Abstract
Purpose :
Krüppel-like Factor 4 (KLF4) is one of the essential transcription factors for maintenance of human corneal epithelial cells (HCECs). The study investigated whether KLF4 suppresses epithelial to mesenchymal transition (EMT) in HCECs, by preventing the TGF-β signaling pathway.
Methods :
HCECs were collected from cadaver donors and cultivated. Once HCECs were confluent, they were subcultured for KLF4 knockdown (KLF4-KD) using siRNA transfection. HCECs were harvested after 7 days and analyzed by qRT-PCR, immunoblotting, immuno-fluorescent staining and immunoassays. KLF4 overexpression was performed using lentiviral KLF4 expression vectors during HCEC subculture. Likewise, subcultured HCECs were harvested after 72 hours and underwent qRT-PCR, immunoblotting and immuno-fluorescent staining analyses. In addition, TGF-β2 treatment was performed on HCECs overexpressing KLF4, which were examined by immunoblotting and immuno-fluorescent staining.
Results :
Under KLF4-KD conditions, HCECs lost their epithelial phenotype, and epithelial markers KRT12 and KRT14 were downregulated, whereas mesenchymal markers FN1, VIM, CDH2 and SLUG were upregulated. Immunocytochemical analysis showed that CDH1 (E-cadherin) positive adherence junction disappeared in KLF4-KD samples. In addition, several TGF-β associated markers (TGFB1, TGFB2, TGFBR1 and TGFBR2) were significantly upregulated, up to 6-fold, and TGF-β2 secretion increased up to 5-fold. Furthermore, SMAD2/3, which act as the main signal transduction molecules within the TGF-β signaling pathway, localized within the nucleus, indicating that KLF4-KD in HCECs promotes EMT through the TGF-β canonical pathway. Conversely, when KLF4 was overexpressed, cultivated HCECs tended to decrease in size by half, and upregulate epithelial genes KRT14, CDH1 and OVOL2; which are morphological and biological characteristics of HCECs in vivo. In support, nuclear-translocation of SMAD2/3 induced by TGF-β2 treatment, was obstructed in KLF4-overexpressed HCECs.
Conclusions :
These data suggest a new role of KLF4 within human corneal epithelium as an inhibitor of SMAD2/3 nuclear translocation, which as a result prevents EMT.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.