Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition
2013; Springer Nature; Volume: 32; Issue: 2 Linguagem: Inglês
10.1038/emboj.2012.336
ISSN1460-2075
AutoresDavid Balli, Vladimir Ustiyan, Yufang Zhang, I‐Ching Wang, Alex J Masino, Xiaomeng Ren, Jeffrey A. Whitsett, Vladimir V. Kalinichenko, Tanya V. Kalin,
Tópico(s)FOXO transcription factor regulation
ResumoArticle4 January 2013free access Source Data Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition David Balli David Balli Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Vladimir Ustiyan Vladimir Ustiyan Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Yufang Zhang Yufang Zhang Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author I-Ching Wang I-Ching Wang Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Alex J Masino Alex J Masino Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Xiaomeng Ren Xiaomeng Ren Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Jeffrey A Whitsett Jeffrey A Whitsett Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Vladimir V Kalinichenko Vladimir V Kalinichenko Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Tanya V Kalin Corresponding Author Tanya V Kalin Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author David Balli David Balli Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Vladimir Ustiyan Vladimir Ustiyan Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Yufang Zhang Yufang Zhang Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author I-Ching Wang I-Ching Wang Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Alex J Masino Alex J Masino Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Xiaomeng Ren Xiaomeng Ren Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Jeffrey A Whitsett Jeffrey A Whitsett Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Vladimir V Kalinichenko Vladimir V Kalinichenko Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Tanya V Kalin Corresponding Author Tanya V Kalin Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Author Information David Balli1, Vladimir Ustiyan1, Yufang Zhang1, I-Ching Wang1, Alex J Masino2, Xiaomeng Ren1, Jeffrey A Whitsett1, Vladimir V Kalinichenko1 and Tanya V Kalin 1 1Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA 2Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA *Corresponding author. Department of Pediatrics, Division of Pulmonary Biology, The Perinatal Institute, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, 3333 Burnet Avenue, MLC 7009, Cincinnati, OH 45229, USA. Tel.:+1 513 803 1201; Fax:+1 513 636 2423; E-mail: [email protected] The EMBO Journal (2013)32:231-244https://doi.org/10.1038/emboj.2012.336 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-β-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-β-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT. Introduction Pulmonary fibrosis includes a heterogeneous group of lung disorders characterized by progressive and irreversible destruction of lung architecture, disruption of gas exchange and death from respiratory failure (Wynn, 2011). Pulmonary fibrosis results from dysregulated repair of damaged tissue following a variety of damaging stimuli, including ionizing radiation (Mehta, 2005). Radiotherapy is often utilized for treatment of solid tumours in the thorax and a variety of metastatic tumours. Approximately 60% of patients with non-small-cell lung cancer (NSCLC) received radiation therapy for their cancer treatment (Kong et al, 2005). Ionizing radiation injures pulmonary epithelial and endothelial cells, and causes the release of pro-inflammatory cytokines that recruit macrophages and lymphocytes to the sites of injury (Wynn, 2011). Myofibroblasts produce collagen and extracellular matrix (ECM) proteins during the repair of the basement membranes following radiation-induced injury. During the normal healing process, alveolar–capillary permeability is restored and inflammation resolves. All too frequently, radiation to the thorax causes pulmonary fibrosis as lung injury, inflammation and remodelling persist (Hardie et al, 2010; Wynn, 2011). While various signalling pathways have been implicated in the pathogenesis of radiation-induced pulmonary fibrosis, the transcriptional programmes that drive disease progression are poorly understood. Activated myofibroblasts play a central role during the pathogenesis of pulmonary fibrosis by synthesizing and depositing ECM proteins. Myofibroblasts are likely derived from various cells, including: (1) resident stromal fibroblasts, (2) bone-marrow-derived ‘fibrocytes’ and (3) alveolar type II epithelial cells, a subset of which undergo epithelial-to-mesenchymal transition (EMT) (Andersson-Sjoland et al, 2011; Wynn, 2011). Recent studies support the role of EMT in pulmonary fibrosis (Willis et al, 2005; Kim et al, 2006; Willis et al, 2006; Kim et al, 2009). During EMT, epithelial cells lose apical–basal polarity, basement membrane attachment and cell–cell contact. Epithelial cells gain mesenchymal characteristics associated with increased migratory behaviour, cytoskeletal rearrangements and their migration into the lung interstitium where they produce excess ECM. EMT is controled by a network of signalling and transcriptional events mediated in part by TGF-β signalling (Thiery and Sleeman, 2006; Chapman, 2011). The TGF-β/Smad signalling pathway is required for both EMT and fibrosis in a variety of organs (Xu et al, 2009). Snail and Twist family members of transcription factors are important regulators of EMT, repressing E-cadherin and activating the mesenchymal transcriptomes (Kalluri and Weinberg, 2009). Forkhead Box M1 (Foxm1) is a member of the Forkhead family of transcription factors that shares homology in the Winged Helix/Forkhead box DNA-binding domain. In proliferating cells, Foxm1 regulates the G2/M transition of the cell cycle through transcriptional activation of Cdc25B phosphatase, Plk1, AuroraB kinase and Cyclin B1 (Costa et al, 2005). Consistent with its role in cell cycle progression, increased expression of Foxm1 was found in a variety of human tumours (Kalin et al, 2006; Yoshida et al, 2007; Wang et al, 2009). FoxM1 plays important roles during embryonic development, monocyte/macrophage recruitment, DNA repair, surfactant production, angiogenesis and formation of tight junctions during LPS-induced acute lung injury (reviewed in (Kalin et al, 2011) and (Kalinichenko et al, 2001; Kalin et al, 2008b; Wang et al, 2008b; Zhang et al, 2008; Wang et al, 2009; Ren et al, 2010; Balli et al, 2011a)). In the present study, we found increased Foxm1 in biopsy samples from human patients with interstitial pulmonary fibrosis (IPF). Since the role of Foxm1 in fibrotic diseases is unknown, we used gain-of-function and loss-of-function mouse models to identify the role of Foxm1 in radiation-induced pulmonary fibrosis. Expression of constitutively active Foxm1-ΔN mutant within type II epithelial cells increased fibrosis, whereas cell-specific ablation of Foxm1 from type II cells protected mice from the radiation-induced pulmonary fibrosis. We identified the Foxm1 as a novel potent mediator of fibrotic events following radiation injury supporting the potential utility of targeting Foxm1 for treatment of pulmonary fibrosis. Results Foxm1 is increased during radiation-induced pulmonary fibrosis Thoracic irradiation causes pulmonary fibrosis in mice (Chiang et al, 2005). Mice were exposed to ionizing radiation directed to the thoracic region. Foxm1 mRNA progressively increased in the lung tissue following irradiation (Figure 1A). Foxm1 staining was increased in type II epithelial cells of the irradiated lungs as demonstrated by colocalization with SP-C, a marker of alveolar type II cells (Figure 1B). Foxm1 staining was also increased in lung tissue from patients with idiopathic pulmonary fibrosis compared to control lung tissue from organ donors (Figure 2A). Increased numbers of FoxM1-positive cells were identified throughout fibrotic regions, in sharp contrast to the paucity of Foxm1-stained cells in normal lungs from organ donors. Foxm1 protein levels were upregulated in type II epithelial cells of the IPF fibrotic lesions, but not in the control lungs (Figure 2B). Figure 1.Thoracic irradiation increased expression of Foxm1 in the lung. The thoracic regions of C57BL/6 mice were exposed to 12 Gy ionizing radiation. (A) Foxm1 mRNA is increased after irradiation. At designated time points, whole-lung RNA was isolated and Foxm1 mRNA levels were evaluated by qRT–PCR. β-actin mRNA was used for normalization. Data represent means±s.d. of three independent determinations using lung tissue from n=3–5 mice/time point. (B) Foxm1 protein levels were upregulated in type II epithelial cells of the irradiated lungs. SPC-positive lung epithelial cells (green) co-expressed Foxm1 (red) 6 months after irradiation. Representative sections from five irradiated samples and three control samples are shown. A P-value <0.01 is shown with asterisks (**). Magnification is × 400. Download figure Download PowerPoint Figure 2.Foxm1 protein is highly expressed in fibrotic lesions of IPF patients. (A) Lung tissue sections from patients with IPF and control organ donors were stained with H&E or with an antibody specific for human Foxm1. Representative sections from 10 IPF and 6 normal control samples are shown. Magnification: left and middle panels, × 200; right panels, × 1000. (B) Foxm1 protein levels were upregulated in type II epithelial cells of the IPF fibrotic lesions, but not in the control lungs. Pro-SPC-positive lung epithelial cells (green) co-expressed Foxm1 (red) in IPF lungs. Representative sections from six IPF and three normal control samples are shown. Magnification is × 2000. Download figure Download PowerPoint Increased expression of Foxm1 in respiratory epithelial cells increased radiation-induced pulmonary fibrosis and inflammation Since not all the patients develop pulmonary fibrosis after the same dose of irradiation, we tested whether increased activity of Foxm1 in type II alveolar cells is sufficient to induce pulmonary fibrosis. Transgenic mice were used in which a constitutively active form of Foxm1 (Foxm1-ΔN) was expressed under control of SP-C promoter (SP-C–rtTAtg/−/ tetO-Foxm1-ΔNtg/− or epiFoxm1-ΔN mice, (Wang et al, 2010)). Doxycycline (Dox) was used to activate epiFoxm1-ΔN transgene in adult lungs (Supplementary Figure 1A and B). To induce lung fibrosis, the thoracic regions of Dox-treated epiFoxm1-ΔN and Dox-treated control mice were exposed to a single dose of 12 Gy ionizing radiation. As early as 3 months post radiation, increased focal collagen deposition was observed in peribronchial regions of Foxm1-ΔN mice (Supplementary Figure 2A). Lung collagen 1α1 (Col1α1), collagen 3α1 (Col3α1) and α-smooth muscle actin (α-SMA) mRNAs were induced in epiFoxm1-ΔN mice, findings consistent with increased ECM production and myofibroblast activation (Supplementary Figure 2B). Six months after irradiation, pulmonary fibrosis in epiFoxm1-ΔN mice was extensive, while few focal fibrotic regions were found in control mice (Figure 3A). Without irradiation, epiFoxm1-ΔN mice did not develop pulmonary fibrosis and inflammation (Figure 3A–C, Supplementary Figure 3C). Figure 3.Aberrant expression of Foxm1 in AECs exacerbated pulmonary fibrosis following thoracic irradiation. The thoracic regions of transgenic mice expressing constitutively active Foxm1-ΔN mutant in AECs (epiFoxm1-ΔN) were irradiated with 12 Gy. (A). At 6 month post radiation, H&E staining (left panels) of lung tissue demonstrated minimal remodelling and fibrosis in control mice, but severe fibrotic lesions in epiFoxm1-ΔN mice (middle panels). Masson's trichrome staining (right panels) showed increased deposition of collagen in lungs of epiFoxm1-ΔN mice. Representative sections from at least seven mice per group are shown. (B) Increased collagen depositions in irradiated epiFoxm1-ΔN lungs were shown by quantitative Sircol collagen assay. (C). Increased mRNAs of α-SMA and Col3α1 were found by qRT–PCR in epiFoxm1-ΔN mice at 6 months post radiation. β-actin mRNA was used for normalization. Data represent mean±s.d. of three independent determinations using lung tissue from n=5–7 mice. (D) Cellular proliferation is elevated in irradiated epiFoxm1-ΔN lungs compared to control lungs. The number of Ki-67+ cells were counted and quantified per 1000 cells using 10 random microscope fields. A P-value <0.01 is shown with (**) and P-value <0.05 is shown with (*). Magnification: panels A, × 100; inserts, × 400; panels D, × 200. Download figure Download PowerPoint Collagen deposition was increased in epiFoxm1-ΔN lungs (Figure 3A, right panels and 3B). α-SMA and Col3α1 mRNAs were increased in the epiFoxm1-ΔN lungs at 6 months after irradiation (Figure 3C). Increased fibrosis in epiFoxm1-ΔN lungs was associated with sustained pulmonary inflammation as demonstrated by increased numbers of interstitial F4/80-positive macrophages, perivascular CD3-positive lymphocytes (Supplementary Figure 3A) and increased bronchoalveolar lavage (BAL) cell counts (Supplementary Figure 3B). mRNAs encoding pro-inflammatory cytokines Ccl2, Cxcl5 and IL-1β, all known mediators of pulmonary inflammation and fibrosis (Rose et al, 2003; Wilson et al, 2010; Liu et al, 2011), were increased while Ccl3 was not changed (Supplementary Figure 3C). Cotransfection of CMV–Foxm1b expression vector significantly increased transcriptional activity of the −1.3 Kb Ccl2 and the −1.14 Kb Cxcl5 promoter regions in a luciferase reporter assay in vitro (Supplementary Figure 3D). Six months after irradiation, the number of Ki-67-positive cells was increased in the lungs of epiFoxm1-ΔN mice (Figure 3D). Since the Foxm1-ΔN was specifically overexpressed in type II cells and is known to regulate cellular proliferation, we performed colocalization experiments to identify proliferating cell types in the fibrotic lungs. The number of proliferating type II cells that were double-positive for both proSPC and Ki-67 was similar in epiFoxm1-ΔN and control mice (Supplementary Figure 4A and B). No changes in Cyclin B1 and Cyclin D1 mRNAs were observed in isolated type II cells (Supplementary Figure 4C), indicating that overexpression of Foxm1 does not influence proliferation of type II cells in the fibrotic lung. In contrast, the number of proliferating myofibroblasts that were positive for Ki-67 and α-SMA was increased in epiFoxm1-ΔN mice (Supplementary Figure 4A and B). Increased proliferation of fibroblasts was likely a result of increased levels of profibrotic mediators IL-1β, Ccl2 and Cxcl5 (Supplementary Figure 3C and D and (Moore et al, 2001; Quan et al, 2006; Ekert et al, 2011; Kawamura et al, 2012)), that contribute to the fibrotic phenotype in irradiated epiFoxm1-ΔN mice. Taken together, these data indicate that increased activity of Foxm1 in type II cells enhanced pulmonary inflammation and fibrosis after thoracic irradiation. Foxm1 regulates genes associated with EMT The role of EMT in the development of pulmonary fibrosis has been supported by a number of recent publications (Willis et al, 2005; Kim et al, 2006; Willis et al, 2006; Demaio et al, 2011; Marmai et al, 2011). Since expression of Foxm1-ΔN in type II alveolar epithelial cells (AECs) exacerbated pulmonary fibrosis, we tested whether Foxm1 influences EMT. Three months after thoracic irradiation, Snail1, Snail2, Zeb1, Zeb2, Twist2 and Foxf1 mRNAs were increased in epiFoxm1-ΔN lungs (Figure 4A). The epithelial-specific marker E-cadherin, the loss of which is a key step during EMT (Thiery and Sleeman, 2006; Kalluri and Weinberg, 2009), was significantly decreased (Figure 4A). Six months post radiation, Snail1, Zeb1, Zeb2, vimentin and fibronectin mRNAs were increased and E-cadherin mRNA was decreased (Figure 4B). Colocalization experiments demonstrated the presence of Foxm1-ΔN transgenic protein in a subset of α-SMA-positive and vimentin-positive cells localized within fibrotic lesions (Figure 4C), suggesting that the Foxm1-ΔN-expressing epithelial cells underwent EMT after irradiation. Figure 4.Foxm1 regulates epithelial-to-mesenchymal transition during radiation-induced pulmonary fibrosis. Aberrant overexpression of Foxm1 in AECs increased mRNA levels of EMT genes in epiFoxm1-ΔN lungs at 3 months (A) and at 6 months (B) following thoracic irradiation as demonstrated by qRT–PCR. β-actin mRNA was used for normalization. Data represent mean±s.d. of three independent determinations using lung tissue from n=5–13 mice per group. A P-value <0.05 is shown with asterisk (*) and P-value <0.01 is shown with (**). (C) Foxm1-positive epithelial cells (red) from irradiated epiFoxm1-ΔN lungs expressed the mesenchymal markers α-SMA and vimentin (green) at 6 months after radiation. Nuclei were stained with DAPI (blue). Magnification is × 1000. Download figure Download PowerPoint Conditional deletion of Foxm1 from alveolar type II cells protects mice from radiation-induced pulmonary fibrosis Foxm1 was conditionally deleted in the respiratory epithelium (Spc-rtTA/tetO-cre/Foxm1fl/fl termed epiFoxm1 KO mice (Kalin et al, 2008b)). epiFoxm1 KO and control mice were treated with Dox to induce Foxm1 deletion. Since we expected that Foxm1 deletion would decrease pulmonary fibrosis, the dose of thoracic irradiation was increased to 18 Gy. Six months after radiation, the collagen depositions in epiFoxm1 KO lungs were considerably smaller compared to controls (Figure 5A). α-SMA, -Col1α1 and -Col3α1 mRNAs were decreased, consistent with decreased ECM production and myofibroblast activation (Figure 5B). Lung collagen content was decreased in irradiated epiFoxm1 KO mice (Figure 5C). Loss of Foxm1 was associated with decreased Snail1, Twist1, Twist2, Foxf1, Zeb1 and Zeb2 mRNAs (Figure 5D). Pro-inflammatory Cxcl5, IL-1β and TGFβ mRNAs were significantly reduced, whereas Ccl2 and Ccl3 mRNAs were not changed (Figure 5E). These data indicate that ablation of Foxm1 from type II cells diminished radiation-induced pulmonary fibrosis and decreased expression of genes associated with EMT and lung inflammation. Figure 5.Ablation of Foxm1 from pulmonary epithelial cells prevented radiation-induced fibrosis. The thoracic regions of conditional knock-out mice (epiFoxm1 KO) and control mice were irradiated with 18 Gy. (A) At 6 month post radiation, Masson's trichrome staining of lung tissue was performed. Increased collagen depositions were found in the lungs of control mice (n=8 mice), but not in epiFoxm1 KO mice (n=5 mice). Magnification is × 400. (B) Decreased mRNA levels of ECM genes in epiFoxm1 KO lungs at 6 months after thoracic irradiation were demonstrated by qRT–PCR. β-actin mRNA was used for normalization. (C) Decreased collagen depositions were found in irradiated epiFoxm1 KO lungs by quantitative Sircol collagen assay. mRNA levels of EMT genes (D) and pro-inflammatory genes (E) were decreased in irradiated epiFoxm1-KO mice as demonstrated by qRT–PCR. β-actin mRNA was used for normalization. Data represent mean±s.d. of three independent determinations using lung tissue from five to eight mice per group. A P-value <0.05 is shown with (*) and P-value <0.01 is shown with (**). Download figure Download PowerPoint Expression of Foxm1 was inhibited by siRNA-mediated knockdown of Foxm1 in mouse lung epithelial MLE15 cells. A 60% reduction in Foxm1 mRNA was associated with decreased expression of Snail1, Zeb2, Twist2 and Foxf1 mRNAs, whereas Zeb1 mRNA was not changed (Figure 6A), implicating Foxm1 in the regulation of EMT in vitro. Figure 6.Foxm1 is required for EMT in vitro. (A) Depletion of Foxm1 from MLE15 cells was performed by siRNA transfection. At 48 h after transfection, RNA was isolated and examined by qRT–PCR. mRNAs of Foxm1, Snail1, Zeb2, Twist2 and Foxf1 were decreased compared to mock-transfected cells. Expression levels were normalized using β-actin mRNA. Data represent mean±s.d. of three independent determinations. (B, C) Foxm1 knockdown by siRNA prevented the TGF-β-induced EMT in A549 epithelial cells. siFoxm1 transfection decreased protein levels of Snail1, Zeb1, Zeb2, fibronectin and α-SMA, and increased E-cadherin, shown by western blot (B). Foxm1 depletion did not affect pSmad2, pAKT and Jnk2. Jnk1 and p-cJun were decreased in Foxm1-depleted cells, shown by western blot (C). Data represent one of three independent experiments. (D) Foxm1 does not show transcriptional synergy with TGF-β. U2OS cells were co-transfected with CMV–Foxm1 and 3TP-luc-containing SREs. Transfected cells were incubated with rTGF-β and with or without the Jnk1 inhibitor SP10064 for 24 h. Triplicate plates were used to calculate the mean±s.d. relative luciferase activity. Data represent one of two independent experiments. A P-value <0.05 is shown with (*) and P-value <0.01 is shown with (**).Source data for this figure is available on the online supplementary information page. Source Data for Figure 6B, C [embj2012336-SourceData-Fig6.pdf] Download figure Download PowerPoint Foxm1 is required for TGFβ-induced EMT To test whether Foxm1 is required for EMT, human epithelial A549 cells were treated with TGF-β1 to induce EMT in vitro (Kasai et al, 2005). Consistent with the previous studies, TGF-β1 increased mesenchymal markers fibronectin, α-SMA, SNAIL1, ZEB1, ZEB2 and decreased epithelial marker E-cadherin (Figure 6B). Knockdown of Foxm1 by siRNA prevented EMT as demonstrated by reduced protein levels of all mesenchymal markers, and increased level of epithelial E-cadherin in TGF-β1-treated A549 cells (Figure 6B). Inhibition of Foxm1 prevented the induction of SNAIL1, ZEB1 and ZEB2 transcription factors critical for EMT (Figure 6B). Foxm1 depletion did not alter SMAD2 or AKT phosphorylation (Figure 6C), suggesting that Foxm1 acts downstream of TGF-β1/Smad and AKT signalling pathways. There were no differences in total levels of AKT and SMAD2 proteins in Foxm1-deficient cells (Figure 6C). Since Foxm1 directly activated transcription of Jnk1 (Wang et al, 2008a) and JNK1 has been shown to be required for TGF-β1-induced EMT both in vitro and in vivo (Javelaud and Mauviel, 2005; Alcorn et al, 2009), JNK1 protein was examined by western blot. JNK1 was decreased in Foxm1-depleted cells, whereas JNK2 did not change (Figure 6C). Decreased phosphorylation of c-JUN (Figure 6C) was consistent with the loss of JNK1. To determine if Foxm1 influenced transcriptional activity of Smad during EMT, a CMV–Foxm1 expression vector was co-transfected with a Smad reporter (3TP-Luc) containing Smad-responsive elements (SRE) (Lange et al, 2009). Neither Foxm1 nor a Jnk1 inhibitor (SP10064) affected Smad transcriptional activity (Figure 6D) induced by TGFβ1. Thus, Foxm1 does not influence the transcriptional activity of Smad during EMT. Altogether, the loss of JNK1 and decreased expression of SNAIL1, ZEB1 and ZEB2 likely contributes to decreased EMT in Foxm1-deficient cells after TGFβ treatment. Foxm1 directly activates the Snail1 promoter Since Foxm1 induced Snail1 mRNA and protein in vivo and in vitro (Figures 4, 5 and 6), we investigated whether Snail1 is a direct transcriptional target of Foxm1. A potential Foxm1-binding site was identified within the −1.0 Kb promoter regions of mouse and human Snail1 genes (Figure 7A). Chromatin immunoprecipitation (ChIP) assay was used to determine whether Foxm1 binds to the promoter region of Snail1 gene in A549 cells. After TGF-β1 treatment, the specific binding of Foxm1 protein to the Snail1 promoter DNA was increased (Figure 7B). CMV–Foxm1b significantly increased the activity of a −720 bp Snail1 promoter region in a luciferase reporter assay (Figure 7C). Site-directed mutagenesis of the Foxm1-binding site decreased the ability of Foxm1 to activate the −720 bp Snail1 promoter in A549 cells (Figure 7C) and U2OS cells (Supplementary Figure 5), indicating that the −483/−473 Snail1 promoter region contains a functional Foxm1-binding site. Thus, Foxm1 was capable of inducing transcriptional activity of the −720 bp Snail1 promoter region in co-transfection experiments. Interestingly, TGF-β did not synergize with Foxm1 to enhance the −720 bp Snail1 promoter activity (Figure 7C), ecause the Snail1 promoter region lacks AP-1/4-binding sites that are required for activation of Snail1 by TGFβ (Peinado et al, 2003; Medici et al, 2006). Foxm1 did not activate the −1.2 kb Zeb2 promoter (Supplementary Figure 5). Thus, Foxm1 directly bound to and induced the transcriptional activity of the Snail1 gene, indicating that Snail1 is the direct Foxm1 target. Finally, expression of Snail1 in Foxm1-deficient A549 cells restored TGFβ-induced decrease in E-cadherin protein (Figure 7D and E), indicating that Foxm1 influenced EMT through Snail1. Figure 7.Snail1 is a direct transcriptional target of Foxm1. (A) A schematic drawings of promoter region of the human Snail1 gene (hSnail1) and mouse Snail1 gene (mSnail1). Location of a potential Foxm1 DNA-binding site is indicated (box). Site-directed mutagenesis was used to mutate the Foxm1-binding site in mouse Snail1 promoter (from TGTTTATTCTG to AGGTCGATACG, −483/−473, mutSnail1-luc) (Littl
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