Abstract
Fusion and apoptosis share a breakdown of the membrane phospholipids asymmetry, modes of which are largely unknown in osteoclastogenesis. Here, we investigated the externalization of phosphatidylserine (PS) and its receptors, and their biological functions in osteoclastogenesis. Strong immunoreactivities in vivo for the PS receptors TIM4, BAI1, and STAB2 were observed in the TRAP-positive multinucleated cells in the alveolar bone that was being remodeled around the developing dental follicles in rats. These receptors were significantly upregulated during M-CSF/RANKL-induced in vitro osteoclastogenesis using mouse bone marrow-derived cells. PS externalization in preosteoclasts was increased by the M-CSF/RANKL treatment. Multinucleation of preosteoclasts was markedly inhibited by antibodies against PS and its receptors. Among the investigated lipid transporter proteins, floppases (Abcb4, Abcc5, and Abcg1) were upregulated, whereas flippases (Atp11c and Atp8a1) downregulated during osteoclastogenesis. Preosteoclast fusion was markedly blocked by the ATPase inhibitor Na3VO4 and siRNAs against Abcc5 and Abcg1, revealing the importance of these lipid transporters in PS externalization. Further, the levels of Cd47 and Cd31, don’t-eat-me signal inducers, were increased or sustained in the early phase of osteoclastogenesis, whereas those of AnnexinI and Mfg-e8, eat-me signals inducers, were increased in the late apoptotic phase. In addition, Z-VAD-FMK, a pan caspase inhibitor, had no effect on preosteoclast fusion in the early phase of osteoclastogenesis, whereas Abs against PS, TIM4, and BAI1 decreased osteoclast apoptosis during the late phase. These results suggest that PS externalization is essential for the whole process of osteoclastogenesis and share PS receptors and transporters in the early stage fusion and late stage apoptosis. Therefore, modulation of PS and its receptors could be a useful strategy to develop anti-bone resorptive agents.
Highlights
Osteoclasts, important for the remodeling of the skeleton, are multinucleated tartrate-resistant acid phosphatase (TRAP)-positive bone-resorbing cells
The receptors could not be detected in the macrophage colony-stimulating factor (M-CSF)-treated bone marrow-derived cells (BMDCs) on day 3, and only weak expression was observed on day 6 by western blotting and immunofluorescence staining (Fig. 2b, c)
In this study, we for the first time demonstrated the mechanism of PS externalization, its modes of action via PS receptors, and its shared role in preosteoclast fusion and apoptosis of matured osteoclasts
Summary
Osteoclasts, important for the remodeling of the skeleton, are multinucleated tartrate-resistant acid phosphatase (TRAP)-positive bone-resorbing cells. The hematopoietic monocyte/macrophage precursors differentiate into osteoclasts by treatment of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL), two essential cytokines for osteoclast formation. M-CSF/RANKL induces a master transcription factor, nuclear factor of activated T cells c1 (NFATc1), which increases the expression of osteoclast-specific genes such as Acp[5] (encoding TRAP) and Cathepsin K1. Lifespan of osteoclasts from the fusion of mononucleated precursors to apoptotic death is 1–2 weeks and quickly removed by phagocytes, while that of osteoblasts is about 3 months[2,3]. The brief lifespan of osteoclasts suggests a common mechanism that conveys signals leading to apoptotic death soon after fusion for multinucleation. A breakdown of the membrane phospholipids asymmetry is detected in both apoptosis and cell fusion, suggesting that understanding of underlying mechanism of phospholipids
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