MAFB基因突变通过调控破骨细胞发育导致骨质溶解综合征的机制研究

Multicentric carpotarsal osteolysis syndrome (MCTO) is a genetic skeletal disorder characterized by the progressive destruction of bone. It is caused by the missense mutations in the transactivation domain of the transcription factor MAFB. However,the molecular pathogenesis of MCTO remains unknown. MAFB is a negative regulator of the OPG/RANKL/RANK pathway and involves in the regulation of the osteoclast development. A systematic analysis of the phenotypes and genotypes of MCTO patients was conducted in our preliminary work.In vitro experiments showed that the missense mutations of the MAFB transactivation domain did not affect its negative regulation of OPG/RANKL/RANK pathway. It is hypothesized that the missense mutations of the MAFB transactivation domain may promote the formation of preosteoclast through the mechanism of gain of function. In this project, a Mafb mutant mouse model will be constructed. The induced pluripotent stem cells from MCTO patients will be established and differentiated into osteoclasts. The pathophysiological process of MCTO and the effects of MAFB mutation on the development and activity of osteoclasts will be studied. Omics-based methods combined with molecular biotechnology will be utilized to explore the target of the mutant MAFB. Thus we will elucidate the molecular mechanisms of MCTO, lay the foundation for the development of MCTO targeted therapy and provide new insights into the research of the pathogenesis and treatment of the skeletal disorders associated with abnormal osteoclasts.

多发性腕骨跗骨骨质溶解综合征（MCTO）是以骨骼进行性破坏为特征的遗传性骨病，由转录因子MAFB转录激活结构域错义突变所致，相应分子发病机制仍未知。MAFB参与破骨细胞发育调控，是OPG/RANKL/RANK信号通路的负调控因子。课题组前期对MCTO患者的临床表型和基因型进行了系统分析，体外实验发现MAFB转录激活结构域错义突变并未影响其对OPG/RANKL/RANK信号通路的负调控作用。推测该结构域的错义突变可能以功能获得性机制促进破骨细胞前体细胞生成。本项目拟构建Mafb突变小鼠模型以及MCTO患者特异性诱导多能干细胞向破骨细胞定向分化的细胞模型，分析MCTO病理生理过程以及MAFB突变对破骨细胞发育和活性的影响，利用组学和分子生物学手段研究突变MAFB的作用靶点。以期阐明MCTO的分子发病机制，为该病靶向治疗奠定基础，并为破骨细胞异常相关骨病的发病机理及治疗研究提供新思路。

多发性腕骨跗骨骨质溶解综合征（MCTO）是以骨骼进行性破坏为特征的遗传性骨病，由转录因子MAFB转录激活结构域错义突变所致，相应分子发病机制仍未知，缺乏有效治疗手段。本项目对MCTO患者表型与基因型进行了系统研究；构建了Mafb转录激活结构域p.S69L突变小鼠模型，并进行表型分析；检测了MAFB突变对其蛋白表达与功能等的影响；解析了MAFB突变对破骨细胞和成骨细胞分化的调控与分子机制。研究发现，MCTO患者存在骨骼与肾脏受累表型，患者骨代谢十分活跃，存在不同程度骨质疏松，患者均携带MAFB转录激活结构域热点突变。Mafb p.S69L突变小鼠模型再现了MCTO患者骨量减少以及肾脏损害表型。MAFB转录激活结构域突变阻碍了MAFB蛋白磷酸化修饰，导致MAFB蛋白不能通过泛素化途径进行降解，从而稳定性增强；突变不影响MAFB蛋白在细胞内的表达定位；荧光素酶报告基因实验显示，突变导致MAFB转录激活结构域调控活性下调。病理分析显示Mafb突变小鼠骨组织中破骨细胞增多；体外分化实验显示，MAFB突变导致破骨细胞分化减少；破骨细胞前体细胞数以及破骨细胞前体细胞增殖能力不受MAFB突变影响；小鼠骨髓原代细胞单细胞转录组测序显示，纯合突变小鼠单核细胞中破骨细胞分化下调；免疫共沉淀实验显示，突变不影响MAFB蛋白与MITF、c-Fos、NFATc1的相互结合；荧光素酶报告基因实验显示，突变MAFB不影响其对RANKL信号通路的负性调控作用。钙黄绿素荧光双标实验显示，Mafb突变小鼠骨形成加速；体外分化实验显示，MAFB突变导致成骨细胞分化亢进；小鼠模型股骨干转录组测序显示MAFB突变影响BMP、Wnt、TGFβ、Hippo、Hedgehog等多条信号通路；免疫共沉淀实验显示，MAFB突变蛋白可与GSK3相互结合。本项目研究结果显示，MAFB热点突变不仅影响了破骨细胞的正常分化，并对成骨细胞分化也产生了干扰，从而打破了骨代谢的平衡；突变阻碍了MAFB蛋白磷酸化修饰从而使得MAFB异常增多是导致MCTO的关键分子基础，提示下调MAFB表达水平可能起到治疗作用。本项目阐明了MAFB突变导致骨骼溶解的机制，为MCTO靶向治疗方法的研发提供了依据。