Dataset Information

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-β 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-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.

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 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.

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.

SUBMITTER: Tiwari A

PROVIDER: S-EPMC6383833 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Publications

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-β Signaling and Upregulating CDK Inhibitors P16 and P27.

Tiwari Anil A Swamynathan Sudha S Alexander Nicholas N Gnalian John J Tian Shenghe S Kinchington Paul R PR Swamynathan Shivalingappa K SK

Investigative ophthalmology & visual science 20190201 2

<h4>Purpose</h4>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.<h4>Methods</h4>We used spatiotemporally regulated ablation of Klf4 wi ...[more]

PMID: 30786277

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Project description: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 via 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-? signalling pathway, which is involved in corneal epithelial wound healing. Methods:HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-? secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-?2 treatment. Results:KLF4-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 SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-? canonical signalling pathway, TGF-?2 secretion by HCECs increased up to 5 folds, and several TGF-?-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-? signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 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 vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-?2 treatment. Conclusions:These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-?2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-? signalling.

Project description:Background: Epithelial ovarian cancer (EOC) is one of the most lethal malignancies in women worldwide. Many studies showed the transcription factor SNAI2-induced Epithelial-Mesenchymal Transition (EMT) through inhibiting E-cadherin (E-cad) expression. Our previous study reported that miR-222-3p was an important tumor-suppressive miRNA for EOC development and dissemination. The present study aimed to acquire a deeper mechanistic understanding of the role of miR-222-3p regulation that might contribute to improving current anti-metastasis strategies in EOC. Methods: A variety of techniques were used to measure mRNA and protein expression levels, including quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, immunohistochemical (IHC) staining, and immunofluorescence (IF). Four different microRNA (miRNA) target prediction databases were used to predict the target genes of miR-222. Luciferase assay was performed to determine the direct binding of miR-222-3p to the untranslated region (3'-UTR) of PDCD10. The biological effects of PDCD10 and miR-222-3p were also investigated in vitro by Transwell and wound healing assays, as well as in vivo by a xenograft mice model. Combining UCSC and JASPAR, as well as ENCODE public databases, we predicted that the transcription factor SNAI2 could affect miR-222-3p expression. Luciferase assay was utilized to examine the validity of putative SNAI2 binding sites for miR-222-3p regulation. Chromatin immunoprecipitation (ChIP) was used to explore the SNAI2's occupancy on the miR-222-3p promoter. Results: We observed the inhibitory effect of SNAI2 on miR-222-3p transcription and confirmed the tumor-suppressive function of miR-222-3p both in EOC cells and tissues. PDCD10 was upregulated and inversely correlated with miR-222-3p, both in vitro and in vivo, which was consistent with the information in bioinformatics databases. Furthermore, We observed direct binding of miR-222-3p to the 3'-UTR of PDCD10 and inhibition of PDCD10 translation, which, in turn, inhibited EOC cell migration in vitro and repressed EOC xenografted tumor metastasis in vivo. We found that genetic overexpression of PDCD10 (OE-PDCD10) increased cancer metastasis by down-regulating E-cad and enhancing Vimentin (VIM) thereby inducing EMT and promoting β-catenin/Wnt-mediated cell migration.

Dataset Information

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-β Signaling and Upregulating CDK Inhibitors P16 and P27.

Purpose

Methods

Results

Conclusions

Publications

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-β Signaling and Upregulating CDK Inhibitors P16 and P27.

Similar Datasets

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