Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells
Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells
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DOI：
10.1167/iovs.03-0851
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发表时间：
2004-03-01
影响因子：
4.4
通讯作者：
Yu, FSX
中科院分区：
文献类型：
--
作者：
Xu, KP;Ding, Y;Yu, FSX
目的。表皮生长因子受体(EGF)受体(EGFR)-配体的相互作用至少部分地以自分泌的方式介导上皮伤口的愈合。方法：在培养的猪角膜和人角膜上皮细胞中，分别在含有或不含有HB-EGF的Tyrphostin AG 1478(EGFR抑制剂)、GM6001(基质金属蛋白酶抑制剂)或CRM197(白喉毒素突变体)的存在下，允许培养的猪角膜上皮清创伤口和SV40永生化的人角膜上皮细胞(THCE)上皮单层划痕伤口愈合。用EGFR抗体免疫沉淀和磷酸化酪氨酸抗体Western印迹分析EGFR和细胞外信号调节蛋白激酶(ERK)的激活情况。从损伤的THCE细胞中分离分泌的HB-EGF，并通过检测表达HB-EGF-AP的THCE稳定细胞微粉中碱性磷酸酶(AP)的释放来评估创伤诱导的HB-EGF脱落。在器官和细胞培养模型中，上皮创面在基础培养液中愈合，AG1478抑制EGFR激活阻止创面闭合，无论是否添加外源性HB-EGF。GM6001延迟伤口闭合。当外源性EGF或HB-EGF存在时，其作用减弱，表明基质金属蛋白酶抑制剂主要阻止EGFR配体的释放。HB-EGF的高度特异性拮抗剂CRM197可抑制上皮损伤创面的闭合，提示HB-EGF是上皮损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功能阻断抗体都延缓了创伤诱导的THCE细胞中EGFR的磷酸化。创伤后HB-EGF的释放通过THCE条件培养液中的肝素结合蛋白刺激EGFR和ERK的磷酸化以及HB-EGF-AP在THCE细胞中的表达来证实，其中创伤以一种对基质金属蛋白酶抑制物敏感的方式诱导AP活性的释放。HB-EGF的脱落和EGFR的激活是角膜上皮损伤愈合过程中的一个关键事件，提示在早期阶段可能存在对伤口愈合的调控。
PURPOSE. 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.METHODS. 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 AG 1478 (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 fines expressing HB-EGF-AP.RESULTS. 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.CONCLUSIONS. 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.