Mature osteoclast-derived apoptotic bodies promote osteogenic differentiation via RANKL-mediated reverse signaling

In bone remodeling, after a lifespan of ∼2 weeks, osteoclasts undergo apoptosis in each bone turnover cycle, resulting in generation of a large number of apoptotic bodies (ABs). However, the biological roles of osteoclast-derived ABs (OC-ABs) in bone remodeling have not been investigated and remain unknown. In this study, we stimulated bone marrow macrophages with receptor activator of NF-κB ligand (RANKL) to obtain both preosteoclasts and mature osteoclasts (mOCs). We then used alendronate to induce apoptosis in preosteoclasts and mOCs and generate the respective ABs and used flow cytometry and immunoblotting to characterize the sizes and immunogenic characteristics of the extracted ABs. We show that mOC-ABs are engulfed by preosteoblastic MC3T3-E1 cells and promote the viability of these cells. Among all osteoclast-derived extracellular vesicles, mOC-ABs had the highest osteogenic potency. We further observed that mOC-ABs had the highest vesicular receptor activator of NF-κB (RANK) levels among all types of osteoclast-derived extracellular vesicles. Of note, masking of vesicular RANK by soluble RANKL strongly abolished the osteogenic potency of osteoclast-derived ABs. Mechanistically, we found that mOC-ABs induce osteoblast differentiation by activatingPI3K/AKT/mechanistic target of rapamycin (mTOR)/ribosomal protein S6 kinase signaling. In conclusion, OC-ABs promote osteogenic differentiation by stimulating osteoblast differentiation via activation of RANKL reverse signaling. These findings provide important insights into the reversal phase between the bone resorption and formation stages during bone remodeling and identify an AB-dependent cellular signaling mechanism in osteoclast-osteoblast coupling.