Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2^Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired β-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of β-catenin phosphorylation, but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active β-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating β-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.

Wnt/β-连环蛋白信号传导对于多种细胞类型的各种细胞过程至关重要，包括成骨细胞 (OB) 分化和功能。 Wnt/β-catenin 信号传导在 OB 中的确切调控方式仍不清楚。 ATP6AP2 是 V-ATP 酶的辅助亚基，在多种细胞类型/器官和多种信号通路中发挥重要作用。然而，OB 中的 ATP6AP2 是否以及如何调节 Wnt/β-catenin 信号传导和骨形成却知之甚少。在这里，我们提供了 OB 谱系细胞中 ATP6AP2 在小梁骨区域选择性促进 OB 介导的骨形成和骨稳态的证据。条件性敲除（CKO）OB谱系细胞中的ATP6AP2（ Atp6ap2 ^Ocn-Cre ）会减少小梁骨形成和骨量，但不会减少皮质骨形成和骨量。蛋白质组学和细胞生化研究表明，LRP6 和 N-钙粘蛋白在ATP6AP2-KO BMSC 和 OB 中减少，但在骨细胞中没有减少。其他体外和体内研究显示，在基础刺激和 Wnt 刺激条件下， ATP6AP2-KO BMSC 和 OB 中的 β-连环蛋白信号传导受损，但骨细胞并未受损，尽管ATP6AP2-KO骨细胞中的 LRP5 降低，但 BMSC 中并未降低。进一步的细胞生物学研究发现，成骨细胞 ATP6AP2 不是 Wnt3a 抑制 β-catenin 磷酸化所必需的，但对于 LRP6/β-catenin 和 N-cadherin/β-catenin 蛋白复合物在细胞膜上的分布是必需的，从而防止其降解。活性β-连环蛋白的表达减少了ATP6AP2-KO BMSC 中的 OB 分化缺陷。 总而言之，这些结果支持了 ATP6AP2 作为 LRP6 和 N-钙粘蛋白蛋白运输和稳定性的关键调节因子，从而调节 β-连环蛋白水平的观点，证明了成骨细胞 ATP6AP2 在促进 Wnt/LRP6/β 方面的未被认识的功能-连环蛋白信号传导和小梁骨形成。