KLF4 Promotes Corneal Epithelial Cell Fate by Suppressing Epithelial-Mesenchymal Transition

Abstract

Purpose : Previously, we reported that corneal epithelial (CE)-specific ablation of Klf4 results in disrupted CE integrity and barrier function. Here we test the hypothesis that KLF4 promotes CE cell fate by suppressing epithelial-mesenchymal transition (EMT).

Methods : CE-specific Klf4 ablation was achieved by feeding ternary transgenic Klf4^LoxP/LoxP/ Krt12^rtTA/rtTA/ Tet-O-Cre (Klf4^Δ/ΔCE) mice with doxycycline-chow for 15 days. The wild type (WT; ternary transgenic littermates fed with normal chow) and Klf4^Δ/ΔCE corneal histology was examined by hematoxylin and eosin-stained sections. The WT and Klf4^Δ/ΔCE CE wound healing rate was compared by CE debridement using Algerbrush. The CE phenotype was evaluated in unwounded WT and Klf4^Δ/ΔCE corneas and those subjected to CE debridement, by quantifying the expression of Krt12, Ki67, cyclin-D1, β-catenin, E-cadherin, survivin, and paxillin. TGF-β1 was used to induce EMT in human corneal limbal epithelial (HCLE) cells, following which KLF4 and EMT markers were quantified by QPCR, immunoblots and immunofluorescent staining.

Results : Klf4^Δ/ΔCE corneas displayed thicker CE with 6-7 cell layers compared with 4-5 in the WT, abnormal stratification, 8.8-fold increased number of proliferative Ki67+ cells, up-regulation of survivin, β-catenin, cyclin-D1 and paxillin, and downregulation of E-cadherin and Krt12, consistent with loss of CE phenotype and EMT. Above features of EMT were exaggerated in Klf4^Δ/ΔCE corneas subjected to epithelial debridement. Klf4^Δ/ΔCE CE migrated faster, filling 93% of the debrided area within 16 hours of wounding, compared with 61% in the WT. HCLE cells undergoing TGF-β1-induced EMT in vitro evidenced by decreased levels of DSG1, E-cadherin, and ZO-1 coupled with increased nuclear localization of β-catenin, also displayed significant down-regulation of KLF4 (70%), consistent with its role in suppressing EMT.

Conclusions : Increased proliferation, migration, repression of CE markers, and up-regulation of mesenchymal markers in Klf4^Δ/ΔCE CE coupled with downregulation of KLF4 in HCLE cells undergoing TGF-β1-induced EMT reveal that KLF4 promotes CE cell fate by suppressing EMT.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.