应用knock-in小鼠模型研究GDF5前结构域突变在近端指（趾）骨间关节粘连发病机制中的作用

GDF5 plays an important role in embryonic development of the phalanges and joint morphogenesis. Mutations of GDF5 are shown to lead to many kinds of skeletal dysplasia in human. We reported a p.Leu373Arg mutation in GDF5 proregion in a large Chinese family with Proximal symphalangism (SYM1). According to the in vitro assay, we found that the mutation caused the mature GDF5 protein increased and also raised the activation of BMP signaling and expressions of the factors that associated with bone developing. What’s more, the expression of the marker for endoplasmic reticulum was detected. All these results suggest that the GDF5-L373R may be an gain-of-function mutation which may change the proteolysis and the folding of GDF5 proprotein. The mutant GDF5 would lead the abnormal transmission of signal pathway during embryonic development and induce endoplasmic reticulum, furthermore, the cartilage and bone cells make difference during the progress of proliferation, differentiation and apoptosis which causing the impediment for informing the normal joints and finally leading the Proximal symphalangism. This study will begin on the Gdf5-L367R (the same mutation with GDF5-L373R in human) knock-in mice, which have been built and observed the same symptom as SYM1 in human already. We would like to find the differences of the ability of cartilage and bone cells in proliferation, differentiation and apoptosis in different periods during embryo between the wild and mutant knock-in mice. We also would try to find the role of endoplasmic reticulum or autophagy plays in this progress which would identify the mechanism that how GDF 5-L373R works in SYM1 and discover the function of the GDF5 proregion.

GDF5蛋白是肢端发育过程中重要的调控蛋白，其突变可导致多种类型的肢端畸形。课题组前期研究首次发现近端指（趾）骨间关节粘连（SYM1）家系在GDF5蛋白前结构域存在致病突变L373R。体外实验发现该突变导致成熟GDF5蛋白增加、BMP信号通路增强、软骨/骨相关因子和内质网应激关键因子表达增加。因此我们推测：GDF5-L373R是一种功能获得性突变，可能通过影响前体蛋白水解及折叠，引起内质网应激效应，导致胚胎肢芽发育过程中相关信号通路异常、软骨/骨细胞增殖分化凋亡自噬等过程发生改变，最终导致SYM1。本项目拟应用已构建的携带与人GDF5-L373R相应的Gdf5-L367R knock-in小鼠模型，通过检测突变型与野生型小鼠肢端在不同发育阶段的差异，探究内质网应激、细胞凋亡自噬在此过程中的作用，明确GDF5-L373R突变导致SYM1的作用方式和途径，揭示GDF5蛋白前结构域的生物学功能。

GDF5蛋白是肢端发育过程中重要的调控蛋白，其编码基因是近端指（趾）骨间关节粘连（SYM1）的致病基因之一。本项目针对前期在SYM1家系中发现的GDF5蛋白前结构域突变L373R构建了野生型和突变型真核表达载体，通过体外实验发现GDF5-L373R突变能够导致成熟GDF5蛋白增加；通过对下游靶基因表达情况的检测，明确了突变型GDF5蛋白能够增强BMP信号通路，并且影响骨/软骨相关因子的表达。进一步，通过构建携带与人GDF5L373R相应的Gdf5L367R knock-in小鼠模型，对SYM1疾病表型在小鼠肢端得到了重复，证实GDF5蛋白前结构域突变能够导致GDF5蛋白功能变化，进而引起疾病，提示了GDF5蛋白前结构域的重要作用。通过对Gdf5L367R knock-in小鼠不同生长阶段的观察，明确了Gdf5突变小鼠肢端发育模式的改变，阿辛蓝-茜素红染色显示突变小鼠趾骨关节强直、粘连；通过对胚胎期小鼠进行趾骨组织包埋，HE染色，证实突变小鼠软骨细胞数量增加，细胞小而密集，无法向野生型小鼠一样正常形成趾骨关节的关节区分化，只存在软骨增殖区；出生后突变型小鼠趾骨间软骨细胞肥大、钙化。分子水平检测显示Gdf5L367R knock-in小鼠肢端组织与体外实验结果一致，Realtime-PCR、Western blot及免疫组织化学检测结果均显示软骨/骨信号通路中相关因子SOX9、Col II、ID1及RUNX2表达增加，验证了突变导致软骨过度生长；同时观察到GDF5蛋白及其受体BMPR1B蛋白在肢芽近端有表达，提示突变改变了GDF5蛋白发挥作用的位置。进一步对小鼠肢端趾骨关节组织进行透射电镜观察和Realtime-PCR、Western blot检测，发现突变小鼠趾骨区软骨细胞出现内质网扩张现象，内质网应激相关因子GRP78和DDIT3蛋白表达增加，提示突变引起内质网应激反应，改变软骨/骨细胞分化过程是GDF5突变导致SYM1发生的可能机制。本项目的完成揭示了GDF5蛋白前结构域对GDF5蛋白发挥正常功能起到至关重要的作用，为SYM1发病机制的研究和深入提供了理论依据。