IOVS Genetics
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Originally published In Press as doi:10.1167/iovs.08-2246 on July 24, 2008
 (Investigative Ophthalmology and Visual Science. 2009;50:132-139.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.08-2246
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ERK1/2 Mediate Wounding- and G-protein-Coupled Receptor Ligands-Induced EGFR Activation via Regulating ADAM17 and HB-EGF Shedding

Jia Yin1,2 and Fu-Shin X. Yu1,2

1From the Department of Ophthalmology, Kresge Eye Institute, and the 2Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.

PURPOSE. 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.






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