Effects of Chrysotile Exposure in Human Bronchial Epithelial Cells ...

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Effects of Chrysotile Exposure in Human Bronchial Epithelial Cells: Insights into the Pathogenic Mechanisms of Asbestos-Related Diseases Giulia Rossana Gulino,1,2 Manuela Polimeni,1,2 Mauro Prato,3 Elena Gazzano,1,2 Joanna Kopecka,2 Sebastiano Colombatto,2 Dario Ghigo,1,2 and Elisabetta Aldieri 1,2 1Interdepartmental 2Department

Center for Studies on Asbestos and Other Toxic Particulates “G. Scansetti,” University of Torino, Torino, Italy; of Oncology, University of Torino, Torino, Italy; 3Department of Neurosciences, University of Torino, Torino, Italy

B ackground : Chrysotile asbestos accounts for > 90% of the asbestos used worldwide, and exposure is associated with asbestosis (asbestos-related fibrosis) and other malignancies; however, the molecular mechanisms involved are not fully understood. A common pathogenic mechanism for these malignancies is represented by epithelial–mesenchymal transition (EMT), through which epithelial cells undergo a morphological transformation to assume a mesenchymal phenotype. In the present work, we propose that chrysotile asbestos induces EMT through a mechanism involving a signaling pathway mediated by tranforming growth factor beta (TGF-β). Objectives: We investigated the role of chrysotile asbestos in inducing EMT in order to elucidate the molecular mechanisms involved in this event. Methods: Human bronchial epithelial cells (BEAS-2B) were incubated with 1 μg/cm2 chrysotile asbestos for ≤ 72 hr, and several markers of EMT were investigated. Experiments with specific inhibitors for TGF-β, glycogen synthase kinase–3β (GSK-3β), and Akt were performed to confirm their involvement in asbestos-induced EMT. Real-time polymerase chain reaction (PCR), Western blotting, and gelatin zymography were performed to detect mRNA and protein level changes for these markers. Results: Chrysotile asbestos activated a TGF-β–mediated signaling pathway, implicating the contributions of Akt, GSK-3β, and SNAIL-1. The activation of this pathway in BEAS-2B cells was associated with a decrease in epithelial markers (E-cadherin and β-catenin) and an increase in mesenchymal markers (α-smooth muscle actin, vimentin, metalloproteinases, and fibronectin). Conclusions: Our findings suggest that chrysotile asbestos induces EMT, a common event in asbestos-related diseases, at least in part by eliciting the TGF-β–mediated Akt/GSK-3β/ SNAIL-1 pathway. Citation: Gulino GR, Polimeni M, Prato M, Gazzano E, Kopecka J, Colombatto S, Ghigo D, Aldieri E. 2016. Effects of chrysotile exposure in human bronchial epithelial cells: insights into the pathogenic mechanisms of asbestos-related diseases. Environ Health Perspect 124:776–784; http://dx.doi.org/10.1289/ehp.1409627

Introduction Asbestos is a nonspecific term that is commonly used to describe any of six types of naturally occurring fibrous silicate minerals that were widely used commercially during the 20th century. Chrysotile asbestos is estimated to account for 90% of the asbestos used worldwide (Qi et al. 2013). Since the beginning of the 20th century, asbestos inhalation has been considered responsible for a number of lung diseases, such as asbestosis (asbestos-induced fibrosis), lung tumors, and malignant mesothelioma (MM) (Kamp 2009). Both fibrosis and epithelial tumors are highly dependent on the ability of epithelial cells to transform into mesenchymal cells through a process called epithelial–mesenchymal transition (EMT). EMT is both a physiological and pathological process: it has been related to early embryonic development and later organogenesis, as well as to wound healing in fibrotic tissues and to tumor development and metastasis in cancer (Kim et al. 2013; Kim and Cho 2014). During EMT, cell–cell adhesion molecules are inactivated and sometimes destroyed while cell–matrix adhesion increases. Cells undergoing EMT lose epithelial marker proteins, such as the adherent junction

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proteins E-cadherin and β-catenin and the tight junction protein zonula occludens, and begin to express mesenchymal proteins such as collagen, vimentin, α-smooth muscle actin (α-SMA), and fibronectin (Barrallo-Gimeno and Nieto 2005; Cannito et al. 2010; Moody et al. 2005; Peinado et al. 2004). These events lead to the acquisition of a fibroblast-like and spindle-shaped morphology, and cells acquire the capacity to degrade the basement membrane and migrate through the extracellular matrix to populate different territories during either embryonic development or cancer progression, or to adopt a profibrotic myofibroblast nature (Acloque et al. 2009; Cannito et al. 2010; Kalluri and Neilson 2003; Kalluri and Weinberg 2009; Moustakas and Heldin 2012). However, the spectrum of changes occurring during EMT may vary significantly depending on the epithelial cell type, the surrounding microenvironment and the type of inducer. Extracellular signals that can trigger EMT include growth factors such as transforming growth factor–β (TGF-β), hepatocyte growth factor, platelet-derived growth factor, fibroblast growth factor, and cytokines such as tumor necrosis factor–α (TNF-α) volume

(Cannito et al. 2010; Chen et al. 2014; Farrell et al. 2014; Moustakas and Heldin 2012). TGF-β is a multifunctional protein capable of regulating cell growth and differentiation as well as stimulating the production of extracellular matrix (Fine and Goldstein 1987). The different roles of TGF-β have been widely explored: TGF-β exerts its biological activity by regulating growth, differentiation, and epithelial transformation in the multistep processes of carcinogenesis, wound healing, and embryogenesis (Bhowmick et al. 2001; Perdue and Brody 1994). Many studies have investigated the effects of asbestos. Asbestos induces lung fibrosis via increased secretion of TGF-β (Manning et al. 2002), particularly in idiopathic pulmonary fibrosis where TGF-β has been localized in association with bronchiolar epithelial cells and their extracellular matrix (Liu and Brody 2001; Pociask et al. 2004). Casarsa et al. (2011) stressed the importance of EMT markers in MM prognosis. Qi et al. (2013) compared the toxicity of two different kinds of asbestos: chrysotile and crocidolite [an amphibole asbestos that is often considered the most oncogenic type of asbestos (Gibbs and Berry 2008)]. In their work, Qi et al. suggested that continuous exposure to crocidolite and chrysotile could cause EMT of human mesothelial cells via High Mobility Address correspondence to E. Aldieri, Department of Oncology, University of Torino, Torino, Italy. Telephone: 39-0116705844. E-mail: elisabetta.aldieri@ unito.it Supplemental Material is available online (http:// dx.doi.org/10.1289/ehp.1409627). We thank C. Costamagna for his technical assistance. G.R.G. is a recipient of a Ph.D. student fellowship from the Italian Ministry of University and Research; M.P. and M.P. were funded by Italian Ministry of University and Research fellowships; E.G. was funded by an Italian Ministry of University and Research (Future in Research FIRB 2012) fellowship; J.K. is a recipient of an FIRC (Fondazione Italiana per la Ricerca sul Cancro) fellowship. This research was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) funding to Chiara Riganti (grant number RIGC02 AP11). This paper is dedicated to the memory of D.G., who passed away on 7 October 2015. We all remember our friend as a master in life and science, and we hope he will continue to help us from the place where he is now. The authors declare they have no actual or potential competing financial interests. Received: 19 December 2014; Accepted: 30 November 2015; Advance Publication: 18 December 2015; Final Publication: 1 June 2016.

124 | number 6 | June 2016  •  Environmental Health Perspectives

Chrysotile effects on human epithelial cells

washed with 0.1% PBS-Tween and then were incubated for 1 hr with peroxidase-conjugated sheep anti-mouse or sheep anti-rabbit IgG antibody (Amersham International) diluted 1:3,000 in 0.1% PBS-Tween with 5% nonfat dry milk. The membranes were washed again with 0.1% PBS-Tween, and proteins were detected by enhanced chemiluminescence (Perkin Elmer). Anti-E-cadherin, β-catenin, tubulin, SNAIL-1, and TATA-binding protein (TBP) antibodies were all provided by Santa Cruz Biotechnology, Inc. Tubulin and TBP were used as loading controls for the cytosol and the nucleus, respectively. The anti-vimentin antibody was provided by Sigma Chemical Co. The anti-α-SMA antibody was from GeneTex. The anti-Smad2 and p-Smad2 ­antibodies were from Abcam.

Materials and Methods

or crocidolite (data not shown). As a consequence of these preliminary results, we chose to seed 3.5 × 10 5 or 1.5 × 10 5 BEAS-2B cells in 100-mm–diameter Petri dishes and incubate them for ≤ 72 hr or for 7 days, respectively, in the absence or presence of 1 μg/cm 2 chrysotile asbestos or 5 μg/cm 2 crocidolite. In the same manner, after preliminary experiments, 1.5 × 105 NuLi-1 cells were seeded in 60-mm–diameter Petri dishes and incubated for 96 hr in the absence or presence of 1 μg/cm2 chrysotile. In addition, 1.5 × 105 A549 cells were seeded in 100-mm–diameter Petri dishes and then incubated for 96 hr with 5 μg/cm2 chrysotile. The protein content in the cells was detected using a bicinchoninic acid assay (BCA) kit (Sigma Chemical Co.). The plasticware for cell culture was provided by Falcon (Corning Incorporated). Ultrapure water (Millipore) was used for all experiments.

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Cell Morphology

UICC (Union International Contre le Cancer) chrysotile and UICC crocidolite were sonicated (100 W, 30 sec, Labsonic Sonicator; Sartorius Stedim Biotech S.A.) before incubation with cell cultures to dissociate fiber bundles and to improve their suspension in the culture medium.

At the end of the incubation period, cells were observed using a light microscope, and images were obtained with the Leica Application Suite program (Leica Microsystems).

The neutralizing anti–TGF-β antibody was purchased from Abcam and was used at a concentration of 5 μg/mL; the GSK-3β inhibitor SB 216763 and the Akt 1/2 kinase inhibitor were from Sigma and were both used at a concentration of 5 μM.

Measurement of Reactive Oxygen Species (ROS)

Quantitative Real-Time PCR (qRT‑PCR)

BEAS-2B cells were incubated for 30 min or 1, 3 or 6 hr in the absence or presence of 1 μg/cm2 of chrysotile; then, cells were incubated for 30 min with 10 μM 2´,7´-dichloro­ dihydrofluorescein diacetate (DCFH-DA). DCFH-DA is a cell-permeable probe that is cleaved intracellularly by nonspecific esterases to form DCFH, which is further oxidized by ROS to form the fluorescent compound dichlorofluorescein (DCF) in a 1:1 stoichiometry (Bass et al. 1983). After incubation with DCFH-DA, the cells were washed twice with PBS to remove excess probe, and DCF fluorescence was determined using a Synergy HT microplate reader (BioTek Instruments) at an excitation wavelength of 504 nm and an emission wavelength of 529 nm. The fluorescence value was normalized to protein concentration and expressed as units of ­arbitrary fluorescence.

Total RNA was obtained by the guanidinium thiocyanate–phenol–chloroform method (Chomczynski and Sacchi 1987), using RiboZol RNA Extraction Reagents (Amresco) according to the manufacturer’s instructions. Total RNA (0.2 μg) was reverse-transcribed into cDNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories AG) according to the manufacturer’s instructions. qRT-PCR was performed using IQ™ SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions. PCR amplification was performed as follows: 1 cycle of denaturation at 94°C for 3 min, 45 cycles of denaturation at 94°C for 30 sec, annealing for 30 sec, and synthesis at 72°C for 30 sec. The relative expression of each target gene was determined by comparing each PCR gene product with the S14 ribosomal subunit product using the Gene Expression Macro (http://www3.bio-rad.com/LifeScience/ jobs/2004/04-0684/genex.xls; Bio-Rad).

Group Box 1 (HMGB1) and TNF-α signaling. In particular, the authors found that repeated exposure to chrysotile and crocidolite led to similar molecular changes and to similar amounts of HMGB1 secretion in vitro and in vivo, with differences in inducing MM-related biological alterations according to their bioper­sist­ence (Qi et al. 2013). For these reasons, interest in the role of asbestos as an inducer of EMT has recently increased. Given the strong association of chrysotile exposure with TGF-β activation (Murthy et al. 2015; Pociask et al. 2004), which is in turn associated with EMT induction, we investigated the role of chrysotile in inducing EMT via TGF-β in a human bronchial epithelial cellular model (BEAS-2B) to increase knowledge of the molecular bases of asbestos-related lung diseases.

Cell Cultures BEAS-2B cells (immortalized human bronchial epithelial cells) were obtained from American Type Culture Collection (ATCC). They were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human bronchial epithelial cells (NuLi-1) were a generous gift from C. Voena (Department of Molecular Biotechnology and Health Sciences, University of Torino). NuLi-1 cells were cultured in serum-free medium (Bronchial Epithelial Cell Growth Medium; Lonza) in Petri dishes that had been precoated with a 60 μg/mL solution of human placental collagen type IV at least 18 hr in advance, then air-dried and rinsed two to three times with phosphate-buffered saline (PBS). Human lung adenocarcinoma alveolar epithelial cells (A549) were provided by Istituto Zooprofilattico Sperimentale “Bruno Ubertini” (Brescia, Italy). The cells were cultured in Ham’s F12 medium (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. All cell cultures were kept in a humidified incubator at 37°C in a 5% CO2 atmosphere.

Experimental Conditions Dose- and time-dependence experiments were performed to determine the appropriate concentration and time, respectively, for incubating BEAS-2B cells with chrysotile

Western Blot Analysis Cytosolic and nuclear extracts were obtained using an Active Motif nuclear extraction kit (Active Motif) according to the manufacturer’s instructions. Cytosolic and nuclear extracts were separated by sodium dodecyl ­sulfate–­​­polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane sheets (Immobilon-P, Millipore) and probed with the required antibody diluted in 0.1% PBS-Tween with 5% nonfat dry milk. After 1 hr of incubation, the membranes were

Environmental Health Perspectives  •  volume 124 | number 6 | June 2016

Specific Inhibitors

Quantification of TGF-β Secretion by ELISA After incubating BEAS-2B cells in the absence or presence of chrysotile, the extra­cellular medium was collected and centrifuged at 4°C at 13,000 × g for 30 min. To determine the concentration of TGF-β in the supernatant, ELISA was performed according to the manufacturer’s instructions (Invitrogen Corporation). Absorbance was measured at 450 nm with a Synergy HT microplate reader.

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The amount of cytokine was determined using a standard curve and was corrected for the content of cell protein. The results were expressed as pg/mg of intracellular protein.

Gelatin Zymography Because FBS contains matrix metallo­ proteinases (MMPs), cells were cultured in 1% serum medium only. Afterwards, the supernatant was collected, supplemented with Laemmli sample buffer, and subjected to 10% SDS-PAGE with 1 mg/mL gelatin under nondenaturing and nonreducing conditions as previously described (Giribaldi et al. 2011).

Effects of Chrysotile on EMT in NuLi and A549 Cells

A549 cells were observed by optical microscopy; then, the expression of epithelial and mesenchymal markers was evaluated by Western blotting. Asbestos-treated cells had lost their organization in compact islets and had taken on a tapered and spindle-like shape with pointed ends and elongated protrusions,

We performed experiments to confirm the effects of chrysotile on a human bronchial epithelial cell line (NuLi-1) and on a human lung adenocarcinoma cell line (A549). After incubation with chrysotile, NuLi-1 and

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Effect of Chrysotile and Crocidolite on EMT in BEAS-2B Cells After 72 hr of incubation with 1 μg/cm 2 chrysotile, BEAS-2B cells acquired a spindleshaped fibroblast-like morphology similar to that observed after stimulation with TGF-β, which was previously documented by Doerner et al. (Doerner and Zuraw 2009) and is typical of EMT (Figure 1A). After 72 hr of incubation with chrysotile, BEAS-2B cells showed significantly decreased levels of the proteins E-cadherin and β-catenin compared with untreated cells. In parallel, markers commonly associated with a mesenchymal phenotype, such as α-SMA, vimentin, and fibronectin, were significantly increased (Figure 1B). All of the markers were also analyzed with qRT-PCR to investigate changes in gene expression; the results of these experiments were similar to those observed for the Western blot experiments (Figure 1C). Because MMPs play a fundamental role in extracellular matrix remodeling and are markers of EMT (Cannito et al. 2010; Kessenbrock et al. 2010), we investigated the secretion and activity of two MMPs. Cells exposed to chrysotile secreted more MMP-2 and MMP-9 than untreated cells (Figure 1B). BEAS-2B cells were also incubated with 5 μg/cm2 crocidolite for 7 days. After incubation, cells observed by optical microscopy had lost their organization and assumed a ­fibroblast-­like appearance with pointed ends and elongated protrusions (Figure 2A). Expression of E-cadherin and β-catenin was significantly decreased in cells exposed to crocidolite, and the mesenchymal proteins (α-SMA and vimentin) were significantly over-expressed under the same experimental conditions compared with untreated cells (Figure 2B,C).

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Where appropriate, data in figures are reported as the mean ± SEM. The results were analyzed by one-way analysis of variance (ANOVA) and Tukey’s test (SPSS 11.0 for Windows, SPSS Inc.); p