破骨细胞是人体内唯一能够吸收骨质的细胞，V-ATPase介导的酸分泌在其骨吸收中发挥重要作用。在前期研究中，我们发现了（1）在哺乳动物细胞内未见功能报道的V-ATPase e1亚单位e1在RANKL诱导的破骨细胞分化晚期上调；（2）破骨细胞内敲除e1，小鼠表现为骨硬化症；（3）e1敲除后破骨细胞骨吸收功能障碍；（4）e1敲除后破骨细胞的细胞外酸分泌功能障碍；（5）e1通过与a3相互作用，调控细胞外酸分泌。在上述研究基础上，提出假说:RANKL通过JUN上调e1，e1与a3直接作用调控破骨细胞外酸分泌及骨吸收，本项目将进一步分析（1）RANKL上调e1的分子机制；（2）e1与a3相互作用调控破骨细胞外酸分泌的分子机制；（3）靶向抑制破骨细胞内e1治疗破骨细胞相关的骨溶解疾病的应用研究。希望通过此研究，我们能明确RANKL上调e1，进而影响a3，调控破骨细胞外酸化及骨吸收功能的机制并转化应用。

Vacuolar-type H+-ATPases (V-ATPases) proton pumps function to acidify a diverse range of intracellular organelles required to sustain cellular homeostasis. In osteoclasts (OCs), V-ATPases are uniquely enriched on the surface of the ruffled border membrane where they serve to acidify the underlying extracellular milieu, a prerequisite for bone resorption. The mammalian V-ATPase complex is composed of at least 14 subunits (organized into two functionally and structurally distinct domains, the cytoplasmic V1 and the membrane-embedded V0), of which the hydrophobic V0 domain ATP6V0e subunit(s) (i.e. e1 and e2 paralogs) remain poorly characterized. Here, using the Cre-LoxP system, we demonstrate that e1 is the major functional isoform expressed in OCs. Whereas mice with conditional knockout (cKO) of the e2 isoform in either mature OCs (Cathepsin K (CtsKCre)) or OC precursors (RANKCre) have normal bone mass, conditional deletion of the e1 counterpart results in severe OC-rich osteopetrosis (CtsKCre-e1fl/fl cKO mice; designated e1OC herein). Histomorphometric assessment revealed that the marrow spaces of femurs from e1ΔOC mice were completely occluded by unresorbed bone. Consistently, deletion of e1 significantly impaired the bone resorptive function of OCs derived from e1ΔOC spleen cells, but did not alter overall OC differentiation. Mechanistically, this impairment is due to a disruption in extracellular and intracellular acidification as evidenced by live cell confocal microscopy and in vitro acidification assays. Interestingly, loss of e1 resulted in decreased a3 protein level in osteoclasts. Based on these preliminary data, we aim to further investigate how RANKL induces e1 upregulation during osteoclastogenesis? In addition, we will further study the interaction between e1 and a3 to unveil how V-ATPase mediated extracellular acidification are carried our. Moreover, we will target osteoclastic e1 for the treatment of osteoclast related osteolytic diseases. We hope this study will strengthen our understanding of V-ATPase mediated osteoclast acidification and explore potential therapeutic strategies for osteolytic diseases.