title: Spatiotemporally Regulated Ablation of Klf4 in Adult Mouse Corneal Epithelial Cells Results in Altered Epithelial Cell Identity and Disrupted Homeostasis.
abstract: OBJECTIVE
  In previous studies, conditional disruption of Klf4 in the developing mouse ocular surface from embryonic day 10 resulted in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells, revealing the importance of Klf4 in ocular surface maturation. Here, we use spatiotemporally regulated ablation of Klf4 to investigate its functions in maintenance of adult corneal epithelial homeostasis.
  
  
  METHODS
  Expression of Cre was induced in ternary transgenic (Klf4(LoxP/LoxP)/Krt12(rtTA/rtTA)/Tet-O-Cre) mouse corneal epithelium by doxycycline administered through intraperitoneal injections and drinking water, to generate corneal epithelium-specific deletion of Klf4 (Klf4(Δ/ΔCE)). Corneal epithelial barrier function was tested by fluorescein staining. Expression of selected Klf4-target genes was determined by quantitative PCR (QPCR), immunoblotting, and immunofluorescent staining.
  
  
  RESULTS
  Klf4 was efficiently ablated within 5 days of doxycycline administration in adult Klf4(Δ/ΔCE) corneal epithelium. The Klf4(Δ/ΔCE) corneal epithelial barrier function was disrupted, and the basal cells were swollen and rounded after 15 days of doxycycline treatment. Increased numbers of cell layers and Ki67-positive proliferating cells suggested deregulated Klf4(Δ/ΔCE) corneal epithelial homeostasis. Expression of tight junction proteins ZO-1 and occludin, desmosomal Dsg and Dsp, basement membrane laminin-332, and corneal epithelial-specific keratin-12 was decreased, while that of matrix metalloproteinase Mmp9 and noncorneal keratin-17 increased, suggesting altered Klf4(Δ/ΔCE) corneal epithelial cell identity.
  
  
  CONCLUSIONS
  Ablation of Klf4 in the adult mouse corneas resulted in the absence of characteristic corneal epithelial cell differentiation, disrupted barrier function, and squamous metaplasia, revealing that Klf4 is essential for maintenance of the adult corneal epithelial cell identity and homeostasis.
authors.affiliation: Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States.
authors.forename: Emili E
authors.initials: EE
authors.lastname: Delp
authors.affiliation: Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States.
authors.forename: Sudha
authors.initials: S
authors.lastname: Swamynathan
authors.affiliation: Department of Ophthalmology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States.
authors.forename: Winston W
authors.initials: WW
authors.lastname: Kao
authors.affiliation: Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States 3McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States 4Department of Cell Biology, U.
authors.forename: Shivalingappa K
authors.initials: SK
authors.lastname: Swamynathan
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D002454:Cell Differentiation; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000473:pathology; D020869:Gene Expression Profiling; D006706:Homeostasis / Q000502:physiology; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction / Q000379:methods; D062725:Tight Junction Proteins / Q000378:metabolism
chemical_list: C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D062725:Tight Junction Proteins