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

Abstract

Edited by Xiao-Fan Wang In bone remodeling, after a lifespan of ϳ2 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-B 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-B (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.

Unleashing the therapeutic potential of apoptotic bodies

“…This local apoptosis-induced proliferation is pivotal to sustain tissue-wide cell number and epithelial homeostasis. Similarly, mature osteoclast-derived ApoBDs also promoted viability and differentiation in preosteoblastic cells, reportedly through receptor activator of NF-κB (RANK)-mediated PI3K/Akt/mTOR/protein S6 kinase signalling for survival and osteoblast differentiation [47]. Together these findings not only uncover the key roles of ApoBDs in both locally and distantly regulating tissue homeostasis, but also highlight the potential of stem cell-derived ApoBD treatment as a novel regenerative therapy.…”

“…However, from available literature, one could infer and anticipate the emerging potential of developing ApoBDs as a therapeutic delivery vehicle. In the previously mentioned studies, ApoBDs were shown to transfer different molecules (DNA, miRNA and protein) and effectively regulating various aspects of the phagocytosed/recipient cells [46][47][48][49][50]. Other studies have also reported the efficiency of ApoBD contents in mounting cellular responses such as vascular protection and cell migration [51,52].…”

“…In addition, ApoBDs may hold unrivalled potency and safety-related advantages, compared with other EV-and cell-based approaches. Strikingly, compared with exosome, microvesicle and healthy cell counterparts, the aforementioned osteoclast-derived ApoBDs had the highest RANK level and osteogenic potency [47]. Furthermore, the use of ApoBDs in regenerative therapy, particularly organ transplantation, may offer better safety advantage as they did not an induce inflammatory response and graft rejection as observed by other EVs [48].…”

“…Secondly, unlike the smaller EVs, the size and rapid phagocytic uptake of ApoBDs may hinder biodistribution and bioavailability. Whilst this concern requires further investigation, the fact that systemic administration of mature osteoclast-derived ApoBDs successfully delivered the anticipated functional outcomes in the preosteoclast cells [47] suggests the possibility of ApoBD-based delivery system for certain tissues. Furthermore, the inherent phagocyte targeting property of ApoBDs should be strategically exploited for the phagocyte-specific drug targeting.…”

Extracellular Vesicles Derived From Apoptotic Cells: An Essential Link Between Death and Regeneration

“…ApoBDs also mediate intercellular communication between osteoclasts and osteoblasts. In bone remodeling, mature osteoclast-derived ApoBDs (mOC-ApoBDs) are taken up by pre-osteoblastic cells and prompt osteogenic differentiation by activating phosphoinositide 3-kinase (PI3K)/AKT Ser–Thr kinase (AKT) signaling ( Ma et al, 2019 ; Table 1 and Figure 2 ). Moreover, previous studies suggested that osteocyte-derived ApoBDs could recruit the osteoclasts and initiate local bone resorption ( Yuan et al, 2018 ), suggesting that ApoBDs derived from different sources play distinct roles in bone remodeling.…”