Xu Ke-Ping, Ding Yu, Ling Jianhua, Dong Zheng, Yu Fu-Shin X
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA.
Invest Ophthalmol Vis Sci. 2004 Mar;45(3):813-20. doi: 10.1167/iovs.03-0851.
Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)-ligand interactions. This study sought to identify the endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells.
Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence of tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a matrix metalloproteinase [MMP] inhibitor), or CRM197 (a diphtheria toxin mutant), with or without HB-EGF. The activation of EGFR and extracellular signal-regulated kinase (ERK) was analyzed by immunoprecipitation using EGFR antibodies and Western blot analysis with phosphotyrosine antibody. Wound induced HB-EGF shedding was assessed by isolation of secreted HB-EGF from wounded THCE cells and by measuring the release of alkaline phosphatase (AP) in THCE stable cell lines expressing HB-EGF-AP.
In THCE cells, wound-induced EGFR phosphorylation and ERK activation. In both organ and cell culture models, epithelial wounds were healed in basal media and inhibition of EGFR activation by AG1478 blocked wound closure with or without exogenously added HB-EGF. GM6001 delayed wound closure. Its effects diminished in the presence of exogenous EGF or HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. CRM197, a highly specific antagonist of HB-EGF, impaired epithelial wound closure, suggesting that HB-EGF is an endogenous ligand released on epithelial wounding. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR phosphorylation in cultured THCE cells. The release of HB-EGF in response to wounding was demonstrated by the fact that heparin-binding proteins isolated from wounded, but not control, THCE-conditioned medium stimulated EGFR and ERK phosphorylation and by the expression of HB-EGF-AP in THCE cells, in which wounding induced the release of AP activity in an MMP-inhibitor-sensitive manner.
HB-EGF released on wounding acts as an autocrine-paracrine EGFR ligand. HB-EGF shedding and EGFR activation represent a critical event during corneal epithelial wound healing, suggesting a possible manipulation of wound healing during the early phases.
上皮伤口愈合至少部分是由表皮生长因子（EGF）受体（EGFR）-配体相互作用以自分泌方式介导的。本研究旨在确定内源性EGFR配体及其在培养的猪角膜和人角膜上皮细胞中因创伤而产生的机制。
在存在 tyrphostin AG1478（一种EGFR抑制剂）、GM6001（一种基质金属蛋白酶 [MMP] 抑制剂）或CRM197（一种白喉毒素突变体）的情况下，让培养的猪角膜上皮清创伤口和SV40永生化人角膜上皮（THCE）细胞单层划痕伤口愈合，添加或不添加HB-EGF。使用EGFR抗体通过免疫沉淀和用磷酸酪氨酸抗体进行蛋白质印迹分析来分析EGFR和细胞外信号调节激酶（ERK）的激活。通过从受伤的THCE细胞中分离分泌的HB-EGF以及通过测量表达HB-EGF-AP的THCE稳定细胞系中碱性磷酸酶（AP）的释放来评估伤口诱导的HB-EGF脱落。
在THCE细胞中，伤口诱导EGFR磷酸化和ERK激活。在器官和细胞培养模型中，上皮伤口在基础培养基中愈合，AG1478抑制EGFR激活可阻断伤口闭合，无论是否添加外源性HB-EGF。GM6001延迟伤口闭合。在存在外源性EGF或HB-EGF的情况下其作用减弱，这表明MMP抑制剂主要阻断EGFR配体的释放。CRM197是HB-EGF的高度特异性拮抗剂，它损害上皮伤口闭合，这表明HB-EGF是上皮受伤时释放的内源性配体。与对上皮迁移的影响一致，所有抑制剂以及HB-EGF功能阻断抗体均延迟培养的THCE细胞中伤口诱导的EGFR磷酸化。从受伤但非对照的THCE条件培养基中分离的肝素结合蛋白刺激EGFR和ERK磷酸化以及THCE细胞中HB-EGF-AP的表达证明了受伤时HB-EGF的释放，其中伤口以MMP抑制剂敏感的方式诱导AP活性的释放。
受伤时释放的HB-EGF作为自分泌-旁分泌EGFR配体发挥作用。HB-EGF脱落和EGFR激活代表角膜上皮伤口愈合过程中的关键事件，这表明在早期阶段可能对伤口愈合进行调控。
