Molecular Regulation of Corneal Wound Healing

描述(申请人提供)：角膜伤口的适当愈合对于保持透明、健康的角膜和保护视力至关重要。该实验室的长期目标是获得有关角膜伤口愈合的分子和细胞生物学的基本信息。最近的研究表明，上皮损伤可导致肝素结合的表皮生长因子(EGF)样生长因子(HB-EGF)胞外脱落。SHEED HB-EGF反过来作为自分泌配体激活EGF受体(EGFR)和随后的调节角膜上皮损伤愈合所需的细胞内信号通路。目前的提议将检验这样的假设，即EGFR配体的脱落是由ADAM(一种去整合素和金属蛋白酶)以严格调控的方式催化的，并且SHEAD HB-EGF激活EGFR引发了几个细胞内信号通路，这些信号通路协同作用调节角膜上皮细胞的迁移和增殖。(I)通过反义寡核苷酸抑制和4个ADAM的显性负性(DN)和结构性活性(Ca)突变体的表达来鉴定参与创伤反应中角膜上皮细胞HB-EGF脱落和EGFR激活的ADAM。(Ii)创伤诱导的HB-EGF脱落和随后的EGFR激活的调节机制将被阐明。参与PKC-Raf1-MEK-ERK信号级联的蛋白激酶(PKC)同工酶将通过酶分析进行鉴定。它在EGFR激活和角膜伤口愈合中的作用将通过dN和ca突变体的表达来评估。(3)研究EGFR诱导的各个信号通路参与角膜上皮损伤修复调控的机制。有丝分裂原激活的蛋白激酶、磷脂酰肌醇3-激酶、磷脂酶CG-蛋白激酶C和粘着斑激酶这四个这样的通路的激活将用生化激酶分析来确定；它们的相互作用和对角膜上皮迁移和增殖的调节作用将用药理激酶抑制剂和ca-和dN-突变表达来评估。了解角膜上皮损伤愈合过程中从信号产生到信号转导的分子事件有助于确定角膜疾病治疗干预的靶点，如复发性糜烂和持续性上皮缺陷。

DESCRIPTION (provided by applicant): Proper healing of corneal wounds is vital to maintaining a clear, healthy cornea and for preserving vision. The long-term goal of the laboratory has been to obtain basic information about the molecular and cellular biology of corneal wound healing. Recent data demonstrated that epithelial injury induces ectodomain shedding of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). Shed HB-EGF, in turn, acts as an autocrine ligand to activate EGF-receptor (EGFR) and subsequent intracellular signaling pathways necessary for regulating corneal epithelial wound healing. The current proposal will test the hypothesis that EGFR ligand shedding is catalyzed by an ADAM (a disintegrin and metalloprotease) in a tightly regulated manner and that EGFR activation by shed HB-EGF elicits several intracellular signaling pathways that work in concert to regulate corneal epithelial migration and proliferation. (i) The ADAM that is involved in HB-EGF shedding and EGFR activation in corneal epithelial cells in response to wounding will be identified by antisense oligonucleotide inhibition and by expression of dominant negative (dn) and constitutively active (ca) mutants of four ADAMs. (ii) The mechanisms by which wound-induced HB-EGF shedding and sub-sequent EGFR activation is regulated will be elucidated. The protein kinase (PKC) isozyme involved in the PKC-Raf1-MEK-ERK signaling cascade will be identified using enzymatic assays. Its role in EGFR activation and corneal wound healing will be assessed by expression of dn and ca mutants. (iii) The mechanisms by which each EGFR-elicited signaling pathway participates in the regulation of corneal epithelial wound healing will be investigated. Activation of four such pathways, mitogen activated protein kinase, phosphatidylinositol 3-kinase, phospholipase Cg-protein kinase C, as well as focal adhesion kinase, will be determined using biochemical kinase assays; their cross-talk and contribution to regulation of corneal epithelial migration and proliferation will be assessed using pharmacological kinase inhibitors and ca- and dn-mutant expression. An understanding of the molecular events from signal generation to signal transduction during corneal epithelial wound healing should help in the identification of targets for therapeutic interventions of corneal diseases like recurrent erosions and persistent defects of the epithelium.