Chemico-Biological Interactions xxx (2015) 1e10
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BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway Di Liang a, b, Yan Wang a, b, Zhonghui Zhu a, b, Gengxia Yang c, Guoliang An a, b, Xiaoli Li a, b, Piye Niu a, b, Li Chen a, b, Lin Tian a, b, * a b c
School of Public Health, Capital Medical University, Beijing 100069, China Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China Oncology Minimally Invasive Interventional Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2015 Received in revised form 13 October 2015 Accepted 9 November 2015 Available online xxx
Objective: To investigate the anti-fibrotic effects and possible mechanisms of bone morphogenetic protein-7 (BMP-7) on silica induced fibrosis in RLE-6TN cells, and compare the preventive treatment of experimental silicosis with BMP-7 with therapeutic treatment of silicosis in vitro models. Methods: RLE-6TN cells were incubated with the supernatant of RAW264.7, treated by 50 mg/mL silica in either presence or absence of BMP-7 in different phases. Morphological changes and the cellular woundhealing assays were used to evaluate the process of EMT. By using Western Blotting, the epithelial marker E-cadherin (E-cad), and the mesenchymal markers Vimentin (Vim), Snail, and fibronectin (FN) were detected as well as the Smad signaling pathway proteins, including phosphorylated Smad1/5(P-Smad1/ 5), phosphorylated Smad2/3(P-Smad2/3), and non-phosphorylated Smad1, Smad8, and Smad2. The progress of fibrosis was assessed by the content of hydroxyproline (Hyp) and collagen I and III protein levels. In addition, MTT assay was used to explore the toxic effects of silica as well as BMP-7. Results: The EMT model of RLE-6TN cells was established successfully, the cells had a fibroblast-like morphology with increasing migration activity. The expressions of Vim, Snail, FN, collagen I and collagen III were up-regulated with the increase of silica concentration. BMP-7 could attenuate the decrease of P-Smad1/5 and the increase of P-Smad2/3, collagen I, collagen III, and FN via Smad signaling pathway. BMP-7 inhibited the mesenchymal-like responses in RLE-6TN cells, including cell migration, expression of fibrosis markers, and secretion of Hyp. Furthermore, the anti-fibrotic effects in the prevention group were more effective than treatment group. Conclusion: The restoration of BMP signaling with BMP-7 is associated with inhibiting silica-induced fibrosis through the mechanisms of activated BMP-7/Smad and suppressed TGF-b/Smad pathways. Preventive treatment of pulmonary fibrosis progression with BMP-7 may expect to be the optimized strategy than therapeutic therapy of fibrosis. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Silica Fibrosis Bone morphogenetic protein-7
1. Introduction Silicosis is a pulmonary fibrosis disease caused by long-term inhale of silica dust [1]. Nowadays, a large number of industrial workers have exposed to silica dust directly or indirectly from their work environment in both developed and developing countries all
* Corresponding author. Department of Occupational Health and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069, China. E-mail address: [email protected] (L. Tian).
over the world. Once people are diagnosed with this disease, the fibrous pathological changes of their lung tissues are almost irreversible [2]. There are no sensitive biomarkers to detect the early pathological changes, no effective treatment is available. More evidences show that fibroblasts originating from alveolar epithelial cells phenotypically switches to myofibroblasts via local epithelialemesenchymal transition (EMT). This process may have manufactured one third of the activated fibroblasts [3e5]. Epithelial injury can lead to EMT which then induces fibrosis. In this transition process, epithelial cells lose epithelial features and
http://dx.doi.org/10.1016/j.cbi.2015.11.012 0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.
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acquire mesenchymal features which make them very likely to transit to fibroblasts or myofibroblasts [6]. EMT has been recognized to play an integral role in the process of fibrosis [7], but most studies have gone no further in that. So it is necessary to investigate this pathway in the final fibrotic processes. The TGF-b family plays the central role in silicosis. They are a large group of cytokines with similar structures and related functions, and mainly include TGF-b, activins and bone morphogenetic proteins (BMPs) [8]. All these actions can be broadly classified as ‘TGF-b like’ and ‘BMP like’ actions [9]. The two branches of the family related to the Smad pathway had different receptors and signaling molecules. After binding to their ligands, receptormediated phosphorylation of Smad2/3 (TGF-b) or Smad1/5/8 (BMPs) proteins were activated and transported to the nucleus altering gene transcription [10]. Recent studies indicated that EMT was an ongoing process in fibrotic lungs in vivo and a potential mechanism towards the accumulation of fibroblasts [3]. In terms of EMT and fibrosis, most of the interested researches were focusing on BMP-7, since it was a member of the BMP family which appeared to have a strong antifibrotic activity [11]. Therein BMP-7 is known as an antagonist against TGF-b-dependent fibrogenic activity in several animal and cell models involving fibrosis of a variety of organs such as kidney, liver, heart and colon [12e20]. Hao et al. also showed that the mechanism of BMP-7-mediated repair was reduction of collagen I and collagen III deposition in extracellular matrix induced by TGF-b in hepatic stellate cells (HSC) [21,22]. However, the relationship between BMP-7 and silica-induced fibrosis was seldom investigated. The potential of anti-fibrotic therapy with BMP-7 will be explored. From these existing researches and their limited findings in this field, it is hypothesized that BMP-7 may have a similar effect on silica-induced fibrosis as other fibrosis models and can be tested in vitro experiments, especially in those with normal alveolar epithelial cells we used. Based on in vitro data, we need to consider the mechanism of BMP-7 countering silica-induced fibrosis, and the pathways' balance subjected to regulation during injury and recovery of silicosis in vitro. We hope to find a practicable strategy for the prevention of fibrosis progression or the treatment of pulmonary fibrosis induced by silica. 2. Materials and methods
mL silica solution so as to collect supernatant after 24 h for next EMT and fibrosis model experiment. The RLE-6TN cells were replanted in six-well tissue-culturing dishes at 1 105 cells/well with full-growth medium. The cells were cultured to an approximately 80% of confluence and then cultured in serum-free DMEM (SF-DMEM) overnight. The cells were then incubated for 48 h in DMEM, each containing one of the following conditions: (1) DMEM only, as a negative control (Control group); (2) RAW supernatant (50 mg/mL) only, as a positive control (Silica group); (3) incubated together with additional RAW supernatant (50 mg/mL) and BMP-7 (800 ng/mL) (BMP-7 group); (4) pretreated with BMP-7 (800 ng/mL) for 24 h followed by RAW supernatant (50 mg/mL) (Prevention group); (5) treated with BMP-7 (800 ng/mL) for 24 h after RAW supernatant (50 mg/mL) was added (Treatment group). These doses were based on preliminary experiment results. Group (1), (2), and (3) were used to investigate the anti-fibrotic effect of BMP-7 and Group (1), (2), (4), and (5) were used as a comparison of the prevention and treatment effect. 2.3. Wound healing assay The RLE-6TN cells were grown to 90% confluence in six-well tissue-culturing dishes and the wounds were induced by a pipettor with sterile plastic micropipette tip. The migration of cells into the wound area were observed and photographed by 100 microscope at specific time points. Then the migration ability of cells between groups were compared. 2.4. MTT assay The measurements of cell's viability after exposure to the RAW supernatant were performed using an MTT ((4, 5-dimethyl-2thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) assay. Cells were exposure to RAW supernatant with the concentration of 0, 25, 50, 100 mg/mL for 24 h to determine the optimal concentration. Cells were labeled by 4-h incubation in MTT at 37 C and then DMSO solution was added flowing the 37 C table concentrator. The plates were read on a Microplate reader photometer at 492-nm wavelength. We used the same method to verify that none of the concentrations of BMP-7(0, 25, 50, 100, 200, 400, 800 ng/mL) used was additionally toxic to RLE-6TN. The relative cell's viability was calculated and compared with the absorbance of the negative control group. All MTT assays were performed at three times.
2.1. Silica particles and BMP-7 2.5. Assay of cell hydroxyproline (Hyp) The crystalline silica particles (provided by SigmaeAldrich Co. LLC)with a nominal size of 0.5e1.0 mm were used. Stock solution was prepared in phosphate-buffered saline (PBS) with final concentration of 2 mg/mL after sonication. Human recombinant BMP-7 (354-BP) was purchased from R&D systems (R&D systems Europe Ltd, Abingdon, UK). All other chemicals used were of the highest grade available commercially.
Reagent kit was obtained from Nanjing Jiancheng biotechnology research institution (Nanjing, China). All indexes were detected respectively incubated with RAW supernatant in different doses. All procedures were done according to the manufacturer's instruction, and the absorbance was determined at 550 nm. The Hyp concentrations of the RLE-6TN cells were expressed as mg/g protein. All Hyp assays were performed at three times.
2.2. Cell culture and treatment 2.6. Western blotting RLE-6TN cells (rat alveolar epithelial cells), purchased from XiangYa Central Experiment Laboratory (Changsha, Hunan province, China), were cultivated in DMEM supplemented with 14% fetal bovine serum (FBS) and 1% (w/v) penicillin/streptomycin. Murine macrophage cell line RAW264.7 cells (RAW) were the same condition with 10% FBS at 37 C in an incubator with 5% CO2 and 95% humidified air. After incubation, cell morphology was observed under light microscope and collected for further analysis. All experiments were carried out using the third-passage cells when they grew in good condition. RAW medium were added 0, 25, 50, 100 mg/
The RLE-6TN cells were washed in ice-cold PBS three times and disrupted in lysis buffer. The concentration of protein was determined by using the bicinchoninic acid (BCA) protein assay (Pierce), total protein was extracted and an equal amount of protein (20 mg) was size-fractionated in 8% poly-acrylamide SDS-gel, and then was transferred to PVDF membranes. Membranes were blocked in 3% BSA solution and then incubated overnight at 4 C respectively with the primary antibodies against E-cadherin (1:500, Santa Cruz Biotechnology, Inc.), Viminten (1:5000, Abcam, CA, USA), Snail
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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(1:1000, Abgent), collagen I (1:5000, Abcam, CA, USA), collagen III (1:5000, Abcam, CA, USA), FN (1:500, Santa Cruz Biotechnology, Inc.), P-Smad1/5 (1:1000; Cell Signaling Technology, Boston, MA, USA), P-Smad2/3 (1:1000; Cell Signaling Technology, Boston, MA, USA), Smad1 (1:500, Santa Cruz Biotechnology, Inc.), Smad8 (1:500, Santa Cruz Biotechnology, Inc.), and Smad2 (1:1000; Cell Signaling Technology, Boston, MA, USA). Proteins of interest were detected using the ECL enhanced chemiluminescence method, and GAPDH (1:1000; Cell Signaling Technology, Boston, MA, USA) was used as loading controls to normalize the objective bands. Western blots were scanned and quantified by using Tanon-5200 (Beijing Yuan Ping Hao Biotech Co., Ltd).
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that RAW supernatant significantly improved the expression of the mesenchymal markers, including Vim, by approximately 150% (P < 0.05; Fig. 3A) and the reduced expression of the epithelial marker E-cad by approximately 40%(P < 0.05; Fig. 3B). The ratio of Snail/GAPDH in silica group was higher than that in control group (P < 0.01; Fig. 3E), and this was considered to have a similar role as mesenchymal markers. Similarly, collagen I and collagen III expression, were also presenting a certain doseeresponse trend, increased 1.4 folds and 1.3 folds at 50 mg/mL silica group in comparison to control group (P < 0.05; Fig. 3C and D). All these changes had dose-dependent trends, which reached the peak or nadir at 50 mg/mL. Thus, these results indicated that the EMT and fibrosis model in vitro has been well established for 24 h in RLE-6TN cells.
2.7. Immunofluorescence RLE-6TN cells were grown on chamber slides for 48 h, then rinsed twice with phosphate-buffered saline(PBS) and fixed with 4% paraformaldehyde for 20 min, followed by 0.3% Triton X-100 for 10 min at room temperature (RT). After rinsed in PBS and blocked with 5% BSA at RT for 10 min, slides were incubated with primary anti-bodies against collagen I (1:500, Abcam, CA, USA) or collagen III (1:100, Abcam, CA, USA) overnight at 4 C. After washed, they were lucifuge-incubated with the secondary anti-bodies conjugated to fluorescein isothiocyanate (FITC) for 1 h at RT and rinsed again. Cells were counterstained with DAPI. Immunolabeling was examined by Eclipse 80i microscope (Nikon, Tokyo, Japan). 2.8. Statistical methods All data were obtained from at least three independent experiments performed in triplicate, and the results were presented as means ± standard deviation (SD). One-way analysis of variance was applied to analyze the difference among groups, Statistical significance was set at P < 0.05. All statistical analyses were done with the SPSS 17.0 software. 3. Results 3.1. Silica-induced EMT and fibrosis in RLE-6TN cells were in a dosedependent manner At the beginning, RLE-6TN cells cultured in complete medium showed typical cobblestone morphology, arranged just like “paving stone” samples. After stimulated with dose levels of RAW supernatant for 24 h, the cells changed to more elongated, spindleshaped and fibroblast-like morphology. These changes were associated with a dose-dependent manner, and the most obvious change occurred in 50 mg/mL silica treatment group (Fig. 1A). A cellular wound healing assay was performed after the sample was treated with RAW supernatant, then time-lapse and dose gradient images were obtained from 0 to 24 h. Silica raised the migration activity with a dose-dependent manner compared with the control group; the 50 mg/mL group was the closest to complete healing by 24 h (Fig. 1B). We exposed RLE-6TN cells to RAW supernatant at doses ranging from 0 mg/mL to 100 mg/mL for 24 h to determine the optimal concentration, 50 mg/mL was the optimal concentration (Fig. 2A). MTT assay also showed that there was no significant difference among groups without RAW supernatant in different BMP-7 concentrations (P > 0.05; Fig. 2B), but a significant increase was seen at 800 ng/mL BMP-7 when 50 mg/mL RAW supernatant was treated (P < 0.05; Fig. 2C). In this study, we observed the up-regulation of Snail and mesenchymal markers in the silica-induced fibrosis model. Parallel with the morphological changes, Western blotting analysis showed
3.2. Silica-induced fibrosis in RLE-6TN cells associated with TGF-b/ Smad and BMP-7/Smad signaling balance Smad proteins have shown to bind directly to the TGF-b/Smad and BMP/Smad pathway. The expression of P-Smad1/5, a marker of BMP/Smad signaling had a doseeresponse trend and significantly down-regulated in silica group (50 mg/mL) in comparison with the control group (P < 0.05; Fig. 4A). On the contrary, as an indicator for TGF-b/Smad signaling, the expression of P-Smad2/3 increased gradually, particularly at a dose of 50 mg/mL where it reached the peak (P < 0.05; Fig. 4B). The reduced expression of nonphosphorylation Smad1 and Smad8, as auxiliary proteins, correlated with the increased expression of non-phosphorylation Smad2. As a matter of fact, there was no clear dose-related change detected for all three proteins (P > 0.05; Fig. 4CeE). 3.3. The anti-fibrotic effect of BMP-7 on silica-induced fibrosis in RLE-6TN cells via BMP-7/Smad signaling pathway The anti-fibrotic effect of BMP-7 on fibrosis resulted from blocking the nuclear accumulation of P-Smad2/3 and increasing the expression of P-Smad1/5 in RLE-6TN cells. The level of P-Smad2/3's expression in the silica group was significantly higher than that in the control group (P < 0.05; Fig. 5D). The expression of P-Smad1/5 in silica group decreased significantly compared with that in control group but were increased when BMP-7 was added (P < 0.05; Fig. 5C). The anti-fibrotic effect of BMP-7 was characterized by lower levels of collagen I and collagen III compared to the silica group (P < 0.05; Fig. 5A and B). The Hyp levels increased in the silica group by approximately 2.5 folds at 50 mg/mL (0.5 ± 0.03 mg/g protein) in comparison with control group (0.2 ± 0.08 mg/g protein) (P < 0.05) (Fig. 6A). The silica induced a considerable elevation level at 48 h which was from 0.19 ± 0.06 mg/g protein to 0.5 ± 0.03 mg/g protein in RLE-6TN cells. After BMP-7 intervened, Hyp had significantly reduced from 0.73 ± 0.05 mg/g protein to 0.39 ± 0.18 mg/g protein (P < 0.05) (Fig. 6B). Immunofluorescence staining revealed that collagen I and collagen III mainly expressed in cytoplasm. The two proteins had low background values, and the expressions significantly increased with induction of silica, but were suppressed when BMP-7 was added (Fig. 7). 3.4. Both preventative and therapeutic effects of BMP-7 on silicainduced fibrosis in RLE-6TN cells via BMP-7/Smad signaling pathway The control group mostly maintained epithelial cell's morphology and the silica group had fibroblast-like changes. In contrast to silica group, BMP-7 treatment group showed no obvious changes in morphology; on the other hand, BMP-7 prevention
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 1. Silica induced EMT and fibrosis in RLE-6TN cells on morphology changes and migration activity. RLE-6TN cells were exposed to DMEM (Control) or 0, 25, 50 or 100 mg/mL silica supernatant for 24 h. Compared to the basal condition, silica induced RLE-6TN cells lost their cobblestone shape and adopted a fibroblast-like, spindle-shaped morphology, and this trend along with the concentration increased(A). Wound healing assay showed that silica supernatant increased the migration activity, and 50 mg/mL silica group closed to completely heal at 24-h time point compared with the control group(B).
Fig. 2. Silica inhibited cell proliferation while BMP-7 promoted cell proliferation without extra toxic effect by MTT assay. RLE-6 TN cells were cultured in 0, 25, 50, 100 mg/mL silica supernatant for 24 h, which made the proliferation inhibition at 50 mg/mL(A). RLE-6 TN cells were cultured in 0, 25, 50, 100, 200, 400 or 800 ng/mL BMP-7 with or without 50 mg/mL silica supernatant for 24 h. There was no significant difference between groups without silica supernatant (B), however, a significant raised at 800 ng/mL BMP-7 when silica supernatant was given (p < 0.05) (C). The data are presented as the means ± SD. *p < 0.05 versus Control group.
group could partly reverse the morphological changes induced by silica in RLE-6TN cells (Fig. 8). BMP-7 induced P-Smad1/5 and reached its peak at the concentration of 800 ng/mL. Beyond this, there was no obvious incremental activation observed in RLE-6TN cells. Therefore, we decided to use 800 ng/mL of BMP-7 in the following experiments to study the preventative and therapeutic effects. Furthermore, the expressions of P-Smad2/3 in BMP-7 prevention and treatment groups were reduced in comparison with silica group (P < 0.05; Fig. 9E). The expression of P-Smad1/5 protein in prevention group was higher than that in silica group at the final time points (P < 0.05; Fig. 9D). The anti-fibrotic effect of BMP-7 was detected with collagen I, collagen III, and FN as indexes of fibrosis. The level of collagen I in the silica group was significantly higher than that in the control group (P < 0.01). Furthermore, BMP-7 prevention and treatment groups were reduced compared with silica group (P < 0.05; Fig. 9B). More consistent properties were shown in collagen III and FN, whose expression levels increased about 1.69 folds and 1.32 folds in comparison with that in control group (P < 0.05). In prevention group, the two proteins had a distinct decrease in expression. However, there was no tendency for BMP-7 to affect the treatment (P > 0.05; Fig. 9A and C).
4. Discussion In our study, we specifically investigated the rat lung epithelialT-antigen negative cells (RLE-6TN), a cell line stemmed from the rat normal alveolar epithelium, which made it quite representative and persuasive in this prospective study in both physiological and pathological ways. When the silica-induced EMT vitro model was successfully established, we observed the expression of Vim rose along with spindle-shaped and fibroblast-like morphological changes. Meanwhile, the loss of E-cad was considered as an important feature of phenotype transition process of epithelial cells [23], and the rising expressions of collagen I, III became the major fiber-forming components for fibrosis [6]. We knew that Snail as a cell-adhesion-suppressing transcription factor actually combined with Smads, and then recognized and combined with E-box to inhibit the expression of E-cad. But studies on Snail-mediated EMT or even fibrosis which were through convergence of Smaddependent signaling had no clear conclusion. Other studies found that the expression of Snail increased in patients and mice models with idiopathic pulmonary fibrosis, while the degree of EMT resulting from TGF-b can be reduced by controlling the expression of Snail in type-II alveolar epithelial cells [24,25]. Fortunately, just
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 3. Silica promoted changes in the expression of EMT and fibrosis markers in a dose-dependent manner. RLE-6 TN cells were incubated with DMEM (Control) or 0, 25, 50,100 mg/ mL silica supernatant for 24 h. Western blotting analyses revealed that expression of Vim(A), Snail(C), collagen I(D) and collagen III(E) were increased while the epithelial markers Ecad(B) was decreased in a dose-dependent manner. When the silica supernatant concentration reached up to 50 mg/mL, all of the markers showed significant changes. The data were presented as the means ± SD. *p < 0.05 versus Control group, **p < 0.01 versus Control group.
like a mesenchymal marker, Snail did increase as the concentration of silica increased. These results indicated that silica promoted the ability of RLE-6TN cells of adopting a fibroblast-like behavior, including increasing migratory ability and the expression of mesenchymal and fibrosis markers, secreting collagen I, III, and decreasing the expression of E-cad. Silica-induced EMT and fibrosis occurred via two specific pathways, the Smad-dependent pathway and the Smad-independent pathway [26]. Smad-dependent pathway represented the dominant one; and the other one might be necessary, but not sufficient [7]. In the context of fibrosis, the signaling activity of BMP seemed to oppose the actions of TGF-b’s profibrotic activity. TGF-b and BMP-7 promoted the activation of phosphorylation of the distinct Smad proteins. With silica as a stimulus, P-Smad1/5 presented a
certain dose-response trend and significantly down-regulated; while the expression of P-Smad2/3 increased gradually. In addition, the expression of auxiliary Smad proteins, including Smad1, Smad2, and Smad8, showed no statistical differences. Myllarniemi et al. emphasized that the balance of TGF-b and BMP signaling was important to regenerative events of lung and could be significantly perturbed in pulmonary fibrosis [10]. As a result, we can draw the conclusion that silica led to the activation of TGF-b/Smad pathway and inhibition of BMP-7/Smad pathway. The impairment of BMP signaling due to BMP-7 has now been proven to be an effective anti-fibrotic approach in experimental models with renal, hepatic, and cardiac fibrosis [17e19]. Weiskirchen R et al. found that adding exogenous BMP-7 can block the progression of fibrotic disease and promote the recovery of
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Fig. 4. Silica promoted changes in the expression of TGF-b/Smads/BMP-7 pathway proteins in a dose-dependent manner. RLE-6 TN cells were incubated with DMEM (Control) or 0, 25, 50, 100 mg/mL silica supernatant for 48 h. Western Blotting analyses revealed that the expression of the P-Smad1/5(A) was decreased while P-Smad2/3(B) was increased in a dose-dependent manner. Smad1(C), Smad8(D) and Smad2(E) showed no statistical differences. The data are presented as the means ± SD. *p < 0.05 versus Control group, **p < 0.01 versus Control group.
epithelial characteristics by either inhibiting a Smad signaling pathway or directly modifying the biological activity of TGF-b [27]. BMP-7 treatment significantly reduced collagen deposition in asbestos-treated mice since the activation of BMP-7/Smad pathway promoted the resolution of fibrosis [28]. Our previous results were in full agreement with the role of BMP-7 in reversing fibrosis in rat models with pulmonary fibrosis [1]. The cell's viability that determined by MTT assay verified that using 800 ng/mL BMP-7 not only had no toxic side effects but also promoted the cellular proliferation of RLE-6TN. Based on those findings, we designed a research to find out if the restoration of impaired BMP signaling would result in an anti-fibrotic effect by adding exogenous BMP-7 in normal lung
epithelial cells, RLE-6TN. To a certain extent, we observed that the degree of morphological change was partially prevented by BMP-7. Although BMP-7 restrained silica-induced elevation of all mesenchymal fibrosis markers, it did not successfully restore them to control levels. When we used Smad proteins as targets to investigate the potential anti-fibrotic mechanism of BMP-7, we found the expression of P-Smad2/3 was restrained by BMP-7, and the PSmad1/5 level was just recovered partly compared with silica group. One plausible explanation of the results was that, to some degree, BMP-7 can protect RLE-6TN cells partly from silica-induced damage, which might be associated with partial with the block TGF-b/Smad signaling. On the other hand, the transcription of
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 5. BMP-7 suppressed silica-induced EMT and fibrosis in RLE-6TN cells associated with inhibition of the TGF-b/Smad signaling pathway and activation of BMP-7/Smad signaling pathway. RLE-6 TN cells were incubated with silica supernatant and 800 ng/mL BMP-7 together for 48 h to observe anti-fibrosis effect. Fibrosis markers and Smad proteins were detected using Western Blotting, results revealed that BMP-7 suppressed the high expression of collagen I (A), III (B) and P-Smad2/3(D) induced by silica, and reversed the low expression of P-Smad1/5(C). The data represent the means ± SD. *p < 0.05 versus Control group, #p < 0.05 versus Silica group.
collagen genes such as collagen I and III gene were triggered by PSmad2/3 [29,30], so the reducing of collagen I and III deposition correlated with decreasing expression of P-Smad2/3. The ‘TGF-b like’ and ‘BMP like’ actions determined the balance between TGF-b and BMP signaling pathway which further influenced the progress of fibrosis, and the anti-fibrotic potential of BMP-7 in the regulation of fibrosis is now recognized based on the counteractive effect against the TGF-b/Smad signaling pathways. Considering that silicosis was a long-term, progressive, and irreversible lung tissue fibrosis disease, and there was no effective measures, finding out the early sensitive biomarkers of pathological changes and effective treatments [31], preventing the onset and development of this disease appeared to be extremely critical. Knowing that BMP-7 was able to act as an effective anti-fibrosis agent, we tried to add exogenous BMP-7 at the early stage of silica-induced fibrosis or even before being exposed to RAW supernatant, to delay or even reverse fibrosis process effectively. In
order to verify which kind of strategy was more effective, we designed an experiment with two groups: prevention group and treatment group. The level of P-Smad1/5 appeared to increase in prevention group in comparison with silica group, and this trend was even more significant than that in treatment group. Additionally, the results of Western blot also showed that P-Smad2/3, collagen I, collagen III, and FN had lower expression in prevention group. Notably, the level of fibrosis was significantly alleviated in RLE-6TN which was pretreated with BMP-7 in comparison with those treated with BMP-7 after the initiation of fibrosis. When the balance was disrupted by silica, BMP-7 always acted as a key regulator of BMP-7/Smad signaling pathways and revealed roles of TGF-b. Moreover, the earlier BMP-7 was used, the better antagonism effect of fibrosis would be. These data enhanced the assumption that BMP-7 is a fibrosis inhibitor which suppresses the progression of silica-induced pulmonary fibrosis and has the potential to be used as a preventive treatment.
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Fig. 6. Silica promoted the secretion of hydroxyproline (HYP) in a dose-dependent manner while BMP-7 inhibited the rise trend. The HYP levels were increased parallel with silica supernatant in a dose-dependent manner(A), the content of silica group was significantly higher than that of Control group, and this trend was reduced when adding with BMP7(B). The data were presented as the means ± SD. *p < 0.05 versus Control group, #p < 0.05 versus Silica group.
Fig. 7. BMP-7 suppressed silica-induced expression of collagen I and III in RLE-6TN cells. Immunofluorescence staining revealed that collagen I and III were mainly expressed in cytoplasm. They were significantly increased following induction by silica, but were reduced by BMP-7.
Fig. 8. Pretreated with BMP-7 could inhibit the fibroblast-like morphology changes on RLE-6TN cells. The morphology changes of different treatment groups were stained by original magnification ( 200). BMP-7 prevention group could partly reverse the morphological changes induced by silica in RLE-6TN cells, but BMP-7 treatment group showed no obvious changes in morphology.
5. Conclusion As discussed above, we successfully established an EMT and a fibrosis model in RLE-6TN cells to study the preventative and therapeutic effects of BMP-7 on this pathological process. The TGFb and BMP signaling balance was important to the development of
pulmonary fibrosis. The strategies to restore BMP signaling such as adding BMP-7 may be more plausible for the prevention of fibrosis progression than for the treatment of fibrosis. Our results were based on the research on protein level in vitro. Next, we are going to explore the omni-directional mechanism by working on silent and over-expressed BMP-7 gene. Meanwhile, the further research on
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 9. BMP-7 had both preventive and therapeutic effects on silica-induced fibrosis in RLE-6TN cells via BMP-7/Smad signaling pathway. Fibrosis markers and Smad proteins were detected using western blotting, results revealed that BMP-7 inhibited the high expression of FN (A), collagen I (B) and III(C) induced by silica. The effects of BMP-7 on the changes in fibrosis-markers were suppressed by mediating the P-Smad2/3(E) and actived by P-Smad1/5(D) in RLE-6TN cells and the effect was more obvious pretreated with BMP-7. The data represent the means ± SD. *p < 0.05 versus Control group, #p < 0.05 versus Silica group.
mRNA level and preventative treatment experiments with BMP-7 will be investigated in vivo. Acknowledgments This work was supported by grants of National Natural Science Foundation of China (No. 81273047). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2015.11.012. References [1] G. Yang, Z. Zhu, Y. Wang, A. Gao, P. Niu, L. Tian, Bone morphogenetic protein-7 inhibits silica-induced pulmonary fibrosis in rats, Toxicol. Lett. 220 (2013) 103e108.
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BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway Di Liang a, b, Yan Wang a, b, Zhonghui Zhu a, b, Gengxia Yang c, Guoliang An a, b, Xiaoli Li a, b, Piye Niu a, b, Li Chen a, b, Lin Tian a, b, * a b c
School of Public Health, Capital Medical University, Beijing 100069, China Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China Oncology Minimally Invasive Interventional Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2015 Received in revised form 13 October 2015 Accepted 9 November 2015 Available online xxx
Objective: To investigate the anti-fibrotic effects and possible mechanisms of bone morphogenetic protein-7 (BMP-7) on silica induced fibrosis in RLE-6TN cells, and compare the preventive treatment of experimental silicosis with BMP-7 with therapeutic treatment of silicosis in vitro models. Methods: RLE-6TN cells were incubated with the supernatant of RAW264.7, treated by 50 mg/mL silica in either presence or absence of BMP-7 in different phases. Morphological changes and the cellular woundhealing assays were used to evaluate the process of EMT. By using Western Blotting, the epithelial marker E-cadherin (E-cad), and the mesenchymal markers Vimentin (Vim), Snail, and fibronectin (FN) were detected as well as the Smad signaling pathway proteins, including phosphorylated Smad1/5(P-Smad1/ 5), phosphorylated Smad2/3(P-Smad2/3), and non-phosphorylated Smad1, Smad8, and Smad2. The progress of fibrosis was assessed by the content of hydroxyproline (Hyp) and collagen I and III protein levels. In addition, MTT assay was used to explore the toxic effects of silica as well as BMP-7. Results: The EMT model of RLE-6TN cells was established successfully, the cells had a fibroblast-like morphology with increasing migration activity. The expressions of Vim, Snail, FN, collagen I and collagen III were up-regulated with the increase of silica concentration. BMP-7 could attenuate the decrease of P-Smad1/5 and the increase of P-Smad2/3, collagen I, collagen III, and FN via Smad signaling pathway. BMP-7 inhibited the mesenchymal-like responses in RLE-6TN cells, including cell migration, expression of fibrosis markers, and secretion of Hyp. Furthermore, the anti-fibrotic effects in the prevention group were more effective than treatment group. Conclusion: The restoration of BMP signaling with BMP-7 is associated with inhibiting silica-induced fibrosis through the mechanisms of activated BMP-7/Smad and suppressed TGF-b/Smad pathways. Preventive treatment of pulmonary fibrosis progression with BMP-7 may expect to be the optimized strategy than therapeutic therapy of fibrosis. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Silica Fibrosis Bone morphogenetic protein-7
1. Introduction Silicosis is a pulmonary fibrosis disease caused by long-term inhale of silica dust [1]. Nowadays, a large number of industrial workers have exposed to silica dust directly or indirectly from their work environment in both developed and developing countries all
* Corresponding author. Department of Occupational Health and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069, China. E-mail address: [email protected] (L. Tian).
over the world. Once people are diagnosed with this disease, the fibrous pathological changes of their lung tissues are almost irreversible [2]. There are no sensitive biomarkers to detect the early pathological changes, no effective treatment is available. More evidences show that fibroblasts originating from alveolar epithelial cells phenotypically switches to myofibroblasts via local epithelialemesenchymal transition (EMT). This process may have manufactured one third of the activated fibroblasts [3e5]. Epithelial injury can lead to EMT which then induces fibrosis. In this transition process, epithelial cells lose epithelial features and
http://dx.doi.org/10.1016/j.cbi.2015.11.012 0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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acquire mesenchymal features which make them very likely to transit to fibroblasts or myofibroblasts [6]. EMT has been recognized to play an integral role in the process of fibrosis [7], but most studies have gone no further in that. So it is necessary to investigate this pathway in the final fibrotic processes. The TGF-b family plays the central role in silicosis. They are a large group of cytokines with similar structures and related functions, and mainly include TGF-b, activins and bone morphogenetic proteins (BMPs) [8]. All these actions can be broadly classified as ‘TGF-b like’ and ‘BMP like’ actions [9]. The two branches of the family related to the Smad pathway had different receptors and signaling molecules. After binding to their ligands, receptormediated phosphorylation of Smad2/3 (TGF-b) or Smad1/5/8 (BMPs) proteins were activated and transported to the nucleus altering gene transcription [10]. Recent studies indicated that EMT was an ongoing process in fibrotic lungs in vivo and a potential mechanism towards the accumulation of fibroblasts [3]. In terms of EMT and fibrosis, most of the interested researches were focusing on BMP-7, since it was a member of the BMP family which appeared to have a strong antifibrotic activity [11]. Therein BMP-7 is known as an antagonist against TGF-b-dependent fibrogenic activity in several animal and cell models involving fibrosis of a variety of organs such as kidney, liver, heart and colon [12e20]. Hao et al. also showed that the mechanism of BMP-7-mediated repair was reduction of collagen I and collagen III deposition in extracellular matrix induced by TGF-b in hepatic stellate cells (HSC) [21,22]. However, the relationship between BMP-7 and silica-induced fibrosis was seldom investigated. The potential of anti-fibrotic therapy with BMP-7 will be explored. From these existing researches and their limited findings in this field, it is hypothesized that BMP-7 may have a similar effect on silica-induced fibrosis as other fibrosis models and can be tested in vitro experiments, especially in those with normal alveolar epithelial cells we used. Based on in vitro data, we need to consider the mechanism of BMP-7 countering silica-induced fibrosis, and the pathways' balance subjected to regulation during injury and recovery of silicosis in vitro. We hope to find a practicable strategy for the prevention of fibrosis progression or the treatment of pulmonary fibrosis induced by silica. 2. Materials and methods
mL silica solution so as to collect supernatant after 24 h for next EMT and fibrosis model experiment. The RLE-6TN cells were replanted in six-well tissue-culturing dishes at 1 105 cells/well with full-growth medium. The cells were cultured to an approximately 80% of confluence and then cultured in serum-free DMEM (SF-DMEM) overnight. The cells were then incubated for 48 h in DMEM, each containing one of the following conditions: (1) DMEM only, as a negative control (Control group); (2) RAW supernatant (50 mg/mL) only, as a positive control (Silica group); (3) incubated together with additional RAW supernatant (50 mg/mL) and BMP-7 (800 ng/mL) (BMP-7 group); (4) pretreated with BMP-7 (800 ng/mL) for 24 h followed by RAW supernatant (50 mg/mL) (Prevention group); (5) treated with BMP-7 (800 ng/mL) for 24 h after RAW supernatant (50 mg/mL) was added (Treatment group). These doses were based on preliminary experiment results. Group (1), (2), and (3) were used to investigate the anti-fibrotic effect of BMP-7 and Group (1), (2), (4), and (5) were used as a comparison of the prevention and treatment effect. 2.3. Wound healing assay The RLE-6TN cells were grown to 90% confluence in six-well tissue-culturing dishes and the wounds were induced by a pipettor with sterile plastic micropipette tip. The migration of cells into the wound area were observed and photographed by 100 microscope at specific time points. Then the migration ability of cells between groups were compared. 2.4. MTT assay The measurements of cell's viability after exposure to the RAW supernatant were performed using an MTT ((4, 5-dimethyl-2thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) assay. Cells were exposure to RAW supernatant with the concentration of 0, 25, 50, 100 mg/mL for 24 h to determine the optimal concentration. Cells were labeled by 4-h incubation in MTT at 37 C and then DMSO solution was added flowing the 37 C table concentrator. The plates were read on a Microplate reader photometer at 492-nm wavelength. We used the same method to verify that none of the concentrations of BMP-7(0, 25, 50, 100, 200, 400, 800 ng/mL) used was additionally toxic to RLE-6TN. The relative cell's viability was calculated and compared with the absorbance of the negative control group. All MTT assays were performed at three times.
2.1. Silica particles and BMP-7 2.5. Assay of cell hydroxyproline (Hyp) The crystalline silica particles (provided by SigmaeAldrich Co. LLC)with a nominal size of 0.5e1.0 mm were used. Stock solution was prepared in phosphate-buffered saline (PBS) with final concentration of 2 mg/mL after sonication. Human recombinant BMP-7 (354-BP) was purchased from R&D systems (R&D systems Europe Ltd, Abingdon, UK). All other chemicals used were of the highest grade available commercially.
Reagent kit was obtained from Nanjing Jiancheng biotechnology research institution (Nanjing, China). All indexes were detected respectively incubated with RAW supernatant in different doses. All procedures were done according to the manufacturer's instruction, and the absorbance was determined at 550 nm. The Hyp concentrations of the RLE-6TN cells were expressed as mg/g protein. All Hyp assays were performed at three times.
2.2. Cell culture and treatment 2.6. Western blotting RLE-6TN cells (rat alveolar epithelial cells), purchased from XiangYa Central Experiment Laboratory (Changsha, Hunan province, China), were cultivated in DMEM supplemented with 14% fetal bovine serum (FBS) and 1% (w/v) penicillin/streptomycin. Murine macrophage cell line RAW264.7 cells (RAW) were the same condition with 10% FBS at 37 C in an incubator with 5% CO2 and 95% humidified air. After incubation, cell morphology was observed under light microscope and collected for further analysis. All experiments were carried out using the third-passage cells when they grew in good condition. RAW medium were added 0, 25, 50, 100 mg/
The RLE-6TN cells were washed in ice-cold PBS three times and disrupted in lysis buffer. The concentration of protein was determined by using the bicinchoninic acid (BCA) protein assay (Pierce), total protein was extracted and an equal amount of protein (20 mg) was size-fractionated in 8% poly-acrylamide SDS-gel, and then was transferred to PVDF membranes. Membranes were blocked in 3% BSA solution and then incubated overnight at 4 C respectively with the primary antibodies against E-cadherin (1:500, Santa Cruz Biotechnology, Inc.), Viminten (1:5000, Abcam, CA, USA), Snail
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(1:1000, Abgent), collagen I (1:5000, Abcam, CA, USA), collagen III (1:5000, Abcam, CA, USA), FN (1:500, Santa Cruz Biotechnology, Inc.), P-Smad1/5 (1:1000; Cell Signaling Technology, Boston, MA, USA), P-Smad2/3 (1:1000; Cell Signaling Technology, Boston, MA, USA), Smad1 (1:500, Santa Cruz Biotechnology, Inc.), Smad8 (1:500, Santa Cruz Biotechnology, Inc.), and Smad2 (1:1000; Cell Signaling Technology, Boston, MA, USA). Proteins of interest were detected using the ECL enhanced chemiluminescence method, and GAPDH (1:1000; Cell Signaling Technology, Boston, MA, USA) was used as loading controls to normalize the objective bands. Western blots were scanned and quantified by using Tanon-5200 (Beijing Yuan Ping Hao Biotech Co., Ltd).
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that RAW supernatant significantly improved the expression of the mesenchymal markers, including Vim, by approximately 150% (P < 0.05; Fig. 3A) and the reduced expression of the epithelial marker E-cad by approximately 40%(P < 0.05; Fig. 3B). The ratio of Snail/GAPDH in silica group was higher than that in control group (P < 0.01; Fig. 3E), and this was considered to have a similar role as mesenchymal markers. Similarly, collagen I and collagen III expression, were also presenting a certain doseeresponse trend, increased 1.4 folds and 1.3 folds at 50 mg/mL silica group in comparison to control group (P < 0.05; Fig. 3C and D). All these changes had dose-dependent trends, which reached the peak or nadir at 50 mg/mL. Thus, these results indicated that the EMT and fibrosis model in vitro has been well established for 24 h in RLE-6TN cells.
2.7. Immunofluorescence RLE-6TN cells were grown on chamber slides for 48 h, then rinsed twice with phosphate-buffered saline(PBS) and fixed with 4% paraformaldehyde for 20 min, followed by 0.3% Triton X-100 for 10 min at room temperature (RT). After rinsed in PBS and blocked with 5% BSA at RT for 10 min, slides were incubated with primary anti-bodies against collagen I (1:500, Abcam, CA, USA) or collagen III (1:100, Abcam, CA, USA) overnight at 4 C. After washed, they were lucifuge-incubated with the secondary anti-bodies conjugated to fluorescein isothiocyanate (FITC) for 1 h at RT and rinsed again. Cells were counterstained with DAPI. Immunolabeling was examined by Eclipse 80i microscope (Nikon, Tokyo, Japan). 2.8. Statistical methods All data were obtained from at least three independent experiments performed in triplicate, and the results were presented as means ± standard deviation (SD). One-way analysis of variance was applied to analyze the difference among groups, Statistical significance was set at P < 0.05. All statistical analyses were done with the SPSS 17.0 software. 3. Results 3.1. Silica-induced EMT and fibrosis in RLE-6TN cells were in a dosedependent manner At the beginning, RLE-6TN cells cultured in complete medium showed typical cobblestone morphology, arranged just like “paving stone” samples. After stimulated with dose levels of RAW supernatant for 24 h, the cells changed to more elongated, spindleshaped and fibroblast-like morphology. These changes were associated with a dose-dependent manner, and the most obvious change occurred in 50 mg/mL silica treatment group (Fig. 1A). A cellular wound healing assay was performed after the sample was treated with RAW supernatant, then time-lapse and dose gradient images were obtained from 0 to 24 h. Silica raised the migration activity with a dose-dependent manner compared with the control group; the 50 mg/mL group was the closest to complete healing by 24 h (Fig. 1B). We exposed RLE-6TN cells to RAW supernatant at doses ranging from 0 mg/mL to 100 mg/mL for 24 h to determine the optimal concentration, 50 mg/mL was the optimal concentration (Fig. 2A). MTT assay also showed that there was no significant difference among groups without RAW supernatant in different BMP-7 concentrations (P > 0.05; Fig. 2B), but a significant increase was seen at 800 ng/mL BMP-7 when 50 mg/mL RAW supernatant was treated (P < 0.05; Fig. 2C). In this study, we observed the up-regulation of Snail and mesenchymal markers in the silica-induced fibrosis model. Parallel with the morphological changes, Western blotting analysis showed
3.2. Silica-induced fibrosis in RLE-6TN cells associated with TGF-b/ Smad and BMP-7/Smad signaling balance Smad proteins have shown to bind directly to the TGF-b/Smad and BMP/Smad pathway. The expression of P-Smad1/5, a marker of BMP/Smad signaling had a doseeresponse trend and significantly down-regulated in silica group (50 mg/mL) in comparison with the control group (P < 0.05; Fig. 4A). On the contrary, as an indicator for TGF-b/Smad signaling, the expression of P-Smad2/3 increased gradually, particularly at a dose of 50 mg/mL where it reached the peak (P < 0.05; Fig. 4B). The reduced expression of nonphosphorylation Smad1 and Smad8, as auxiliary proteins, correlated with the increased expression of non-phosphorylation Smad2. As a matter of fact, there was no clear dose-related change detected for all three proteins (P > 0.05; Fig. 4CeE). 3.3. The anti-fibrotic effect of BMP-7 on silica-induced fibrosis in RLE-6TN cells via BMP-7/Smad signaling pathway The anti-fibrotic effect of BMP-7 on fibrosis resulted from blocking the nuclear accumulation of P-Smad2/3 and increasing the expression of P-Smad1/5 in RLE-6TN cells. The level of P-Smad2/3's expression in the silica group was significantly higher than that in the control group (P < 0.05; Fig. 5D). The expression of P-Smad1/5 in silica group decreased significantly compared with that in control group but were increased when BMP-7 was added (P < 0.05; Fig. 5C). The anti-fibrotic effect of BMP-7 was characterized by lower levels of collagen I and collagen III compared to the silica group (P < 0.05; Fig. 5A and B). The Hyp levels increased in the silica group by approximately 2.5 folds at 50 mg/mL (0.5 ± 0.03 mg/g protein) in comparison with control group (0.2 ± 0.08 mg/g protein) (P < 0.05) (Fig. 6A). The silica induced a considerable elevation level at 48 h which was from 0.19 ± 0.06 mg/g protein to 0.5 ± 0.03 mg/g protein in RLE-6TN cells. After BMP-7 intervened, Hyp had significantly reduced from 0.73 ± 0.05 mg/g protein to 0.39 ± 0.18 mg/g protein (P < 0.05) (Fig. 6B). Immunofluorescence staining revealed that collagen I and collagen III mainly expressed in cytoplasm. The two proteins had low background values, and the expressions significantly increased with induction of silica, but were suppressed when BMP-7 was added (Fig. 7). 3.4. Both preventative and therapeutic effects of BMP-7 on silicainduced fibrosis in RLE-6TN cells via BMP-7/Smad signaling pathway The control group mostly maintained epithelial cell's morphology and the silica group had fibroblast-like changes. In contrast to silica group, BMP-7 treatment group showed no obvious changes in morphology; on the other hand, BMP-7 prevention
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 1. Silica induced EMT and fibrosis in RLE-6TN cells on morphology changes and migration activity. RLE-6TN cells were exposed to DMEM (Control) or 0, 25, 50 or 100 mg/mL silica supernatant for 24 h. Compared to the basal condition, silica induced RLE-6TN cells lost their cobblestone shape and adopted a fibroblast-like, spindle-shaped morphology, and this trend along with the concentration increased(A). Wound healing assay showed that silica supernatant increased the migration activity, and 50 mg/mL silica group closed to completely heal at 24-h time point compared with the control group(B).
Fig. 2. Silica inhibited cell proliferation while BMP-7 promoted cell proliferation without extra toxic effect by MTT assay. RLE-6 TN cells were cultured in 0, 25, 50, 100 mg/mL silica supernatant for 24 h, which made the proliferation inhibition at 50 mg/mL(A). RLE-6 TN cells were cultured in 0, 25, 50, 100, 200, 400 or 800 ng/mL BMP-7 with or without 50 mg/mL silica supernatant for 24 h. There was no significant difference between groups without silica supernatant (B), however, a significant raised at 800 ng/mL BMP-7 when silica supernatant was given (p < 0.05) (C). The data are presented as the means ± SD. *p < 0.05 versus Control group.
group could partly reverse the morphological changes induced by silica in RLE-6TN cells (Fig. 8). BMP-7 induced P-Smad1/5 and reached its peak at the concentration of 800 ng/mL. Beyond this, there was no obvious incremental activation observed in RLE-6TN cells. Therefore, we decided to use 800 ng/mL of BMP-7 in the following experiments to study the preventative and therapeutic effects. Furthermore, the expressions of P-Smad2/3 in BMP-7 prevention and treatment groups were reduced in comparison with silica group (P < 0.05; Fig. 9E). The expression of P-Smad1/5 protein in prevention group was higher than that in silica group at the final time points (P < 0.05; Fig. 9D). The anti-fibrotic effect of BMP-7 was detected with collagen I, collagen III, and FN as indexes of fibrosis. The level of collagen I in the silica group was significantly higher than that in the control group (P < 0.01). Furthermore, BMP-7 prevention and treatment groups were reduced compared with silica group (P < 0.05; Fig. 9B). More consistent properties were shown in collagen III and FN, whose expression levels increased about 1.69 folds and 1.32 folds in comparison with that in control group (P < 0.05). In prevention group, the two proteins had a distinct decrease in expression. However, there was no tendency for BMP-7 to affect the treatment (P > 0.05; Fig. 9A and C).
4. Discussion In our study, we specifically investigated the rat lung epithelialT-antigen negative cells (RLE-6TN), a cell line stemmed from the rat normal alveolar epithelium, which made it quite representative and persuasive in this prospective study in both physiological and pathological ways. When the silica-induced EMT vitro model was successfully established, we observed the expression of Vim rose along with spindle-shaped and fibroblast-like morphological changes. Meanwhile, the loss of E-cad was considered as an important feature of phenotype transition process of epithelial cells [23], and the rising expressions of collagen I, III became the major fiber-forming components for fibrosis [6]. We knew that Snail as a cell-adhesion-suppressing transcription factor actually combined with Smads, and then recognized and combined with E-box to inhibit the expression of E-cad. But studies on Snail-mediated EMT or even fibrosis which were through convergence of Smaddependent signaling had no clear conclusion. Other studies found that the expression of Snail increased in patients and mice models with idiopathic pulmonary fibrosis, while the degree of EMT resulting from TGF-b can be reduced by controlling the expression of Snail in type-II alveolar epithelial cells [24,25]. Fortunately, just
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 3. Silica promoted changes in the expression of EMT and fibrosis markers in a dose-dependent manner. RLE-6 TN cells were incubated with DMEM (Control) or 0, 25, 50,100 mg/ mL silica supernatant for 24 h. Western blotting analyses revealed that expression of Vim(A), Snail(C), collagen I(D) and collagen III(E) were increased while the epithelial markers Ecad(B) was decreased in a dose-dependent manner. When the silica supernatant concentration reached up to 50 mg/mL, all of the markers showed significant changes. The data were presented as the means ± SD. *p < 0.05 versus Control group, **p < 0.01 versus Control group.
like a mesenchymal marker, Snail did increase as the concentration of silica increased. These results indicated that silica promoted the ability of RLE-6TN cells of adopting a fibroblast-like behavior, including increasing migratory ability and the expression of mesenchymal and fibrosis markers, secreting collagen I, III, and decreasing the expression of E-cad. Silica-induced EMT and fibrosis occurred via two specific pathways, the Smad-dependent pathway and the Smad-independent pathway [26]. Smad-dependent pathway represented the dominant one; and the other one might be necessary, but not sufficient [7]. In the context of fibrosis, the signaling activity of BMP seemed to oppose the actions of TGF-b’s profibrotic activity. TGF-b and BMP-7 promoted the activation of phosphorylation of the distinct Smad proteins. With silica as a stimulus, P-Smad1/5 presented a
certain dose-response trend and significantly down-regulated; while the expression of P-Smad2/3 increased gradually. In addition, the expression of auxiliary Smad proteins, including Smad1, Smad2, and Smad8, showed no statistical differences. Myllarniemi et al. emphasized that the balance of TGF-b and BMP signaling was important to regenerative events of lung and could be significantly perturbed in pulmonary fibrosis [10]. As a result, we can draw the conclusion that silica led to the activation of TGF-b/Smad pathway and inhibition of BMP-7/Smad pathway. The impairment of BMP signaling due to BMP-7 has now been proven to be an effective anti-fibrotic approach in experimental models with renal, hepatic, and cardiac fibrosis [17e19]. Weiskirchen R et al. found that adding exogenous BMP-7 can block the progression of fibrotic disease and promote the recovery of
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Fig. 4. Silica promoted changes in the expression of TGF-b/Smads/BMP-7 pathway proteins in a dose-dependent manner. RLE-6 TN cells were incubated with DMEM (Control) or 0, 25, 50, 100 mg/mL silica supernatant for 48 h. Western Blotting analyses revealed that the expression of the P-Smad1/5(A) was decreased while P-Smad2/3(B) was increased in a dose-dependent manner. Smad1(C), Smad8(D) and Smad2(E) showed no statistical differences. The data are presented as the means ± SD. *p < 0.05 versus Control group, **p < 0.01 versus Control group.
epithelial characteristics by either inhibiting a Smad signaling pathway or directly modifying the biological activity of TGF-b [27]. BMP-7 treatment significantly reduced collagen deposition in asbestos-treated mice since the activation of BMP-7/Smad pathway promoted the resolution of fibrosis [28]. Our previous results were in full agreement with the role of BMP-7 in reversing fibrosis in rat models with pulmonary fibrosis [1]. The cell's viability that determined by MTT assay verified that using 800 ng/mL BMP-7 not only had no toxic side effects but also promoted the cellular proliferation of RLE-6TN. Based on those findings, we designed a research to find out if the restoration of impaired BMP signaling would result in an anti-fibrotic effect by adding exogenous BMP-7 in normal lung
epithelial cells, RLE-6TN. To a certain extent, we observed that the degree of morphological change was partially prevented by BMP-7. Although BMP-7 restrained silica-induced elevation of all mesenchymal fibrosis markers, it did not successfully restore them to control levels. When we used Smad proteins as targets to investigate the potential anti-fibrotic mechanism of BMP-7, we found the expression of P-Smad2/3 was restrained by BMP-7, and the PSmad1/5 level was just recovered partly compared with silica group. One plausible explanation of the results was that, to some degree, BMP-7 can protect RLE-6TN cells partly from silica-induced damage, which might be associated with partial with the block TGF-b/Smad signaling. On the other hand, the transcription of
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 5. BMP-7 suppressed silica-induced EMT and fibrosis in RLE-6TN cells associated with inhibition of the TGF-b/Smad signaling pathway and activation of BMP-7/Smad signaling pathway. RLE-6 TN cells were incubated with silica supernatant and 800 ng/mL BMP-7 together for 48 h to observe anti-fibrosis effect. Fibrosis markers and Smad proteins were detected using Western Blotting, results revealed that BMP-7 suppressed the high expression of collagen I (A), III (B) and P-Smad2/3(D) induced by silica, and reversed the low expression of P-Smad1/5(C). The data represent the means ± SD. *p < 0.05 versus Control group, #p < 0.05 versus Silica group.
collagen genes such as collagen I and III gene were triggered by PSmad2/3 [29,30], so the reducing of collagen I and III deposition correlated with decreasing expression of P-Smad2/3. The ‘TGF-b like’ and ‘BMP like’ actions determined the balance between TGF-b and BMP signaling pathway which further influenced the progress of fibrosis, and the anti-fibrotic potential of BMP-7 in the regulation of fibrosis is now recognized based on the counteractive effect against the TGF-b/Smad signaling pathways. Considering that silicosis was a long-term, progressive, and irreversible lung tissue fibrosis disease, and there was no effective measures, finding out the early sensitive biomarkers of pathological changes and effective treatments [31], preventing the onset and development of this disease appeared to be extremely critical. Knowing that BMP-7 was able to act as an effective anti-fibrosis agent, we tried to add exogenous BMP-7 at the early stage of silica-induced fibrosis or even before being exposed to RAW supernatant, to delay or even reverse fibrosis process effectively. In
order to verify which kind of strategy was more effective, we designed an experiment with two groups: prevention group and treatment group. The level of P-Smad1/5 appeared to increase in prevention group in comparison with silica group, and this trend was even more significant than that in treatment group. Additionally, the results of Western blot also showed that P-Smad2/3, collagen I, collagen III, and FN had lower expression in prevention group. Notably, the level of fibrosis was significantly alleviated in RLE-6TN which was pretreated with BMP-7 in comparison with those treated with BMP-7 after the initiation of fibrosis. When the balance was disrupted by silica, BMP-7 always acted as a key regulator of BMP-7/Smad signaling pathways and revealed roles of TGF-b. Moreover, the earlier BMP-7 was used, the better antagonism effect of fibrosis would be. These data enhanced the assumption that BMP-7 is a fibrosis inhibitor which suppresses the progression of silica-induced pulmonary fibrosis and has the potential to be used as a preventive treatment.
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Fig. 6. Silica promoted the secretion of hydroxyproline (HYP) in a dose-dependent manner while BMP-7 inhibited the rise trend. The HYP levels were increased parallel with silica supernatant in a dose-dependent manner(A), the content of silica group was significantly higher than that of Control group, and this trend was reduced when adding with BMP7(B). The data were presented as the means ± SD. *p < 0.05 versus Control group, #p < 0.05 versus Silica group.
Fig. 7. BMP-7 suppressed silica-induced expression of collagen I and III in RLE-6TN cells. Immunofluorescence staining revealed that collagen I and III were mainly expressed in cytoplasm. They were significantly increased following induction by silica, but were reduced by BMP-7.
Fig. 8. Pretreated with BMP-7 could inhibit the fibroblast-like morphology changes on RLE-6TN cells. The morphology changes of different treatment groups were stained by original magnification ( 200). BMP-7 prevention group could partly reverse the morphological changes induced by silica in RLE-6TN cells, but BMP-7 treatment group showed no obvious changes in morphology.
5. Conclusion As discussed above, we successfully established an EMT and a fibrosis model in RLE-6TN cells to study the preventative and therapeutic effects of BMP-7 on this pathological process. The TGFb and BMP signaling balance was important to the development of
pulmonary fibrosis. The strategies to restore BMP signaling such as adding BMP-7 may be more plausible for the prevention of fibrosis progression than for the treatment of fibrosis. Our results were based on the research on protein level in vitro. Next, we are going to explore the omni-directional mechanism by working on silent and over-expressed BMP-7 gene. Meanwhile, the further research on
Please cite this article in press as: D. Liang, et al., BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-b/Smad and BMP-7/Smad signaling pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.11.012
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Fig. 9. BMP-7 had both preventive and therapeutic effects on silica-induced fibrosis in RLE-6TN cells via BMP-7/Smad signaling pathway. Fibrosis markers and Smad proteins were detected using western blotting, results revealed that BMP-7 inhibited the high expression of FN (A), collagen I (B) and III(C) induced by silica. The effects of BMP-7 on the changes in fibrosis-markers were suppressed by mediating the P-Smad2/3(E) and actived by P-Smad1/5(D) in RLE-6TN cells and the effect was more obvious pretreated with BMP-7. The data represent the means ± SD. *p < 0.05 versus Control group, #p < 0.05 versus Silica group.
mRNA level and preventative treatment experiments with BMP-7 will be investigated in vivo. Acknowledgments This work was supported by grants of National Natural Science Foundation of China (No. 81273047). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2015.11.012. References [1] G. Yang, Z. Zhu, Y. Wang, A. Gao, P. Niu, L. Tian, Bone morphogenetic protein-7 inhibits silica-induced pulmonary fibrosis in rats, Toxicol. Lett. 220 (2013) 103e108.
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