Bre调控破骨细胞分化的效应与分子机制研究

Osteoporosis is a major health hazard and osteoclasts are important functional bone cells involved in the pathology of osteoporosis. Therefore, further exploration of the molecular mechanisms of osteoclast differentiation is essential for the discovery of novel therapeutic targets for osteoporosis. Our preliminary study found that the expression of Bre was significantly decreased during osteoclast differentiation. Knockout or knockdown of Bre expression significantly enhanced osteoclast differentiation, while overexpression of Bre inhibited osteoclast differentiation. However, the in vivo regulatory effect and molecular mechanism of Bre that affect osteoclast differentiation remained unclear. In the present study, we intend to use the method of bone morphometry and histology to investigate the difference in bone resorption and bone mass of Bre knockout mice and Bre transgenic mice and determine the in vivo regulative effect of Bre on osteoclast differentiation. Basis of the physically interacting between Bre and Hey1 and the inhibitory effect of Hey1 on osteoclast differentiation, we intend to combine methods in cell and molecular biology to investigate the molecular mechanism of Bre regulating osteoclast differentiation. Furthermore, changes of Bre and Hey1 in osteoporosis were studied by constructing mouse OVX osteoporosis model. The study intends to clarify the role of Bre in osteoclast differentiation and its involvement in osteoporosis and will provide an experimental basis and theoretical support for Bre-targeted drug design.

骨质疏松症是危害健康的重大疾病，破骨细胞是参与骨质疏松的重要功能细胞，深入探索破骨细胞分化的调控机制，对发现骨质疏松治疗新靶点至关重要。课题组前期研究发现：Bre在破骨细胞分化过程中显著下调；敲除或沉默Bre可显著增强破骨细胞分化，而过表达Bre则抑制破骨细胞分化，但Bre对体内破骨细胞分化的调控作用及分子机制尚不清楚。本研究拟利用已构建完成的Bre基因敲除小鼠和转基因小鼠，运用骨形态计量学和组织学方法分析小鼠的骨吸收状态和骨量差异，以确定Bre对体内破骨细胞分化的调节作用。综合运用细胞与分子生物学研究方法，围绕Bre与破骨细胞分化抑制分子Hey1的结合，开展Bre调控破骨细胞分化的分子机制研究，并通过构建小鼠OVX骨质疏松模型探索Bre及Hey1在骨质疏松中的变化特征。以期明确Bre在破骨细胞分化和骨质疏松中的功能，从而为基于Bre靶点的药物设计提供实验基础和理论支撑。

骨质疏松症是严重危害老龄人健康的重大疾病，我国是全球骨质疏松症人口最多的国家，开展骨质疏松症发病机理的研究意义重大。破骨细胞介导的骨吸收激活是骨质疏松症的关键发病机制之一，深入探索破骨细胞分化的调控机制，对于发现骨质疏松症治疗新靶点至关重要。申请人前期研究发现骨组织高表达的基因Bre在破骨细胞分化过程中表达显著下调，同时Bre在骨吸收激活的OVX骨质疏松模型中表达显著下调，并且干预Bre基因的表达可影响破骨细胞分化。通过本项目的实施，我们发现在BMMs及RAW264.7体外破骨细胞分化模型中，沉默Bre基因的表达可显著促进破骨细胞的分化及骨吸收活性，相反过表达Bre基因则显著抑制破骨细胞的分化和骨吸收活性，说明Bre是破骨细胞分化的负向调控基因。进而，我们构建了Bre转基因小鼠（Bre-TG）和Bre基因敲除小鼠（Bre-KO）从体内验证Bre的分子功能，我们发现Bre-TG小鼠骨吸收活性被显著抑制同时骨量增加，而Bre-KO小鼠则骨吸收激活并且骨量显著丢失，说明Bre可以在体内负向调控骨吸收进而影响小鼠骨量。更进一步，我们构建了LPS诱导的头盖骨损伤模型，我们发现Bre-TG小鼠可以显著降低LPS诱导的骨吸收激活进而降低头盖骨损伤。分子机制研究层面我们通过蛋白质互作质谱分析，发现破骨细胞分化负向调控分子Hey1是Bre的结合蛋白，并通过蛋白质免疫共沉淀和免疫荧光进一步证实了Bre和Hey1蛋白的相互结合。此外，通过开展功能回复实验，我们发现沉默Hey1可以显著减弱Bre对破骨细胞分化的抑制作用，说明Bre的分子功能依赖于Hey1。最后，通过Nfatc1启动子荧光素酶报告实验，我们发现Bre可增强Hey1对Nfatc1启动子转录活性的抑制作用。综上所述，我们明确了Bre对破骨细胞分化的调节作用和分子机制，为将来的转化应用提供了理论和实验支撑。在本项目的资助下，申请人于Nature Communications、Bone Research等国际知名刊物发表研究论文6篇，授权发明专利1件。