Abstract
IntroductionThis study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling.MethodsThe expression of RANKL, JAK2, STAT3, and SOCS3 proteins was assessed by western blot analysis, real-time PCR and ELISA in IL-6 combined with soluble IL-6 receptor (sIL-6R)-stimulated rheumatoid arthritis (RA)-FLS with or without tacrolimus treatment. The effects of tacrolimus on synovial inflammation and bone erosion were assessed using mice with arthritis induced by K/BxN serum. Immunofluorescent staining was performed to identify the effect of tacrolimus on RANKL and SOCS3. The tartrate-resistant acid phosphatase staining assay was performed to assess the effect of tacrolimus on osteoclast differentiation.ResultsWe found that RANKL expression in RA FLS is regulated by the IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. Inhibitory effects of tacrolimus on RANKL expression in a serum-induced arthritis mice model were identified. Tacrolimus inhibits RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing STAT3. Among negative regulators of the JAK/STAT pathway, such as CIS1, SOCS1, and SOCS3, only SOCS3 is significantly induced by tacrolimus. As compared to dexamethasone and methotrexate, tacrolimus more potently suppresses RANKL expression in FLS. By up-regulating SOCS3, tacrolimus down-regulates activation of the JAK-STAT pathway by IL-6/sIL-6R trans-signaling, thus decreasing RANKL expression in FLS.ConclusionsThese data suggest that tacrolimus might affect the RANKL expression in IL-6 stimulated FLS through STAT3 suppression, together with up-regulation of SOCS3.
Highlights
This study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of nuclear factor-B (NF-B) ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling
We found that IL-6/soluble IL-6 receptor (sIL-6R) increased receptor activator of NF-κB ligand (RANKL) expression in a dose-dependent manner, whereas OPG expression after IL-6/sIL-6R treatment was decreased compared to untreated cells (Figure 1A)
Enhanced expression of suppressor of cytokine signaling 3 (SOCS3) and RANKL might be induced by activation of the JAK-STAT signaling pathway, which is stimulated by IL-6/sIL-6R
Summary
This study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-B ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling. Receptor activator of NF-B ligand (RANKL) is a transmembrane protein of the TNF superfamily, which is an important molecule in bone metabolism [1]. The IL-6 and IL-6R complex leads to homodimerization of the cell surface molecule, gp130, which subsequently transduces a signal that activates intracytoplasmic Janus activated kinase (JAK) tyrosine kinase. JAK tyrosine kinase preferentially induces tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) [6]. The roles of suppressor of cytokine signaling/cytokine-inducible SH2 (SOCS/CIS) have been retained, both SOCS1 and SOCS3 negatively regulate JAK tyrosine kinase as feedback inhibitors [6]. Regulation of STAT3 and SOCS3 in the FLS of patients with RA through the IL-6/gp130/STAT3 signaling pathway might be a potent therapeutic strategy in the treatment of RA
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