Krüppel-like factor 4 (KLF4) Controls Corneal Epithelial Stratification by Regulating Cell Polarity and Plane of Cell Division

Abstract

Purpose : Epithelial cells all over the body, including those in the cornea must attain specific types of polarity to perform specialized functions. Previously, we have shown that KLF4 plays an essential role in corneal epithelial (CE) homeostasis by suppressing epithelial-mesenchymal transition (EMT), frequently associated with loss of polarity. Here, we have examined the role of KLF4 in CE cell polarity and plane of division.

Methods : Klf4 was ablated in a spatiotemporally regulated manner in the CE by feeding Klf4^Δ/ΔCE (Klf4^LoxP/LoxP/Krt12^rtTA/rtTA/Tet-O-Cre) mice with doxycycline chow for at least a month. Age- and sex-matched littermates fed with normal chow served as controls. Expression of apicobasal polarity markers and Rho family GTPases was examined by QPCR, immunofluorescent staining and immunoblots. Organization of actin bundles was examined using fluorescently tagged phalloidin. The pattern of immunofluorescent stain with anti-survivin and anti-phospho-histone 3 antibodies relative to the basement membrane was used to determine the plane of cell division as horizontal (0°-45°), or vertical (60°-90°).

Results : QPCR, immunoblots and immunofluorescence revealed decreased expression and mis-localization of apical Pals1 and Crumbs1, apicolateral Par3, and basolateral Scribble in the Klf4^Δ/ΔCE cells. Cdc42 was upregulated, while Rac and Rho were mislocalized in the Klf4^Δ/ΔCE cytoplasm unlike their membrane expression in the control. Phalloidin staining revealed disrupted actin cytoskeleton in the Klf4^Δ/ΔCE cells. Survivin and phospho-histone H3 staining revealed a tilt in the Klf4^Δ/ΔCE plane of cell division favoring vertical axis, with 61.4% vertical and 38.5% horizontal, compared with 36.8% vertical and 63.2% horizontal in the control.

Conclusions : Spatiotemporally regulated ablation of Klf4 within the Klf4^Δ/ΔCE cells resulted in (i) a loss of apico-basal polarity, (ii) altered expression of Rho family GTPases, (iii) decreased expression and disrupted organization of actin bundles, and (iv) a tilt in the plane of cell division favoring vertical axis. Together, these results reveal the critical role of KLF4 in establishing CE cell polarity and stratification.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Fig. 1. Klf4^Δ/ΔCE cells display altered expression of polarity markers and a tilt in favor of vertical plane of division, revealing the critical role of KLF4 in establishing CE cell polarity and stratification