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Nihal Kaplan, Han Peng, Rosa Ventrella, Constadina Arvanitis, Robert M Lavker, Spiro Getsios; EphA2/ephrinA1 signaling helps to set the limbal-corneal epithelial boundary as revealed by a novel cell confrontational co-culture model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5833.
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© ARVO (1962-2015); The Authors (2016-present)
Juxtamembrane signaling through Eph receptors and ephrin ligands regulate tissue patterning and boundary formation. Interestingly, ephrinA1 is expressed in the limbal epithelium whereas EphA2 is concentrated in the corneal epithelium. What regulates the boundary between the stem cell-enriched limbal epithelium and the more differentiated corneal epithelium is a major unanswered question in corneal epithelial biology. Given the differential distribution of ephrinA1 and EphA2 in the cornea, we examined whether these signaling complexes regulate tissue patterning events that contribute to functionally integrated limbal-corneal epithelial compartments.
Retroviral transduction was used to manipulate ephrinA1 and EphA2 levels in a human corneal epithelial cell line, hTCEpi. EphrinA1- and EphA2- hTCEpi cells were differentially labeled with fluorescent cell trackers and seeded in discrete culture compartments to mimic their limbal and corneal expression patterns, respectively. Time lapse imaging of ephrin-A1 and EphA2 hTCEpi cells confronting one another was used to examine the role of this ligand-receptor system in the formation and organization of the limbal-corneal epithelial boundary.
We found that ephrinA1-hTCEpi cells destabilized E-cadherin-based cell-cell junctions when confronted by EphA2-hTCEpi cells. Upon formation of a heterotopic ephrinA1/EphA2 hTCEpi junction, ephrinA1 expressing cells reversed the direction of migration of EphA2 expressing cells leading to coordinated migration of the two cell populations. EphA2 signaling was required for the switch in directional migration since stable silencing of this receptor or expression of a dominant negative EphA2 mutant failed to alter E-cadherin membrane localization. Pharmacological inhibition of ADAM-10 implicates this protease as a key regulator of ephrinA1/EphA2 limbal-corneal boundary formation, E-cadherin destabilization, and coordinated cell migration.
Our novel corneal epithelial cell confrontational co-culture model suggests that ephrinA1 helps organize the limbal epithelium interface with EphA2 expressing corneal epithelium. Thus, EphA2/ephrinA1 signaling complexes likely play critical roles in normal limbal-corneal epithelial compartmentalization as well as in response to injury.
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