MOLECULAR REGULATION OF CORNEAL WOUND HEALING

描述：角膜上皮损伤后的快速愈合 对于维持其屏障功能是必不可少的。的长期目标是 实验室的目的是获得有关分子和细胞的基本信息 角膜伤口愈合生物学。最近的数据表明，一个很大的 淀粉样蛋白B/A4前体蛋白2的表达上调 与创伤后角膜再上皮化和细胞的关系 过表达APLP2促进细胞外基质(ECM)迁移 蛋白质。APLP2与淀粉样蛋白b/a4前体关系非常密切。 阿尔茨海默病的蛋白质。在角膜伤口愈合过程中， APLP2的胞外结构域被迁移的上皮细胞脱落，并被整合 进入前缘周围的伤口床。 APLP2的kunitz型丝氨酸蛋白酶抑制器域(KPI)抑制 一种未知的蛋白水解酶与角膜上皮伤口闭合 器官培养。角膜中的APLP2可通过添加氯代苦参碱进行修饰 硫酸盐链。这些数据表明，APLP2在角膜中起作用(S) 伤口修复。目前的提案将检验APLP2的假设 控制Erine蛋白水解酶活性，调节细胞黏附，促进 角膜再上皮化过程中的细胞迁移。(I)该机制由 哪些生长因子调节角膜中APLP2的合成和脱落 上皮细胞将通过代谢标记和Western 印迹；蛋白激酶C和酪氨酸的共同参与 APLP2脱发中依赖于磷酸化的信号转导途径 将使用特定的封闭剂进行调查。(Ii)机制 APLP2介导上皮细胞黏附和迁移的机制 用几种方法进行评估，包括纯化APLP2亚型， 重组APLP2嵌合蛋白的基因转染和表达 抗蛋白水解性切割，体外细胞黏附和细胞 迁徙分析。特别是，APLP2胞外结构域脱落的作用 作为一种打破黏附和促进细胞迁移的手段，以及 细胞外基质相关APLP2在上皮细胞黏附和迁移中的作用 调查过了。(3)KPI抑制蛋白水解酶(S)的鉴定 含有APLP2的将使用亲和纯化和cDNAs 该酶与APLP2的相互作用及其在细胞周期中的作用 生长因子的处理将通过器官培养和一种 体外无细胞试验。这些实验将为开始提供基础 构建分子路径和互连的详细地图 蛋白质网络在伤口修复和开发治疗中的作用 用于治疗角膜疾病，如复发性糜烂和持续性 上皮细胞的缺陷。

DESCRIPTION: Rapid healing of the corneal epithelium in response to injury is essential for maintenance of its barrier function. The long-term goal of the laboratory is to obtain basic information about the molecular and cell biology of corneal wound healing. Recent data demonstrated that a greatly elevated expression of amyloid b/A4 precursor-like protein (APLP2) correlates with post-wound corneal re-epithelization and that cells overexpressing APLP2 have enhanced migration over extracellular matrix (ECM) proteins. APLP2 is very closely related to the amyloid b/A4 precursor protein of Alzheimer's disease. During corneal wound healing, the ectodomain of APLP2 is shed by migrating epithelial cells, and incorporated into the wound bed around the leading edge. The Kunitz type serine proteinase inhibitor domain (KPI) of APLP2 inhibits an uncharacterized proteinase and blocks epithelial wound closure in corneal organ culture. APLP2 in the cornea is modified by addition of chrondroitin sulfate chains. These data suggest that APLP2 plays a role(s) in corneal wound repair. The current proposal will test the hypotheses that APLP2 controls erine proteinase activity, mediates cell adhesion, and promotes cell migration during corneal re-epithelialization. (i) The mechanism by which growth factors regulate synthesis and shedding of APLP2 in corneal epithelial cells will be evaluated by metabolic labeling and Western blotting; the involvement of both protein kinase C and tyrosine phosphorylation dependent signal transduction pathways in APLP2 shedding will be investigated using specific blocking reagents. (ii) The mechanism by which APLP2 mediates epithelial cell adhesion and migration will be assessed using several methods including purification of APLP2 isoforms, cDNA transfection and expression of chimeric APLP2 proteins that are resistant to proteolytic cleavage, and in vitro cell adhesion and cell migration assays. In particular, the role of ectodomain shedding of APLP2 as a means to break adhesion and to facilitate cell migration, and of ECM-associated APLP2 in epithelial cell adhesion and migration will be investigated. (iii) The identify of proteinase(s) that is inhibited by KPI containing APLP2 will be determined using affinity purification and cDNA cloning, and the role of the interplay of this proteinase and APLP2 in the growth factor processing will be elucidated using organ culture and an in vitro cell-free assay. These experiments will provide the basis to begin constructing a detailed map of the molecular pathways and interconnecting networks of proteins functioning in wound repair and to develop therapeutics for treatment of corneal diseases like recurrent erosions and persistent defects of the epithelium.