Dataset Information

Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas.

ABSTRACT:

Purpose

Corneal epithelial (CE) homeostasis requires coordination between proliferation and differentiation. Here we examine the role of cell proliferation regulator Krüppel-like factor 5 (Klf5) in adult mouse CE homeostasis.

Methods

Klf5 was ablated in a spatiotemporally restricted manner by inducing Cre expression in 8-week-old ternary transgenic Klf5LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre (Klf5?/?CE) mouse CE by administering doxycycline via chow. Normal chow-fed ternary transgenic siblings served as controls. The control and Klf5?/?CE corneal (1) histology, (2) cell proliferation, and (3) Klf5-target gene expression were examined using (1) periodic acid Schiff reagent-stained sections, (2) Ki67 expression, and (3) quantitative PCR and immunostaining, respectively. The effect of KLF4, KLF5, and OCT1 on gastrokine-1 (GKN1) promoter activity was determined by transient transfection in human skin keratinocyte NCTC-2544 cells.

Results

Klf5 expression was decreased to 23% of the controls in Klf5?/?CE corneas, which displayed increased fluorescein uptake, downregulation of tight junction proteins Tjp1 and Gkn1, desmosomal Dsg1a, and basement membrane Lama3 and Lamb1, suggesting defective permeability barrier. In transient transfection assays, KLF5 and OCT1 synergistically stimulated GKN1 promoter activity. Klf5?/?CE CE displayed significantly fewer cell layers and Ki67+ proliferative cells coupled with significantly decreased cyclin-D1, and elevated phospho(Ser-10) p27/Kip1 expression. Expression of Krt12, E-cadherin, and ?-catenin remained unaltered in Klf5?/?CE corneas.

Conclusions

Klf5 contributes to adult mouse CE homeostasis by promoting (1) permeability barrier function through upregulation of Tjp1, Gkn1, Dsg1a, Lama3, and Lamb1, and (2) basal cell proliferation through upregulation of cyclin-D1 and suppression of phospho(Ser-10) p27/Kip1, without significantly affecting the expression of epithelial markers Krt12, E-cadherin, and ?-catenin.

SUBMITTER: Loughner CL

PROVIDER: S-EPMC5598321 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Publications

Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas.

Loughner Chelsea L CL Tiwari Anil A Kenchegowda Doreswamy D Swamynathan Sudha S Swamynathan Shivalingappa K SK

Investigative ophthalmology & visual science 20170901 11

<h4>Purpose</h4>Corneal epithelial (CE) homeostasis requires coordination between proliferation and differentiation. Here we examine the role of cell proliferation regulator Krüppel-like factor 5 (Klf5) in adult mouse CE homeostasis.<h4>Methods</h4>Klf5 was ablated in a spatiotemporally restricted manner by inducing Cre expression in 8-week-old ternary transgenic Klf5LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre (Klf5Δ/ΔCE) mouse CE by administering doxycycline via chow. Normal chow-fed ternary transgenic ...[more]

PMID: 28910443

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Project description:BackgroundKlf5 plays an important role in maturation and maintenance of the mouse ocular surface. Here, we quantify WT and Klf5-conditional null (Klf5CN) corneal gene expression, identify Klf5-target genes and compare them with the previously identified Klf4-target genes to understand the molecular basis for non-redundant functions of Klf4 and Klf5 in the cornea.Methodology/principal findingsPostnatal day-11 (PN11) and PN56 WT and Klf5CN corneal transcriptomes were quantified by microarrays to compare gene expression in maturing WT corneas, identify Klf5-target genes, and compare corneal Klf4- and Klf5-target genes. Whole-mount corneal immunofluorescent staining was employed to examine CD45+ cell influx and neovascularization. Effect of Klf5 on expression of desmosomal components was studied by immunofluorescent staining and transient co-transfection assays. Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases and 72 concordant decreases. PN56 Klf5CN corneas shared 241 increases and 98 decreases with those previously described in Klf4CN corneas. Xenobiotic metabolism related pathways were enriched among genes decreased in Klf5CN corneas. Expression of angiogenesis and immune response-related genes was elevated, consistent with neovascularization and CD45+ cell influx in Klf5CN corneas. Expression of 1574 genes was increased and 1915 genes decreased in WT PN56 compared with PN11 corneas. Expression of ECM-associated genes decreased, while that of solute carrier family members increased in WT PN56 compared with PN11 corneas. Dsg1a, Dsg1b and Dsp were down-regulated in Klf5CN corneas and their corresponding promoter activities were stimulated by Klf5 in transient co-transfection assays.Conclusions/significanceDifferences between PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in functions of Klf5 during corneal maturation. Klf5 contributes to corneal epithelial homeostasis by regulating the expression of desmosomal components. Klf4- and Klf5-target genes are largely distinct, consistent with their non-redundant roles in the mouse cornea.

Dataset Information

Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas.

Purpose

Methods

Results

Conclusions

Publications

Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas.

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