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J. K. Klarlund, E. R. Block; Dual Mechanisms for Activating the Receptor for Epidermal Growth Factor in Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5042.
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Wounding the corneal epithelium induces activation of the epidermal growth factor receptor (EGFR), an event that is absolutely required for induction of motility. Signaling by ATP and Phospholipase D (PLD) has been implicated as upstream events in activation of the EGFR. The purpose of our experiments was to characterize the roles of these signaling molecules in healing wounds in sheets of corneal epithelial cells.
Multiple wounds were induced in confluent sheets of a human corneal epithelial cell line (HCLE). PLD activities were measured by labeling cells with [3H]myristic acid and assaying production of buturyl phosphatidic acid, release of ATP was measured by a bioluminescent assay, release of EGFR ligands was measured by the DuoSet® ELISA (R&D Systems), and activation of the EGFR was determined by immunoblotting with an antibody that recognizes the receptor phosphorylated on tyrosine 1173.
Knockdown with siRNAs demonstrated that the PLD2 isoform is activated upon wounding. PLD2 was activated by addition of exogenous ATP. Wounding resulted in ATP release form the cells, and inclusion of apyrase, an enzyme that degrades ATP, in the in the tissue culture medium inhibited PLD2 activation after wounding. This, together with previously published data (Mazie et al. (2006) J. Cell Sci. 119, 1645), suggests that these signaling molecules are linked in the following sequence: wounding→ATP release→PLD2 →EGFR activation. To test the importance of ATP signaling for wound healing, cells were incubated with apyrase under conditions where extracellular ATP is quantitatively degraded, and that allows retention of the enzyme activity. Surprisingly, this treatment had no effect on the rates of wound healing in HCLE and primary rabbit corneal epithelial cells. We therefore examined whether an alternative mechanism for EGFR activation exists, which lead to the identification of a pathway that is independent of ATP/PLD2 signaling. Immunoblotting of extracts of cells at a distance from wounds and immunofluorescence studies revealed that the ATP/PLD2-dependent pathway acts far from the wounds (at least 0.5 cm) whereas activation through the ATP/PLD2-independent mechanism is phased out approximately 250 µm from the wounds.
The EGFR is activated by two distinct mechanisms: one depends on ATP/PLD2 signaling and acts far from wounds, another is independent on ATP/PLD2 signaling and acts at short distances. Activation of the latter pathway is sufficient for wound healing.
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