Corneal Homeostasis and Repair

描述(申请人提供)：为了更好地诊断和处理角膜伤口愈合问题，了解在伤口愈合过程中调节细胞增殖和决定角膜透明度的细胞表型特征变化的基本机制是很重要的。角膜基质细胞是在正常成人角膜中静止的角膜基质细胞，在伤口愈合过程中被激活，成为表型特征改变的增殖性成纤维细胞或肌成纤维细胞。本项目的目的是阐明控制和调节的信号机制：1)角膜上皮细胞和激活的角膜基质细胞的细胞增殖(细胞周期进程)；2)角膜基质细胞激活时的表型变化。我们以前的研究表明，小的GTP酶RhoA通过其下游的靶点，调节培养中的角膜上皮细胞和激活的角膜基质细胞的细胞周期进程。我们还确定，在角质形成细胞激活时，丝裂原激活蛋白激酶(MAPK)、INK和ERK的激活可以导致几种对维持角膜透明度至关重要的蛋白质的表达下调。这个相互竞争的更新方案的具体目的是测试关于角膜上皮细胞和激活的基质细胞中细胞周期调节的信号机制的特定假设和预测。在组织培养中，我们将通过siRNAs抑制信号蛋白的表达，并通过结构性活性或显性负性重组蛋白来调节它们的活性，从而对信号蛋白进行功能分析。通过这些途径，我们将解决以下问题：目的1)RhoA的下游效应者MDIA和ROCK的下游效应者LIMK是否参与了RhoA/ROCK介导的细胞周期调节蛋白、细胞周期蛋白、细胞周期蛋白依赖性激酶(CDK)和CDK抑制物(GDI)的变化？；目的2)缝隙连接蛋白43是否调节细胞周期进展？目的3)小GTP酶RhoA、CDC42和RAC以及MAPK、INK和ERK是否调节角质形成蛋白多糖(角质形成蛋白和腔细胞)、角膜晶体蛋白(ALDH1和KDTT)和D3(IV)胶原的表达？这一新信息将进一步加深我们对角膜动态平衡和伤口愈合机制的了解。这将有助于更好地诊断和治疗与细胞增殖失调有关的持续性角膜伤口愈合问题，并防止损伤、角膜移植或屈光手术后不透明瘢痕组织的形成。

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.