Molecular Regulation of Corneal Wound Healing

描述（由申请人提供）：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。最近的数据表明，上皮损伤诱导肝素结合表皮生长因子（EGF）样生长因子（HB-EGF）的胞外区脱落。脱落的HB-EGF，反过来，作为一个自分泌配体激活EGF受体（EGFR）和随后的细胞内信号通路调节角膜上皮伤口愈合所必需的。目前的建议将测试的假设，EGFR配体脱落是由一个ADAM（解整合素和金属蛋白酶）在一个严格的监管方式催化，EGFR激活脱落HB-EGF elevates几个细胞内信号通路，协同工作，以调节角膜上皮细胞的迁移和增殖。(i)将通过反义寡核苷酸抑制和通过四种亚当斯的显性负性（dn）和组成型活性（ca）突变体的表达来鉴定应答创伤的角膜上皮细胞中参与HB-EGF脱落和EGFR活化的ADAM。(ii)将阐明创伤诱导的HB-EGF脱落和亚表皮生长因子受体活化的调节机制。将使用酶测定法鉴定参与PKC-Raf 1-MEK-ERK信号级联的蛋白激酶（PKC）同工酶。其在EGFR活化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。(iii)将研究每种EGFR引发的信号通路参与调节角膜上皮伤口愈合的机制。将使用生化激酶试验确定四种此类途径（丝裂原活化蛋白激酶、磷脂酰肌醇3-激酶、磷脂酶Cg-蛋白激酶C以及粘着斑激酶）的激活;将使用药理学激酶抑制剂和ca-和dn-突变体表达评估其串扰和对角膜上皮迁移和增殖调节的贡献。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（如复发性糜烂和上皮持续性缺损）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.