukin-6 receptor.