破骨细胞空泡ATP酶V0结构域e1亚单位的转录调控机制研究

Bone is a dynamic organ under the sophisticated regulation of osteoblasts, osteoclasts and osteocytes. The imbalance of cellular crosstalk can result in several bone metabolism disorders such as osteoporosis, osteopetrosis, etc. The increase of existing osteoclast number or activity is the direct cause of osteoporosis. Thought to be the only acid-secreting machinery in osteoclasts, vacuolar ATPase (V-ATPase)-mediated acidification is crucial for osteoclastic bone resorption. In recent years, the applicant mainly focused on the functional study of osteoclast V-ATPase complex. To examine the role of V-ATPase V0 domain e1 subunit in bone metabolism, the applicant has established an osteoclast-specific Atp6v0e1 knockout mouse line. By evaluating the mouse phenotype and investigating the cellular function of osteoclast, the mechanism of how e1 subunit is involved in osteoclastic bone resorption has been elucidated. However, little is learned regarding its transcriptional regulation mechanisms. In the initial study, the applicant demonstrated the up-regulation of e1 subunit during osteoclast differentiation. In this study, the applicant proposes to investigate the transcriptional regulation of osteoclast V-ATPase e1 subunit, by applying pharmacological inhibition, retroviral infection, luciferase report assay screening, ChIP, putative binding sites prediction, point mutagenesis, etc. This study will advance the understanding of osteoclastic bone resorption, providing insights into the novel treatment of bone resorption related disorders.

骨骼是由成骨细胞、破骨细胞及骨细胞共同精确调控的，处于动态平衡的器官。若平衡被打破，则可表现为骨质疏松、骨硬化等疾病。破骨细胞数量增多或活性增强是导致骨质疏松的直接原因。空泡ATP酶作为破骨细胞唯一的泌酸机制，对破骨细胞骨吸收起关键作用。近年来，申请者针对破骨细胞空泡ATP酶V0结构域e1亚单位，构建了破骨细胞特异的e1亚单位基因敲除小鼠，通过系统研究基因敲除小鼠的骨骼表型、破骨细胞功能，揭示了e1亚单位在破骨细胞空泡ATP酶介导的骨吸收过程中的生物学功能。然而，作为破骨细胞空泡ATP酶重要亚单位之一，其转录调控机制有待发掘。前期试验中，申请者证实e1亚单位在破骨细胞分化过程中表达大幅上调。在此基础上，本研究将采用药物抑制、逆转录病毒转染、荧光素酶筛选、计算机模拟预测、构建点突变质粒等分子生物学手段，旨在阐明破骨细胞分化过程中e1亚单位转录调控机制，以期为溶骨性疾病的治疗开拓新思路。

骨骼是由成骨细胞、破骨细胞及骨细胞共同精确调控的，处于动态平衡的器官。若平衡被.打破，则可表现为骨质疏松、骨硬化等疾病。破骨细胞数量增多或活性增强是导致骨质疏松的直接原因。空泡ATP酶作为破骨细胞唯一的泌酸机制，对破骨细胞骨吸收起关键作用。作为破骨细胞空泡ATP酶重要亚单位之一，v0结构域e1亚单位的表达调控机制有待发掘。通过本项目的研究，我们利用药物抑制、基因沉默、PCR、免疫电泳、免疫共沉淀、体外细胞测试、免疫荧光-共聚焦显像、微型CT等手段，证明巨噬细胞迁移抑制因子与cAMP 反应原件结合蛋白共同调控破骨细胞的分化，为溶骨性疾病的治疗开拓新思路。同时我们还通过共聚焦显微镜记录破骨细胞在不同环境中形态变化，结合RNA测序，推导极化状态、多核分布对细胞功能的可能影响，为进一步理解破骨细胞生理功能进行了有益的探索。