Akt and Notch pathways mediate polyhexamethylene guanidine phosphate-induced epithelial-mesenchymal transition via ZEB2

Toxicology and Applied Pharmacology 380 (2019) 114691

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Akt and Notch pathways mediate polyhexamethylene guanidine phosphateinduced epithelial-mesenchymal transition via ZEB2 Mi Ho Jeonga, Ha Ryong Kimb, Yong Joo Parka,

⁎,1

a b

T

, Kyu Hyuck Chunga,

⁎,1

School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of Korea College of Pharmacy, Daegu Catholic University, Gyeongsan, Gyeongsangbuk-do 38430, Republic of Korea

ARTICLE INFO

ABSTRACT

Keywords: PHMG-p Epithelial-mesenchymal transition Akt Notch ZEB2

Polyhexamethylene guanidine phosphate (PHMG-p), an antimicrobial additive, was used as a humidifier disinfectant in Korea and caused severe lung injuries, including lung fibrosis, in hundreds of victims. As PHMG-pinduced lung fibrosis is different from that induced by known fibrogenic agents such as bleomycin, it is important to understand the molecular mechanisms underlying this effect. A recent study showed that epithelialmesenchymal transition (EMT) could play key roles in PHMG-p-induced pulmonary fibrosis. Therefore, we aimed to characterize the molecular mechanisms associated with PHMG-p-induced EMT. We observed EMT, macrophage infiltration, and fibrosis in mouse lung tissues after intratracheal instillation of PHMG-p. Furthermore, PHMG-p-induced EMT was observed in A549 cells by the evaluation of cell morphology and quantitation of mRNA and protein expression. The use of EMT inhibitors revealed that PHMG-p induced EMT through the activation of Akt and Notch signaling. Moreover, the transcription factor ZEB2 was observed in PHMG-p-treated A549 cells and mouse lungs. The results indicated that upstream regulators, including Akt and Notch 1, acted as intracellular effectors that triggered ZEB2 expression after exposure to PHMGp. Attenuation of PHMG-p-induced EMT following inhibition or silencing of Akt and Notch signaling or ZEB2 implied that PHMG-p-induced EMT was a result of Akt, Notch, and ZEB2 activation. Our findings showed that PHMG-p induced EMT through Akt/Notch signaling pathways and that ZEB2 played an important role in PHMG-p-induced lung toxicity. This study will help to understand the mechanisms of action of PHMG-p associated with lung fibrogenesis.

1. Introduction Polyhexamethylene guanidine phosphate (PHMG-p) has been used as an antibacterial, antifungal, and antiviral additive in plastics, fabric softeners, paints, swimming pools, and papers and for sanitation in food processing plants and cooling towers (Vitt et al., 2015; Kim et al., 2016). It contains a guanidine structure common to many disinfectants and is widely used as a base structure for the synthesis of biocides (Qian et al., 2011). In South Korea (hereafter referred to as Korea), commercial guanidine-based disinfectants, including PHMG-p, were used in humidifiers to prevent microbial growth without any review of inhalation toxicity. The aerosol form of PHMG-p released by humidifiers can penetrate and accumulate deep in the lungs (Heyder, 2004; Lee and Yu, 2017). Recent toxicological studies have shown that PHMG-p induces inflammatory and fibrotic responses in a manner that is different from that of known fibrogenic agents such as bleomycin (Kim et al.,

2015; Kim et al., 2016; Kim et al., 2018; Song et al., 2014). Therefore, it is important to characterize the molecular mechanisms involved in PHMG-p-induced lung fibrosis. Lung fibrosis is a chronic lung disease associated with poor prognosis characterized by the accumulation of excessive extracellular matrix (ECM) and progressive decline of lung function. During fibrogenesis, the number of myofibroblasts increases, and the balance between ECM deposition and turnover skews toward excessive ECM accumulation (Todd et al., 2012). Chronic inflammation had been thought to play an important role in the development of lung fibrosis. However, recent findings showed that lung fibrosis results from multiple cycles of epithelial cell injury and activation that provoke the proliferation of mesenchymal cell. The epithelial-mesenchymal transition (EMT) is a biological event that contributes to the production of myofibroblasts (Willis et al., 2005). EMT is a dynamic cellular process in which epithelial cells lose their typical phenotype and acquire mesenchymal cell-

Corresponding authors at: School of Pharmacy, Sungkyunkwan University, 2066, Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do 16419, Republic of Korea. E-mail addresses: [email protected] (Y.J. Park), [email protected] (K.H. Chung). 1 Equal contributors. ⁎

https://doi.org/10.1016/j.taap.2019.114691 Received 9 June 2019; Received in revised form 19 July 2019; Accepted 22 July 2019 Available online 23 July 2019 0041-008X/ © 2019 Elsevier Inc. All rights reserved.

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like (fibroblast-like) phenotypes (Tanjore et al., 2009; Vaughan and Chapman, 2013). Dissociation of cell-cell junctions (e.g., E-cadherin, claudins, and cytokeratins) and acquisition of mesenchymal markers [e.g., N-cadherin, vimentin, and alpha smooth muscle (α-SMA)] are important markers of EMT (Bartis et al., 2014). Chang et al. (2012) reported that > 40% of fibroblasts in vivo were derived from epithelial cells through EMT. EMT is regulated by various signaling pathways, including the transforming growth factor (TGF)-β/Smad, Wnt, and Notch (Guo et al., 2014) pathways. These signaling pathways can activate transcription factors of EMT, which are able to destabilize adherens junctions and degrade E-cadherin by binding to the E-cadherin promoter (Yilmaz and Christofori, 2009). TGF-β, the most widely-studied EMT-inducing factor, is activated by ligand binding to its cell surface receptor, resulting in phosphorylation of the Smad family of proteins. Upon ligand binding, the Smad complex translocates to the nucleus, resulting in the upregulation of EMT-related transcription factors such as Snail, Twist, and ZEB (Shi and Massague, 2003). The PI3K/Akt and Wnt signaling pathways stabilize β-catenin by inhibiting glycogen synthase kinase-3β (GSK-3β), which is a negative regulator of β-catenin (Fukumoto et al., 2001; Cong et al., 2013). Stabilized β-catenin enters the nucleus and initiates EMT through binding to the promoter region of T-cell factor/ lymphoid enhancer factor (TCF/LEF) (Bachelder et al., 2005; Chua et al., 2007). Cleavage of the Notch intracellular domain (NICD) by the metalloprotease tumor necrosis factor-α-converting enzyme (TACE) and γ-secretase complex following ligand binding to the Notch receptor results in nuclear translocation and binding to the DNA-binding protein CSL (Wang et al., 2010). The NICD/CSL complex activates downstream EMT-related genes, such as genes in the Hairy and enhancer of split (Hes) family (Gutierrez and Look, 2007). Toxicogenomic data obtained using lung epithelial cells exposed to PHMG-p indicated that PHMG-p acts as an EMT-inducing chemical (Shin et al., 2018). The present study aimed to elucidate the molecular mechanisms of PHMG-p-induced EMT. Mice and an epithelial cell line were used to examine PHMG-p-induced EMT and the associated mechanisms underlying this effect. Our study has furthered the understanding of the mechanisms involved in PHMG-p with regard to lung fibrogenesis.

tertiary amyl alcohol; 0.02 ml/g body weight), and each mouse was then administered a single 50-μl dose of PHMG-p diluted in saline (1.2 mg/kg) intratracheally, as described in previous studies (Song et al., 2014; Kim et al., 2018). Each group consisted of three mice. On day 7 after exposure, the mice were sacrificed, and the lungs were fixed in 10% formalin or stored in liquid nitrogen until analysis. 2.3. Quantitative real-time PCR (qPCR) assay A549 cells were seeded in 6-well plates at 6 × 104 cells/well, incubated for 24 h at 37 °C and then treated with PHMG-p. After the indicated time periods, the cells were washed once with PBS, and the total RNA was extracted using RNAiso reagent (Takara Bio Inc., Kyoto, Japan) in accordance with the manufacturer's instructions. The relative expression levels of mRNAs were quantified using a CFX Connect RealTime System (Bio-Rad) with SYBR Premix Ex TaqII (Takara Bio Inc.). The threshold cycle was determined for each sample during the exponential growth phase by plotting the baseline fluorescence signal versus cycle number. The primer sequences used in this study are listed in Supplementary Table S1. The mRNA values for each gene were normalized to GAPDH. 2.4. Western blotting analysis A549 cells were seeded in 6-well plates at 6 × 104 cells/well, incubated for 24 h at 37 °C and then treated with PHMG-p. After the indicated time periods, the cells were washed twice with phosphate buffered saline (PBS) and lysed with radioimmunoprecipitation assay buffer (Thermo Scientific) with protease inhibitor cocktail (GenDEPOT, Barker, TX, USA), phosphate inhibitor (BioVision, Nilpitas, CA, USA), and 0.1% SDS. Equal amounts of protein were denatured in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA), and the denatured total proteins (15 μg) were loaded onto a 10% acrylamide gel. Following separation, the proteins were transferred to 0.2-μm immune-blot PVDF membranes (Bio-Rad). The membranes were blocked with 5% skim milk/TBS-T for 1 h at 20–25 °C and then incubated for 12 h at 4 °C with primary antibodies. Following 1 h of incubation with secondary antibodies, the blots were then developed with enhanced chemiluminescence (ECL) reagents (Bio-Rad) using an automatic X-ray film processor (JP-33; JPI Healthcare, Seoul, Korea). Antibodies used in this study are listed in Supplementary Table S2. Image J software (NIH, Bethesda, MD, USA) was used for quantification. The density of each band was normalized to that of GAPDH.

2. Material and methods 2.1. Cell culture and reagents A549 human lung adenocarcinoma cells were obtained from Korean Cell Line Bank (Seoul, Korea) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Scientific, Rockford, IL, USA) supplemented with 5% fetal bovine serum (Biotechnics Research Inc., Lake Forest, CA, USA), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in an atmosphere of 5% CO2/95% air under saturation humidity. PHMG-p was provided by the Korea Institute of Toxicology (Jeongeup, Korea). To determine the roles of the Akt/GSK3β and Notch signaling pathways in the EMT process, we used the Akt1/2 inhibitor A6730 (Sigma-Aldrich, St. Louise, MO, USA) and the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT (Sigma-Aldrich)), used to inhibit Notch signaling, dissolved in DMSO.

2.5. Immunocytochemistry A549 cells were seeded onto coverslips placed in 12-well plates at 1 × 105 cells/well, incubated for 24 h at 37 °C, and then treated with 2 μg/ml PHMG-p for 48 h. The cells were washed twice with cold 1× PBS and fixed with 4% paraformaldehyde for 30 min at 37 °C. Following fixing, the cell membranes were permeabilized with 0.1% Triton X-100 in PBS for 10 min at 37 °C and blocked with bovine serum albumin (BSA; 1%, w/v) in PBS (0.2 μm filtered) for 1 h at 37 °C. After blocking, the cells were incubated with primary antibodies diluted in 1% (w/v) BSA in PBS for 1 h at 37 °C, and then incubated with secondary antibodies under the same conditions as those used for the incubation with primary antibodies. The antibodies used in this study are listed in Supplementary Table S2. After washing with PBS, the coverslips were removed from the plates and mounted onto glass slides using Prolong™ Diamond Antifade Mountant with DAPI (Thermo Scientific) for 5 min at 20–25 °C to stain the nuclei. Immunofluorescence was analyzed with an LSM 700 confocal laser scanning microscope (Carl Zeiss, Jena, Germany).

2.2. Animal studies All animal experiments were approved by the Sungkyunkwan University Animal Care Committee and conducted in accordance with the guidelines of the National Institutes of Health. Male C57BL/6 mice (8 weeks old; 20–22 g) were purchased from Daehan BioLink (Chungbuk, Korea). Water and food were provided ad libitum. After acclimatization for a week, the mice were anesthetized by an intraperitoneal injection of 1.25% avertin (2,2,2-tribromoethanol in 2

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Fig. 1. Histopathology and immunohistochemistry analyses of mouse lung instilled with PHMG-p. (A) Representative histological sections of tissues stained with hematoxylin and eosin (H&E) and Masson'a trichrome (black arrow: collagen; yellow arrow: infiltrated macrophages; scale bar = 40 μm) and (B) immunohistochemistry analysis (blue: nuclear; green: E-cadherin; red: α-SMA; white scale bar = 100 μm) at 7 days after intratracheal instillation with PHMG 1.2 mg/ kg. (C) Expression of proteins related with EMT (E-cadherin and α-SMA) was measured by western blotting analysis using lysates of mouse lungs. Each value represents mean ± standard deviation. *p < .05 versus the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.6. Histopathological examination

2.10. Statistical analysis

Seven days after exposure to PHMG-p, mouse lung tissue was fixed with 10% formalin for 12 h and then embedded in paraffin. The paraffin blocks were sectioned into 4 μm-thick slices and then stained with hematoxylin and eosin (H&E) and Masson's trichrome. The sections were visualized using an inverted microscope (CK40-F100; Olympus Optical Corp., Tokyo, Japan) equipped with a Leica MC170 HD camera (Leica Microsystems, Wetzlar, Germany). The Leica application suite program was used to adjust the images.

Data were analyzed using Excel® 2013 (Microsoft, Redmond, WA, USA) and Sigmaplot® 12.0 software (Systat Software, Inc., San Jose, CA, USA). Data from each assay are expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS version 21.0 (SPSS, Chicago, IL, USA). Differences between groups were assessed by Duncan's post-hoc test after one-way analysis of variance. Statistical significance was accepted for values of p < .05 and p < .01. 3. Results

2.7. Immunohistochemistry

3.1. PHMG-p induced EMT in mouse lung and A549 cells

Immunohistochemical analysis was performed to detect EMT in the lungs of the mice exposed to PHMG-p (1.2 mg/kg) for 7 days. Formalinfixed lungs were embedded in optimal cutting temperature compound, sectioned using a microtome cryostat (Leica Microsystems) at −20 °C, and then placed onto glass slides. The sections were washed, blocked with 3% BSA in PBS, and incubated in a humid chamber with primary antibodies for 12 h at 4 °C. The slides were then incubated with secondary antibodies for 90 min and mounted using Prolong™ Diamond Antifade Mountant with DAPI (Thermo Scientific) for 5 min at 20–25 °C to stain the nuclei. Immunofluorescence was analyzed using an LSM 700 laser scanning confocal microscope (Carl Zeiss).

PHMG-p (1.2 mg/kg) was intratracheally instilled into the mice at 8 weeks of age. The concentration of PHMG-p used in this experiment (1.2 mg/kg) was six times lower than the estimated exposure experienced by patients with severe lung fibrosis (Song et al., 2014). The body weights of mice were decreased, and the weights of the lungs were increased in the mice treated with PHMG-p compared to the control mice (Supplementary Fig. S1). Seven days after intratracheal instillation with PHMG-p, foamy macrophages and collagen fibers were observed in the H&E and Masson's trichrome-stained lung tissues (Fig. 1A). In the immunostained results, increased red fluorescence (mesenchymal marker, α-SMA) was observed in the PHMG-p-exposed tissue compared to that in control tissue, which indicated that PHMG-p induced EMT in the mouse lungs (Fig. 1B). Moreover, at 7 days after treatment with PHMG-p, the protein levels of E-cadherin were decreased, whereas the expression of α-SMA was significantly increased, as determined by western blotting (Fig. 1C). For the in vitro experiments, the concentrations of PHMG-p used for treatment were determined based on cytotoxicity (data not shown). When A549 cells were exposed to PHMG-p at 0–2 μg/ml for 48 h, morphological analysis revealed that the cells became spindle-like, which is a characteristic of mesenchymal cells (Fig. 2A). At both the mRNA and protein levels, E-cadherin expression decreased and α-SMA expression increased in a dose-dependent manner in response to PHMGp treatment (Fig. 2B and C). Immunofluorescence results showed that PHMG-p treatment led to decreasing of E-cadherin expression while increasing the expression of the mesenchymal marker α-SMA (Fig. 2D). These results indicated that PHMG-p induced EMT in both in vivo and in vitro models.

2.8. Luciferase reporter assay To examine TCF/LEF-dependent transcriptional activity, A549 cells were transfected with a luciferase reporter vector containing TCF/LEFresponsive element, as previously reported (Korinek et al., 1997). To evaluate the PHMG-p-induced γ-secretase activity, the UAS-responsive element reporter gene construct MH100 and the C99-Gal4VP16 (ΔEGVP) plasmid were transfected into A549 cells, as previously described (Karlstrom et al., 2002). Activated γ-secretase released the transcription factor by cleaving ΔE-GVP, which activated the UAS luciferase vector. Transcriptional regulation of E-cadherin by the Zinc finger transcription factor was analyzed by measuring luciferase activity after transfection with the proE-cad178-luc luciferase reporter vector containing the Ecadherin promoter region from −178 to +92, which has an E-box to allow analysis of promoter activity (Mazda et al., 2011). Transient transfection of the reporter construct was performed using LipofectAMINE (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The cells were exposed to PHMG-p at 1, 2, and 4 μg/ml for 24 h. Cell lysates were prepared using luciferase lysis buffer (Promega, Fitchburg, WI, USA). Luciferase activity in the cell lysates was determined using a Dual-Luciferase® Reporter Assay System kit (Promega) according to the manufacturer's instructions. Renilla luciferase activity from co-transfected pRL-TK plasmid vector was used as control for transfection efficiency.

3.2. The Akt/β-catenin and Notch pathways contributed to PHMG-pinduced EMT To elucidate the upstream mechanisms of PHMG-p-induced EMT, we screened known EMT-related pathways using various inhibitors. Because only two inhibitors of Akt and γ-secretase affected the expression of EMT markers (data not shown), the Akt and Notch signaling pathways were investigated in A549 cells after exposure to PHMG-p. As shown in Fig. 3A, Akt phosphorylation increased in a dose-dependent manner in response to PHMG-p treatment. PHMG-p treatment also increased the levels of phosphorylated GSK-3β and the active form of βcatenin. To determine whether PHMG-p could induce β-catenin transcriptional activity in A549 cells, we performed a luciferase reporter gene assay using a TCF/LEF luciferase vector, which contained the TCF/LEF responsive element to which active β-catenin binds. Luciferase

2.9. Transfection procedures Cells at 70% confluence were exposed to a mixture of Opti-MEM, Lipofectamine RNAiMAX (Invitrogen), and Silencer® (Thermo Scientific) siRNAs in accordance with the manufacturers' guidelines. Silencer® negative control No. 1 siRNA (Thermo Scientific) was used as a control. After 24 h of transfection, the media was aspirated, and A549 cells were treated with 2 μg/ml PHMG-p for 24 or 48 h. 4

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Fig. 2. PHMG-p-induced EMT in human alveolar epithelial A549 cells. (A) Morphology of A549 cells exposed to PHMG-p was observed by light microscopy. (B) mRNA levels of E-cadherin (epithelial marker) and α-SMA (mesenchymal marker) were quantified with qPCR assay. Data were normalized to that of GAPDH. (C) Expression of proteins related to EMT was detected by western blotting analysis. GAPDH was used as a loading control for the same amount of cell lysates. Bar graphs were obtained from ImageJ quantification values. (D) Expression of EMT markers in A549 cells exposed to 2 μg/ml of PHMG-p was analyzed by immunofluorescence. Each value represents mean ± standard deviation from three separate experiments. *p < .05; **p < .01 versus control.

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Fig. 3. The Akt/β-catenin and Notch pathways were involved in PHMG-p-induced EMT. (A) Following treatment of A549 cells for 24 h with PHMG-p at various concentrations, the expression of proteins associated with Akt/β-catenin and Notch were analyzed by western blotting analysis. (B) To elucidate whether β-catenin translocates into the nucleus and binds to TCF/LEF-responsive element, A549 cells were transfected with TCF/LEF luciferase reporter vector and treated with PHMGp, and intensity of luciferase activity was measured. (C) In Notch signaling, γ-secretase activity was analyzed by transfecting ΔE-GVP plasmid and UAS luciferase reporter vector to A549 cells, and intensity of luciferase activity was measured. The firefly luciferase activity was normalized to Renilla luciferase activity. (D) A6730 (Akt inhibitor), DAPT (γ-secretase inhibitor), and (E) siRNAs against Akt and Notch were applied to A549 cells exposed to 2 μg/ml of PHMG-p. GAPDH was used as a loading control for the same amount of cell lysates. Bar graphs were obtained from Image J quantification values. Each value represents mean ± standard deviation from three separate experiments. *p < .05; **p < .01 versus control. ##p < .01 versus the PHMG-p-treated group.

activity was significantly increased in the PHMG-p-treated A549 cells, which indicated that PHMG-p stimulated the binding of active β-catenin to the TCF/LEF binding site (Fig. 3B). In addition, Fig. 3A shows that cleaved NICD and its downstream protein, Hes-1, were upregulated by PHMG-p in a dose-dependent manner. Treatment of A549 cells transfected with ΔE-GVP plasmid and UAS reporter vector with PHMGp resulted in significantly increased luciferase activity (Fig. 3C), which indicated that overexpression of NICD and Hes-1 proteins resulted from increased γ-secretase activity. The inhibitors or siRNAs of Akt and Notch were used to evaluate the roles of the Akt/β-catenin and NICD signaling pathways in PHMG-pinduced EMT. Both the Akt1/2 inhibitor A6730 and the γ-secretase inhibitor DAPT blocked phosphorylation of Akt and cleavage of Notch, respectively, at 24 h. Furthermore, each inhibitor blocked PHMG-p-induced EMT at 48 h (Fig. 3D). Similarly, silencing of Akt and Notch with siRNAs attenuated the PHMG-p-induced decrease in E-cadherin expression and increase in α-SMA (Fig. 3E). These results showed that the Akt/β-catenin and Notch pathways contributed to PHMG-p-induced EMT.

et al., 2018). The pathological characteristics of PHMG-p exposure include continuous and irreversible induction of inflammatory and fibrotic responses, which was different from the pathological characteristics associated with bleomycin exposure (Kim et al., 2018; Song et al., 2014). Therefore, it is necessary to characterize the molecular mechanisms of action of PHMG-p to develop a better understanding of PHMG-p-induced lung fibrosis and develop treatment and mitigation strategies. The present study demonstrated that PHMG-p induced EMT by activating the Akt/β-catenin and Notch pathways. In addition, the transcription factor ZEB2, a downstream effector of both pathways, played an important role in PHMG-p-induced EMT. Understanding the mechanisms underlying PHMG-p-induced EMT may provide insights into chemical-induced lung fibrosis at the molecular level. PHMG-p treatment promoted EMT in vivo and in vitro (Figs. 1 and 2). Among the various signaling pathways of EMT, TGF-β/Smad pathway is the canonical EMT induction pathway. When activated by TGF-β, Smad2/3 forms complexes with Smad4, which translocate to the nucleus and regulate the transcription of target genes associated with EMT induction (Kasai et al., 2005). However, the importance of additional pathways, including the Akt/β-catenin, Notch, MAPK, and STAT3 pathways have recently received increased attention (Lamouille et al., 2014). Recent studies have shown that lung fibrosis-inducing chemicals, such as bleomycin and paraquat, can induce EMT through the Akt/ β-catenin and/or Notch pathways (Zhu et al., 2016; Li et al., 2017; Zhang et al., 2017). Our preliminary study with TGF-β receptor inhibitor showed that TGF-β signaling pathway has little relationship with PHMG-p-induced EMT (Supplementary Fig. 2). Instead, PHMG-p triggered the activation of the Akt/β-catenin and Notch pathways, resulting in EMT induction in A549 cells (Fig. 3). A previous report showed that Akt/β-catenin and Notch pathways cross-communicate through shared upstream and downstream regulators or through mutually-influenced expression (AndroutsellisTheotokis et al., 2006; Meurette et al., 2009; Wang et al., 2014). For example, Notch signaling in breast epithelial cells activates the Akt/βcatenin pathway, resulting in resistance to apoptosis (Meurette et al., 2009). Furthermore, β-catenin interacts with NICD, resulting in the upregulation of Hes-1 (Jin et al., 2009). β-catenin may also bind to CSL, which is the DNA binding site of NICD, resulting in transcription of genes downstream of the Notch pathway (Yamamizu et al., 2010). As shown in Fig. 3E, Notch reduced Akt phosphorylation. However, Akt knockdown did not alter the expression of NICD. In addition, α-SMA expression was sharply attenuated as a result of Notch knockdown. Thus, we propose that NICD partially regulated the Akt/β-catenin pathway directly or indirectly through effectors upstream of Akt. Several studies have reported that inhibition of Notch signaling prevented fibrogenesis in the peritoneum (Zhu et al., 2010), kidney (Bielesz et al., 2010), skin (Dees et al., 2011), and liver (Chen et al., 2012) in animal models. Therefore, Notch may be a potential therapeutic target for the mitigation of PHMG-p-induced lung fibrosis. As shown in Fig. 4A, the PHMG-p-induced decrease in E-cadherin occurred at the transcriptional level. It is known that various transcription factors such as Snail, Slug, ZEB1 and ZEB2 regulate EMT by repressing E-cadherin expression (Lamouille et al., 2014). Our results showed that TGF-β increased ZEB1, ZEB2, Snail, Slug and Twist 1 expression in A549 cells (Fig. 4B and C). However, PHMG-p only induced the expression of ZEB2, but not Snail and Slug, which are typical

3.3. ZEB2 played a key role in PHMG-p-induced EMT To determine whether PHMG-p-induced EMT was regulated at the transcriptional or translational level, the E-cadherin luciferase reporter vector was transfected into A549 cells prior to treatment with PHMG-p. PHMG-p significantly reduced luciferase activity, which indicated that PHMG-p decreased the expression of E-cadherin at the transcriptional level (Fig. 4A). The protein and mRNA expression of various transcription factors was analyzed. TGF-β, known to contribute to EMT, was used to evaluate the mechanism of EMT induction. A549 cells were exposed to TGF-β or PHMG-p for 48 h. ZEB2, Snail, and Slug were significantly increased by both PHMG-p and TGF-β, as determined by qPCR (Fig. 4B and C). Protein levels of ZEB2, ZEB1, Snail, and Slug, but not Twist1, were significantly increased following TGF-β treatment. However, PHMG-p only increased the expression of ZEB2 at the protein level (Fig. 4C). Furthermore, in vivo data showed that the protein expression of ZEB2 was upregulated seven days after PHMG-p instillation, suggesting a relationship between ZEB2 and PHMG-p-induced EMT (Fig. 4D). We also evaluated whether the PHMG-p-induced increases in ZEB2 expression directly contributed to EMT. Transfection with ZEB2 siRNA attenuated PHMG-p-induced EMT through increased E-cadherin expression and reduced α-SMA expression, as compared to control siRNAtransfected cells (Fig. 5A). Pretreatment with inhibitors of Akt and Notch followed by incubation with PHMG-p for 48 h resulted in attenuated ZEB2 expression compared to that in cells treated with PHMG-p only (Fig. 5B). In addition, siRNAs of Akt and Notch downregulated the expression of ZEB2 following exposure to PHMG-p (Fig. 5C). These data indicated that ZEB2 was a downstream target of the Akt/β-catenin and Notch pathways in PHMG-p-induced EMT. 4. Discussion In Korea, adding a humidifier disinfectant containing PHMG-p to humidifier water introduced aerosolized particles into the air, which were able to enter human lungs, resulting in pulmonary fibrosis (Kim 7

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Fig. 4. Expression of transcription factors associated with EMT in A549 cells and lung tissues exposed to PHMG-p. (A) The transcriptional expression of E-cadherin by PHMG-p was measured after transfection of E-cadherin luciferase reporter vector to A549 cells. A549 cells were exposed to PHMG-p 2 μg/ml for 48 h, and then the expression of EMT-related transcription factors was analyzed by (B) qPCR assay and (C) western blotting. TGF-β (2 ng/ml) was used as positive control of EMT induction (D) Western blotting was conducted at 7 days after instillation to confirm the expression of ZEB2 in mouse lung tissues. GAPDH was used as a loading control at the same amount of tissue lysates. Each value represents mean ± standard deviation from three separate experiments. *p < .05; **p < .01 versus control.

transcription factors induced by TGF-β. The result of knockdown of ZEB2 demonstrated that up-regulated ZEB2 played important role in PHMG-p induced EMT (Fig. 5A). Moreover, PHMG-p-induced ZEB2 expression was attenuated by the knockdown of Akt and Notch (Fig. 5B and C). These results suggested that ZEB2 played an important role as a downstream effector of the Akt/β-catenin and Notch pathways in PHMG-p-induced. ZEB2 is known to repress E-cadherin by binding at Eboxes and recruiting C-terminal-binding protein co-repressor (Peinado et al., 2007). Vandewalle et al. (2005) demonstrated that, during the EMT, morphological changes and expression of junction proteins, including E-cadherin, were determined by ZEB2, not by Snail or Slug in various cancer cells, which indicated that ZEB2 is a key factor in epithelial phenotype determination. Moreover, Skrypek et al. (2018) and Yalim-Camci et al. (2019) suggested that ZEB2 can be a therapeutic target to prevent EMT. Taken together, ZEB2 is not only important to understand the mechanism of PHMG-p induced EMT, but also can be a therapeutic target in PHMG-p induced pulmonary fibrosis. However, further studies are needed to determine the mechanisms by which EMT occurs in patients with PHMG-p-induced lung fibrosis. In conclusion, we showed for the first time that PHMG-p activated the Akt/β-catenin and Notch signaling pathways, resulting in increased ZEB2 expression. The interplay between these pathways induced EMT, resulting in PHMG-p-induced lung fibrogenesis. This research provided

an understanding of fibrosis pathology by PHMG-p as a fibrogenic agent with interesting mechanism of action. Author contributions Mi Ho Jeong designed and performed the experiments; Ha Ryong Kim and Yong Joo Park analyzed the data and wrote the manuscript; and Kyu Hyuck Chung conceived the design and idea of the study. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2019R1C1C1008645) and the Korea Environmental Industry and Technology Institute (No. 2018002490005).

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Fig. 5. ZEB2 as downstream of Akt/β-catenin and Notch pathways in PHMG-p-induced EMT. (A) ZEB2 as a transcription factor of EMT was silenced to confirm its effect on PHMGp-induced EMT. (B) A549 cells were exposed to PHMG-p 2 μg/ ml following inhibition of Akt and NICD. A6730 (Akt inhibitor) and DAPT (γ-secretase inhibitor) were used to explore the roles of Akt or NICD in the expression of ZEBb2. (C) Akt and/or Notch siRNAs were transfected to A549 cells before treatment with PHMG-p. GAPDH was used as a loading control for the same amount of cell lysates. Data was determined by three separate experiments.

Appendix A. Supplementary data

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