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A. Fatima, R. M. Lavker, S. Getsios; Ligand Activation of EphA2 Restricts Human Corneal Keratinocyte Migration. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2498.
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© ARVO (1962-2015); The Authors (2016-present)
Eph receptor tyrosine kinases (RTKs) become activated by ephrin ligands present on neighboring cells. Several members of this large family, including EphA2, are expressed in corneal epithelial cells where their functions remain to be defined. EphA2 expression is regulated by the MAPK-Erk1/2 pathway and this RTK also serves as a direct target of PI3K-AKT signaling. Interestingly, ephrin-induced activation of EphA2 is capable of inhibiting these same signaling effectors, which have each been shown to drive cell migration. We reasoned that ligand activation of EphA2 might play a key role in restricting human corneal keratinocyte (HCK) migration by inhibition of Erk1/2 and/or AKT.
We localized EphA2 immunohistochemically in the human cornea in concert with linear scratch wound healing studies using ephrin-A1 ectodomain peptides to trigger EphA2 phosphorylation in primary and immortalized HCKs. EphA2 knockdown and genetic reconstitution was combined with pharmacological inhibition of Erk1/2 or AKT during wound healing.
EphA2 was localized to human corneal epithelial cells in vivo and in vitro. Ephrin-A1 peptide treatment enhanced EphA2 phosphorlyation and further inhibited the ability of primary or immortalized HCKs to seal linear scratch wounds. The ephrin-A1-mediated restriction of migration was specific for this RTK subtype as this response was lost upon EphA2 silencing. A corresponding decrease in Erk1/2 and AKT activation was found following ephrin-A1 peptide treatment, consistent with the role of these signaling pathways in promoting HCK migration. Interestingly, pharmacological inhibition of MEK1/2 or PI3K restricted HCK migration but this delay in wound healing could only be potentiated by ephrin-A1 peptide treatment under conditions where the Erk1/2 but not the AKT pathway was blocked. Reconstituted expression of full-length but not cytodomain truncated mouse EphA2 restored the ability of ephrin-A1 peptides to inhibit these signaling pathways, highlighting the importance of the cytoplasmic kinase domain for EphA2 functions in wound healing.
EphA2 activation by ephrin-A ligands is capable of inhibiting HCK migration as well as Erk1/2 and AKT signaling during wound healing in vitro. Loss of the normally restricting effects of ephrin ligands on EphA2 following cell depletion in wounds likely serves as a contextual cue to guide migrating keratinocytes into cell-free areas, thereby facilitating corneal reepithelialization.
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