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Objective: To explore the influence of chemokine, CXCL16, on the expression of the receptor activator nuclear factor κB ligand (RANKL) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS). Methods: The expression of CXCL16/CXCR6 and RANKL in RA or osteoarthritis (OA) patient synovia was examined by Western blot and immunohistochemistry. The serum concentration of CXCL16 and RANKL was measured by enzyme-linked immunosorbent assay (ELISA). RA-FLS were treated with recombinant CXCL16, and RANKL mRNA and protein were measured using PCR, Western blot and ELISA. Results: The synovial expression of CXCL16, CXCR6, and RANKL was higher in RA patients than in patients with OA. The serum CXCL16 and RANKL levels were higher in RA patients compared with OA patients and healthy controls. CXCL16 correlated with erythrocyte sedimentation rate, C reactive protein, disease activity, serum rheumatoid factor, and RANKL. RA-FLS treated with CXCL16 showed markedly increased expression of RANKL. When STAT3 or p38 activation was blocked by an inhibitor, CXCL16 failed to upregulate RANKL expression. In contrast, inhibiting the Akt or Erk pathway did not achieve the same effect. Conclusions: CXCL16 upregulates RANKL expression in RA-FLS and these effects are mainly mediated by the JAK2/STAT3 and p38/MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

Highlights: [•] AG490 inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells. [•] AG490 affects cell proliferation and cell cycle distribution. [•] AG490 reduces NFATc1 expression during RANKL-induced osteoclastogenesis. [•] AG490 disrupts the activation of RANKL-mediated JAK2/STAT3 signaling pathway. [•] STAT3 depletion partly mimics the effect of AG490 on RANKL-induced osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Tofacitinib, a small molecule JAK inhibitor, has been widely used to reduce inflammation and inhibit progression of bone destruction in rheumatoid arthritis. STAT3, a downstream signaling molecule of JAK, plays a key role in the activation of signaling in response to inflammatory cytokines. Thus, targeting STAT3 may be an inspiring strategy for treating osteoclast-related diseases such as rheumatoid arthritis. In this study, we first investigated the effects of Stattic, a STAT3 inhibitor, on receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. Stattic inhibited osteoclast differentiation and bone resorption in RANKL-induced RAW264.7 cells in a dose-dependent manner. Stattic also suppressed RANKL-induced upregulation of osteoclast-related genes tartrate-resistant acid phosphatase, matrix metalloproteinase 9, cathepsin K, RANK, tumor necrosis factor receptor-associated factor 6, and osteoclast-associated receptor in RAW264.7 cells. Moreover, Stattic exhibited an inhibitory effect on cell proliferation and cell cycle progression at higher dosages. At the molecular level, Stattic inhibited RANKL-induced activation of STAT3 and NF-κB pathways, without significantly affecting MAPK signaling. In addition, Stattic inhibited RANKL-induced expression of osteoclast-related transcription factors c-Fos and NFATc1. Importantly, Stattic also prevented bone loss caused by ovariectomy. Together, our data confirm that Stattic restricts osteoclastogenesis and bone loss by disturbing RANKL-induced STAT3 and NF-κB signaling. Thus, Stattic represents a novel type of osteoclast inhibitor that could be useful for conditions such as osteoporosis and rheumatoid arthritis. [ABSTRACT FROM AUTHOR]