June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Spatiotemporally regulated ablation of Klf5 results in dysregulated epithelial homeostasis in mature mouse corneas
Author Affiliations & Notes
  • Chelsea L. Loughner
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Doreswamy Kenchegowda
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
    Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Sudha Swamynathan
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Shivalingappa K Swamynathan
    Ophthalmology and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
    Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Chelsea Loughner, None; Doreswamy Kenchegowda, None; Sudha Swamynathan, University of Pittsburgh School of Medicine (P); Shivalingappa Swamynathan, University of Pittsburgh School of Medicine (P)
  • Footnotes
    Support  NEI, NIH Grant R01EY026533; NEI, NIH Grant P30 EY08098; Unrestricted grants from ‘Research to Prevent Blindness’ and the ‘Eye and Ear Foundation of Pittsburgh’
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 960. doi:
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    • Get Citation

      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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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