Notch signaling mediates TGF-β1-induced epithelial–mesenchymal transition through the induction of Snai1
Introduction
Epithelial–mesenchymal transition (EMT) is a process by which epithelial cells undergo phenotypic transition to mesenchymal cells (Thiery and Sleeman, 2006). In this process, epithelial cells downregulate the expression of cell adhesion molecule including E-cadherin, dissolve cell–cell junctions, lose their apical–basal polarity, deposit ECM proteins, and acquire migratory and invasive behavior. EMT occurs during normal embryonic development in response to physiologic signals (Acloque et al., 2009), whereas in the adult, EMT is induced in pathological states such as cancer and organ fibrosis. In cancer, tumor cells become more invasive after undergoing EMT and access the blood vessel through intravasation, resulting in distant metastasis (Thiery, 2002, Kalluri and Weinberg, 2009), the major cause of death from cancer. Indeed, loss of E-cadherin expression, which is considered a hallmark of EMT (Peinado et al., 2007), was found to be an independent prognostic factor in cancer patients (Bremnes et al., 2002, Sulzer et al., 1998).
Several factors have been shown to induce EMT in vitro and in vivo, including hypoxia (Sahlgren et al., 2008), reactive oxygen species (Rhyu et al., 2005), transforming growth factor β1 (TGF-β1) (Jayachandran et al., 2009, Zavadil et al., 2004, Kasai et al., 2005), connective tissue growth factor (Gore-Hyer et al., 2002) and insulin-like growth factor-2 (Morali et al., 2001). Among these, TGF-β1 is one of the potent and well-studied factors and has been shown to induce EMT in various cell types including alveolar type II epithelial cells (Jayachandran et al., 2009, Kasai et al., 2005). TGF-β1-induced EMT is regulated by transcriptional mechanisms that are mediated by Snail, ZEB and bHLH families of transcription factors, which repress the expression of epithelial marker genes and concomitantly enhance mesenchymal genes expression (Xu et al., 2009). Suppression of E-cadherin expression is mediated by the interaction of these transcription factors with E-boxes within the E-cadherin promoter. In alveolar epithelial cells, the Snail family of transcription factors, Snai1 and Snai2, are involved in TGF-β1-induced EMT in vitro (Jayachandran et al., 2009). These transcription factors were detected in vivo in hyperplastic alveolar epithelial cells in the lungs of patients with Idiopathic Pulmonary Fibrosis (Jayachandran et al., 2009).
Notch signaling is a highly conserved mechanism that regulates intercellular communication and directs individual cell fate decisions through physical cell contact. In mammals, there are four Notch receptor proteins (Notch1–4) and 5 ligands (Delta-like [Dll]-1, Dll-3, Dll-4, Jagged1 and Jagged2). The receptor–ligand interaction induces a series of proteolytic cleavages of Notch receptors that free the notch intracellular domain (NICD) from the cell membrane. The NICD translocates to the nucleus, complexes with the transcriptional repressor CSL (RBP-Jκ) and co-activators, and activates transcription of target genes such as Hey and Hes families of transcriptional repressors. The NICD acts as a constitutive transcriptional activator and has been used to examine the role of Notch signaling activation through each receptor.
Several lines of evidence indicate the critical role of Notch signaling in EMT (Niessen et al., 2008, Sahlgren et al., 2008, Zavadil et al., 2004). Zavadil et al. (2004) demonstrated that blocking Notch signaling by knocking-down either Hey1 or Jagged1 expression or by γ-secretase inhibitor attenuated EMT. γ-Secretase is a protease active in cleavage of Notch receptors. Hypoxia-induced EMT was mediated by Notch signaling through direct induction of Snai1 and indirect induction of lysyl oxidase, which stabilizes Snai1 (Sahlgren et al., 2008). Moreover, Notch signaling directly modulated Snai2 expression that was required for EMT of endothelial cells in cardiac cushion morphogenesis (Niessen et al., 2008). However, the role Notch plays in TGF-β1-induced EMT of lung alveolar epithelial cells remains unknown. In the current study, using A549 cells, we demonstrated activated Notch signaling promoted TGF-β1-induced EMT through direct modulation of Snai1 expression.
Section snippets
Antibodies
The primary monoclonal antibodies directed against E-cadherin, N-cadherin and Snai1 were purchased from Cell Signaling Technology (Danvers, MA). Vimentin, fibronectin, Notch2, Notch4, Jagged1, and GAPDH specific antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Delta-like4 monoclonal antibody was obtained from Abcam (Cambridge, UK) and V5 monoclonal antibody was obtained from Invitrogen (Carlsbad, CA). HA monoclonal antibody was obtained from Sigma Aldrich (St. Louis,
TGF-β1 induces phenotypic change indicative of EMT via Snai1 dependent manner in A549 cells
To examine the role of Notch signaling in EMT, first, we investigated the morphology, the migration capacity and the expression of EMT markers in A549 cells treated with TGF-β1. A549 cells exhibited a cobblestone-like morphology in the absence of TGF-β1, while after exposure to TGF-β1, the A549 cells changed into spindle-shaped fibroblast-like cells (Fig. 1A). A549 cells treated with TGF-β1 exhibited greater motility as determined by both a wound healing assay and a Transwell cell migration
Discussion
EMT is a process by which epithelial cells undergo phenotypic transition to mesenchymal cells. Mounting evidence has demonstrated that EMT is associated with invasive and migratory ability of cancer cells, conferring enhanced metastatic properties to these cells (Thiery, 2002, Kalluri and Weinberg, 2009). Also, evidence is emerging showing the contribution of EMT to the development of organ fibrosis (Kim et al., 2006). In the current study, we investigated the molecular mechanism underlying
Acknowledgements
We thank Dr. Igor Prudovsky (Maine Medical Center Research Institute) for the generous gift of pcDNA4-N2ICD-V5. We are also grateful to Dr. Aly Karsan (British Colombia Cancer Research Center) for the generous gift of pLNCX-N4ICD-HA. We also thank Dr. Antonio García de Herreros (Universitat Pompeu Fabra) for Snai1 promoter construct.
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