Smurf1对BMP7介导的晶状体上皮细胞生物学行为的调控作用

The pathological mechanism of posterior capsule opacification (PCO) includes proliferation and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). Transforming growth factor (TGF-β) is the most significant cytokine that induces the EMT. It was indicated in our previous study that bone morphogenetic protein-7 (BMP-7), a member of TGF-β superfamily, can inhibit the EMT induced by TGF-β and the proliferation of LECs probably through activating Smad1/5/8. Ubiquitin-proteasome pathway (UPP) can regulate Smurf1, one of the ubiquitin-protein ligating enzymes (E3) which can selectively degrade Smad1/5, and inactivate the BMP signaling pathway, so as to regulate the proliferation, migration and EMT of LECs at the post-translation level. We hypothesize that Smurf1 may be a key regulator of UPP pathway to regulate the activity of TGF-β/BMP signaling pathways. We intend to study the interaction of Smurf1 and BMP-7 signaling pathway and their influences on the biological behavior of mouse LECs by using CRISPR/Cas9 gene editing technique and rAAV. We will also analyze the key signaling molecules in the TGF-β/BMP signaling pathway regulated by Smurf1 though bioinformatics and protein microarray technique. Moreover, we will establish injury-induced anterior subcapsular cataract models of mouse and observe the influences of Smurf1 on cataract formation histologically and pathologically, as well as the influences on the activity of TGF-β/BMP signaling pathway. The purpose of this project is to explore a new target in the prevention and treatment of posterior capsule opacification.

晶状体上皮细胞（LECs）增殖、上皮间质转分化（EMT）是后发性白内障（PCO）的病理基础。转化生长因子（TGF-β）是导致EMT最重要的细胞因子。我们发现TGF-β超家族成员骨形成蛋白7（BMP7）和泛素蛋白酶体途径（UPP）抑制剂MG132都能抑制TGF-β的生物学作用，并且MG132可以下调E3泛素连接酶Smurf1激活BMP途径，由此我们提出：UPP通路中的Smurf1可能是TGF-β/BMP信号通路的关键调节因子，选择性降解Smad蛋白，在翻译后水平调控LECs的增殖和EMT。本项目拟：1）应用CRISPRI/Cas9及腺相关病毒载体调控Smurf1及BMP7的表达水平，观察对LECs生物学行为的影响2）蛋白芯片技术分析Smurf1调控TGF-β/BMP信号通路中的关键信号分子3）构建外伤诱导小鼠前囊下白内障模型，从体内实验进一步验证，以期为后发性白内障的预防和治疗提供新的靶点。

后发性白内障（PCO）是白内障术后最常见的并发症，其病理机制与白内障术后残留的晶状体上皮细胞(LECs)的增殖、迁移、上皮间质转分化相关（EMT）。导致EMT最重要的细胞因子是转化生长因子β（TGF-β），TGF-β超家族成员骨形成蛋白（BMP）与TGF-β起到拮抗作用。目前，Smurf1对BMP介导的LECs生物学行为的影响对PCO形成机制尚未阐明。本项目选取与晶状体发育最为密切的两个BMP家族成员BMP-4和BMP-7为研究对象。首先，我们发现BMP-4和BMP-7都可以在细胞水平抑制LECs的增殖、迁移、EMT，其可能机制为抑制了细胞间信号Notch通路。同时，在动物水平我们进一步证实了两者都可以抑制小鼠外伤性前囊下白内障模型中前囊下浑浊的形成。其次，小鼠晶状体的转录组测序结果显示，在BMP-4及BMP-7组中， E3泛素连接酶Smurf1的表达量降低，且与Smurf1相关的差异基因主要集中在TGF-β/BMP信号通路及UPP途径，提示Smurf1可能是连接TGF-β/BMP通路与UPP途径的重要因子。根据上述结果，我们认为BMP通路是抑制后发性白内障形成的有效药物靶点，其可能机制为Notch通路的抑制和Smuf1信号的低表达。