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.

融合和凋亡共享膜磷脂不对称性的破坏，破骨细胞生成的模式在很大程度上是未知的。在这里，我们调查了磷脂酰丝氨酸（PS）及其受体的外在化及其在破骨细胞形成中的生物学功能。在肺泡骨骼中的TRAP阳性多核细胞中观察到了PS受体TIM4，BAI1和STAB2在体内的强免疫反应性，该细胞在大鼠发育中的毛囊周围被重塑。在使用小鼠骨髓衍生细胞在M-CSF / RANKL诱导的体外破骨细胞形成过程中，这些受体显着上调。通过M-CSF / RANKL处理，破骨细胞中的PS外部化增加。抗破骨细胞及其受体的抗体显着抑制破骨细胞的多核化。Abcb4，Abcc5和Abcg1）被上调，而在破骨细胞形成过程中，flippases（Atp11c和Atp8a1）被下调。破骨细胞融合被ATPase抑制剂Na ₃ VO ₄和针对Abcc5和Abcg1的siRNA显着阻断，揭示了这些脂质转运蛋白在PS外化中的重要性。此外，在破骨细胞形成的早期阶段，不吃我的信号诱导物Cd47和Cd31的水平升高或持续，而AnnexinI和Mfg-e8的水平则升高或持续。在细胞凋亡后期，“我吃”信号诱导剂增加。此外，泛半胱氨酸蛋白酶抑制剂Z-VAD-FMK在破骨细胞形成的早期阶段对破骨细胞融合没有影响，而针对PS，TIM4和BAI1的Abs在晚期则减少破骨细胞凋亡。这些结果表明PS外部化对于破骨细胞形成的整个过程至关重要，并且在早期融合和晚期细胞凋亡中共享PS受体和转运蛋白。因此，调节PS及其受体可能是开发抗骨吸收剂的有用策略。