Toxicology 370 (2016) 116–126
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Aryl hydrocarbon receptor–ligand axis mediates pulmonary fibroblast migration and differentiation through increased arachidonic acid metabolism Hsiang-Han Sua , Hsin-Ting Linb , Jau-Ling Suena,c,h , Chau Chyun Sheui,j, Kazunari K. Yokoyamaa,c,d,e,f,g, Shau-Ku Huangc,k,**,1, Chih Mei Chengb,c,* ,1 a
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Taiwan c Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan d Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan e Center for Infectious Diseases and Cancer, Kaohsiung Medical University, Kaohsiung, Taiwan f Faculty of Science and Engineering, Department of Pharmacological Science, Tokushima Bunri University, Sanuki, Japan g Department of Molecular Prevention Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan h Center for Research Resources and Development, Kaohsiung Medical University, Taiwan i Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan j Department of Internal Medicine, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan k National Institute of Environmental Health Sciences, National Health Research Institutes, Miaoli County, Taiwan b
A R T I C L E I N F O
Article history: Received 18 August 2016 Received in revised form 29 September 2016 Accepted 29 September 2016 Available online 30 September 2016 Keywords: Aryl hydrocarbon receptor Arachidonic acid Alpha-SMA Myocardin-related transcription factor Fibrosis
A B S T R A C T
Pulmonary fibroblast migration and differentiation are critical events in fibrogenesis; meanwhile, fibrosis characterizes the pathology of many respiratory diseases. The role of aryl hydrocarbon receptor (AhR), a unique cellular chemical sensor, has been suggested in tissue fibrosis, but the mechanisms through which the AhR-ligand axis influences the fibrotic process remain undefined. In this study, the potential impact of the AhR-ligand axis on pulmonary fibroblast migration and differentiation was analyzed using human primary lung fibroblasts HFL-1 and CCL-202 cells. Boyden chamber-based cell migration assay showed that activated AhR in HFL-1cells significantly enhanced cell migration in response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), and a known AhR antagonist, CH223191, inhibited its migratory activity. Furthermore, the calcium mobilization and subsequent upregulated expression of arachidonic acid metabolizing enzymes, including cyclooxygenase2 (COX-2) and 5-lipoxygenase (5-LOX), were observed in TCDD-treated HFL-1 cells, concomitant with elevated levels of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) secretion. Also, significantly increased expression of a-smooth muscle actin a-SMA), a fibroblast differentiation marker, was also noted in TCDD-treated HFL-1 cells (p < 0.05), resulting in a dynamic change in cytoskeleton protein levels and an increase in the nuclear translocation of the myocardin-related transcription factor. Moreover, the enhanced levels of a-SMA expression and fibroblast migration induced by TCDD, PGE2 and LTB4 were abrogated by selective inhibitors for COX-2 and 5-LOX. Knockdown of AhR by siRNA completely diminished intracellular calcium uptake and reduced a-SMA protein verified by promoter-reporter assays and chromatin immunoprecipitation. Taken together, our results suggested the importance of the AhR-ligand axis in fibroblast migration and differentiation through its capacity in enhancing arachidonic acid metabolism. ã 2016 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author at: Department of Biomedical Science and Environmental Biology, 100 Shih Chuan 1st Rd, Kaohsiung Medical University, Kaohsiung, Taiwan. ** Corresponding author at: National Institute of Environmental Health Sciences, National Health Research Institutes, Miaoli County, Taiwan. E-mail addresses:
[email protected] (S.-K. Huang),
[email protected] (C.M. Cheng). 1 These two authors contribute equally to the manuscript. http://dx.doi.org/10.1016/j.tox.2016.09.019 0300-483X/ã 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Common pulmonary diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are the major publichealth problem worldwide (Papaiwannou et al., 2014). Both genetic and environmental factors play major roles in their pathogenesis (Wynn, 2011). Respiratory diseases are often
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associated with pulmonary fibrosis and are characterized by the progressive and irreversible destruction of lung tissues in severe cases. Moreover, these diseases are triggered by multiple factors, including exposure to environmental contaminants (Chen and Stubbe, 2005). Although the major pathways associated with pulmonary fibrosis have been recognized, the sequence of events causing pulmonary fibrosis, particularly in response to environmental stimuli, remains undefined. Fibroblasts are critical in excessive scar tissue formation or increased extracellular matrix accumulation in response to longterm damage or environmental stimuli (Hinz, 2012). Fibrogenic gene expression (Huang et al., 2012) and wound healing process (Xu and Chisholm, 2011) are closely associated with the cytoskeleton remodeling of actin filament. Dynamic changes in actin filaments initiated by calcium signaling activation (Follonier Castella et al., 2010) or various intracellular and environmental stimuli (Olson and Nordheim, 2010) may contribute to various physiological functions and serve as a platform for signal transduction (Chhabra and Higgs, 2007; Coso et al., 1995, 1996; Fisher et al., 2012). Furthermore, recent evidence has suggested that the exposure to environmental contaminants is closely associated with activation of arachidonic acid cascade (Dong and Matsumura, 2008; Lupo et al., 2007), and that bioactive eicosanoids not only mediate inflammatory responses but also regulate fibrogenesis (Charbeneau and Peters-Golden, 2005). Arachidonic acid metabolites, such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), are essential mediators in both acute and chronic inflammation. Increased PGE2 synthesis as well as cyclooxygenase-2 (COX-2) and a-smooth muscle actin (a-SMA) protein colocalization have been observed in lesions of the stromal area of the cornea, suggesting that PGE2 is involved in accelerating the corneal wound-healing process (Kawamura et al., 2008). The assembly of actin filaments after the activation of a-SMA promoter activity by the cooperative regulation of FAK and myocardin-related transcription factor (MRTF)-A was observed in NIH 3T3 cells (Chan et al., 2009). Collectively, these results, suggest the possible crosstalk between arachidonic acid metabolism and MRTF axis activation. However, in the context of exposure to environmental stimuli, the association between the generation of arachidonic acid metabolites and fibrogenesis remains unclear. Aryl hydrocarbon receptor (AhR) is a ligand-activated receptor for various environmental contaminants and endogenous metabolites. Environmental contaminant-induced AhR activation influences inflammatory, fibrotic responses and lung functions in various disease models (Chiba et al., 2011; Ramirez et al., 2010), although the detailed mechanisms remain to be elucidated. Particularly, the potential role of the AhR–ligand axis in regulating pulmonary fibroblast migration and differentiation and its underlying mechanisms has not been completely defined. We hypothesize that AhR–ligand axis activation is involved in the imbalance of arachidonic acid metabolism, pulmonary fibroblast migration and subsequent fibrotic progression. Herein, we provide evidence supporting the involvement of the AhR–ligand axis in regulating pulmonary fibroblast migration and differentiation through, at least in part, its ability to increase the generation of arachidonic acid metabolites.
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MD, USA). Cell Titer 96 Aqueous One Solution Reagent was purchased from Promega (San Luis Obispo, CA, USA). The Abs used for Western blotting analysis were as follows: anti-a-SMA (Millipore), anti-cPLA2 (Santa Cruz Biotechnology), anti-COX-2 (Santa Cruz Biotechnology), anti-5-LOX (BD), anti-MRTF-A (Santa Cruz), anti- b-actin (Santa Cruz Biotechnology), anti-GAPDH (GeneTex), anti-PARP (Santa Cruz), anti-mouse IgG–HRP (Cell Signaling Technology), anti-rabbit IgG–HRP (Cell Signaling Technology), anti-goat IgG–HRP Abs; for immunofluorescence staining, anti a-SMA (Sigma–Aldrich Co.), anti-AhR (Santa Cruz Biotechnology), anti-mouse IgG2a and IgG2b isotype control (e-Bioscience), goat anti-mouse IgG-conjugated Alexa Fluor 568 (Cell Signaling Technology), anti-rabbit IgG-conjugated Alexa Fluor 568 (Cell Signaling Technology), and Alexa Fluor 488-conjugated phalloidin (Invitrogen) Abs were used. siRNAs for AhR and scrambled control siRNAs were purchased from Invitrogen. Anti-AhR Abs, used for the chromatin immunoprecipitation assay, was purchased from Genetex.
2.2. Cell culture Human normal fetal lung fibroblast, HFL-1 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan), and normal male CCL-202 human lung fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA). For sub culturing, HFL-1 cells were maintained in Ham’s F12K medium with 2 mM L-glutamine and 10% fetal bovine serum (FBS). The CCL-202 cells were maintained in Eagle’s minimal essential medium with Earle’s balanced salts solution (BSS), supplemented with 10% FBS, 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate, and 1% penicillium/streptomycin. Cells were cultured at 37 C in a humidified atmosphere containing 5% CO2. 2.3. Proliferation assay Proliferation of HFL-1 cells was assessed by MTT assay. HFL-1 cells (6 103 cells/well) were seeded in a 96 well microplate for 24 h. Cells were incubated in the serum free medium before TCDD treatment. After treatment with TCDD, cells were then incubated with MTT (5 mg/ml) at 37 C for 4 h. Proliferation of HFL-1 cell was monitored by measuring the formation of formazan at the O.D. 570 nm by spectrophotometer. 2.4. Migration assay Boyden chamber-based cell migration assay was performed using a 24-well micro-chemotaxis chamber and polycarbonate filters (BD) (8-mm diameter pores). HFL-1 cells (80% confluence) were seeded on the upper chamber of a trans well chamber containing Ham’s F12 serum-free medium, medium containing TCDD or FICZ was replaced in the lower chamber. The chambers were incubated at 37 C under 5% CO2 for 24 h. Cell migration was examined following the manufacture’s protocol. The top of filters were mechanically scraped, and the fibroblasts, which migrated to the undersurface of the filter, were fixed in 100% methanol and stained with 1% crystal violet. The number of migrated cells was examined under a microscope.
2. Materials and methods 2.5. Western blotting 2.1. Reagents and antibodies Ham’s F12K medium, minimal essential medium, L-glutamine, fetal bovine serum (FBS), sodium pyruvate, and a nonessential amino acid solution were purchased from Gibco (Gaithersburg,
Cells (1 105 cells/mL) were seeded in 100-mm2 dishes. At 90% confluence, the cells were starved with serum-free medium for 24 h and treated with various experimental conditions. Subsequently, the cells were harvested and homogenized in RIPA cell
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lysis buffer (150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.6 in ddH2O. For nuclear protein extraction, the nucleus was disrupted with a high-salt solution containing 20 mM HEPES, pH7.9, 25% glycerol, 1.5 mM MgCl2, 0.6 M KCl, 0.2 mM EDTA, 0.5 mM DTT, a cocktail of protease inhibitors (Roche), and PMSF. Protein concentration was measured by the use of BCA protein assay kit (Thermo Scientific). Equal amounts of protein were separated by electrophoresis on 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis, the separated proteins were transferred to a nitrocellulose paper (Amersham Biosciences), blocked in 5% nonfat dry milk, probed first with indicated specific primary Abs and then HRP-conjugated secondary Abs in 1% milk, and developed through chemiluminescence (ECL, Amersham Biosciences). 2.6. Intracellular calcium measurement Intracellular calcium was measured as previously described with a slight modification (Ikeda et al., 2013). Briefly, HFL-1 cells were seeded in 3-mm2 glass bottom dishes (Ibidi) 16–18 h before the experiments. Fluo-4AM (Invitrogen) was loaded in the cells in Ham’s F12K (Gibco, Invitrogen) medium at 37 C for 20 min before the experiments. The cells were washed with Balanced Salt Solution (BSS) containing 130 mM NaCl, 5.5 mM d-glucose, 3 mM MgSO47H2O, 5.4 mM KCl, 20 mM HEPES, pH = 7.4 and immersed with BSS buffer. For recording intracellular calcium mobilization, calcium thermal images were acquired using fluorescent confocal microscopy (Olympus FV1000). The fluorescent signal of Fluo-4AM was detected using an argon laser operated at 488 and 510 nm for excitation and emission, respectively. The cells were pre-incubated in BSS for 2–3 min before TCDD treatment. Subsequently, images were acquired every 3.247 s. Changes in the fluorescence intensity (F) were expressed as follows: (F–F0)/F0, where F0 indicates the resting fluorescence intensity. Treatment of the cells with an AhR antagonist, CH223191 (Sigma, 10 mM), was performed 1 h before loading of Fluo-4AM. 2.7. AhR siRNA knockdown 4
2
HFL-1 cells (5 10 cells/mL) were seeded on 18-mm coverslips for fluorescent confocal microscopy 16–18 h before siRNA transfection. AhR-specific siRNA: 50 - GAGGCUCAGGUUAUCAGUUUAUUCA-30 , 50 - UGAAUAAACUGAUAACCUGAGCCUC-30 (Invitrogen) or scrambled control siRNAs (Catalog number: 12935-110, Invitrogen) were incubated with TransIT-LT1 transfection reagent (Mirus, Cat. No. 2225) in Opti-MEM medium (GIBCO) to a final concentration of 0.1 mM. The complex was added to the cells and incubated for another 24 h and harvested for analysis. 2.8. Enzyme-linked immunosorbent assay HFL-1 cells (7 103 cells/mL) were cultured in 96-well plates for 16–18 h. The cells were treated with TCDD at 37 C for 24 h. After TCDD treatment, the cells were centrifuged at 2000 rpm at 4 C for 10 min. The treated supernatant was collected for measurement of PGE2 and LTB4 by Enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (R&D).
additional 24 h. After washing with phosphate-buffered saline (PBS) and fixing in 4% paraformaldehyde (Sigma–Aldrich) for 30 min, the cells were perforated with 0.1% Triton X-100 (Sigma– Aldrich) for 10 min and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h. The slides were incubated overnight at 4 C with anti-a-SMA and anti- MRTF-A Abs in 1% BSA. After washing with PBS and blocking with 1% BSA in PBS for 20 min, the slides were incubated with a secondary antibody conjugated with goat anti mouse IgG Alexa Fluor 568 (Cell Signaling Technology) for 1 h at 4 C. For actin filament staining, the cells were washed with PBS and incubated with Alexa Fluor 488-conjugated phalloidin (Invitrogen) for 20 min. After a final wash, the slides were incubated with 40 , 6-diamidino-2-phenylindole at a final concentration of 2 mg/mL for 5 min and covered with a mounting medium (Dako). Fluorescent images of the z axis were scanned by 0.45 mm for each section viewed through laser confocal microscopy (Olympus FV1000) and analyzed using FV10 2.1 viewer. The fluorescence intensities of actin filaments in 10–12 random areas of the obtained images were analyzed using FV10-ASW. 2.10. Plasmid construction and luciferase assay An expression construct (pGL3- a-SMA) was made with a firefly luciferase gene and the promoter sequence spanning the 50 flanking region from 1099 to +102 of the gene encoding a-SMA. Primers used for PCR amplification of the a-SMA gene promoter were 50 -GGTACCACTCATGGCAAAAGGGGAAG-30 and 50 0 AAGCTTCGGGTAATTAAAAGAGCCACTG-3 . HFL-1 cells (5 104 cells/mL) were seeded in 24-well plates and grown for 24 h before transfection with pGL3-a-SMA plasmids. The cells were transfected with 2.5 mg of pGL3-a-SMA plasmid DNA by TransIT-LT1 transfection reagent (Mirus) in OPTI-MEM medium (Gibco) for 24 h. The cells were then treated with a vehicle control, DMSO, TCDD (0.01 mM) or TGF-b (1 ng/mL) for 24 h. After treatments, the cells were harvested and the luciferase activities were measured using the Steady-Glo Luciferase Assay System (Promega). 2.11. Chromatin immunoprecipitation assay For chromatin immunoprecipitation assay, HFL-1 cells were exposed to TCDD for 0.5 h and 3 h, fixed with 1% formaldehyde solution and washed with cold PBS before collection by centrifugation. Cell pellets were suspended in SDS lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH = 8.1)] and incubated on ice for 10 min. The cell lysates were sonicated on ice using an ultrasonic sonicator (Misonix Inc.) at setting 30 with 30-s pulses for 4 h to an average length of 100–1000 bp. A soluble chromatin solution was incubated with either anti-AhR Abs or preimmune IgG. Immunoprecipitation was performed at 4 C with overnight rotation. After washing and eluting, DNAs were purified through phenol chloroform extraction and ethanol precipitation, and resuspended in ddH2O for additional Realtime-PCR experiments. Specific primer pairs used for amplifying the a-SMA promoter regions were as follows. Primer 1: 50 -GCAAGGACTACACAGACACTGC-30 , 50 -GTTGGGCATTTGGGGTTAAT-30 ; primer 2: 50 TCTTAGTCCATTTTCACATTGCT-30 , 50 -GTTTTCCCTGCTCTTGCTTG-30 . D Ct was calculated by Ct of experimental value minus Ct of negative control value. Folds of enrichment of DRE binding was calculated by 2DCt and normalized to input control.
2.9. Immunofluorescent microscopy
2.12. Statistics
Cells were seeded on 18-mm2 coverslips in 12-well plates at a density of 5 104 cells/mL. At 90% confluence, the cells were starved for 24 h and treated with TCDD, PGE2, or LTB4 for an
Statistical analyses were performed using non parametric one way ANOVA and student’s t test. Data are expressed as mean SEM, and p < 0.05 was considered statistically significant.
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3. Results 3.1. TCDD induces migration of pulmonary fibroblasts through AhR Pulmonary fibroblast migration is generally considered as a marker for fibroblast differentiation. A Boyden chamber-based cell migration assay was performed to assess the effect of AhR ligands in pulmonary fibroblast HFL-1 cells. TCDD (0.0 5mM; an exogenous ligand) and FICZ (0.1 5mM; an endogenous ligand) enhanced cell migration by 1.8- to 2-fold, respectively, for HFL-1 cells (Fig. 1a, b). The activation of AhR in response to TCDD and FICZ was confirmed by the activation of a known AhR-targeted gene, CYP1B1, encoding a member of the phase I enzyme cytochrome P450 family (Supplementary Fig. 1). Effect of TCDD on the proliferation of HFL-1 cell was analyzed by MTT assay, and no significant effect was observed among the groups treated with TCDD (Fig. 1c). To confirm the role of AhR in fibroblast migration further, CH223191, a known AhR antagonist, was used to examine the AhR specificity in cell migration. First, CH223191 alone reduced HFL-1 migration (p < 0.05, Fig. 1d), suggesting the role of AhR in basal migratory activity of pulmonary fibroblasts. Second, TCDD significantly enhanced HFL-1 migration as compared with untreated cells (p < 0.001). Third, TCDD-mediated migration was inhibited in HFL-1 cells in the presence of CH223191 (p < 0.001). These data suggested that AhR also plays a role in TCDD-induced pulmonary fibroblast migration. 3.2. Calcium mobilization and arachidonic acid metabolism are induced by TCDD It has been reported that the intracellular calcium mobilization regulates cPLA2 activity and arachidonic acid metabolism (Giurdanella et al., 2011), and that AhR activation is critical for COX2 activation and PGE2 expression in lung fibroblasts by cigarette smoke extracts (Martey et al., 2005). For investigating whether
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calcium flow plays a role in TCDD-induced AhR activation and arachidonic acid metabolism in pulmonary fibroblasts, TCDD-induced intracellular calcium mobilization was examined. The thermal images of calcium mobilization were captured through inverted confocal microscopy. Intracellular calcium mobilization stimulated by TCDD increased rapidly after stimulation of HFL-1 cells with 0.05 mM of TCDD. Calcium mobilization period was short and the calcium level returned to the baseline rapidly. Also, knockdown of AhR completely diminished the calcium mobilization induced by TCDD (Fig. 2a, p < 0.05). Similar results were verified by the use of an AhR antagonist, CH223191 (Supplementary Fig. 2a). Next, in an attempt to examine whether TCDD-induced AhR activation was associated with arachidonic acid metabolism in pulmonary fibroblasts, expressions of COX-2 and 5-LOX were analyzed by western blotting. TCDD at 0.001 mM and 0.1 mM induced upregulation of COX-2 and 5-LOX by 1.5- to 2-fold, respectively (p < 0.05, Fig. 2b–e). Moreover, significant increases in PGE2 levels (Fig. 2f; p < 0.05) were observed, whereas the level of LTB4 in HFL-1 cells was increased when the cells were treated with a higher concentration (0.1 mM) of TCDD (Fig. 2g; p < 0.05). To determine whether the effect of AhR-induced fibroblast migration was mediated through arachidonic acid metabolites, selective COX-2 and 5-LOX inhibitors, NS398 and zileuton, respectively, were used. Compared with HFL-1 cells treated with TCDD alone, those directly treated with PGE2 or LTB4 exhibited significantly increased fibroblast migration (p < 0.05, Fig. 3), and the elevated levels were abrogated in presence of the COX-2 and 5-LOX inhibitors (p < 0.05), suggesting that PGE2 and LTB4 may be involved in TCDD-induced migration. 3.3. Actin polymerization and a-SMA expression are mediated by activation of AhR and arachidonic acid metabolism Expression of a-SMA is a characteristic of fibroblast activation in many fibrotic tissues (Fagone et al., 2011). To test whether
Fig. 1. TCDD and AhR induced migration of pulmonary fibroblasts. HFL-1cells were seeded in 24-well Boyden chamber culture dishes for16–24 h before treatment. (a) Staining of HFL-1 cells by crystal violet with or without treatment with TCDD or FICZ is shown by purple color. (b) Migration of fibroblasts after 24 h of treatment with TCDD (0.05 mM) and FICZ (0.1 mM) and the number of migrated cells is shown as fold increase over the control culture. (c) Proliferation of HFL-1 cells treated with 0.1 m M to 0.001 mM of TCDD was assessed by MTT assay. (d) Antagonistic effect of CH223191 (10 mM). Data represent mean SEM (n = 3–4).*p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. TCDD-induced calcium mobilization and arachidonic acid metabolism. AhR knockdown was performed using transient transfection of siRNA specific for AhR (siAhR37) by using the Trans IT transfection reagent. (a) Calcium thermal images of HFL-1 cells acquired through fluorescent confocal microscopy (Olympus FV1000). Lines of different colors representing the average of fluorescent intensity acquired from different treatment groups, black: control, red: TCDD, orange: siAhR37, blue: siAhR37 plus TCDD, green: scramble siRNA plus TCDD. The levels of (b-c) COX-2, (d-e) LOX-5 gene expression, and the protein levels for (f) COX-2 and (g) LOX-5 in cells treated with medium control or TCDD (0.1–0.001 mM) in a serum-free culture medium for 24 h. Intracellular calcium mobilization was measured as described in Materials and Methods. The relative levels of COX-2 and 5-LOX were normalized to GAPDH. The levels of (f) PGE2 and (g) LTB4. Supernatants were collected after 24 h of TCDD (0.1–0.001 mM) treatment. Data shown are the representative of three experiments. *p < 0.05, **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
TCDD-induced pulmonary fibroblast migration was associated with a-SMA expression, TCDD-stimulated a-SMA expression was analyzed. TCDD (0.001 or 0.01 mM) significantly increased a-SMA expression (p < 0.05) in HFL-1 cells after 24 h exposure (Fig. 4a, b). For determining the role of AhR in a-SMA expression further, knock-down experiment by siRNA-AhR was carried out to examine the a-SMA protein expression by fluorescent confocal microscopy. The treatment of siRNA-AhR reduced TCDD-induced a-SMA expression, while a set of scrambled control siRNA had no effect on TCDD-induced a-SMA expression (Fig. 4c). Because actin stress fiber formation was known to be significant in activated fibroblasts (Sandbo et al., 2011), we next investigated whether both AhRmediated a-SMA expression and fibroblast migration was associated with a dynamic change in cytoskeleton protein levels. Indeed, the AhR activation induced significant stress fiber formation and actin filament polymerization in both TCDD-treated CCL-202 (Fig. 4d) and HFL-1 (Fig. 4e) cells. Statistical difference in
the level of actin polymerization was observed in HFL-1 cells but not in CCL-202cells (Fig. 4f) (p < 0.05); however, the degree of polymerization of actin stress fibers was more intense in CCL-202 cells than HFL-1 cells after exposure to TCDD. Next, we examined whether the arachidonic acid metabolites, PGE2 and LTB4, were involved in TCDD-induced a-SMA expression. Fig. 5a shows that a-SMA protein expression was significantly increased by TCDD, PGE2, and LTB4 (Fig. 5a), but in the presence of selective COX-2 and 5-LOX inhibitors, TCDD-induced expression of a-SMA protein was inhibited as compared to the control level (Fig. 5b). 3.4. TCDD induces nuclear localization of myocardin-related transcription factor Actin filament polymerization is known to release Myocardiarelated transcription factor (MRTF) family and the coactivator of
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4. Discussion
Fig. 3. Arachidonic acid metabolites induced pulmonary fibroblast migration through AhR. HFL-1 cells were seeded in 24-well Boyden chamber culture dishes for 16–24 h before treatment. The cells were fixed and stained with crystal violet. The number of migrated cells was photographed, counted, and shown as the fold increase of control. The inhibitory effect of NS398 (10 mM) or zileuton (5 mM) was assessed in the presence of TCDD (0.1 mM). Data shown are the average of three independent experiments. *p < 0.05, ns: not significant.
serum response factor, as well as to modulate subsequent cytoskeletal gene expression (Olson and Nordheim, 2010). To examine whether both TCDD-induced actin polymerization and a-SMA expression could coincide with nuclear translocation of MRTF, nuclear translocation of MRTF-A was determined in cells after TCDD treatment (Fig. 6a). Fluorescence confocal microscopy revealed that nuclear translocation of MRTF-A was evident in the cells treated with TCDD. Expression of MRTF in nuclear fraction was also revealed by westen blot analysis (Fig. 6b). 3.5. Recruitment of AhR to a-SMA promoter AhR is known to form heterodimer with AhR nuclear translocator (ARNT) after ligand binding and regulate target genes by binding to the cis-element of dioxin response elements (DREs) (Ma, 2001). The nucleotide sequence analysis of a-SMA gene (NM_001613.2) revealed two consensus DRE binding sites (DRE1: 1780 to 1770 and DRE2: 1080 to 1773, relative to the transcription start site; Fig. 7a). To determine whether a-SMA expression in response to TCDD is caused by the recruitment of AhR to the a-SMA promoter region, ChIP assays were performed using HFL-1 cells treated with TCDD for 0.5 h and 3 h. Soluble chromatin preparations of formaldehyde fixed nuclei were used for immunoprecipitation with anti-AhR Abs. Fig. 7b shows the results, where two fold enrichment of AhR binding to DRE2 site was noted at 0.5 h after TCDD treatment (p < 0.05), whereas DRE1 site showed no significant difference in AhR binding (Fig. 7b). To determine whether TCDD activated the a-SMA promoter, a luciferase reporter assay was also performed using a transient transfection construct containing the 50 -flanking region spanning 1099 to +102 of the a-SMA promoter region. The luciferase activity was enhanced 3-fold in cells stimulated by TCDD (Fig. 7c). These results suggested the importance of AhR in regulating a-SMA expression in pulmonary fibroblasts.
In this study, we demonstrated that the AhR-ligand axis through, in part, the generation of arachidonic acid metabolites induced pulmonary fibroblast migration and differentiation. Pulmonary fibroblast migration is known to be essential in pulmonary fibroblast differentiation (Cigna et al., 2012). Our results showed that pulmonary fibroblast migration was enhanced through AhR-mediated generation of arachidonic acid metabolites, such as LTB4 and PGE2. The enhanced migration was not due to the overgrowth of fibroblasts, as the proliferation of HFL-1 was not affected by TCDD treatment (Fig. 1c). Further, increased intracellular calcium in cells treated with AhR’s ligand has been noted in other experimental models (Kawasaki et al., 2014; Mayati et al., 2012), which, together with ERK kinase activation, elicits activation of cPLA2 and subsequent generation of arachidonic acids (Kawasaki et al., 2014). In addition, activation of COX2 enzyme and subsequent generation of PGE2 are known to be involved in AhR signaling (Martey et al., 2005). Kim et al. also demonstrated that, TCDD-induced calcium influx via T-type channels was evident in rat insulin-secreting beta cell line by using a T-type calcium channel blocker (Kim et al., 2009). In pulmonary epithelial cells, Tsai et al. also revealed a non-canonical AhR signaling pathway in enhancing the levels of cytosolic calcium and activated calcium/ calmodulin-dependent protein kinase II (CaMKII), leading to increased expression of MMP-1 through mitogen-activated protein kinase (MAPK) (Tsai et al., 2014). Our results showed that TCDD treatment induced a transient calcium fluctuation and activation of arachidonic acid pathway in HFL-1 cells (Fig. 2a). Importantly, this event appeared to be required for the subsequent migration of fibroblasts, as the blockade of COX2 and 5-LOX enzymes abrogated the migratory activity of HFL-1 cells. Similarly, results from the AhR knockdown experiments supported a direct link between AhR signaling, calcium mobilization and arachidonic acid metabolism. As a corollary, the role of fibroblast-derived LTB4 and PGE2 has been demonstrated in the wound closure model (Green et al., 2004; Iwanaga et al., 2012; Kawamura et al., 2008). Also, in a medaka embryo model and in lung fibroblasts, the activation of AhR caused by TCDD or cigarette smoke extracts activated the arachidonic acid pathway (Dong et al., 2010; Martey et al., 2005), which is triggered by calcium signaling and subsequent cPLA2 activation (Follonier Castella et al., 2010; Korbecki et al., 2013). Bui et al. utilized a mouse model and showed that an increased level of phospholipase A2 was shown to correlate with TCDD-induced increases in eicosanoid levels in some organs (lung and liver) through the upregulation of cytochrome P450 and lipoxygenase pathways (Bui et al., 2012). Further, a recent study also indicated that knockout of AhR reduced COX-2 and PGE2 expression through the regulation of oxidative stress in mesangial cells (Lee et al., 2016). Collectively, these studies support the link between the AhR-ligand axis and arachidonic acid metabolism. AhR is involved in mediating cell morphology, migration, and adhesion (Fernandez-Salguero 2010; Zhang et al., 2012), which indicates its importance in maintaining cell homeostasis. Previously, Bozyk and Moore have suggested that endogenous COX-2derived PGE2 inhibits pulmonary fibrosis (Bozyk and Moore, 2011); in addition, Penke et al. indicated the inhibitory role of PGE2 in TGF-b-induced a-SMA expression (Penke et al., 2014). In this study, we demonstrated, for the first time, that the AhR-ligand axis mediated both a-SMA expression (Fig. 4a, b) and induction of arachidonic acid metabolites (Fig. 5a, b), wherein the expression of a-SMA may be, in part, amplified by AhR-mediated PGE2 and LTB4, as evidenced by the use of inhibitors of arachidonic acid metabolism, such as NS398 and zileuton. It is likely, therefore, that the various effects of AhR expression for enhancing or
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Fig. 4. TCDD induced actin polymerization and a-SMA expression in pulmonary fibroblasts. Western blotting analysis of (a) a-SMA expression and (b) its relative levels normalized by GADPH. Equal amounts of proteins were loaded and separated using electrophoresis on 10% SDS-PAGE gels and probed with anti a-SMA Abs. Bar graphs depict the results of densitometry analysis. Data represent mean SEM (n = 3–4). (c) Confocal microscope analysis of a-SMA expression in HFL-1 cells after 0.01 mM of TCDD treatment. For fluorescent confocal microscopy, anti a-SMA Abs were used, Fluorescent conjugate of aa-SMA protein expression was shown in red. AhR knockdown was performed using transient transfection of siRNA specific for AhR. All cells were visualized through laser confocal microscopy (Olympus FV1000) at 400 magnifications. (4d, 4e) Actin filament polymerization in HFL-1 cells under different conditions as indicated. Actin filament stained with phalloidin-conjugated Alexa Fluor 488 was shown in green. (4f) Actin filament polymerization was measured as described in materials and methods. Bar graphs depict the results of fluorescent intensity measured, which was analyzed using a laser confocal microscope (Olympus FV1000) unde 400 magnification. *p < 0.05. Data shown are the average of three or more experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
inhibiting cell proliferation, migration, and adhesion may be dependent on different cell types (Barouki and Coumoul, 2010). a-SMA is considered as the hallmark of fibroblast to myofibroblast differentiation, which is associated with cytoskeleton
rearrangement (Follonier Castella et al., 2010), and cytoskeleton protein dynamics are not only the key elements in modulating cell migration but also serving as a platform for signal transduction. Carvajal-Gonzalez et al. reported that AhR-regulated cell migration
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Fig. 5. Arachidonic acid metabolites are involved in a-SMA expression in pulmonary fibroblasts. (a) Confocal microscope analysis of a-SMA expression in HFL-1 cells after PGE2 (50 mM) or LTB4 (100 nM) treatment, or (b) the cells were pretreated with NS398 (10 mM) or zileuton (5 mM) before TCDD(0.01 mmM) stimulation. Actin filaments were stained with phalloidin-conjugated Alexa Fluor 488. All cells were visualized through laser confocal microscopy (Olympus FV1000) at 600 magnification (Olympus FV1000). Data shown are the representative of three experiments.
was mediated through cytoskeleton remodeling and proto-oncogene Vav3 through RhoA/ROCK signaling pathway (CarvajalGonzalez et al., 2009). Similarly, in our study, the expression of actin stress fiber and polymerization of actin fiber were significantly different in cell-type specific manner; for example, HFL-1 cells
induced to express F-actin predominantly by exposure to TCDD, but not in the case of CCL202 cells. This observation is supported by the finding that an increase actin polymerization is not only caused by the increased amount of actin but also caused by an increase in its polymerization (Low et al., 1984).
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Fig. 6. TCDD induced MRTF nuclear localization in pulmonary fibroblasts. (a) HFL-1 cells were grown in 18-mm2 cover slides and cultured in 12-well culture dishes for 16– 24 h before the treatments. The nuclear localization of MRTF was observed 1 h after TCDD (0.01 mM) treatment. Analysis of anti-MRTF-A were used for the nuclear expression. Fluorescent conjugate of MRTF-A protein expression was shown in red. Actin filament stained with phalloidin-conjugated Alexa Fluor 488 was shown in green. Nuclei were stained by DAPI as shown in blue. (b) Nuclear protein was extracted for the examination of nuclear translocation of MRTF, expression of poly ADP ribose polymerase (PARP) was used as an internal control. Data and images shown are the representative of three experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
It has been reported that actin dynamics in response to environmental stimuli may initiate the expression of the MRTF/SRF pathway, subsequently result in a-SMA expression (Olson and Nordheim, 2010). We demonstrated the role of TCDD in mediating MRTF nuclear translocation and a-SMA expression, suggesting the potential role of MRTF in mediating a-SMA expression. It is evident that the regulation of a-SMA promoter through the MRTF/SRF pathway is critical for progression of pulmonary fibrosis (Olson and Nordheim, 2010; Poncelet and Schnaper, 2001; Thannickal and Horowitz, 2006). In an attempt to understand the mechanism of TCDD-induced a-SMA expression, we first identified two possible DRE binding sites (Fig. 7a) on the a-SMA promoter and the chromatin immunoprecipitation and promoter assays showed that only one of the DRE elements (DRE2) was critical for AhR recruitment to the a-SMA promoter. Our data supported the direct regulation of a-SMA expression by AhR activation through transcriptional regulation, where the
subsequent effects of arachidonic acid metabolites are closely integrated, suggesting that AhR may mediate a-SMA expression through a combined non genomic calcium signaling event and a de novo transcriptional process. Thus, the homeostasis of arachidonic acid metabolism appears to be essential for initiating subsequent pathologic effects, such as fibroblast migration and differentiation. Therefore, chronic exposure to xenobiotic, such as TCDD, may disrupt the homeostasis maintained by AhR in pulmonary fibroblasts. Whether AhR is involved in altering the EP receptor profiles or in crosstalk between transcription factors mediated by AhR or arachidonic metabolites in mediating a-SMA expression requires additional studies for confirmation. Further understanding of the mechanisms of long-term exposure effects of environmental contaminants and subsequent AhR-mediated arachidonic acid metabolism may provide a niche and therapeutic target for modulating xenobiotic-induced pulmonary fibrosis.
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Fig. 7. Recruitment of AhR to the a-SMA promoter region. (a) Sequence analysis of 50 UTR of the a-SMA promoter revealed two DRE sites. (b) ChIP assay of AhR’s binding to a-SMA promoter. HFL-1 cells were treated with TCDD (0.01 mmM) for indicated times. The cells were subjected to ChIP assay using anti-AhR Abs. Mouse IgG1 antibody (Iso) was used as the negative control. The amounts of DNA after ChIP were quantified and normalized to an input DNA. The fold enrichment of DRE1 and DRE2 are shown. (c) a-SMA promoter activity. The relative luciferase activities are expressed as a ratio of the pGL3 reporter activity to that of the control plasmid. Data shown represent the mean S.E. of three independent measurements.
Disclosure The authors have no financial conflict of interest. Acknowledgements This paper is supported partially by Kaohsiung Medical University Aim for the Top Universities Grant (KMU-TP104A05, KMU-TP104A10) of Kaohsiung Medical University. We would like to express our special thanks to the Center for Research Resources and Development of Kaohsiung Medical University for the technical help of Fluorescent confocal microscope. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2016.09.019. References Barouki, R., Coumoul, X., 2010. Cell migration and metastasis markers as targets of environmental pollutants and the Aryl hydrocarbon receptor. Cell Adhes. Migration 4, 72–76.
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