Corneal Homeostasis and Repair

描述（由申请人提供）：为了更好地诊断和管理角膜伤口愈合问题，重要的是要了解调节细胞增殖的基本机制和伤口愈合期间决定角膜透明度的细胞表型特征的改变。角膜基质细胞是正常成人角膜中静止的角膜基质细胞，在伤口愈合期间被激活，成为具有改变的表型特征的增殖性成纤维细胞或肌成纤维细胞。本项目的目标是阐明控制和调节以下信号传导机制：1）角膜上皮细胞和活化角膜基质细胞的细胞增殖（细胞周期进程）和2）角膜基质细胞活化后的表型变化。我们以前的研究表明，小GTdR RhoA，通过其下游目标ROCK，调节细胞周期进程中的角膜上皮细胞和激活的角膜基质细胞培养。我们还确定，在角膜细胞活化时，促分裂原活化蛋白激酶（MAPK）、INK和ERK的活化可导致对维持角膜透明度至关重要的几种蛋白质的表达下调。这一竞争性更新提案的具体目标测试了关于角膜上皮细胞和活化基质细胞中细胞周期调节的信号传导机制的具体假设和预测。在组织培养中，我们将进行功能分析的信号蛋白，通过抑制它们的表达与siRNA和调节它们的活动与组成型活性或显性负重组蛋白。目的1）RhoA/ROCK介导的细胞周期调控蛋白、细胞周期蛋白、细胞周期蛋白依赖性激酶（cyclin dependent kinases，CDK）和CDK抑制剂（CDK inhibitors，GDIs）的变化是否与RhoA/ROCK的下游效应因子mDia和下游效应因子LIMK有关？目的2）缝隙连接蛋白Cx43是否调控细胞周期进程？目的3）小GTP酶RhoA、Cdc 42和Rac以及MAPK、INK和ERK是否调节角膜细胞活化后硫酸角质素蛋白聚糖（keratocan和lumican）、角膜晶体蛋白（ALDH 1和DTKT）和D3（IV）胶原的表达？这些新的信息将进一步加深我们对角膜稳态和伤口愈合机制的认识。它将导致更好地诊断和治疗与失调的细胞增殖相关的持续性角膜伤口愈合问题，并预防损伤、角膜移植术或屈光手术后的不透明瘢痕组织形成。

DESCRIPTION (provided by applicant): For better diagnosis and management of corneal wound healing problems it is important to understand the basic mechanisms that regulate cellular proliferation and alterations in the phenotypic characteristics of the cells that determine corneal transparency during wound healing. Keratocytes, corneal stromal cells that are quiescent in the normal adult cornea, are activated during wound healing to become proliferative fibroblasts or myofibroblasts with altered phenotypic characteristics. The goal of this project is to elucidate the signaling mechanisms that control and modulate: 1) the cellular proliferation (cell cycle progression) of corneal epithelial cells and activated corneal keratocytes and 2) the phenotypic changes upon the activation of keratocytes. Our previous studies demonstrated that the small GTPase RhoA, through its downstream target ROCK, regulates cell cycle progression in both corneal epithelial cells and activated corneal stromal cells in culture. We have also determined that upon keratocyte activation, the activation of mitogen activated protein kinases (MAPKs), INK and ERK, can lead to downregulation of the expression of several proteins that are critical to the maintenance of corneal transparency. The Specific Aims of this competing renewal proposal test specific hypotheses and predictions about the signaling mechanism in cell cycle regulation in corneal epithelial cells and activated stromal cells. In tissue culture, we will perform functional analyses of the signaling proteins by inhibiting their expression with siRNAs and by modulating their activities with constitutively active or dominant negative recombinant proteins. Using these approaches we will address the following questions: Aim 1) Are mDia, a downstream effector of RhoA, and LIMK, a downstream effector of ROCK, involved in the RhoA/ROCK mediated changes in cell cycle regulatory proteins, cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (GDIs)?; Aim 2) Does connexin 43, a gap junction protein regulate cell cycle progression?; and Aim 3) Do small GTPases RhoA, Cdc42 and Rac, and the MAPKs, INK and ERK, regulate the expression of keratan sulfate proteoglycans (keratocan and lumican), corneal crystallins (ALDH1 and DTKT) and D3(IV) collagen upon keratocyte activation? This new information will further our knowledge of corneal homeostasis and wound healing mechanisms. It will lead to better diagnoses and treatments of persistent corneal wound healing problems that are related to dysregulated cellular proliferation, and to prevention of nontransparent scar tissue formation following injury, keratoplasty or refractive surgery.