KLF4 Plays an Essential Role in Corneal Epithelial Homeostasis by Promoting Epithelial Cell Fate and Suppressing Epithelial–Mesenchymal Transition

Abstract

Purpose

The purpose of this study was to test the hypothesis that KLF4 promotes corneal epithelial (CE) cell fate by suppressing the epithelial–mesenchymal transition (EMT), using spatiotemporally regulated CE-specific ablation of Klf4 in Klf4 ^Δ/ΔCE ( Klf4 ^LoxP/LoxP / Krt12 ^rtTA/rtTA / Tet-O-Cre) mice.

Methods

CE-specific ablation of Klf4 was achieved by feeding Klf4 ^Δ/ΔCE mice with doxycycline chow. The wild-type (WT; normal chow-fed littermates) and the Klf4 ^Δ/ΔCE histology was compared by hematoxylin and eosin–stained sections; EMT marker expression was quantified by quantitative PCR, immunoblots, and immunofluorescent staining; and wound healing rate was measured by CE debridement using Algerbrush. KLF4 and EMT markers were quantified in human corneal limbal epithelial (HCLE) cells undergoing TGF-β1–induced EMT by quantitative PCR, immunoblots, and immunofluorescent staining.

Results

The epithelial markers E-cadherin, Krt12, claudin-3, and claudin-4 were down-regulated, whereas the mesenchymal markers vimentin, β-catenin, survivin, and cyclin-D1 and the EMT transcription factors Snail, Slug, Twist1, Twist2, Zeb1, and Zeb2 were up-regulated in the Klf4 ^Δ/ΔCE corneas. The Klf4 ^Δ/ΔCE cells migrated faster, filling 93% of the debrided area within 16 hours compared with 61% in the WT. After 7 days of wounding, the Klf4 ^Δ/ΔCE cells that filled the gap failed to regain epithelial characteristics, as they displayed abnormal stratification; down-regulation of E-cadherin and Krt12; up-regulation of β-catenin, survivin, and cyclin-D1; and a 2.5-fold increase in the number of proliferative Ki67 ⁺ cells. WT CE cells at the migrating edge and the HCLE cells undergoing TGF-β1–induced EMT displayed significant down-regulation of KLF4.

Conclusions

Collectively, these results reveal that KLF4 plays an essential role in CE homeostasis by promoting epithelial cell fate and suppressing EMT.