The effect of tacrolimus on JAK2, STAT3, and SOCS3 in IL-6/sIL-6R-stimulated FLS. (A) Stimulation with IL-6/sIL-6R (100 ng of both) induced phosphorylation of JAK2 and STAT3. However, tacrolimus reversed these changes, thereby significantly reducing the expression of p-JAK2 and p-STAT3. (B) Tacrolimus treatment of IL-6/sIL-6R-stimulated FLS potently suppressed IL-6 expression. Among negative regulators of the JAK-STAT signaling pathway, prominent induction of SOCS3 mRNA expression was induced by tacrolimus (†P <0.05 at 100 and 1,000 nM) in comparison to IL-6/sIL-6R-stimulated FLS. Expression of SOCS1 and CIS1 mRNA was not similarly induced. (C) Tacrolimus enhanced the level of SOCS3 protein in IL-6/sIL-6R-treated FLS in a dose-dependent manner. (D) In SOCS3 knockdown FLS, IL-6/sIL-6R induced overexpression of RANKL, p-NF-κB and NFATc1. In contrast, addition of tacrolimus induced SOCS3 expression and attenuated RANKL expression. The protein levels of p-NF-κB and NFATc1 were significantly reduced, in comparison to those in SOCS3 knockdown FLS without tacrolimus. (E) The immunofluorescence assay indicated the presence of an increased number of SOCS3-positive cells after treatment with tacrolimus compared to controls. Data are determined in three independent experiments. CIS1, cytokine-inducible SH2; IL-6, interleukin-6; JAK2, Janus activated kinase; RANKL, receptor activator of NF-κB ligand; sIL-6R, soluble interleukin-6 receptor; SOCS1, suppressor of cytokine signaling 1; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3.