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Anil Tiwari, Sudha Swamynathan, Nicholas Alexander, John Gnalian, Shenghe Tian, Paul R. Kinchington, Shivalingappa K. Swamynathan; 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(2):731-740. doi: https://doi.org/10.1167/iovs.18-26423.
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Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) cell fate while suppressing mesenchymal properties. TGF-β plays a crucial role in cell differentiation and development, and if dysregulated, it induces epithelial-mesenchymal transition (EMT). As KLF4 and TGF-β regulate each other in a context-dependent manner, we evaluated the role of the crosstalk between KLF4 and TGF-β-signaling in CE homeostasis.
We used spatiotemporally regulated ablation of Klf4 within the adult mouse CE in ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/ Krt12rtTA/rtTA/ Tet-O-Cre) mice and short hairpin RNA (shRNA)-mediated knockdown or lentiviral vector-mediated overexpression of KLF4 in human corneal limbal epithelial (HCLE) cells to evaluate the crosstalk between KLF4 and TGF-β-signaling components. Expression of TGF-β signaling components and cyclin-dependent kinase (CDK) inhibitors was quantified by quantitative PCR, immunoblots, and/or immunofluorescent staining.
CE-specific ablation of Klf4 resulted in (1) upregulation of TGF-β1, -β2, -βR1, and -βR2; (2) downregulation of inhibitory Smad7; (3) hyperphosphorylation of Smad2/3; (4) elevated nuclear localization of phospho-Smad2/3 and Smad4; and (5) downregulation of CDK inhibitors p16 and p27. Consistently, shRNA-mediated knockdown 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. Furthermore, overexpression of KLF4 in HCLE cells resulted in downregulation of TGF-β1, -βR1, and -βR2 and upregulation of SMAD7, p16, and p27.
Collectively, these results demonstrate that KLF4 regulates CE cell cycle progression by suppressing canonical TGF-β signaling and overcomes the undesirable concomitant decrease in TGF-β–dependent CDK inhibitors p16 and p27 expression by directly upregulating them.
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