Xu Ke-Ping, Yu Fu-Shin X
Kresge Eye Institute, Departments of Ophthalmology and of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Invest Ophthalmol Vis Sci. 2007 May;48(5):2242-8. doi: 10.1167/iovs.06-0560.
The authors sought to determine how hepatocyte growth factor (HGF) receptor c-Met and epidermal growth factor receptor (EGFR) cross talk in response to injury in human ARPE-19 cells.
A scratch wound was made on a cell monolayer of ARPE-19 cells using a sequence-comb or a pipet tip, and it was allowed to heal in the presence or absence of HGF and heparin-binding EGF-like growth factor (HB-EGF). The activation of EGFR was analyzed by immunoprecipitation with EGFR antibody, followed by Western blotting with phosphotyrosine-specific antibody. Phosphorylation of extracellular signal-regulated kinase (ERK) and AKT (a major substrate of phosphatidylinositol 3'-kinase (PI3K) was assessed by Western blotting. The release of c-Met ectodomain into the culture media was determined by Western blotting using an antibody against the extracellular region. Cell migration was assessed by Boyden chamber migration assay.
ARPE-19 cells underwent spontaneous wound healing in basal medium, and exogenously added HB-EGF and HGF significantly enhanced wound closure. Basal and growth factor-enhanced wound closures were attenuated but not slowed by hydroxyurea, a cell proliferation inhibitor. RPE cells expressed all four erbBs, and wounding induced EGFR transactivation and downstream ERK and PI3K phosphorylation in ARPE-19 cells. HGF also induced EGFR tyrosine phosphorylation. The EGFR kinase inhibitor AG1478 blocked wound- and HGF-stimulated EGFR transactivation and attenuated spontaneous and growth factor-induced wound closure. Wounding and EGFR ligands induced the release of c-Met into the culture media. Moreover, pretreatment of cells with HB-EGF impaired ARPE-19 migration toward HGF in a matrix metalloproteinase inhibitor-sensitive manner.
EGFR modulates HGF/c-Met activity by inducing c-Met ectodomain shedding, and HGF/c-Met transactivates EGFR, leading to an enhanced activation of downstream signaling pathways. Cross talk between EGFR and c-Met may play a key role in regulating RPE cell migration, proliferation, and wound healing.
作者试图确定肝细胞生长因子（HGF）受体c-Met与表皮生长因子受体（EGFR）如何在人ARPE-19细胞中响应损伤而相互作用。
使用梳齿或移液器吸头在ARPE-19细胞单层上制造划痕伤口，并在有或无HGF和肝素结合表皮生长因子样生长因子（HB-EGF）的情况下使其愈合。通过用EGFR抗体进行免疫沉淀，然后用磷酸酪氨酸特异性抗体进行蛋白质印迹分析EGFR的激活情况。通过蛋白质印迹评估细胞外信号调节激酶（ERK）和AKT（磷脂酰肌醇3'-激酶（PI3K）的主要底物）的磷酸化。使用针对细胞外区域的抗体通过蛋白质印迹法测定c-Met胞外域释放到培养基中的情况。通过Boyden小室迁移试验评估细胞迁移。
ARPE-19细胞在基础培养基中进行自发伤口愈合，外源性添加的HB-EGF和HGF显著增强伤口闭合。羟基脲（一种细胞增殖抑制剂）减弱但未减缓基础和生长因子增强的伤口闭合。RPE细胞表达所有四种erbB，并且创伤诱导ARPE-19细胞中的EGFR反式激活以及下游ERK和PI3K磷酸化。HGF也诱导EGFR酪氨酸磷酸化。EGFR激酶抑制剂AG1478阻断伤口和HGF刺激的EGFR反式激活，并减弱自发和生长因子诱导的伤口闭合。创伤和EGFR配体诱导c-Met释放到培养基中。此外，用HB-EGF预处理细胞以基质金属蛋白酶抑制剂敏感的方式损害ARPE-19向HGF的迁移。
EGFR通过诱导c-Met胞外域脱落来调节HGF/c-Met活性，并且HGF/c-Met反式激活EGFR，导致下游信号通路的激活增强。EGFR和c-Met之间的相互作用可能在调节RPE细胞迁移、增殖和伤口愈合中起关键作用。
