CDK5调控角膜上皮创伤愈合在分子水平上的机制研究

Proper healing of corneal wounds is vital for maintaining a clear, healthy cornea and preserving vision. Cdk5, a proline-directed protein kinase, strengthens cell- extracellular matrix (ECM) matrix adhesion, reorganizes cytoskeleton and retards closure of an in vitro scrape wound in a mouse corneal epithelial cell . Thereby, Cdk5 can have an active role in the therapeutically effect in corneal epithelial wound healing. While it is evident that Cdk5 is essential for focal adhesion formation and stress fiber, it is still entirely unclear how Cdk5 affect these processes. Our long-term goal is to figure out how Cdk5 can be modulated on corneal epithelial wound healing for preventive and therapeutic purpose. The objective of this application, which is the next step in pursuit of that goal, is to determine how Cdk5 enhances cell-ECM adhesion, that is, focal adhesion assembly and cytoskeletal reorganization. The central hypothesis of current application is that Cdk5 an essential function required for cell-ECM adhesion via Src family kinases (SFKs) signaling. we will explore the function of Cdk5 on FAs assembly,and identify a reduction of FA formation upon Cdk5 deficiency that is associated with a delayed cell spreading on fibronetin. Both effects will go along with a decreased tyrosine phosphorylation level of FA associated proteins and an interrupted Src/FAK binding, which is caused by impaired periphery targeting of Src as well as a diminished level of Src autophophorylation. The rationale for the proposed research is that, once it is clear how Cdk5 enhances cell-ECM adhesion via affecting Src activity and which one, Cdk5 can be pharmacologically used in new and innovative approaches to the prevention and treatment of corneal epithelial defect.

细胞的黏附移行是角膜上皮创伤愈合的关键步骤。它的调节主要是通过整合素介导的细胞与胞外基质通过阶梯式信号传导来完成的。Cdk5属独特细胞周期依赖性蛋白激酶 (CDK)家族成员，在哺乳动物非神经元的组织和细胞中广泛表达。我们前期发现Cdk5促进小鼠角膜上皮细胞黏附并伴有Cdk5/Src复合体形成。然而目前依然不清楚除Src外，Cdk5是否参与细胞黏附信号传导的另一些步骤。本课题就此将采用永生化人角膜上皮细胞系，拟明确的是Cdk5 1.是否调控Src的上游因子FAK(Y397)的活性磷酸化；2.是否调控活性Src以及介导其由胞浆转移至细胞膜；3. 是否参与了FAK/Src复合体的形成；4. Cdk5/Src复合体是直接或间接作用。基于前期工作，我们的假说是Cdk5也许通过直接调控Src活性表达并将其转移至细胞膜上，进而影响人角膜上皮细胞的粘附移行。该课题为促进角膜上皮创伤愈合提供了新思路。

CDK5参与整合素介导的角膜上皮细胞与基质粘附和移行； 本课题研究进一步揭示CDK5通过调控Src磷酸化位点(pY416) 从而参与角膜上皮细胞粘附和移行作用。我们的研究结果显示CDK5以及它的磷酸化形式Cdk5 (pY15)位于角膜上皮细胞的局部细胞粘附斑 FAs（与vinculin共染色）。免疫共沉降和Western Blot技术检测显示siCdk5（对照siRNA ）阻止FAK\Src 复合体结合。并且siCdk5降低细胞中早期重要细胞黏附启动蛋白：FAK 和其活性磷酸化FAK (pY397) 表达， 以及Src和其活性磷酸化Src activation (pY416 ) 和Src 下游活性磷酸化蛋白FAK（pY527）， Paxillin(pY118)的表达。免疫荧光组化实验显示siCdk5更减少了角膜上皮细胞边缘的粘附斑Vinculin 的形成。我们的这些研究数据提示Cdk5是局部细胞粘附斑 FAs复合体的结构组成成分. Src 磷酸化活性以及移行至细胞膜边缘的FAs是Cdk5依赖决定性的 .Cdk5在整合素介导的细胞粘附信号传导中将Src转移至细胞膜边缘的局部细胞粘附斑复合体中起关键作用。因为在角膜上皮细胞粘附过程中Cdk5是细胞信号从跨膜的整合素到细胞膜边缘的FAs的传导过程的重要因素，所以我们的研究结果为角膜上皮创伤愈合的治疗提供潜在药物作用靶点。