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J. Yin, K.-P. Xu, F.-S. X. Yu; Identification of Early Signals Triggering EGFR Activation in Response to Wounding in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3475. doi: https://doi.org/.
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The aim of the present study is to identity the roles of adenosine triphosphate (ATP) and Lysophosphatidic Acid (LPA), two initial mediators released from injured corneal epithelial cells, in transactivating epidermal growth factor receptor (EGFR) and in regulating epithelial cell migration, proliferation and wound healing.
ATP released into culture media from injured HCECs was assessed by luciferase/luciferin bioluminescent assay. Heparin-binding EGF-like growth factor (HB-EGF) shedding was assessed by measuring the release of alkaline phosphatase (AP) in a stable HCEC line that expresses HB-EGF with AP inserted in the heparin binding site. The activation of EGFR was analyzed by immunoprecipitation and phosphorylation of ERK and AKT (a major substrate of phosphatidylinositol 3'-kinase [PI3K]) was analyzed by Western blotting. Epithelial wound healing was assessed by porcine cornea organ culture and human corneal epithelial cell (HCEC) scratch wound models.
Mechanical injury greatly increased HCEC extracellular ATP concentration, which was required for early cell activation, assessed by AKT and ERK phosphorylation. ATP-γ-S (a non-hydrolyzable ATP analog) accelerated epithelial wound closure via enhancing HB-EGF shedding and EGFR transactivation in an ADAM-dependent manner. LPA, known to be released from injured corneas in vivo, acted in a similar fashion to transactivate EGFR and promote wound healing. Consistent with EGFR activation, wounding, ATP-γ-S and LPA induced ERK and AKT phosphorylation. However, early ERK activation (5 minutes) was EGFR independent and wound-induced HB-EGF shedding and EGFR activation were attenuated by ERK inhibition, suggesting that ERK may function upstream of EGFR to transduce injury signals to the cells.
Cellular components, ATP and LPA, released from injured corneal epithelial cells, act as mediators to transactivate EGFR via ERK and EGFR represents a convergent point of cell signaling accessible to a variety of stimuli in response to pathophysiological challenge in HCECs. Manipulation of signaling pathways leading to EGFR activation might be used as therapeutic approaches to treat corneal wound healing related diseases.
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