MOLECULAR REGULATION OF CORNEAL WOUND HEALING

描述：角膜上皮对损伤的快速愈合反应 对于维持其屏障功能至关重要。 的长期目标 该实验室是为了获得有关分子和细胞的基本信息， 角膜伤口愈合生物学 最近的数据显示， 淀粉样蛋白B/A4受体样蛋白（APLP 2）表达升高 与创伤后角膜上皮再生有关， 过表达APLP 2增强了细胞外基质（ECM）的迁移 proteins. APLP 2与淀粉样蛋白B/A4前体非常密切相关 阿尔茨海默病的蛋白质。 在角膜伤口愈合期间， APLP 2的胞外域被迁移的上皮细胞脱落，并被掺入 在前缘周围的伤口里 APLP 2的Kunitz型丝氨酸蛋白酶抑制剂结构域（KPI）抑制 一种未表征的蛋白酶，可阻止角膜上皮伤口闭合 器官培养 角膜中的APLP 2通过添加软骨素而被修饰 硫酸盐链 这些数据表明，APLP 2在角膜内皮细胞中起作用。 伤口修复 本提案将检验以下假设： 控制蛋白酶活性，介导细胞粘附，并促进 角膜上皮再生过程中的细胞迁移。 (i)的机制 哪些生长因子调节角膜中APLP 2的合成和脱落 上皮细胞将通过代谢标记和Western印迹进行评价。 印迹法;蛋白激酶C和酪氨酸 APLP 2脱落中的磷酸化依赖性信号转导途径 将使用特定的封闭试剂进行研究。 (ii)机制 APLP 2介导的上皮细胞粘附和迁移将是 使用包括APLP 2同种型的纯化在内的几种方法进行评估， 嵌合APLP 2蛋白的cDNA转染和表达， 抗蛋白水解裂解，以及体外细胞粘附和细胞 迁移分析。 特别地，APLP 2的胞外结构域脱落的作用 作为破坏粘附和促进细胞迁移的手段， ECM相关的APLP 2在上皮细胞粘附和迁移中的作用将是 研究了 (iii)KPI抑制蛋白酶的鉴定 含有APLP 2的细胞将使用亲和纯化和cDNA 克隆，以及这种蛋白酶和APLP 2在 生长因子的加工过程将使用器官培养和免疫组织化学方法来阐明。 体外无细胞测定。 这些实验将为开始 构建分子通路的详细地图， 在伤口修复中发挥作用的蛋白质网络， 用于治疗角膜疾病，如复发性糜烂和持续性 上皮缺陷。

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.