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.