Cross–Talk Between Hepatocyte Growth Factor Receptor c–Met and Epidermal Growth Factor Receptor During RPE Wound Healing

Abstract

Abstract: : Purpose: Hepatocyte growth factor (HGF) has been implicated in cell migration and proliferation of retinal pigment epithelial (RPE) cells and in the development of proliferative vitreous retinopathy. The study sought to determine how HGF receptor c–Met and epidermal growth factor receptor (EGFR) cross–talk in response to wounding in a human ARPE19 cell line. Methods: Scratch wound was made on cell monolayer using sequence–comb and allowed to heal in the presence or absence of HGF, EGF and heparin–binding EGF–like growth factor (HB–EGF). Cell migration was assessed by Boyden chamber and scratch wound assays. Proteolytic cleavage and release of c–Met (ectodomain shedding) into culture media was determined by Western blotting using antibody against extracellular region of c–Met. The activation of EGFR was analyzed by immunoprecipitation with EGFR antibody and Western blotting with phosphotyrosine antibody. Phosphorylations of extracellular signal–regulated kinase (ERK) and phosphatidylinositol 3'–kinase (PI3K) were assessed by Western blotting with antibodies specific to phosphorylated proteins. Cell proliferation was determined by CellTiter 96 Aqueous One Solution. Results: Wounding induced ectodomain shedding of c–Met into culture media from cell surface in a matrix metalloproteinase dependent manner. Exogenous HGF greatly enhanced wound closure and attenuated wound–induced c–Met shedding. Stimulation of cells with EGFR ligands, EGF or HB–EGF also increased c–Met shedding. Pretreatment of cells with HB–EGF significantly impaired HGF induced cell migration in a Boyden chamber assay, suggesting that HB–EGF–stimulated c–Met shedding is one of the mechanisms modulating HGF/c–Met activity in RPE cells. Furthermore, treatment of cells with HGF induced EGFR tyrosine phosphorylation as well as ERK and PI3K activation in a Src tyrosine kinase dependent manner. HGF–induced RPE cell proliferation was retarded by a mitogen–activated protein kinase (MAPK) kinase or a Src tyrosine kinase inhibitor. Conclusions: Our results suggest that EGFR modulates HGF/c–Met activity by generating c–Met ectodomain shedding, which reduces the availability of the receptor on the cell surface. HGF/c–Met may transactivate EGFR, leading to an enhanced activation of downstream signaling pathways. The cross–talk between EGFR and c–Met, likely through Src tyrosine kinase, may play a key role in regulating RPE cell migration, proliferation and wound healing.

Keywords: retinal pigment epithelium • wound healing • signal transduction