Inhibitor of DNA-Binding 3 Is a Novel Regulator of Limbal Epithelial Cell Migration Via the EphA2/Akt Signaling Pathway.

Purpose: Upon corneal injury, early and late transit amplifying (TA) cells, the progeny of epithelial stem cells, migrate to the site of the wound, which facilitates its closure. Understanding the targetable signals that guide such cell migration is essential for the development of novel wound-healing strategies. Methods: Single-cell RNA sequencing (scRNA-seq) was conducted in wild-type cornea. To investigate the role of inhibitor of DNA binding 3 (ID3) in the limbal epithelium, bulk RNA-seq was conducted in limbal epithelial cells with ID3 knockdown. hTCEpi cells were transfected with small interfering RNAs (siRNAs) against ID3, and cell migration was assessed using scratch wound assays. Results: The scRNA-seq revealed that ID3, a transcription factor, was preferentially expressed in the stem/early TA population of limbal epithelium. Bulk RNA-seq in limbal epithelial cells with ID3 knockdown suggested a role of ID3 in cell migration. Scratch wound assays confirmed that loss of ID3 in human limbal epithelial cells markedly accelerated wound sealing, indicating an inhibitory role of ID3 in cell migration. Gene Ontology analysis of the RNA-seq data suggested that ID3 negatively regulates EphA2 (a receptor tyrosine kinase) and Akt signaling, both of which have a critical role in cell migration. Consistently, loss of ID3 induced ligand-independent activation of EphA2, as well as enhanced phosphorylation of Akt proteins. Scratch wound assays demonstrated that both ligand-independent activation of EphA2 and phosphorylation of Akt were required for enhanced cell migration in cells lacking ID3. Conclusions: Our findings indicate that ID3, EphA2, and Akt form a novel signaling axis, which plays a critical role in corneal epithelial wound healing.