SNAI Transcription Factors Mediate Epithelial-Mesenchymal Transition in Lung Fibrosis

Thorax Online First, published on October 22, 2009 as 10.1136/thx.2009.121798 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from SNAI transcription factors mediate epithelial-mesenchymal transition in lung fibrosis

1Aparna Jayachandran, 2Melanie Königshoff, 1Haiying Yu, 1Ewa Rupniewska, 1Matthias Hecker, 3Walter Klepetko, 1Werner Seeger and 2Oliver Eickelberg

1Department of Medicine, University of Giessen Lung Center, University of Giessen, Giessen, Germany and 2Comprehensive Pneumology Center, Institute of Lung Biology and Disease (iLBD), Ludwig-Maximilians-University and Helmholtz Zentrum München, Neuherberg / Munich, Germany, and 3Department of Thoracic Surgery, University Hospital Vienna, Vienna, Austria

*Address Correspondence to: Prof. Dr. Oliver Eickelberg Comprehensive Pneumology Center Institute of Lung Biology and Disease (iLBD) Helmholtz Zentrum München Ingolstädter Landstraße 1 Tel.: +49 89 3187 2319 85764 Neuherberg / München Fax: +49 89 3187 2400 Germany Email: [email protected]

Keywords: TGF-β signalling, SNAI, EMT, alveolar epithelial cells Word count: 3353

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Copyright Article author (or their employer) 2009. Produced by BMJ Publishing Group Ltd (& BTS) under licence. Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from ABSTRACT

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterised by accumulation of activated (myo)fibroblasts and excessive extracellular matrix (ECM) deposition. The enhanced accumulation of (myo)fibroblasts may be attributed, in part, to the process of TGF-β1-induced epithelial-mesenchymal transition (EMT), the phenotypic switching of epithelial to fibroblast-like cells. Although alveolar epithelial type II (ATII) cells have been shown to undergo EMT, the precise mediators and mechanisms remain to be resolved. The objective of this study is to investigate the role of SNAI transcription factors in the process of EMT and in IPF. Methods: Using quantitative RT-PCR, immunofluorescence, immunohistochemistry, Western blotting, as well as gain- and loss-of- function studies and functional assays, we assessed the role of SNAI1 and SNAI2 in TGF-β1- induced EMT in ATII cells in vitro; and analysed the expression of SNAI transcription factors in experimental and idiopathic pulmonary fibrosis in vivo. Results: TGF-β1 treatment increased expression and nuclear accumulation of SNAI1 and SNAI2, in concert with induction of EMT in ATII cells. SNAI overexpression was sufficient to induce EMT and siRNA-mediated SNAI depletion attenuated TGF-β1-induced ATII cell migration and EMT. SNAI expression was elevated in experimental and human idiopathic pulmonary fibrosis and localised to hyperplastic ATII cells in vivo. Conclusions: Our results demonstrate that TGF- β1-induced EMT in ATII cells is essentially controlled by the expression and nuclear translocation of SNAI transcription factors. Increased SNAI1 and SNAI2 expression in experimental and idiopathic pulmonary fibrosis in vivo suggest that SNAI-mediated EMT may contribute to the fibroblast pool in IPF.

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2 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from INTRODUCTION

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder of major concern due to its unresolved pathogenesis and limited responsiveness to currently available therapies.1 The hallmark lesions of IPF are fibroblast foci, which are sites featuring α-smooth muscle actin (αSMA)-positive, activated (myo)fibroblasts.2 3 Currently, three major theories attempt to explain the accumulation of activated (myo)fibroblasts in the lungs of IPF patients. First, local fibroproliferation of resident pulmonary fibroblasts in response to fibrogenic cytokines and growth factors, increase the fibroblast pool.4 Second, bone marrow-derived circulating fibrocytes trafficking to the lung may serve as progenitors for interstitial fibroblasts.5-9 Third, alveolar epithelial type II (ATII) cells, via the process of epithelial-mesenchymal transition (EMT), can undergo a phenotypic, reversible switching to fibroblast-like cells.10-12 The orchestrated series of events during EMT includes remodeling of epithelial cell- cell and cell-matrix adhesion contacts, reorganisation of the actin cytoskeleton, induction of mesenchymal gene expression, and the acquisition of motile capacity.13 During development and disease pathogenesis, EMT is under tight transcriptional control maintained by factors such as Twist, NF-κB, Rho, Rac, Snai, or GSK-3β.13 14 The transcription factors eliciting EMT in IPF are yet to be identified. In this context, the zinc finger transcription factor SNAI1 (also called Snail) and SNAI2 (also called Slug), have been reported to act as regulators of EMT during development and disease, including cancer and organ fibrosis.15 16 Transforming growth factor (TGF)-β1 represents a main inducer and regulator of EMT in multiple organ systems, including the lung.10 17 While the ability of TGF-β1 to induce EMT has recently been described in ATII cells in vitro11 18-22 and in vivo in transgenic mice12; the molecular mechanisms that control these dynamics and their precise role in other experimental models of lung fibrosis and IPF remain to be elucidated. This study was performed to examine the hypothesis that TGF-β1 induces EMT via SNAI transcription factors in ATII cells and that SNAI transcription factors contribute to the β development of IPF. In detail, we examined the characteristics of TGF- 1-induced EMT, http://thorax.bmj.com/ modulated via SNAI transcription factor in vitro, and evaluated the expression pattern of SNAI transcription factors in experimental as well as human idiopathic pulmonary fibrosis in vivo. (word count: 358)

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3 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from MATERIALS AND METHODS

Reagents The following antibodies were used in this study: anti-pro-surfactant protein (proSP-C) (Chemicon International, Temecula, USA), anti-α-smooth muscle actin (αSMA) (Sigma, St. Louis, USA), anti-e-Cadherin (ECAD) (BD Biosciences, San Jose, USA), anti-tight junction protein (TJP)1, anti-Occludin (OCCL) (Zymed Laboratories, San Francisco, USA), anti- SNAI1 and anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, USA), anti-SNAI2 (Cell Signaling Technology, Beverly, USA), and anti-SNAI1 (a gift from Dr. Becker, Institute of Pathology, Technical University of Munich, Germany). Recombinant human transforming growth factor-β1 (TGF-β1) was purchased from R&D Systems (Minneapolis, USA).

Cell culture The human lung epithelial cell line A549 (ATCC CCL-185; Manassas, USA) was maintained in DMEM (Invitrogen, Pasching, Austria), supplemented with 10% FBS (PAA Laboratories, Carlsbad, USA). Primary mouse ATII cells were isolated from adult male C57BL/6N mice as previously described.23

Human Tissues Lung tissue biopsies were obtained from twelve patients with IPF (mean age 51.3 ± 11.4 years; six females, six males) and nine transplant donors (serving as control subjects) (mean age 47.5 ± 13.9 years; four females, five males).24 The study protocol was approved by the Ethics Committee of the Justus-Liebig-University School of Medicine (AZ 31/93). Informed consent was obtained from each subject for the study protocol.

Animal Tissues All animal studies were performed in accordance with the guidelines of the Ethic’s

Committee of University of Giessen School of Medicine and approved by the local http://thorax.bmj.com/ authorities. Adult male C57BL/6N mice were treated with bleomycin and were sacrificed at the indicated time points.

RNA extraction and RT-PCR Total RNA was extracted using RNeasy columns (Qiagen, Hilden, Germany) according to the manufactures protocol and PCR amplification was performed with human and mouse primers (Tables S1 and S2, online supplement). on October 1, 2021 by guest. Protected copyright.

Quantitative RT-PCR Quantitative RT-PCR was performed as previously described.23 All results were normalised to the relative expression of the constitutively expressed gene porphobilinogen deaminase (Pbgd). Relative transcript abundance of target gene is expressed in ∆Ct values (∆Ct=Ct reference-Ct target). Relative changes in transcript levels compared to controls are expressed as ∆∆Ct values (∆∆Ct=∆Ct treated-∆Ct control). All ∆∆Ct values correspond approximately to the binary logarithm of the fold change. The human and mouse primer sequences are depicted in Table S3 and S4, online supplement, respectively. siRNA transfection The siRNA oligonucleotides specific for human SNAI1 and SNAI2 mRNA (Table 1) were obtained from Dharmacon Inc. (Lafayette, USA). A549 cells were transiently transfected with 100 nM SNAI or non-specific siRNA using LipofectamineTM2000 reagent (Invitrogen). 4 h

4 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from post transfection, cell were treated with TGF-β1. Cells were lysed after 24 h and efficiency of gene knockdown was monitored.

Table 1: siRNA sequences

Gene name Sense Sequence Antisense Sequence SNAI1-si#1 ACUCAGAUGUCAAGAAGUAUU PUACUUCUUGACAUCUGAGUUU

SNAI1-si#2 GCAAAUACUGCAACAAGGAUU PUCCUUGUUGCAGUAUUUGCUU

SNAI1-si#3 GCUCGGACCUUCUCCCGAAUU PUUCGGGAGAAGGUCCGAGCUU

SNAI1-si#4 GCUUGGGCCAAGUGCCCAAUU PUUGGGCACUUGGCCCAAGCUU

SNAI2-si#1 GGACACACAUACAGUGAUUUU PAAUCACUGUAUGUGUGUCCUU

SNAI2-si#2 UAAAUACUGUGACAAGGAAUU PUUCCUUGUCACAGUAUUUAUU GAAUGUCUCUCCUGCACAAUU SNAI2-si#3 PUUGUGCAGGAGAGACAUUCUU

PCUACACAGCAGCCAGAUUCUU SNAI2-si#4 GAAUCUGGCUGCUGUGUAGUU

Migration Assay Cell migration was determined using Boyden chamber assay (ThinCertsTM Tissue Culture Inserts, 24 well, pore size 3.0 μm from Kremsmunster, Austria), as described previously.25 A549 cells were transfected with SNAI1, SNAI2 or non-specific siRNA at a total concentration of 75 nM, detached, and 5 × 104 cells were seeded into Boyden chamber insert. Cells were cultured for 8 h to allow their attachment to the membrane and migration was induced by adding TGF-β1 (2 ng/ml) to the media in the lower wells. After 24 h, cells were

fixed and stained using crystal violet solution, and non-migrated cells were removed by cotton http://thorax.bmj.com/ swabbing. The membranes were carefully separated from the insert wall, and optical densities of migrated cells were measured with a GS-800 Calibrated Densitometer and analysed with the Quantity One software.

Statistical analysis of data Values are presented as mean ± SEM. All ΔCt values obtained from qRT-PCR were analysed for normal distribution using the Shapiro-Wilk test, with the assignment of a normal on October 1, 2021 by guest. Protected copyright. distribution with p > 0.05. All ΔΔCt values were analyzed using the two-tailed, one-sample t- test. Normality of data was confirmed using quantile-quantile plots. Intergroup differences of DCt values from patients and bleomycin-treated mice were derived using a one-tailed, two- sample t-test. A one-way analysis of variance (ANOVA) with Tukey HSD post hoc test for studies with more that two groups. A level of p<0.05 was considered statistically significant.

Further experimental procedures The detailed procedures for Western blotting, densitometric analysis, immunohistochemistry, immunofluorescence, laser-assisted microdissection, and transfection of human SNAI1 and SNAI2, as well as the sequences of all primers used in this study, are provided in the online supplement.

5 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from RESULTS

TGF-β1-induced EMT in alveolar epithelial type II (ATII) cells. Initially, a comprehensive analysis of TGF-β1-induced EMT in ATII cells was performed. We assessed the gene and protein expression as well as localisation of epithelial and mesenchymal marker in primary mouse alveolar epithelial type II (ATII) cells (fig E1) and the human ATII cell line A549. TGF-β1 treatment for 24 h induced changes in α-smooth muscle actin (αSMA), e-cadherin (ECAD), and tight junction protein (TJP) 1 expression and/or localisation indicative of EMT. While ECAD and TJP1 staining decreased, an appreciable number of cells expressing αSMA increased from 5.9 ± 2.8 % in vehicle treated, to 28.2 ± 8.1 % in TGF-β1-treated cells (fig 1A,B). The presence of αSMA and TJP1 double- positive cells in TGF-β1-treated, but not in vehicle-treated cells, further corroborated the occurrence of TGF-β1-induced EMT in primary ATII cells (Fig 1A, bottom panels).

SNAI transcriptions factor expression in TGF-β1-induced EMT in ATII cells. We subsequently quantified the mRNA expression patterns of several EMT marker genes in ATII cells, using quantitative (q)RT-PCR. ATII cells were treated with TGF-β1 in a dose-and time-dependent manner as indicated (fig 2, Fig E2). Although the magnitude of EMT marker gene expression in both cell types investigated, differs, most markers were differentially regulated after 8h. TGF-β1 treatment led to a decreased expression of the epithelial cell marker ECAD and occludin (OCCL), concomitant with increased expression of the mesenchymal marker vimentin (VIM) and αSMA, 8h after treatment of ATII cells. Notably, vimentin was strongly induced in primary ATII cells, but not in A549 cells (fig 2A and B). Importantly, we observed a significant upregulation of both SNAI1 and SNAI2, along with the alterations in EMT marker gene expression (fig 2A and B). Since the SNAI transcription factors were regulated at the mRNA level, we next wanted to assess the protein expression and function in TGF-β1-induced EMT in ATII cells.

Immunofluorescence analysis of TGF-β1-treated ATII cells revealed increased nuclear http://thorax.bmj.com/ translocation of endogenous SNAI1 and SNAI2 upon TGF-β1 treatment in A549 and primary ATII cells, respectively (fig 2C and D, fig E3). In addition, SNAI1 and SNAI2 protein expression was increased by TGF-β1 in a time-dependent manner, as assessed by Western blot analysis. These changes were in concert with time-dependent decrease of ECAD and increased αSMA expression in response to TGF-β1 treatment (fig3A and B).

EMT induction by SNAI transcription factor overexpression in ATII cells. on October 1, 2021 by guest. Protected copyright. To further investigate whether SNAI has a functional role in inducing EMT in the lung, the full-length SNAI1 and SNAI2 cDNA were cloned into mammalian expression vectors and transiently transfected into A549 cells (transfection efficiency is depicted in fig E4). We examined the gene and protein expression pattern of different EMT marker post SNAI transfection. SNAI1 overexpression led to a significant increased αSMA expression (fig 4A), which was further confirmed on the protein level (fig 4B, fig E5A). Interestingly, SNAI2 overexpression revealed significant changes on the gene and protein level of the epithelial cell targets ECAD and OCCL, however, no significant changes were observed for αSMA expression (fig 4C,D and fig E5B). Taken together, overexpression of SNAI transcription factors are able to induce EMT in ATII cells, even in the absence of TGF-β1.

Effect of SNAI transcription factor silencing on TGF-β1-induced EMT in ATII cells. In order to determine the effect of SNAI1 or SNAI2 on the process of TGF-β1-induced EMT in ATII cells, we next characterised SNAI mRNA knock-down using four different sequences of siRNA oligonucleotides targeting SNAI1 and SNAI2, respectively (fig E6).

6 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from Quantitative and semi-quantitative RT-PCR analysis revealed that three out of four SNAI1 (fig E6 A and B) and SNAI2 (fig E6 C and D) siRNA oligonucleotides were effective in reducing the respective mRNA level in response to TGF-β1 treatment. The most effective siRNA oligonucleotides targeting SNAI1 and SNAI2 were then used to examine the effect of SNAI depletion on TGF-β1-induced EMT. Both, SNAI1 and SNAI2 depletion effectively attenuated TGF-β1-induced EMT in ATII cells (fig 5). SNAI1-depleted ATII cells exhibited a significant attenuated decrease in OCCL and TJP1, as well as reduced increase in αSMA mRNA levels, as assessed by qRT-PCR and semi-quantitative PCR 24 h after TGF-β1 treatment compared with non-specific scrambled siRNA-treated ATII cells (fig 5A and B). Similarly, SNAI2 depletion in ATII cells demonstrated an increase in OCCL and TJP1 mRNA level compared with TGF-β1-treated non-specific scrambled siRNA-depleted ATII cells, whereas no significant changes have been observed in mesenchymal marker expression (fig 5C and D).

Effect of SNAI transcription factor silencing on TGF-β1-induced migration in ATII cells. One of the key requirements for the complete process of EMT is the capability of ATII cells to migrate. TGF-β1 induces ATII cell migration in a dose-dependent manner (figE7). In order to elucidate the impact of SNAI transcription factor on TGF-β1-induced ATII cell migration, we performed an in vitro migration assay on SNAI1- and SNAI2-depleted cells stimulated with TGF-β1. After 24 h of TGF-β1 treatment, cells transfected with non-specific siRNA, exhibited a four-fold induction of cell migration (fig 6). Both, SNAI1- and SNAI2- depleted cells exhibited significant reduced migration under baseline and TGF-β1-induced condition (fig 6).

SNAI transcription factor expression in experimental lung fibrosis. To analyse whether expression of SNAI transcription factors were regulated during experimental lung fibrosis, we subjected mice to bleomycin treatment for 7 or 14 days,

respectively. On the mRNA level, a significant increase in SNAI1 has been observed in lung http://thorax.bmj.com/ homogenates from bleomycin-treated mice compared with saline-treated mice, as assessed by qRT-PCR analysis (fig 7A). Interestingly, significantly elevated level of both SNAI1 and SNAI2 were observed in freshly isolated ATII cells from bleomycin-treated lungs compared with ATII cells from saline-treated lungs, as early as 7 days after treatment (fig 7B). Using immunohistochemistry, we next determined the localisation of SNAI1 and SNAI2 in bleomycin-treated lungs. While SNAI1 was predominantly localised to perivascular mesenchymal cells and alveolar macrophages in saline-treated lungs, it also demonstrated on October 1, 2021 by guest. Protected copyright. localization in the epithelium and, particularly, in subepithelial areas, after bleomycin treatment (fig 7C). Although little or no staining for SNAI2 was detectable in saline-treated lungs, weak SNAI2 staining localised to the lung interstitium after bleomycin treatment (fig 7C). αSMA served as a well-characterised marker of the lung interstitium after bleomycin treatment.

SNAI transcription factor expression in idiopathic pulmonary fibrosis (IPF). Finally, we asked whether SNAI expression was also increased in the lungs of patients with IPF. To this end, we determined the mRNA expression in lung homogenates obtained from IPF patients and transplant donors. The qRT-PCR analysis revealed increased levels of SNAI2 gene expression in both lung homogenates as well as in microdissected alveolar septae from IPF patients compared with transplant donors (fig 8A and B). Immunohistochemistry further confirmed the expression and localisation of SNAI1 and SNAI2 predominantly in hyperplastic alveolar epithelial cells in IPF, whereas only weak SNAI1 and SNAI2 expression was detected in lung specimen of transplant donors (fig 8C). Western blot analysis further

7 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from corroborated elevated protein level of both SNAI1 and SNAI2 in lung homogenates from IPF patients compared with transplant donors (fig 8D).

DISCUSSION IPF is the most common form of idiopathic interstitial pneumonias (IIP), which exhibits a poor prognosis and unresponsiveness to currently available therapies, reflecting our limited understanding of the basic mechanisms and mediators implicated in the pathogenesis of this disease.1 26 27 While the initial injury in IPF is affecting the alveolar epithelium, the interstitial fibroblast / activated (myo)fibroblast represents the key effector cell accumulating in fibroblast foci and responsible for the increased ECM deposition that is characteristic for IPF.28-30 The epithelial-mesenchymal transition (EMT), a process were alveolar epithelial cells turn into fibroblast-like cells, has recently been reported to serve as a source for the fibroblast pool in tissue fibrosis of the liver, kidney, and lung.11 12 31 EMT has been initially described in embryonic development.14 The orchestrated series of events during EMT involve changes in cell polarity, loss of epithelial cell markers, induction of mesenchymal gene expression, and enhanced cell migration. 13 TGF-β1, known as a key profibrotic growth factor, has emerged as a potent inducer of EMT. 17 32 In the present study, we report that SNAI transcription factors are key regulators of TGF-β1-induced EMT in the lung. Elevated expression of SNAI1 and SNAI2 were observed in ATII cells in response to TGF-β1 in vitro. Depletion of SNAI1 and SNAI2 using siRNA knockdown in ATII cells inhibited TGF-β1- induced alterations in EMT marker gene expression and ATII cell migration in response to TGF-β1. Interestingly, ectopic overexpression of SNAI transcription factors promotes EMT even in the absence of TGF-β1. Finally, increased level of SNAI1 and SNAI2 in experimental and human idiopathic pulmonary fibrosis in vivo further indicated a significant contribution of SNAI transcriptions factors in the process of EMT in lung fibrosis. At the onset of our studies, we performed a detailed analysis of the occurrence of EMT in ATII cells in response to TGF-β1, determining marker gene and protein expression and β localisation in primary mouse ATII cells and A549 cells. Upon exposure to TGF- 1, ATII http://thorax.bmj.com/ cells demonstrated an increased expression of mesenchymal marker (such as αSMA) with a corresponding decrease in epithelial markers (ECAD and TJP1), suggestive of EMT. EMT was further corroborated by the presence of αSMA and TJP1 double-positive cells in TGF-β1- treated, but not in untreated cells. These results are in accordance to previous studies, reporting that TGF-β1 induces EMT in lung epithelial cells in vitro.11 19-21 Recently, TGF-β1- induced EMT has also been demonstrated in vivo in triple transgenic mouse model.12 13 17 Several regulatory molecules have been implicated in the process of EMT. Here, on October 1, 2021 by guest. Protected copyright. we demonstrated increased expression and nuclear translocation of the zinc finger transcription factors SNAI1 and SNAI2 along with EMT in ATII cells. Elevated mRNA level of SNAI1 have been reported in TGF-β1-treated A549 cells22, which has been further confirmed in this study. In addition, SNAI1 induction upon TGF-β1 has been recently demonstrated in MDCK cells, a canine renal epithelial cell line.33 Furthermore, it has been demonstrated that SNAI1 deficient mice die at the gastrulation stage34; because of the inability to undergo EMT, reinforcing the importance of the SNAI transcription factors in the process of embryonic development. The impact of SNAI-mediated EMT in pathophysiological conditions such as cancer or tissue fibrosis, however, is less well substantiated and requires further investigations. Recently, high SNAI expression has been associated with poor prognosis and tumor recurrence in lung cancer patients.35 In addition, SNAI1 has been reported to induce chemo- resistance of cancer cells.36 In tissue fibrosis, SNAI-mediated EMT has been proposed to be involved in kidney fibrosis.37 The comprehensive analysis presented herein strongly suggest that SNAI1 and SNAI2 are essential mediators involved in the initiation and perpetuation of

8 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from TGF-β1-mediated EMT in experimental and human idiopathic pulmonary fibrosis, but by no means proof EMT in IPF in vivo. Since we were able to inhibit EMT in ATII cells with siRNAs targeting SNAI1 and SNAI2, it will have to be demonstrated whether in vivo interference with these factors may lead to an attenuation of fibrosis. We also present evidence that both SNAI1 and SNAI2, induce EMT even in the absence of TGF-β1, hence recapitulating their role as potent inducers of EMT. It has to be pointed out that SNAI1 and SNAI2 may differ in their respective target genes. Our results suggest that SNAI1 preferable regulated αSMA, whereas SNAI2 alters epithelial targets. This is of special interest, as cell- specific SNAI expression in vivo may lead to distinct cell fates. Given that SNAI2 was the dominant transcription factor regulated in human IPF tissue, we propose that some ATII cells undergoing EMT in IPF may exhibit a αSMA-negative fibroblast phenotype mediated by SNAI2. In IPF, recent studies were able to demonstrate evidence of EMT in lung tissue biopsies, suggesting that this process contributes to the increased pool of (myo)fibroblasts in lung fibrosis11 12, as well as in allografts after lung transplantation.38 In contrast, another recent study failed to supply evidence for EMT in pulmonary fibrosis.39 This discrepancy may be due to the transient nature and complexity of the EMT process, and further highlights the challenge in this research field. Taken together, we demonstrated that SNAI transcription factors mediate EMT in ATII cells in vitro and are increased expressed in experimental and human idiopathic pulmonary fibrosis in vivo. We speculate that EMT is an early event in IPF and that activation and nuclear translocation of SNAI transcription factors constitute an important early regulator of EMT in ATII cells. http://thorax.bmj.com/ on October 1, 2021 by guest. Protected copyright.

9 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from Acknowledgements We thank Dr. Becker (Institute of Pathology, Technical University of Munich, Germany) for the generous gift of anti-rat monoclonal SNAI1 antibody. We are indebted to all members of the Eickelberg Lab for stimulating discussions, and Andreas Jahn and Simone Becker for excellent technical assistance.

Competing interests None

Funding The authors are supported by the Helmholtz Association, the German Research Foundation (DFG) KliFo 118, the International Graduate Program “Signaling Mechanisms of Lung Physiology and Disease” GRK1062, and a career development award by the University of Giessen School of Medicine to M.K.

Licence for publication The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article to be published in THORAX editions and any other BMJPG Ltd products to exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals /instructions-for-authors/licence-forms). . http://thorax.bmj.com/ on October 1, 2021 by guest. Protected copyright.

10 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from REFERENCES 1. American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. June 2001. Am J Respir Crit Care Med 2002;165(2):277-304. 2. King TE, Jr., Schwarz MI, Brown K, Tooze JA, Colby TV, Waldron JA, Jr., et al. Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality. Am J Respir Crit Care Med 2001;164(6):1025-32. 3. Visscher DW, Myers JL. Histologic spectrum of idiopathic interstitial pneumonias. Proc Am Thorac Soc 2006;3(4):322-9. 4. Phan SH. The myofibroblast in pulmonary fibrosis. Chest 2002;122(6 Suppl):286S- 89S. 5. Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113(2):243-52. 6. Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, et al. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 2004;114(3):438-46. 7. Moeller A, Gilpin SE, Ask K, Cox G, Cook D, Gauldie J, et al. Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2009;179(7):588-94. 8. Moore BB, Murray L, Das A, Wilke CA, Herrygers AB, Toews GB. The Role of CCL12 in the Recruitment of Fibrocytes and Lung Fibrosis. Am J Respir Cell Mol Biol 2006. 9. Andersson-Sjoland A, de Alba CG, Nihlberg K, Becerril C, Ramirez R, Pardo A, et al. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol 2008;40(10):2129-40. 10. Willis BC, Dubois RM, Borok Z. Epithelial Origin of Myofibroblasts during Fibrosis in the Lung. Proc Am Thorac Soc 2006;3(4):377-82.

11. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, du Bois RM, et http://thorax.bmj.com/ al. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol 2005;166(5):1321-32. 12. Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Nat Acad Sci

2006;103(35):13180-5. on October 1, 2021 by guest. Protected copyright. 13. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7(2):131-42. 14. Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 2003;15(6):740-6. 15. Nieto MA. The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 2002;3(3):155-66. 16. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005;132(14):3151- 61. 17. Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 2005;24(37):5764-74. 18. Ando S, Otani H, Yagi Y, Kawai K, Araki H, Fukuhara S, et al. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells. Respir Res 2007;8:31.

11 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from 19. Kasai H, Allen JT, Mason RM, Kamimura T, Zhang Z. TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res 2005;6(1):56. 20. Yao HW, Xie QM, Chen JQ, Deng YM, Tang HF. TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro. Life Sci 2004;76(1):29-37. 21. Xu GP, Li QQ, Cao XX, Chen Q, Zhao ZH, Diao ZQ, et al. The Effect of TGF-beta1 and SMAD7 gene transfer on the phenotypic changes of rat alveolar epithelial cells. Cell Mol Biol Lett 2007. 22. Kim JH, Jang YS, Eom KS, Hwang YI, Kang HR, Jang SH, et al. Transforming growth factor beta1 induces epithelial-to-mesenchymal transition of A549 cells. J Korean Med Sci 2007;22(5):898-904. 23. Königshoff M, Kramer M, Balsara N, Wilhelm J, Amarie OV, Jahn A, et al. WNT1- inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J Clin Invest 2009;119(4):772-87. 24. Königshoff M, Balsara N, Pfaff EM, Kramer M, Chrobak I, Seeger W, et al. Functional Wnt signaling is increased in idiopathic pulmonary fibrosis. PLoS ONE 2008;3(5):e2142. 25. Yu H, Königshoff M, Jayachandran A, Handley D, Seeger W, Kaminski N, et al. Transgelin is a direct target of TGF-{beta}/Smad3-dependent epithelial cell migration in lung fibrosis. FASEB J 2008. 26. Thannickal VJ, Flaherty KR, Hyzy RC, Lynch JP, 3rd. Emerging drugs for idiopathic pulmonary fibrosis. Expert opinion on emerging drugs 2005;10(4):707-27. 27. Walter N, Collard HR, King TE, Jr. Current perspectives on the treatment of idiopathic pulmonary fibrosis. Proc Am Thorac Soc 2006;3(4):330-8. 28. Phan SH. Biology of fibroblasts and myofibroblasts. Proc Am Thorac Soc 2008;5(3):334-7. 29. Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med

2001;134(2):136-51. http://thorax.bmj.com/ 30. Scotton CJ, Chambers RC. Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest 2007;132(4):1311-21. 31. Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renal fibrosis. J Mol Med 2004;82(3):175-81. 32. Willis BC, Borok Z. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Amer Journal Physiol 2007;293(3):L525-34.

33. Peinado H, Quintanilla M, Cano A. Transforming growth factor beta-1 induces snail on October 1, 2021 by guest. Protected copyright. transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J Bio Chem 2003;278(23):21113-23. 34. Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2000;2(2):84-9. 35. Shih JY, Tsai MF, Chang TH, Chang YL, Yuan A, Yu CJ, et al. Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma. Clin Cancer Res 2005;11(22):8070-8. 36. Zhuo W, Wang Y, Zhuo X, Zhang Y, Ao X, Chen Z. Knockdown of Snail, a novel zinc finger transcription factor, via RNA interference increases A549 cell sensitivity to cisplatin via JNK/mitochondrial pathway. Lung Cancer 2008;62(1):8-14. 37. Lange-Sperandio B, Trautmann A, Eickelberg O, Jayachandran A, Oberle S, Schmidutz F, et al. Leukocytes induce epithelial to mesenchymal transition after unilateral ureteral obstruction in neonatal mice. Am J Pathol 2007;171(3):861-71.

12 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from 38. Ward C, Forrest IA, Murphy DM, Johnson GE, Robertson H, Cawston TE, et al. Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients. Thorax 2005;60(10):865-71. 39. Yamada M, Kuwano K, Maeyama T, Hamada N, Yoshimi M, Nakanishi Y, et al. Dual-immunohistochemistry provides little evidence for epithelial-mesenchymal transition in pulmonary fibrosis. Histochem Cell Biol 2008;129(4):453-62.

http://thorax.bmj.com/ on October 1, 2021 by guest. Protected copyright.

13 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from Figure Legends

Figure 1: TGF-β-induced epithelial-mesenchymal transition (EMT) in alveolar epithelial type II (ATII) cells. (A) Immunofluorescence detection of αSMA, ECAD, and TJP1 was performed after treatment with TGF-β1 (2 ng/ml) or vehicle for 24 h. Co-localisation of αSMA (red) and TJP1 (green) was assessed by immunofluorescence in ATII cells treated with TGF-β1 for 24 h. Nuclei are visible by 4’6-diamidino-2-phenylindole (DAPI) staining. Original magnification is 40×. Panel (B) depicts the quantification of the percentage of αSMA-positive cells 24 h after TGF-β1 (2 ng/ml) treatment. Original magnification of a representative figure showing immunofluorescence detection of αSMA (green) with TGF-β1 (2 ng/ml) or vehicle for 24 h is 10×. Results are representative of ten independent experiments and data are expressed as mean ± SEM; * p<0.05, n= 10.

Figure 2: SNAI transcription factor expression in TGF-β-induced EMT in ATII cells. Using qRT-PCR analysis, the expression pattern of the indicated EMT marker was detected in A549 (A) and primary mouse ATII cells (B), after TGF-β1 (2 ng/ml) treatment for 2 and 8 h, as indicated. Data are expressed as mean ± SEM; * p<0.05, n= 5. (C,D) Immunofluorescence analysis was performed using a primary antibody directed against SNAI1 and SNAI2 in both cells. The original magnification of representative image from three independent experiments is 40×.

Figure 3: Protein expression of EMT marker in TGF-β1-treated ATII cells. Protein expression of ECAD, αSMA, SNAI1 and SNAI2 was assessed by Western blot analysis. A549 cells were treated with TGF-β1 (2 ng/ml) for several time points as indicated. Western blot analysis (A) was performed using primary antibody directed against ECAD, αSMA, SNAI1 and SNAI2. Lamin A/C served as a loading control. (B) Western blots were scanned and intensity is represented as fold change. Data are representative of three independent ∗ experiments and are expressed as mean ± SEM; p<0.05, n= 3. http://thorax.bmj.com/

Figure 4: EMT induction by SNAI transcription factor overexpression in ATII cells. EMT marker expression was analysed by qRT-PCR (A,C) and Western blot (B,D). A549 cells overexpressing human SNAI1 (A,B) or SNAI2 (C,D) protein for 24 and 48 h and analysis was done compared with the empty vector (EV) control. The α-tubulin served as the loading control. Data are representative of five independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n= 5. on October 1, 2021 by guest. Protected copyright.

Figure 5: Effect of SNAI transcription factor silencing on TGF-β1-induced EMT in ATII cells. (A,C) Using qRT-PCR analysis, the expression patterns of EMT marker genes was assessed in A549 cells after siRNA treatment against SNAI1 (A) and SNAI2 (C), with TGF- β1 exposures for 24 h and compared with control non-specific scrambled siRNA treatment with TGF-β1 exposures for 24 h. (B,D) semi-quantitative RT-PCR analysis, the expression patterns of EMT marker genes was detected in A549 cells after siRNA treatment against SNAI1 (B) and SNAI2 (D), with TGF-β1 exposures for 24 h (lane 4) and compared with controls- i. non-specific scrambled siRNA treatment with (lane 3) or without (lane 1) TGF-β1 for 24 h and ii. siRNA against SNAI without TGF-β1 treatment (lane2). Hspa8 was employed as loading control. scr; scrambled siRNA oligonucleotide. Data are representative of three independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n= 3.

Figure 6: Effect of SNAI transcription factor silencing on TGF-β1-induced migration in ATII cells. The siRNA treated A549 cells where treated with or without TGF-β1 for 24 h. The

14 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from relative migration potential was assessed using Boyden chamber assay. scr; scrambled siRNA oligonucleotide. Membranes were scanned and the intensity is represented as bars. Data are representative of three independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n=3.

Figure 7: SNAI transcription factor expression in experimental lung fibrosis. Mice were exposed to bleomycin, and lungs were harvested after 7 or 14 days, as indicated. (A) RNA was isolated and qRT-PCR was performed for SNAI genes in 7 or 14 days bleomycin- or saline-treated lung homogenates. (B) SNAI gene expression was quantified in primary ATII cells freshly isolated from 7 or 14 days bleomycin-treated lungs, using qRT-PCR. Data are representative of five independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n= 5. (C) Immunohistochemical analysis of αSMA, SNAI1, and SNAI2 localisation was performed in paraffin-embedded tissue from bleomycin- or saline-treated lungs after 14 days.

Figure 8: SNAI transcription factor expression in IPF. (A) Gene expression analysis of SNAI1 and SNAI2 genes was performed by qRT-PCR analysis of total RNA derived from lung homogenates of twelve IPF patients and nine transplant donors. (B) SNAI1 and SNAI2 mRNA levels were quantified using qRT-PCR in laser-assisted microdissected septae from five IPF patients and five transplant donors. Data are expressed as mean ± SEM; ∗ p < 0.05, n= 5. (C) Immunohistochemical analysis of SNAI1 and SNAI2 localisation was performed in paraffin-embedded lung specimen from IPF patients or transplant donors. (D) Western blot analysis of SNAI1 and SNAI2 was performed thrice in lung homogenates obtained form six IPF patients and six transplant donors. Lamin A/C served as the loading control.

http://thorax.bmj.com/ on October 1, 2021 by guest. Protected copyright.

15 Figure 1

A B 40 * 35

30 αSMA 25

15 αSMA-positive cells

% 10 ECAD 5

0 Vehicle TGF-β1 TJP1

αSMA

αSMA TJP1 + + TJP1

Vehicle TGF-β1 Vehicle TGF- 1

Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from from Downloaded 2009. October 22 on 10.1136/thx.2009.121798 as published first Thorax: http://thorax.bmj.com/ on October 1, 2021 by guest. Protected by copyright. by Protected guest. by 2021 1, October on Figure 2

A B 4 2 h TGF-β1 2 2 h TGF-β1 * * 8 h TGF-β1 * 8 h TGF-β1 * 3 * * CT) CT) * Δ Δ Δ 2 Δ 1 * * 1

0 0 log-fold change ( log-fold change ( -1 * * * -2 -1 ECADOCCL VIM αSMA SNAI1 SNAI2 eCadOccl Vim αSMA Snai1 Snai2

A549 cells ATII cells

C D SNAI1 SNAI1 SNAI2 SNAI2

Vehicle TGF-β1 Vehicle TGF-β1

ECAD 120 kDa

αSMA 42 kDa

SNAI1 29 kDa

SNAI2 30 kDa

LAMIN A/C 50/70 kDa

0h 2h 8h 12h 24h 48h

B 5 2h * 8h 0h 2h 8h 12h 24h 48h 12h * 24h 4 48h

3 * 2 *

foldchange (OD) foldchange * 1 *

ECADαSMA SNAI1

A B 2 * 24 h Snai1 SNAI1 29 kDa 48 h Snai1

ΔCT) ECAD 120 kDa Δ 1 OCCL 65 kDa

α 0 SMA 42 kDa

log-fold change ( α-tubulin 55 kDa

-1 EVSNAI1 EV SNAI1 ECAD OCCL αSMA 24 h 48 h

C D 2 24 h Snai2 SNAI2 30 kDa 48 h Snai2 1 ΔCT) ECAD 120 kDa Δ

0 OCCL 65 kDa

αSMA 42 kDa -1 * *

log-fold change ( α-tubulin 55 kDa -2 EV SNAI2 EV SNAI2 ECAD OCCL αSMA

24 h 48 h

A B 4 si scr + TGF-β1 SNAI1 616 bp si Snai1 + TGF-β1 3 * ECAD 631 bp ΔCT) 2 * * 1 OCCL 683 bp

0 αSMA 1000 bp

-1 HSPA8 330 bp relative mRNA level ( -2 scr si scr si

-3 Vehicle TGF-β1 ECAD OCCL TJP1 VIM αSMA

C D 4 si scr + TGF-β1 β SNAI2 631 bp 3 si Snai2 + TGF- 1 ECAD 631 bp ΔCT) 2 *

OCCL 683 bp 1 *

0 αSMA 1000 bp

-1 HSPA8 330 bp relative mRNA level ( -2 scr si scr si

-3 Vehicle TGF-β1

ECAD OCCL TJP1 VIM αSMA

A 5 vehicle * TGF-β1 4

3 *#

2 *# relative migration 1

scr siSNAI1 siSNAI2

A B 5 3 * Saline * 7 days 2 4CT) CT) Δ 14 days CT) Δ Δ * 1 3 0 2 -1 1 relative mRNA level ( relative mRNA level ( relative mRNA level ( -2

0 -3 Snai1 Snai2 Snai1 Snai2

C αSMA SNAI1 SNAI2 Saline

Bleomycin

A B 8 8 Donor * Donor 7 IPF IPF

ΔCT) 6 ΔCT) 6 * 5 4 4 3 2 2 relative mRNA level ( relative mRNA level ( 1 0 0 SNAI1 SNAI2 SNAI1 SNAI2

C SNAI1 SNAI2 D

SNAI1 29 kDa

Lamin A/C 50/70 kDa Donor SNAI2 30 kDa

Lamin A/C 50/70 kDa

Donor IPF

IPF

A Fresh fix Day 2 B

Smad2 409 bp

Smad3 565 bp Pro-SPC Smad4 500 bp

Smad6 234 bp

Smad7 166 bp TJP1 Alk1 735 bp

Alk5 429 bp

TβrII 410 / 500 bp

αSMA Gapdh 225 bp

1234

A 3 0.02ng 0.2ng 2ng * 2 20ng

ΔCT) Δ 1

* * -1

log-fold change ( * -2

eCad SMA

-3

A A549 cells B ATII cells SNAI1 +DAPI SNAI2 +DAPI SNAI1 +DAPI SNAI2 +DAPI

Vehicle TGF-β1 Vehicle TGF-β1

efficiency = 17±3 %

A 2 *

1,5 *

fold change change fold (OD) * 0,5

0 0024 48 0024 48 0024 48 ECAD OCCL SMA

B 1,5

* *

fold change change fold (OD) 0,5

0 0024 48 0024 48 0024 48

ECAD OCCL SMA

A B 120

100

80 Snai1 616 bp 60 * * Hspa8 330 bp 40 si #1si #2 si #3si #4 scr % remaining expression 20

0 si scr si #1 si #2 si #3 si #4

C D

120

100

80 * Snai2 631 bp 60

Hspa8 330 bp * 40 * si #1si #2 si #3si #4 scr % remaining expression 20

si scr si #1 si #2 si #3 si #4

3 *

2 * relative migration 1

0ng/ml 0,02ng/ml 0.2ng/ml 2ng/ml 20ng/ml

TGF-β1 TGF-β1 TGF-β1 TGF-β1 TGF-β1

Cloning and transfection of human SNAI1 The human SNAI1 expression construct was generated by amplifying full-length SNAI1 cDNA from total lung RNA by PCR (using the forward and reverse primers 5´-CAG TGC CTC ACC ACT ATG C-3´ and 5´- AGG ATC CTC GAG GGT CAG-3´, respectively). SNAI1 cDNA was cloned into pcDNA3.1(-) and transiently transfected into A549 cells using LipofectamineTM2000 reagent at a 1:3 DNA:Lipofectamine ratio in OptiMEM (Invitrogen).

Western Blot A549 cells were grown on six-well culture dishes and following transient transfection with SNAI1, cells were harvested in lysis buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM sodium vanadate, and Complete inhibitor (Roche Diagnostics)]. 15-min following incubations on ice, lysates were centrifuged for 20 min and supernatants collected into fresh tubes. Protein content was determined using the Protein DC Assay (Bio-Rad, Hercules, USA) and 20 µg of each sample separated on 10% SDS-polyacrylamide gels. Following electrophoresis, proteins were blotted on nitrocellulose membranes (GE Osmonics, Inc., Minnetonka, USA). Blocking of membranes was performed using 5% nonfat dry milk in PBS for 1 h, after which incubations with primary antibodies were performed overnight. After incubation with the respective horseradish peroxidase-conjugated secondary antibodies (Pierce Chemical, Rockford, USA), proteins were detected using ECL plus reagents (Amersham Biosciences; USA) according to the manufacturer's instructions.

Densitometric analysis Densitometric analysis of autoradiographies was performed using a GS-800 Calibrated Densitometer and 1-D analysis software Quantity One (Bio-Rad, USA). Protein expression

was normalized to the appropriate house-keeping protein used in the experiment. http://thorax.bmj.com/

Immunohistochemistry Expression and localization of αSMA, Snai1 and Snai2 (dilution 1:100) was assessed in paraffin-embedded tissue sections using the Histostain Plus Kit from Zymed. 3 μm mouse or human sections mounted on poly-L-lysine-coated slides were dewaxed and rehydrated by immersion in ethanol (100%, 95%, and 70%) and PBS. After antigen retrieval, endogenous peroxidase activity was blocked with 3% H2O2 for 20 min. Negative controls for on October 1, 2021 by guest. Protected copyright. immunostaining were performed with species-matched pre-immune serum in each run.

Immunofluorescence analyses Cells were plated on 8-well chamber slides incubated in the absence or presence of TGF-β1 (2 ng/ml) for 24 h, and fixed with ice-cold methanol. After blocking nonspecific binding sites with 5% FBS in PBS, cells were incubated with primary antibodies at 4 °C overnight, washed 3X in PBS and incubated with FITC or Alexa Fluor 546 (Zymed and Molecular Probes, Eugene, USA respectively). Nuclei were visualized by 4’6-diamidino-2-phenylindole staining (DAPI; Roche Diagnostics, Basel, Switzerland). The staining was analyzed by deconvolution fluorescence microscope (Leica Microsystems, Bensheim, Germany). For quantification of αSMA-positive cells, 10 randomly-chosen fields of αSMA and DAPI stained cells were counted. Ratio of total αSMA positive cells and total DAPI stained cells was calculated. Data represents the mean ± SD (n= 10) and statistics was performed by two-tailed Student’s t-test, with a p-value of <0.001 regarded as significant. Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from Laser-assisted microdissection In brief, 10 μm cryosections were mounted on glass slides, stained with hemalaun for 45 s, immersed in 70% and 96% ethanol, and stored in 100% ethanol until use. Alveolar septae were selected and microdissected with a sterile 30 G needle under optical control using the Laser Microbeam System (PALM, Bernried, Germany). Microdissected tissues were transferred into reaction tubes containing 200 l RNA lysis buffer and samples were processed for RNA analysis.

Table 1: Human RT-PCR primers

Gene Bank Annealing Cycle Amplicon Accession Forward primer (5´- 3´) Reverse primer (5´- 3´) Temp. Number Size (bp) Number (°C)

SNAI1 TTTACCTTCCAGCAG TGACATCTGAGTGGG 55 28 616 NM_005985 CCCTA TCTGG SNAI2 CCATGCCTGTCATAC TTGGAGCAGTTTTTG 55 28 631 NM_003068 CACAA CACTG αSMA AGTTATGGTGGGTAT GAGGGAAGGTGGTTT 62 30 1000 NM_001613 GGGTCAGAA GGGAGA VIM CGAAAACACCTGCA TCCAGCAGCTTCCTG 55 28 693 NM_003380 ATCTT TAGGT ECAD GGTTCAAGCTGCTGA CTCAAAATCCTCCCT 55 28 631 NM_004351 CCTTC GTCCA OCCL TATGGAGGAAGTGGC TCATTCACTTTGCCA 62 30 683 NM_002538 TTTGG TTGGA HSPA8 TTACCCGTCCCCGATT TGTGTCTGCTTGGTA 55 22 330 NM_006597 TGAAGAAC GGAATGGTGGTA http://thorax.bmj.com/

Table 2: Mouse RT-PCR primers

Gene Bank Annealing Cycle Amplicon Accession Forward primer (5´- 3´) Reverse primer (5´- 3´) Temp. Number Size (bp)

Smad2 CTCCGGCTGAACTGT TTACAGCCTGGTGGG 60 25 409 NM_010754 CTCCTACT ATCTTACA

Smad3 AGAACGGGCAGGAG GGATTCGGGGAGAGG 60 25 565 NM_016769 GAGAAGTGGT TTTGGAGA

Smad4 ACAGAGAACATTGGA AGTAGCTGGCTGAGC 55 28 500 NM_008540 TGGAC AGTAA

Smad6 GAGCACCCCCATCTT AACAGGGGCAGGAGG 60 25 234 NM_008542 CGTCAA TGATG

Smad7 CCTCCTCCTTACTCC ACGCACCAGTGTGAC 60 28 166 NM_001042660 AGATA CGATC

ALK1 AGGGCCGATATGGTG GCCGGTTAGGGATGG 58 24 735 NM_009612 AGGTGTGG TGGGTGTC

ALK5 AGAGCGTTCATGGTT GGGGCCATGTACCTT 429 59 25 NM_009370 CCGAGAG TTAGTGC

2 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from TβrII GAGAGGGCGAGGGCG GTGGTAGGTGAGCTT 410/500 60 24 NM_031132 AGGAGTAAAGG GGGGT

Snai1 CACCCTCATCTGGGA GCCAGACTCTTGGTG 604 58 30 NM_011427 CTCTC CTTGT

Snai2 AACATTTCAACGCCT CAGTGAGGGCAAGAG 631 58 32 NM_011415 CCAAG AAAGG

αSMA CTGACAGAGGCACCA CTTCTGCATCCTGTC 490 60 25 NM_007392 CTGAA AGCAA

Vim CGCAGCCTCTATTCC AGCCACGCTTTCATA 693 58 30 NM_0117013 TCATC CTGCT eCad AGTTTACCCAGCCGG AGGGTTCCTCGTTCT 602 58 30 NM_009864 TCTTT CCACT

Occl GCTCTCTCAGCCAGC AATCATGAACCCCAG 640 58 30 NM_008756 GTACT GACAA

Gapdh ACACATTGGGGGTAG AACTTTGGCATTGGA 225 60 21 NM_008084 GAACA AGG

Table 3: Human quantitative RT-PCR primers

Gene Bank Forward primer (5´- 3´) Reverse primer (5´- 3´) Accession Number

SNAI1 TGGGCGCTCCGTAAA ACGAGGGAAACGCAC NM_005985 CAC ATCA SNAI2 GGCAAGGCGTTTTCC CTCTGTTGCAGTGAG NM_003068 AGAC GGCAA αSMA CGAGATCTCACTGAC AGAGCTACATAACAC http://thorax.bmj.com/ NM_001613 TACCTCATGA AGTTTCTCCTTGA VIM GAGAACTTTGCCGTT TCCAGCAGCTTCCTG NM_003380 GAAGC TAGGT ECAD ATACACTCTCTTCTC ATACACTCTCTTCTC NM_004351 TCACGCTGTGT TCACGCTGTGT OCCL GCCGAGGAGCCGGTC CAGGATGAGCAATGC

NM_002538 TAG CCTTT on October 1, 2021 by guest. Protected copyright. TJP1 GAGGAAACAGCTATA TGACGTTTCCCCACT NM_003257 TGGGAACAAC CTGAAA PBGD CCCACGCGAATCACT TGTCTGGTAACGGCA NM_000190 CTCAT ATGCG

Table 4: Mouse quantitative RT-PCR primers

Gene Bank Forward primer (5´- 3´) Reverse primer (5´- 3´) Accession Number

Snai1 AGCCCAACTATAGCG GGGGTACCAGGAGAG NM_011427 AGCTG AGTCC Snai2 GAAGCCCAACTACAG AGGAGAGTGGAGTGG NM_011415 CGAAC AGCTG

3 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from αSMA GCTGGTGATGATGCT GCCCATTCCAACCAT NM_007392 CCCA TACTCC

Vim TGAAGGAAGAGATGG TCCAGCAGCTTCCTG NM_0117013 CTCGT TAGGT eCad CCATCCTCGGAATCC TTTGACCACCGTTCT NM_009864 TTGG CCTCC Occl CCGCCAAGGTTCGCT TCAGGTCTGTAAGGA NM_008756 TATC GGTGGACTT Tjp1 ACTATGACCATCGCC GGGGATGCTGATTCT NM_009386 TACGG CAAAA Pbgd GGTACAAGGCTTTCA ATGTCCGGTAACGGC NM_001110251 CGATCGC GGC

ONLINE SUPPLEMENT: FIGURE LEGENDS

Figure E1: Gene expression patterns of TGF-β signaling molecules in primary ATII cells. (A) Expression and localisation of pro-SPC, TJP1 and αSMA in freshly isolated mouse ATII cells and two and three days after plating was determined by immunofluorescence analysis. Original magnification is 20×. (B) Expression of TGF-β1 signalling components in ATII cells. Expression analysis of Smad2-4, Smad6-7, ALK1, ALK5, TβRII, and Gapdh was performed by RT-PCR of whole lung RNA derived from two different mice (lanes 1, 2), and from two different primary ATII cell preparations (lanes 3, 4). TβRII amplification reveals the two known isoforms of this receptor. Product sizes are indicated on the right.

Figure E2: TGF-β1 dose dependent EMT in ATII cells. The qRT-PCR analysis shows expression level of ECAD and αSMA upon treatment with increasing doses of TGF-β1 as http://thorax.bmj.com/ indicated for 24 h. Data are representative of three independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n= 3.

Figure E3: SNAI1 and SNAI2 localization in A549 and ATII cells. Immunofluorescence analysis shows double staining of SNAI1 and SNAI2 (green) with nuclear DAPI (blue) in both the cell types. Original magnification is 40×.

on October 1, 2021 by guest. Protected copyright. Figure E4: Transfection efficiency of A549 cells. Cells were seeded in an eight-well chamber slide and transiently transfected with EGFP for 24 h. Nuclei are visible by DAPI staining. Original magnification is 20×. Results are representative of three independent experiments. Figure E5: Densitometric analysis of SNAI1 (A) and SNAI2 (B) overexpression in A549 cells. Data are representative of three independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n= 3.

Figure E6: siRNA-mediated downregulation of SNAI1 and SNAI2 expression in A549 cells. The mRNA knockdown of four different SNAI1-si#1-#4 (A and B) and SNAI2-si#1-#4 (C and D) siRNA oligonucleotides was assayed by quantitative and semi-quantitative RT-PCR. In A,C the relative expression of SNAI1 and SNAI2 is analysed against PBGD and expressed as log-fold change. In B,D HSPA8 was used as the loading control. Data are representative of three independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n= 3.

4 Thorax: first published as 10.1136/thx.2009.121798 on 22 October 2009. Downloaded from Figure E7: TGF-β1 dose dependent migration of A549 cells. A549 cells where treated for 24 h with or without TGF-β1 (0.02, 0.2, 2 and 20ng/ml) The relative migration potential of A549 cells was assessed using Boyden chamber assay as described in Material and Methods. Data are representative of three independent experiments and are expressed as mean ± SEM; ∗ p<0.05, n=3.