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

Abstract

PurposeThe 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 Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mice.MethodsCE-specific ablation of Klf4 was achieved by feeding Klf4Δ/ΔCE mice with doxycycline chow. The wild-type (WT; normal chow-fed littermates) and the Klf4Δ/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.ResultsThe 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 Klf4Δ/ΔCE corneas. The Klf4Δ/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 Klf4Δ/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.ConclusionsCollectively, these results reveal that KLF4 plays an essential role in CE homeostasis by promoting epithelial cell fate and suppressing EMT.

KLF4 coordinates corneal epithelial apicobasal polarity and plane of cell division, and is downregulated in ocular surface squamous neoplasia

“…Apicobasal polarity is disrupted in Klf4 Δ/ΔCE CE. As reported previously (14,17,19,20), CEspecific ablation of Klf4 resulted in hyperplasia concurrent with downregulation of CE markers keratin-12 (Krt12), tight junction protein-1 (Tjp1), and E-cadherin, reminiscent of EMT (Supplemental Fig. 1).…”

“…Previously, we reported that CE-specific ablation of Klf4 results in (i) EMT coupled with loss of CE barrier function and hyperplasia (14,17,20), and (ii) activation of canonical TGF-KLF4, corneal epithelial polarity and plane of division β signaling and downregulation of cell cycle inhibitors favoring increased proliferation (21). The data presented in this report elucidate the crucial role of Klf4 in orchestrating the CE stratification and homeostasis by coordinating the expression and localization of ABP determinants, and plane of CE cell division ( Fig.…”

“…Previously, we reported that CE-specific ablation of Klf4 results in defects that resemble OSSN (17,20,21). To determine if OSSN is indeed accompanied by EMT, we obtained surgically excised human tissues suspected of OSSN, and ascertained OSSN by histology ( Fig.…”

“…Previous studies from our lab and others identified Krüppel-like factor-4 (KLF4), one of the most abundantly expressed transcription factors in the CE, as a major determinant of CE properties (14,15). Corneal Klf4-target genes collectively promote CE structural stability and barrier functions while suppressing epithelialmesenchymal transition (EMT) and TGF-β signaling (14,(16)(17)(18)(19)(20)(21). Though Klf4 is known to influence the intestinal epithelial cell ABP (22), its involvement in regulating the core ABP determinants is unexplored in stratified tissues like the CE.…”

“…Though Klf4 is known to influence the intestinal epithelial cell ABP (22), its involvement in regulating the core ABP determinants is unexplored in stratified tissues like the CE. Given that (i) Klf4 is abundantly expressed in the CE where it upregulates the expression of tight and adherence junction components that play a key role in ABP (19), (ii) Klf4 ablation results in EMT and increased TGFβ signaling commonly associated with compromised ABP and epithelial tumors (20,21), (iii) TGF-β-induced EMT is invariably associated with a loss of ABP (23), and (iv) decreased expression or mutations in Klf4 are commonly associated with tumors (24,25) that display loss of core polarity components and altered plane of cell division (26), we predicted that Klf4 contributes to CE homeostasis by coordinating CE cell ABP and plane of division. Data presented in this report reveal that spatiotemporally regulated ablation of Klf4 in the adult mouse CE affects (i) the expression and sub-cellular localization of core ABP markers Pals1, Crumbs1, Par3 and Scribble, (ii) expression and/or localization of Rho family GTPases, (iii) cytoskeletal F-actin organization, and (iv) the plane of cell division, elucidating the key integrative role of Klf4 in coordinating CE cell ABP and plane of division.…”