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I. Boucher, V. Trinkaus-Randall; Injury Induced Purinergic Receptor Stimulation Leads to Epidermal Growth Factor Receptor Activation and Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1958.
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Corneal epithelial injury induces the release of nucleotides leading to G-protein coupled purinergic receptor (P2YR) activation. G-protein coupled receptors have been shown to stimulate release of heparin binding-epidermal growth factor (HB-EGF), which activates the epidermal growth factor receptor (EGFR). The aim of this study was to investigate the role of P2Y signaling cascades in mediating activation of the EGFR and other downstream events including wound repair.
Human corneal epithelial cells (HCET) were used. In addition, EGFR deficient cells (PAE) and those stably transfected with EGFR (E1-PAE) were used as a model to investigate the role of the EGFR. Scratch wounds were made, and live-cell imaging using an Zeiss LSM510 was used to monitor wound healing over time. Cells were maintained at 37ºC and 5% CO2 for 20 hours in a Zeiss environmental chamber, and change in wound area was measured. Transwell migration assays were performed in parallel. Western blot Aaalysis was used to examine downstream signaling.
Reactive Blue-2, an inhibitor of purinergic signaling, reduced the injury induced phosphorylation of EGFR and ERK. The use of an antibody directed against HB-EGF reduced EGFR phosphorylation after injury. Wound healing is compromised when cells are incubated in the presence of CRM197, which inhibits the release of HB-EGF. The migration was restored to control levels by addition of exogenous HB-EGF. E1-PAE cells showed increased migration upon stimulation with UTP over unstimulated cells, while PAE cells lacking the EGFR did not. In the Transwell migrations, PAE cells migrated significantly less to UTP than E1-PAE cells.
P2Y stimulation induces release of HB-EGF and subsequent EGFR phosphorylation. Inhibiting HB-EGF release decreases cell migration. EGFR was necessary for nucleotide induced scratch wound migration. However, other compensatory pathways may exist as shown by partial inhibition in the Transwell migration assay. Together these data indicate P2Y receptors act upstream of the EGFR after corneal epithelial injury.
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