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Chelsea L. Loughner, Doreswamy Kenchegowda, Sudha Swamynathan, Shivalingappa K Swamynathan; Spatiotemporally regulated ablation of Klf5 results in dysregulated epithelial homeostasis in mature mouse corneas. Invest. Ophthalmol. Vis. Sci. 2017;58(8):960.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we reported that Le-Cre-mediated conditional ablation of Krüppel-like transcription factor-5 (Klf5) in the developing murine ocular surface results in abnormal cornea, conjunctiva, and the eyelids. Here, we employ Tet-On system for spatiotemporally regulated ablation of Klf5 to examine its functions in mature mouse corneal epithelial (CE) homeostasis.
Cre expression was induced in 8-week-old ternary transgenic Klf5LoxP/LoxP/ Krt12rtTA/rtTA/ Tet-O-Cre (Klf5Δ/ΔCE) mouse corneas by doxycycline administered through chow, to ablate Klf5 in the CE. The wild type (WT) and Klf5Δ/ΔCE corneal (i) histology, (ii) cell proliferation, and (iii) Klf5-target gene expression were examined using (i) hematoxylin and eosin (H&E), and periodic acid-Schiff’s reagent (PAS)-stained sections, (ii) Ki67 expression, and (iii) qPCR and immunostaining, respectively. The effect of Klf5, Klf4 and Oct1 on gastrokine-1 (Gkn1) promoter activity was determined by transient transfection in human corneal limbal epithelial (HCLE) cells.
Klf5 was efficiently ablated within 15 days of doxycycline administration in Klf5Δ/ΔCE corneas (23% of WT), resulting in disrupted epithelial basement membrane, decreased cell proliferation (51% of WT), and irregular stratification. Klf5Δ/ΔCE corneas displayed decreased expression of basement membrane laminin (55% of WT), corneal crystallin transketolase (Tkt; 36% of WT), Gkn1 (17% of WT), uroplakin-1b (Upk1b; 14% of WT), and Upk3b (11% of WT) that promote permeability barrier, and desmosomal protein desmoglein-1a (Dsg1a; 13% of WT). Gkn1 promoter activity was stimulated 7-fold by Klf4, 19-fold by Klf5, 2.75-fold by Oct1, and 41-fold in a synergistic manner by Klf5 and Oct1.
Spatiotemporally regulated ablation of Klf5 in mature mouse CE results in disrupted epithelial basement membrane, dysregulated cell proliferation and stratification, accompanied with decreases in Gkn1, Dsg1a, Upk1b, Upk3b, and Tkt expression, consistent with a prominent role for Klf5 in mature CE homeostasis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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