KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-β2 suppression
KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-β2 suppression
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DOI：
10.1016/j.reth.2019.08.003
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发表时间：
2019-12-01
影响因子：
4.3
通讯作者：
Nishida, Kohji
中科院分区：
文献类型：
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作者：
Fujimoto, Satoko;Hayashi, Ryuhei;Nishida, Kohji
简介：Kruppel样因子4（KLF 4）被认为是Yamanaka因子之一，最近，我们和其他人已经表明，KLF 4是重编程非人角膜上皮细胞（HCECs）成为HCECs所必需的转录因子之一。由于上皮间质转化（EMT）的抑制是至关重要的HCECs通过调节转录因子的稳态，在这项研究中，我们的目的是调查是否KLF 4防止EMT HCECs和阐明内的经典TGF-β信号通路，这是参与角膜上皮伤口愈合的潜在机制。我们使用siRNA转染产生KLF 4敲低（KD）HCEC，并分析形态学、基因或蛋白质表达和内源性TGF-β分泌。结果：KLF 4-KD HCECs呈成纤维细胞样形态，上皮标志物角蛋白12和角蛋白14表达下调，间充质标志物纤维连接蛋白1、波形蛋白、N-钙粘蛋白和SLUG表达上调。尽管KLF 4-KD HCECs中E-钙粘蛋白表达保持不变，但免疫细胞化学分析显示，KLF 4-KD HCECs中E-钙粘蛋白阳性粘附连接减少，免疫印迹分析显示E-钙粘蛋白总蛋白水平降低。此外，在TGF-β经典信号传导途径内，HCEC分泌的TGF-β 2增加高达5倍，并且在KLF 4-KD HCEC中几种TGF-β相关标志物（TGFB 1、TGFB 2、TGFBR 1和TGFBR 2）显著上调高达6倍。SMAD 2/3是TGF-β信号通路的主要信号转导分子，发现其定位于KLF 4-KD HCEC的细胞核中。当KLF 4过表达时，培养的HCECs显示上皮标志物角蛋白14和E-钙粘蛋白的上调，表明KLF 4在体内人角膜上皮的稳态中起着贡献作用。此外，即使在TGF-β 2处理后，HCECs中KLF 4过表达也会导致SMAD 2磷酸化水平降低，SMAD 2/3的核定位改变。这些结果表明，KLF 4可预防HCEC中的EMT，并提示KLF 4在人角膜上皮中作为内源性TGF-β 2抑制剂的新作用，从而强调了KLF 4通过减弱TGF-β信号传导来预防EMT和随后的角膜纤维化疤痕形成的潜力。(C)2019年，日本再生医学学会。制作和主办：Elsevier B. V.
Introduction: Kruppel-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-beta 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-beta secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-beta 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-beta canonical signalling pathway, TGF-beta 2 secretion by HCECs increased up to 5 folds, and several TGF-beta-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-beta 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-beta 2 treatment.Conclusions: These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-beta 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-beta signalling. (C) 2019, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.