Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Xu
Ke-Ping
KP
Kresge Eye Institute, Departments of Ophthalmology and of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Yu
Fu-Shin X
FS
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002460:Cell Line; D002465:Cell Movement; D015194:Diffusion Chambers, Culture; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D018739:Oncogene Proteins v-erbB / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D020239:Receptor Cross-Talk / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation; D014945:Wound Healing / Q000187:drug effects / Q000502:physiology*
C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D018739:Oncogene Proteins v-erbB; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases