KLF4 regulates corneal epithelial cell cycle progression by suppressing canonical TGF-b signaling and upregulating cdk inhibitors P16 and P27

Abstract

PURPOSE. Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) cell fate while suppressing mesenchymal properties. TGF-b plays a crucial role in cell differentiation and development, and if dysregulated, it induces epithelial-mesenchymal transition (EMT). As KLF4 and TGF-b regulate each other in a context-dependent manner, we evaluated the role of the crosstalk between KLF4 and TGF-b-signaling in CE homeostasis. METHODS. 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 TGFb- signaling components. Expression of TGF-β signaling components and cyclin-dependent kinase (CDK) inhibitors was quantified by quantitative PCR, immunoblots, and/or immunofluorescent staining. RESULTS. 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. CONCLUSIONS. 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. Keywords: KLF4, TGF-b, SMAD, EMT, corneal epithelium, squamous neoplasia © 2019 The Authors.