July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
KLF4 regulates corneal epithelial cell cycle progression by suppressing canonical TGF-β signaling, and upregulating CDK inhibitors P16 and P27
Author Affiliations & Notes
  • Anil Tiwari
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Sudha Swamynathan
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Nicholas Alexander
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • John Sabastian Gnalian
    Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Shenghuo Tien
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Paul R Kinchington
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Swamynathan Shivalingappa K
    Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Anil Tiwari, None; Sudha Swamynathan, None; Nicholas Alexander, None; John Gnalian, None; Shenghuo Tien, None; Paul Kinchington, None; Swamynathan Shivalingappa K, None
  • Footnotes
    Support  NIH Grant RO1 EY026533, NIH core grantP30 EY08098 to the Department of Ophthalmology,the Ear and Eye Foundation of Pittsburgh,and unrestricted grant from Research to Prevent Blindness, New York, NY.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5106. doi:https://doi.org/
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      Anil Tiwari, Sudha Swamynathan, Nicholas Alexander, John Sabastian Gnalian, Shenghuo Tien, Paul R Kinchington, Swamynathan Shivalingappa K; KLF4 regulates corneal epithelial cell cycle progression by suppressing canonical TGF-β signaling, and upregulating CDK inhibitors P16 and P27. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5106. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Krüppel-like factor 4 (KLF4) is highly expressed in the corneal epithelial (CE) cells, promoting epithelial cell fate and suppressing mesenchymal properties. Transforming growth factor-β (TGF-β)-signaling plays a crucial role in cell growth, differentiation, and induces epithelial-mesenchymal transition (EMT). Here we examined that KLF4 contributes to CE homeostasis by suppressing TGF-β-signaling and upregulating cell cycle inhibitors to create an anti-tumorigenic environment.

Methods : We used adult mouse CE-specific spatiotemporally regulated ablation of Klf4 using ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mice, and human corneal limbal epithelial (HCLE) cells stably expressing KLF4 or anti-KLF4 shRNA to determine the role of KLF4 in maintaining CE phenotype. Selected target-gene expression was evaluated using QPCR, immunoblots and/or immunofluorescent staining.

Results : CE-specific ablation of Klf4 resulted in (i) upregulation of TGF-β1, -β2, TGF-βR1 and -βR2, (ii) downregulation of Smad7, (iii) hyperphosphorylation of Smad2/3, and (iv) elevated nuclear localization of phospho-Smad2/3 and Smad4. ShRNA-mediated suppression of KLF4 in HCLE cells resulted in upregulation of TGF-β1 and -β2, hyperphosphorylation and nuclear localization of SMAD2/3, downregulation of SMAD7 and elevated SMAD4 nuclear localization. Over-expression of KLF4 in HCLE cells (HCLE-KLF4) resulted in downregulation of TGF-β1, βR1, -βR2, and upregulation of SMAD7. Cyclin-dependent kinase (CDK) inhibitors p16 and p27 were decreased in Klf4Δ/ΔCE corneas and increased in HCLE-KLF4 cells. TGF-β treatment resulted in significant upregulation of EMT transcription factors (TFs) Slug, Zeb1 and Zeb2 in HCLE, but not HCLE-KLF4 cells. HCLE-KLF4 also suppressed the EMT TF Snail and prevented its activation upon TGF-β treatment.

Conclusions : These results shows that KLF4 promotes CE phenotype by suppressing canonical TGF-β-signaling, and overcomes the undesirable concomitant decrease in TGF-β-dependent p16 and p27 expression by directly upregulating them. KLF4 contributes to CE homeostasis by suppressing TGF-β-signaling and upregulating cell cycle inhibitors to create an anti-tumorigenic environment, establishing the importance of the crosstalk between KLF4 and TGF-β-signaling for CE homeostasis.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

KLF4 and TGF-β cross talk regulates CE cell cycle and homeostasis.

KLF4 and TGF-β cross talk regulates CE cell cycle and homeostasis.

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