Scholarly article on topic 'Bleomycin induced epithelial–mesenchymal transition (EMT) in pleural mesothelial cells'

Bleomycin induced epithelial–mesenchymal transition (EMT) in pleural mesothelial cells Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Li-Jun Chen, Hong Ye, Qian Zhang, Feng-Zhi Li, Lin-Jie Song, et al.

Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis. Recent studies revealed that pleural mesothelial cells (PMCs) undergo epithelial–mesenchymal transition (EMT) and play a pivotal role in IPF. In animal model, bleomycin induces pulmonary fibrosis exhibiting subpleural fibrosis similar to what is seen in human IPF. It is not known yet whether bleomycin induces EMT in PMCs. In the present study, PMCs were cultured and treated with bleomycin. The protein levels of collagen-I, mesenchymal phenotypic markers (vimentin and α-smooth muscle actin), and epithelial phenotypic markers (cytokeratin-8 and E-cadherin) were measured by Western blot. PMC migration was evaluated using wound-healing assay of culture PMCs in vitro, and in vivo by monitoring the localization of PMC marker, calretinin, in the lung sections of bleomycin-induced lung fibrosis. The results showed that bleomycin induced increases in collagen-I synthesis in PMC. Bleomycin induced significant increases in mesenchymal phenotypic markers and decreases in epithelial phenotypic markers in PMC, and promoted PMC migration in vitro and in vivo. Moreover, TGF-β1-Smad2/3 signaling pathway involved in the EMT of PMC was demonstrated. Taken together, our results indicate that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis.

Academic research paper on topic "Bleomycin induced epithelial–mesenchymal transition (EMT) in pleural mesothelial cells"

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Toxicology and Applied Pharmacology

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Bleomycin induced epithelial-mesenchymal transition (EMT) in pleural mesothelial cells

Li-Jun Chen a,1 Hong Ye b,c'\ Qian Zhang a, Feng-Zhi Lia, Lin-Jie Song a, Jie Yang a, Qing Mu a, Shan-Shan Rao b, Peng-Cheng Caid, Fei Xiang a,c, Jian-Chu Zhang a,c, Yunchao Su e, Jian-Bao Xin a,c* Wan-Li Ma a,c'*

a Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China b Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China c Key Laboratory of Pulmonary Diseases, Ministry of Health of China, Wuhan, Hubei, China

d Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China e Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA, USA

ARTICLE INFO ABSTRACT

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis. Recent studies revealed that pleural mesothelial cells (PMCs) undergo epithelial-mesenchymal transition (EMT) and play a pivotal role in IPF. In animal model, bleomycin induces pulmonary fibrosis exhibiting subpleural fibrosis similar to what is seen in human IPF. It is not known yet whether bleomycin induces EMT in PMCs. In the present study, PMCs were cultured and treated with bleomycin. The protein levels of collagen-I, mesenchymal phenotypic markers (vimentin and a-smooth muscle actin), and epithelial phenotypic markers (cytokeratin-8 and E-cadherin) were measured by Western blot. PMC migration was evaluated using wound-healing assay of culture PMCs in vitro, and in vivo by monitoring the localization of PMC marker, calretinin, in the lung sections of bleomycin-induced lung fibrosis. The results showed that bleomycin induced increases in collagen-I synthesis in PMC. Bleomycin induced significant increases in mesenchymal phenotypic markers and decreases in epithelial phenotypic markers in PMC, and promoted PMC migration in vitro and in vivo. Moreover, TGF-|1 -Smad2/3 signaling pathway involved in the EMT of PMC was demonstrated. Taken together, our results indicate that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis.

© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Article history: Received 25 October 2014 Revised 22 December 2014 Accepted 5January2015 Available online 14 January 2015

Keywords: Bleomycin

Epithelial-mesenchymal transition (EMT)

Pleural mesothelial cell

Fibrosis

Collagen

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown cause that typically leads to respiratory failure and death within 3-5 years of diagnosis (King et al., 2011). There are no effective pharmacological therapies yet for IPF. The pathological feature of IPF includes fibroproliferative foci, depositions of extracellular matrix (ECM) including collagen and alveolar collapse. Interestingly, these his-topathological alterations are predominantly located in subpleural areas (Raghu et al., 2011). The cause for the subpleural distribution of fibrotic plague in IPF is not known. Recently, Mubarak et al. (2012) reported that pleural mesothelial cells (PMCs) trafficking into lung parenchyma contributed to the subpleural fibrotic foci in idiopathic pulmonary

Abbreviations: IPF, idiopathic pulmonary fibrosis; PMC, pleural mesothelial cell; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix; AEC, alveolar epithelial cell; a-SMA, a-smooth muscle actin.

* Corresponding authors at: 1277 Jie Fang Da Dao, Wuhan, Hubei 430022, China. Fax: +86 27 85726527.

E-mail addresses: 814643835@qq.com (J.-B. Xin), whmawl@aliyun.com (W.-L. Ma). 1 These authors contributed equally to this manuscript.

fibrosis. Nevertheless, it remains unclear how PMCs contribute to the pathogenesis of subpleural pulmonary fibrosis.

In IPF, myofibroblasts are hallmark and prominent components of lung tissue fibrosis. They may be derived from several sources of cells which undergo epithelial-mesenchymal transition (EMT). Myofibroblasts in pulmonary fibrosis have been shown to arise from resident pulmonary fibroblasts or blood-borne fibrocytes through transdifferentiation (Hashimoto et al., 2004). Alveolar epithelial cells (AECs) may undergo transdifferentiation and turn into myofibroblasts (Willis et al., 2005). PMCs can undergo EMT and play a pivotal role in IPF (Decologne et al., 2010; Mubarak et al., 2012; Zolak et al., 2013). PMCs undergoing EMT express distinct mesenchymal markers, lose epithelial markers and apicobasal polarity as well as intercellular junctions. Changes in cell polarity and adhesion help epithelial cells disrupt basement membrane and penetrate into the ECM-rich compartment (Lim and Thiery, 2012).

Bleomycin is widely used as an inducer in the animal models of pulmonary fibrosis. Very interestingly, lung fibrosis induced by bleomycin delivered to animals via different routes has different pattern of foci distributions. For example, lung fibrosis induced by intratracheal

http: //dx.doi.org/10.1016/j.taap.2015.01.004

0041-008X/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

administration of single dose bleomycin is predominantly around the bronchus and is self-limiting (Chung et al., 2003; Moore and Hogaboam, 2008). However, lung fibrosis induced by multiple doses of bleomycin by intraperitoneal injection exhibits a more chronic fibrotic state and subpleural fibrotic distribution similar to what is seen in human IPF (Mouratis and Aidinis, 2011). It has been reported that PMCs are more susceptible to H2O2- and SiO2-induced oxidative stress and damage than lung epithelial cells (Berg et al., 2013). These studies suggest that PMCs should be more sensitive to the toxicity of bleomycin than other lung cells. Nevertheless, it remains unknown whether bleomycin can induce EMT in PMC. In the present study, we used cell culture and animal models and studied the effects of bleomycin on EMT in PMC. We found, for the first time, that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis.

Materials and methods

Materials and reagents. TGF-(31 was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kit for TGF-(31 and TGF-(31 receptor inhibitor (SB431542) were obtained from R&D Systems (Minneapolis, MN, USA). Anti-collagen-I antibody was obtained from Novus Biologicals (Littleton, CO, USA). Antibodies against GAPDH and total-Smad2/3 (t-Smad2/3) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against vimentin, a-smooth muscle actin (aSMA), cytokeratin-8, and E-cadherin were purchased from Epitomics (Burlingame, CA, USA). Antibody against calretinin was purchased from BD Transduction Laboratories (Lexington, KY, USA). Antibody against phosphorylated-Smad2/3 (p-Smad2/3) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Bromodeoxyuridine (BrdU) kit was obtained from Roche (Mannheim, Germany). Belomycin was obtained from Tianjin Taihe Pharmaceutical Co., Ltd (Tianjin, China).

PMC culture and treatments. Human PMC line (MeT-5A) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The PMCs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, Logan, UT, USA) supplemented with 20% fetal calf serum and 5% CO2, 95% air at 37 °C. The cells were 1:3 subcultured when cells grow to confluence, and culture medium was changed every 2 days. The cells equilibrated in 2% serum medium overnight were used for all experiments. PMCs were incubated with different concentrations ofbleomycin (0.1-2 ^g/ml) for 12-72 h.

Western blot analysis. EMT was monitored by measuring the protein contents of collagen-I, vimentin, a-SMA, cytokeratin-8, and E-cadherin, which are commonly used molecular markers for EMT. The samples (10-20 |ag of protein) of cell lysates were denatured and electrophoresed on SDS-PAGE gel. Separated proteins were electro-transferred to nitrocellulose membranes. The membranes were incubated with 5% fat-free milk for 1 h, and then incubated with antibodies against collagen-I, vimentin, a-SMA, cytokeratin-8, E-cadherin, or GAPDH overnight at 4 °C, and then washed with 0.1% Tween-20, 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl (TTBS) three times for 10 min. Secondary antibody IgG conjugated to alkaline phosphatase was diluted in TTBS plus 5% nonfat milk and incubated with the membranes at room temperature for 1 h. The membranes were washed with TTBS and then developed with Supersignal West Pico (Pierce, Rockford, IL, USA) for 3 min before exposure to film (Kodak, Rochester, NY, USA). Films were developed using Kodak Medical X-ray processor 102 (Kodak, Rochester, NY, USA) to visualize the reactive proteins followed by densitometric quantification using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).

immunofluorescence staining of PMC. To determine the intracellular localization and changes of vimentin, a-SMA, cytokeratin-8, and E-cadherin, PMCs were incubated with bleomycin (0.2 ^g/ml) for

72 h. Then the cells were stained using antibodies against vimentin, a-SMA, cytokeratin-8, or E-cadherin overnight at 4 °C, and then with tetramethyl rhodamin isothiocyanate (TRITC)-conjugated goat anti-mouse, or goat anti-rabbit antibody for 30 min. The nuclei were stained for DAPI for 10 minutes in dark. The fluorescence-labeled cells were examined using a fluorescent microscope (Olympus FV500, Olympus, Tokyo, Japan).

¡n vitro wound-healing assay of PMC migration. Confluent monolayers of PMCs in 6-well plates were scratched using the tip of a p-200 pipette to create a uniform cell-free zone in each well. Cellular debris was removed by washing with PBS. Wounded monolayers were then incubated in the presence or absence ofbleomycin. Microscopic images were taken with a digital camera at 12 h, 24 h, and 48 h after wounding. The recovered area was measured with a computerassisted image analysis system (Image-Pro Plus software) and expressed as a percentage of the initial scratched area. At the same time, morphologic changes in PMCs were observed by microscopy when the cells were treated by bleomycin for 12 h, 24 h, and 48 h, respectively.

Cell proliferation assay. Proliferation of PMCs was assayed using a kit from Roche that monitors the incorporation of BrdU into newly synthesized DNA. The BrdU was detected using anti-BrdU-peroxidase conjugated in accordance with the manufacturer's instructions. After reactions were stopped, the absorbance at 450 nm was measured using a Synergy 2 Multi-Mode microplate reader (Biotek Instruments, Winooski, VT, USA).

Pulmonary fibrosis animal model. Male C57BL/6 mice with ages of 12 weeks were purchased from Animal Center at Wuhan University. The study protocol was approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Pulmonary fibrosis was induced by bleomycin as previously described with minor modification (Savani et al., 2000). Briefly, C57BL/6 mice (16-20 g; n = 5 per group) were housed under standard conditions with free access to water and rodent laboratory food. Bleomycin dissolved in saline solution (concentration, 5 mg/ml) was administered by intraperitoneal injection at a dose of 50 mg/kg at 1,5,8,11, and 15 days, respectively. In the control group, saline solution without bleomycin was administered by intraperitoneal injection at the same time. Mice were euthanized at 40 days, and then lung tissues were taken for histological analysis.

Histological analysis. Mice lungs were removed and fixed in 4% paraformaldehyde for 24 h. The fixed lungs were then sliced midsagittally and embedded in paraffin. The slides of 7 |am thickness were stained with Masson staining for morphological analysis and evaluation of collagen deposition. The slides were examined by using a microscope connected with a digital camera and Image-Pro Plus software was used to analyze the images.

¡mmunofluorescence staining of lung tissue. The localization of calretinin, a specific PMC marker, in lung sections of bleomycin-induced lung fibrosis was monitored as an indication of PMC migration in vivo. Frozen mouse lung sections were fixed with 4% parafor-maldehyde for 10 min at room temperature. The slides were incubated with mouse anti-calretinin antibody (1:50 dilution) for 1 h and then with tetramethyl rhodamin isothiocyanate (TRITC)-conjugated goat anti-mouse antibody for 30 min. The nuclei were stained for DAPI for 10 minutes in dark. The slides were mounted with anti-fade reagent and examined using a fluorescent microscope (Olympus FV500, Olympus, Tokyo, Japan). The p-Smad2/3 protein was also stained using same method. The goat anti-p-Smad2/3 antibody dilution was 1: 50, and the second antibody was fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat antibody.

Fig. 1. TGF-ß1 induces EMT in human PMC line, MeT-5A cell. PMCs were incubated with TGF-ß1 (5 ng/ml) for 24 h, after which intracellular protein contents of collagen-I, vimentin, aSMA, cytokeratin-8 and E-cadherin were measured by Western blot as described in the methods. (A) Showing the image of immunoblots of collagen-I, vimentin, a-SMA, cytokeratin-8, E-cadherin and GAPDH proteins from PMCs treated with or without TGF-ß1. (B) Bar graphs depicting changes in relative density of collagen-I (n = 4), vimentin (n = 7), a-SMA (n = 4), cytokeratin-8 (n = 6), and E-cadherin (n = 7) proteins. The density values of blots have been normalized to the control value. *P < 0.05 versus control.

ELiSAfor TGF-fillevel in PMC. TGF-(311evels in PMCs were measured using ELISA kit from R&D Systems (Minneapolis, MN, USA). The procedures were performed according to manufacturer's instructions.

Statistical analysis. In each experiment, experimental and control cells were matched for cell line, age, seeding density, number of

passages, and number of days postconfluence to avoid variation in tissue culture factors that can influence the result. Results are shown as the mean ± SD for n experiments. Differences between groups were analyzed using unpaired t tests or two-way analysis of variance. P value less than 0.05 was considered to be statistically significant.

Fig. 2. Effect ofbleomycin on collagen-I synthesis in PMCs. PMCs were incubated with different concentration ofbleomycin (0-2 |g/ml) for 24 h or 0.2 |g/ml bleomycin for 24 h, 48 h, and 72 h, after which intracellular collagen-I protein contents were measured by Western blot as described in the methods. (A) Showing the image of immunoblots of col-lagen-I, and GAPDH proteins from PMCs treated with different concentration ofbleomycin (0-2 |g/ml). (B) Bar graphs depicting changes in relative density of collagen-I protein. n = 3, *P < 0.05 versus control. (C) Showing a representative image of immunoblots of collagen-I proteins from four separate experiments in PMCs treated with 0.2 |g/ml bleomycin for 24 h, 48 h, and 72 h. (D) Bar graphs depicting changes in relative density of collagen-I protein. n = 4, *P < 0.05 versus control. (B and D) The density values of blots have been normalized to the control value.

Results

TGF-ß1 induces EMT in human PMC line, MeT-5A cell

MeT-5A cell is a human PMC line. To understand the properties of MeT-5A cell and confirm that EMT can be induced in MeT-5A cell, TGF-ß1 was used in cells culture. After treating MeT-5A cells with TGF-ß1 (5 ng/ml) for 24 h, we found that protein levels of collagen-I, vimentin, and a-SMA in MeT-5A cells were increased (Fig. 1). On the contrary, protein levels of cytokeratin-8 and E-cadherin in MeT-5A cells were decreased (Fig. 1). These data suggested that human PMC line, MeT-5A cell can be induced undergoing EMT process.

Bleomycin induces increases in collagen-I synthesis in PMCs

To investigate the effects of bleomycin on collagen-I synthesis in PMCs, collagen-I protein in PMCs was measured by Western blot. We found that incubation of PMCs with bleomycin (0.1-2 ^g/ml) for 24 h caused a dose-dependent increase in the protein level of collagen-I (Figs. 2A and B). PMCs were then incubated with 0.2 ^g/ml bleomycin for 24 h, 48 h, and 72 h. We found that bleomycin-induced increase in the protein level of collagen-I occurred as early as 24 h and continued as long as 72 h (Figs. 2C and D).

for 24-72 h. Mesenchymal phenotypic marker proteins, vimentin and a-SMA, were measured by Western blot. As shown in Figs. 3A, B, D, and E, incubation of PMCs with bleomycin for 24-72 h caused significant increases in protein levels of vimentin and a-SMA. To show the expression of vimentin and a-SMA in situ, immunofluorescence staining was done after cells were treated with bleomycin (0.2 |ag/ml) for 72 h. As shown in Figs. 3C and F, expressions of vimentin and a-SMA were much stronger in bleomycin treated cells than in control cells. These results suggest that bleomycin induced PMC transformation into fibroblasts or myofibroblasts.

Bleomycin induces decreases in cytokeratin-8 and E-cadherin protein in PMCs

To further confirm mesenchymal phenotypic transformation induced by bleomycin in PMCs, epithelial phenotypic marker proteins, cytokeratin-8 and E-cadherin, were monitored by Western blot and immunofluorescence staining. As shown in Fig. 4, incubation of PMCs with bleomycin resulted in decreases in protein levels of cytokeratin-8 and E-cadherin, suggesting that bleomycin-treated PMCs lose the epithelial phenotypic features.

Bleomycin increases cell migration of PMCs

Bleomycin induces increases in vimentin and a-SMA protein in PMCs

To confirm the mesenchymal phenotypic transformation in PMCs, cells were incubated with or without bleomycin (0.2 ^g/ml)

Increased motility is one of the characteristics of cells undergoing EMT. To determine whether bleomycin induces an increase in PMC motility, the migration of PMCs was assessed using an in vitro wound-healing assay on 6-well plates. As shown in

Fig. 3. Effect of bleomycin on vimentin and a-SMA protein in PMCs. PMCs were incubated with bleomycin (0.2 ng/ml) for 24 h, 48 h, and 72 h, after which intracellular vimentin and a-SMA protein contents were measured by Western blot as described in the methods. Immunofluorescence staining of vimentin and a-SMA in situ was done after cells treated with bleomycin (0.2 ng/ml) for 72 h. (A) is a representative image of immunoblots of vimentin protein. (B) is bar graphs depicting changes in relative density of vimentin protein according to A. (C) is a representative image of immunofluorescence staining of vimentin. (D) is a representative image of immunoblots of a-SMA protein. (E) Bar graphs depicting changes in relative density of a-SMA protein according to D. (F) is a representative image of immunofluorescence staining of a-SMA. (B and E) The density values of blots have been normalized to the control value. n = 4, *P < 0.05 versus control.

Fig. 4. Effect ofbleomycin on cytokeratin-8 and E-cadherin protein in PMCs. PMCs were incubated with bleomycin (0.2 |g/ml) for 24 h, 48 h, and 72 h, after which intracellular cytokeratin-8 and E-cadherin protein contents were measured by Western blot. Immunofluorescence staining of cytokeratin-8 and E-cadherin in situ was done after cells treated with bleomycin (0.2 |og/ml) for 72 h. (A) is a representative image of immunoblots of cytokeratin-8 protein. (B) is bar graph depicting changes in relative density of cytokeratin-8 according to A. (C) is a representative image of immunofluorescence staining of cytokeratin-8. (D) is a representative image of immunoblots of E-cadherin protein. (E) is bar graph depicting changes in relative density of E-cadherin according to D. (F) is a representative image of immunofluorescence staining of E-cadherin. (B, E) The density values of blots have been normalized to the control value. n = 4, *P < 0.05 versus control.

Figs. 5A and B, incubation of PMCs with bleomycin (0.2 ^g/ml) caused an increase in PMC migration. Recovered area was about 40% in bleomycin treated group compared with 25% in control group at 12 h. It was about 70% in bleomycin treated group compared with 40% in control group at 24 h. The space of wound was completely filled with migrating PMCs after incubation with bleomycin for 48 h. To distinguish cell migration from cell proliferation, cell proliferation was detected by using a BrdU incorporation assay. Bleomycin did not change cell proliferation in PMCs at 12 h, and 48 h, slightly induced cell proliferation at 24 h (Fig. 5D). These data suggest that bleomycin induces an increase in PMC migration rather than cell proliferation.

Bleomycin causes changes in morphology of PMCs into fibroblasts-like

In the experiments of in vitro wound-healing assay, morphologic changes of PMCs were documented by using microscope at the same time. As shown in Fig. 5C, PMCs cultured in the absence of bleomycin (control) showed epithelial morphology of typical polygonal and cobblestone monolayer. PMCs treated with bleomycin for 12-48 h showed remarkable phenotypic changes (Figs. 5C-f, g, and h). Bleomycin-treated PMCs showed elongated and spindle shaped morphology of fibroblasts-like feature, also suggesting that bleomycin induced mesenchymal phenotypic transformation in PMCs.

PMCs migrate into the fibrotic lung parenchyma in bleomycin-induced pulmonary fibrosis model

We further evaluated bleomycin-induced PMC migration in vivo by monitoring location of calretinin, a marker of mesothelial cells in mouse lungs in a pulmonary fibrosis model. Forty days after first administration ofbleomycin, lung sections from control mice and mice receiving bleomycin were subjected to immunofluorescence staining for calretinin. As shown in Figs. 6B, C, and D, there were abundant collagen fibers deposited in the fibrotic plague in subpleural lung tissue (Masson staining, blue color). In the control mice, as there is no adhesion of pleura with lung parenchyma, so when the lung was taken from the thorax, the pleural membrane was separated from the lung surface. Thus, there was no pleura in the normal lung tissue section, calretinin staining was negative (Fig. 6E). In fibrotic lungs, calretinin protein was expressed in the pleura and parenchyma (Figs. 6F, G, and H), indicating that PMCs migrate into lung parenchyma in bleomycin-induced pulmonary fibrosis.

TGF-fil-Smad2/3 signaling pathway is involved in the EMT of PMC

To know whether TGF-(31-Smad2/3 signaling pathway is involved in the EMT of PMC, we detected TGF-(31 in PMCs by using ELISA kit, and investigated p-Smad2/3 by using Western blot. Furthermore, we did im-munofluorescence staining of p-Smad2/3 in the mouse lung sections.

Fig. 5. Effect of bleomycin on in vitro PMC migration. PMCs were subcultured in the 6-well plate. Confluent monolayers of PMCs were scratched with the tip of a p-200 pipette to create a uniform cell-free zone in each well. Wounded monolayers were then incubated in the presence or absence of bleomycin (0.2 ng/ml). Microscopy pictures were taken with a digital camera at different time points (12 h, 24 h, and 48 h) after scratching. The recovered area was measured with a computer-assisted image analysis system and expressed as a percentage of the initial scratched area. (A) is representative image of PMC migration from three separate experiments. (B) is line graph depicting changes of recovered area in percentage of the initial scratched area. n = 3, *P < 0.05 versus control. (C) is one part of image from A to show morphologic changes of PMCs. (D) is bar graph depicting changes of cell proliferation. The values have been normalized to the control value. n = 20, *P < 0.05 versus control.

We found that bleomycin induced increases in TGF-(31 levels and p-Smad2/3 in PMCs (Figs. 7A and B). In the animal model, the protein levels of p-Smad2/3 were also dramatically increased in the mouse lungs of bleomycin-induced pulmonary fibrosis (Fig. 7C, green color is p-Smad2/3 staining). Then we used TGF-(31 receptor inhibitor, SB431542 to block the TGF-(31-Smad2/3 pathway, and investigated changes of EMT marker proteins in PMCs. As shown in Figs. 7D and E, SB431542 prevented increases of vimentin and decreases of cytokeratin-8 which induced by bleomycin. Taken together, these data suggested that the TGF-(31-Smad2/3 signaling pathway is involved in EMT induced by bleomycin in PMC.

Discussion

According to the description about MeT-5A cell line from ATCC, we know that the cells were isolated from pleural fluids obtained from

non-cancerous individuals. The cells were transfected with p-RSV-T plasmid (an SV40 ori-construct containing the SV40 early region and the Rous sarcoma virus long terminal repeat) and cloned. Previous publications have indicated that TGF-(31 induces EMT in PMC (Nasreen et al., 2009). In order to confirm that EMT can be induced in MeT-5A cell, TGF-(31 was used to treat the cells. Not surprisingly, TGF-(31 induced increases in collagen-1, vimentin, and a-SMA protein expression, and decreases in cytokeratin-8 and E-cadherin protein expression in MeT-5A cells. Thus, we used the MeT-5A cell line as a PMC model for EMT study.

In the present study, we confirmed for the first time that bleomycin induces EMT in PMC, and TGF-(3-Smad2/3 signaling pathway is involved in the underling mechanism. We found that bleomycin induced dose-and time-dependent increases in collagen-1 synthesis in PMCs. Bleomycin-treated PMCs have significantly higher protein levels of mesenchymal phenotypic markers, vimentin and a-SMA, and lower protein

Fig. 6. PMC migration in the flbrotic lungs from bleomycin-induced pulmonary fibrosis model. Pulmonary fibrosis was induced by multiple intraperitoneal injections of bleomycin in mice. At day 40 after first injection, mice were euthanized and lung tissues were taken. The lung sections were stained with Masson staining for morphological analysis. Calretinin was also stained for immunofluorescence. (A) is representative image of Masson staining of lung sections from control group. (B, C, and D) are representative images of Masson staining of lung sections from bleomycin-induced pulmonary fibrosis group. (E) is representative image of calretinin immunofluorescence staining of lung sections from control group. (F, G, and H) are representative images of calretinin immunoflu-orescence staining of lung sections from bleomycin-induced pulmonary fibrosis group. (IF: immunofluorescence; A-D: blue color is collagen; E-H: red color is calretinin, blue color is DAPI. n = 5).

level of epithelial phenotypic markers, cytokeratin-8 and E-cadherin. Moreover, bleomycin promoted PMC migration in vitro and in vivo. Bleomycin-treated PMCs became elongated and spindle shaped, showing characteristic morphology of fibroblasts or myofibroblasts. Lastly, we got evidences indicating that bleomycin induces characteristic changes of EMT through TGF-(31-Smad2/3 pathway in PMCs.

Bleomycin is a chemotherapeutic agent used in the treatment of a variety of tumors including lymphoma, squamous cell carcinoma and testicular carcinoma. Although bleomycin has been claimed to have minimal immunosuppressive activity and low hematopoietic

toxicity, chemotherapy using bleomycin is often complicated by interstitial pulmonary fibrosis (Kawai and Akaza, 2003; Kim et al., 2010). Thus, bleomycin has been widely used in making pulmonary fibrosis animal models (Lee et al., 2014). Repetitive administrations of bleomycin by intraperitoneal or intravenous injection induce pleural and subpleural fibrosis, which is similar to what is seen in human IPF (Mouratis and Aidinis, 2011). Thus, researchers consider that PMCs are more sensitive to the toxicity of bleomycin or profibrotic growth factors than other lung cells (Berg et al., 2013), which should be the mechanism of pleural and subpleural fibrosis. Vimentin is a major intermediate filament protein in mesenchymal cells (Eriksson et al., 2009). a-SMA is a marker of mesenchymal cells and mainly expressed in myofibroblasts (Kage and Borok, 2012). E-cadherin is a cellular junctional protein and maintains the epithelial integrity. Cytokeratin is a cytoskeletal intermediate filament protein, and there are about 20 subtypes expressed in various types of human epithelial cells (Yi and Ku, 2013). Both cytokeratin-8 and E-cadherin are epithelial phenotypic marker proteins in PMCs (Nasreen et al., 2009; Kim et al., 2011). In the current study, we found that bleomycin-treated PMCs progressively lose the expression of cytokeratin-8 and E-cadherin and gradually acquire increased expression of vimentin and a-SMA. This is direct evidence indicating that bleomycin induces EMT in PMCs.

After losing of the cellular junctions and changes in the cytoskel-eton, PMCs acquire increased ability for migration. Our finding shows that bleomycin-treated PMCs migrate much faster in an in vitro wound repair study. Calretinin is a marker of mesothelial cells (Kachali et al., 2006). After demonstrating that calretinin is stably expressed in bleomycin-treated PMCs (Supplemental Materials, Fig. S1), we detected the localization of calretinin in lung tissue, and found that calretinin is mainly located in the subpleural area of lung parenchyma in fibrotic lungs of mice receiving repetitive intraperitoneal injection of bleomycin. These data suggest that PMCs migrate into subpleural area of lung parenchyma. Moreover, our results indicate that bleomycin induced increases in collagen-I protein in PMCs. Increasing ability of collagen-I synthesis in PMC is not only a sign of EMT, but also a sign of remodeling disorder. Thus, PMC produces more collagen-I and then contributes to the deposition of extracellular matrix for subpleural fibrosis when the cell is undergoing EMT.

The mechanism for bleomycin-induced EMT of PMCs is not known. TGF-ß1-Smad2/3 signaling pathway is considered as a typical pathway in EMT (Chapman, 2011). We found that bleomycin induced increases in TGF-ß1 levels and p-Smad2/3 in PMCs, and TGF-ß1 receptor inhibitor prevented EMT in PMCs. We also found that p-Smad2/3 level is much higher in lungs of bleomycin-treated mice. These results indicate that bleomycin induces characteristic changes of EMT through TGF-ß1-Smad2/3 pathway in PMCs, and the latter contributes to subpleural fibrosis.

In summary, bleomycin induces an increase in collagen-I synthesis in PMCs. Bleomycin-treated PMCs acquire mesenchymal phenotypic markers and lose epithelial phenotypic markers and exhibit an increased motility. We conclude that bleomycin induces EMT in PMCs, which contribute to subpleural fibrosis.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2015.01.004.

Conflict of interest

The authors declare that there are no conflicts of interest. Acknowledgments

This work was supported in part by grants from National Nature Science Foundation of China (Nos. 31271490 and 81370186 to W.L.M.; No. 81200020 to P.C.C.; No. 81300047 to FX.; No. 81470257 toJ.B.X.), and in

Fig. 7. TGF-(J1-Smad2/3 signaling pathway is involved in the EMT of PMC. PMCs were incubated with bleomycin (0.2 |og/ml) for 48 h in the absence or presence of TGF-(J1 receptor inhibitor (SB431542), after which TGF-(J1 levels were detected by EL1SA, intracellular p-Smad2/3, t-Smad2/3, cytokeratin-8 and E-cadherin proteins were measured by Western blot. Immunofluorescence staining for p-Smad2/3 in mouse lung sections was done as described in the methods. (A) is bar graph depicting changes of TGF-(J1 levels in PMCs. n = 4, *P < 0.05 versus control. (B) is a representative image of immunoblots of p-Smad2/3 and t-Smad2/3, and a bar graph depicting changes in relative density of p-Smad2/3. The density values of blots have been normalized to the control value. n = 7, *P < 0.05 versus control. (C) is representative images of immunofluorescence staining of p-Smad2/3 in mouse lung sections. (n = 5, blue color is DAP1, green color is p-Smad2/3). (D) is a representative image of immunoblots of vimentin, and a bar graph depicting changes in relative density of vimentin protein. The density values of blots have been normalized to the control value. n = 4, *P < 0.05 versus control, #P < 0.05 versus bleomycin. (E) is a representative image of immunoblots of cytokertin-8, and a bar graph depicting changes in relative density of cytokertin-8 protein. The density values of blots have been normalized to the control value. n = 4, *P < 0.05 versus control, #P < 0.05 versus bleomycin.

part by a grant from the 12th Five-Year National Science and Technology Program of Social Development, Ministry of Science and Technology, China (No. 2012BA105B02 toJ.C.Z.).

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