ERK1/2 mediate wounding- and G-protein-coupled receptor ligands-induced EGFR activation via regulating ADAM17 and HB-EGF shedding.
OBJECTIVE
Previous studies have shown that wounding of human corneal epithelial cells (HCECs) results in the release of G-protein-coupled receptor ligands such as ATP and lysophosphatidic acid (LPA), which in turn transactivate epidermal growth factor (EGF) receptor (EGFR) through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). In the present study, the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in regulating EGFR transactivation was investigated.


METHODS
SV40-immortalized HCECs were wounded or stimulated with ATP and LPA. EGFR and ADAM17 activation was analyzed by immunoprecipitation followed by Western blot analysis with phospho-tyrosine or phospho-serine antibodies, respectively. Phosphorylation of ERK and AKT was analyzed by Western blot analysis. HB-EGF shedding was assessed by measuring the release of alkaline phosphatase (AP) in a stably transfected human corneal epithelial (THCE) cell line expressing HB-EGF-AP. ADAM17 and ERK interaction was determined by coimmunoprecipitation.


RESULTS
Early, but not late, ERK1/2 phosphorylation in response to wounding, LPA, and ATP was EGFR independent, but sensitive to the inhibitors of calcium influx, protein kinase C and Src kinase. Wounding-, LPA-, and ATP-induced HB-EGF shedding and EGFR activation were attenuated by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, as well as by ADAM10 and -17 inhibitors. ADAM17 was found to be physically associated with active ERK and phosphorylated at serine residues in an ERK-dependent manner in wounded cells.


CONCLUSIONS
Taken together, our data suggest that in addition to functioning as an EGFR downstream effector, ERK1/2 also mediates ADAM-dependent HB-EGF shedding and subsequent EGFR transactivation in response to a variety of stimuli, including wounding and GPCR ligands.
Yin
Jia
J
Department of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, 4717 St. Antoine Boulevard, Detroit, MI 48201, USA.
Yu
Fu-Shin X
FS
D051722:ADAM Proteins / Q000378:metabolism*; D000072198:ADAM17 Protein; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000187:drug effects / Q000293:injuries* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008024:Ligands; D008246:Lysophospholipids / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015533:Transcriptional Activation; D014945:Wound Healing / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism
D004791:Enzyme Inhibitors; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D008246:Lysophospholipids; D043562:Receptors, G-Protein-Coupled; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C512478:EGFR protein, human; D066246:ErbB Receptors; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C032881:lysophosphatidic acid; D002118:Calcium