Molecular Regulation of Corneal Wound Healing

描述（由申请人提供）：角膜伤口的适当愈合对保持角膜的透明、健康和视力至关重要。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，上皮损伤可诱导肝素结合的表皮生长因子（EGF）样生长因子（HB-EGF）的外畴脱落。反过来，Shed HB-EGF作为一种自分泌配体，激活egf受体（EGFR）和随后的细胞内信号通路，这是调节角膜上皮伤口愈合所必需的。目前的提案将验证EGFR配体脱落是由ADAM（一种解体素和金属蛋白酶）以严格调控的方式催化的假设，以及脱落HB-EGF激活EGFR引发几种细胞内信号通路，这些信号通路协同调节角膜上皮的迁移和增殖。(i)在角膜上皮细胞对损伤的反应中，参与HB-EGF脱落和EGFR激活的ADAM将通过反义寡核苷酸抑制和四种ADAMs的显性阴性（dn）和组成性活性（ca）突变体的表达来识别。（ii）伤口诱导的HB-EGF脱落和随后的EGFR激活被调控的机制将被阐明。参与PKC- raf1 - mek - erk信号级联的蛋白激酶（PKC）同工酶将使用酶分析鉴定。它在EGFR激活和角膜创面愈合中的作用将通过dn和ca突变体的表达来评估。（iii）研究各egfr诱导的信号通路参与角膜上皮创面愈合调控的机制。有丝分裂原活化蛋白激酶、磷脂酰肌醇3-激酶、磷脂酶cg -蛋白激酶C以及黏附病灶激酶这四种途径的激活将通过生化激酶检测来确定；它们的相互作用和对角膜上皮迁移和增殖的调节作用将通过药理激酶抑制剂和ca-和dn-突变体的表达来评估。了解角膜上皮伤口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（如复发性糜坏和上皮持续缺陷）治疗干预的靶点。

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.