KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells endogenous TGF-β2 suppression.

Introduction: Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-β signalling pathway, which is involved in corneal epithelial wound healing.

Methods: HCECs were collected from cadaver donors and cultivated. We generated knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-β secretion. was overexpressed using lentiviral expression vectors and underwent protein expression analyses after TGF-β2 treatment.

Results: -KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and . Although expression remained unchanged in -KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in -KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-β canonical signalling pathway, TGF-β2 secretion by HCECs increased up to 5 folds, and several TGF-β-associated markers (, , , and ) were significantly upregulated up to 6 folds in the -KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-β signalling pathway, were found to be localised in the nucleus of -KD HCECs. When was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium In addition, overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-β2 treatment.

Conclusions: These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-β2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-β signalling.