Effects of inositol 1,4,5-triphosphate on osteoclast differentiation in RANKL-induced osteoclastogenesis

The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca ²⁺ signaling. However, the intra-cellular levels of inositol 1,4,5-trisphosphate (IP ₃) and IP ₃-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP ₃ and evaluated IP ₃-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca ²⁺ signaling proteins such as IP ₃ receptors (IP ₃RS), plasma membrane Ca ²⁺ ATPase, and sarco/endoplasmic reticulum Ca ²⁺ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP ₃ was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP ₃ levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP ₃RS with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP ₃Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP ₃ levels and the IP ₃-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.