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

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.