Background/Purpose:  Bruton’s tyrosine kinase (Btk) is primarily expressed in B cells, mast cells, platelets, myeloid cells and osteoclasts. Osteoclast differentiation is regulated by signaling pathways activated by receptor activator of nuclear factor-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of ITAM results in the activation of phospholipase C-γ through activation of non-receptor tyrosine kinases Btk and Syk. However, the functional hierarchy of these two kinases has not been fully elucidated. To explore the distinct functions of Btk and Syk, we examined their relative contributions to osteoclast differentiation and activation using inhibitors to both Btk and Syk.

Methods: Human osteoclast precursors were differentiated with 33 ng/mL of M-CSF and 66 ng/mL of RANKL for 7 days. Increasing concentrations (1 nM to 1000 nM) of ONO-4059 and Syk inhibitors were incubated with the cells for 7 days. Osteoclast differentiation was evaluated by TRAP staining followed by light microscopy and quantitation of TRAP+ cells containing at least three nuclei. To evaluate osteoclast function, cytokine production and Btk and Syk signaling were determined by ELISA and Western Blot analysis, respectively.

Results: Both ONO-4059 and the Syk inhibitor specifically inhibited the activity of Btk and Syk, respectively. ONO-4059 dose-dependently inhibited the M-CSF and RANKL-driven osteoclast differentiation by 70% (IC₅₀: 0.853 nmol/L). Surprisingly, the effect of the Syk inhibitor on osteoclast differentiation was much lower (n=3, 30% inhibition). ONO-4059 also reduced the secretion of MIP-1α and RANTES in  bone marrow cell culture, with a weaker effect observed in bone marrow with the Syk inhibitor.

Conclusion: Btk, but not Syk, appears to play a pivotal role in osteoclast differentiation and activation. Rheumatoid arthritis (RA) is often complicated by generalized osteopenia due to increased bone resorption by osteoclasts. These preliminary results suggest that the selective, oral Btk inhibitor, ONO-4059 may be a novel therapeutic target for rheumatoid arthritis (RA) to suppress bone erosion and inflammation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ono-4059-a-novel-small-molecule-brutons-tyrosine-kinase-btk-inhibitor-suppresses-osteoclast-differentiation-and-activation/