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Abstract

The epithelial–mesenchymal transition (EMT) is a critical process in the pulmonary fibrosis. It has been reported that bone morphogenetic protein 7 (BMP-7) was able to reverse EMT in proximal tubular cells. Therefore, we test the hypothesis that EMT contributes to silica-induced pulmonary fibrosis and BMP-7 inhibits EMT in silica-induced pulmonary fibrosis. Progressive silica-induced pulmonary fibrosis in the rat was used as a model of silicosis. Epithelial and mesenchymal markers were measured from rat fibrotic lungs. Then the effects of BMP-7 on the EMT were further confirmed in A549 cells. There are increases of vimentin as a mesenchymal marker and decreases of E-cadherin as an epithelial marker in the silica-exposed rat lungs, which is in agreement with the A549 cells data. However, BMP-7 treatment significantly reduced expression of vimentin in the rat pulmonary fibrosis model and in A549 cells. In conclusion, EMT contributes to silica-induced pulmonary fibrosis. Meanwhile, the treatment of BMP-7 can inhibit silica-induced EMT in vitro and in vivo.

Keywords

Bone morphogenetic protein-7 epithelial–mesenchymal transition pulmonary fibrosis silica

Introduction

The epithelial–mesenchymal transition (EMT), transdifferentiation of epithelial cells into mesenchymal cells, is a prevailing phenomenon that plays crucial roles in organic fibrosis and cancer metastasis.¹ During this process, epithelial cells lose their markers, such as E-cadherin and cytokeratin, as well as acquisition of markers of mesenchymal cells, such as vimentin and α-smooth muscle actin (α-SMA).^2,3 Silicosis, caused by silica particles, is characterized by a persistent inflammatory response and progressive pulmonary fibrosis.⁴ In addition, pulmonary fibrosis is related to fibroblast proliferation and transdifferentiation and excess extracellular matrix generation in the development of silicosis.⁵ However, the origin of fibroblasts has not been explored in great detail.

The mechanism of EMT may account for a fraction of the origin of fibroblasts. The number of cells undergoing EMT, reported in the literature, varies from 10% to 25%.⁶ During the process of EMT, cells gradually become isolated and motile through a functional transition of polarized epithelial cells into migratory mesenchymal cells.⁷ Transforming growth factor β (TGF-β), a strong pro-fibrogenic cytokine, can induce the alveolar epithelial cell transition to fibroblasts via EMT in vivo and in vitro.^8,9 RLE-6TN cells of TGF-β-induced EMT show increased expression of α-SMA and vimentin and decreased expression of E-cadherin and cytokeratin.¹⁰ The underlying molecular mechanisms for EMT have not been elucidated in the pulmonary fibrosis. The canonical Smad-dependent pathway is one of the main pathways in the process of TGF-β-induced EMT.¹¹

Bone morphogenetic protein 7 (BMP-7), belonging to the TGF-β superfamily, is a negative regulator of EMT. BMP-7 can be in the preservation of the epithelial phenotype and reverse TGF-β-induced EMT in renal epithelial cells.^12,13 Growing evidences demonstrated that BMP-7 could inhibit renal, liver, and peritoneal fibrosis in animal models.^14
–16 In addition, studies have confirmed that BMP-7 were able to reverse EMT in proximal tubular cells.¹⁷ Therefore, BMP-7 seems to inhibit TGF-β-induced fibrosis involving EMT via Smad-dependent pathway. The counterbalance of TGF-β and BMP-7 is able to control EMT.¹⁸

We assume that EMT may contribute to silica-induced pulmonary fibrosis. Considering the above functions of BMP-7, we hypothesized that BMP-7 may be involved in EMT in silica-induced pulmonary fibrosis. In this study, we determine whether BMP-7 regulates EMT in the silica-induced fibrosis and assess BMP-7 as a potential therapy for silicosis.

Materials and methods

Animals and reagents

A total of 48 male Wistar rats, aged 8 weeks and weighing 200–240 g, were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). The rats were maintained in a controlled temperature (24 ± 1°C) room with 12-h light:12-h dark cycle and provided with free access to water and food. Silica dust was purchased from the Center of Occupational Health and Poisoning Control, Chinese Centers for Disease Control and Prevention (Beijing, China). Silica dust characterization and suspension preparation was as previously described by Yang et al.¹⁹ Recombinant human BMP-7 (rhBMP-7) was purchased from QuantoBio Co. Ltd. (Beijing, China).

Animal model of pulmonary fibrosis

Rats were instilled with intratracheal silica suspension to induce silica-induced pulmonary fibrosis, as previously described by Yang et al.¹⁹ Briefly, rats were anesthetized in ether followed by intratracheal instillation of 50 mg of silica in 1 ml saline. To evaluate the effect of BMP-7 on silica-induced pulmonary fibrosis, animals exposed to silica were divided into two groups where they received rhBMP-7 (300 µg/kg, intraperitoneally) or vehicle control (1 ml saline), respectively, on day 8 post instillation every other day. Control rats received intratracheal 1 ml saline followed by intraperitoneal injection of 1 ml of saline on day 8 post instillation every other day. The animals were killed to collect the lung tissues for further analysis on day 15 or day 30 after silica instillation. The right lung was used for biochemical analysis, and the left lung was used for histopathology. The total protein was extracted with the tissue total protein extraction kit (KeyGEN, Nanjing, China) from lung homogenates of all groups of rats. All animal experimental procedures were approved by the Laboratory Animal Care and Use Committee at Capital Medical University with a permissive number of 2012-X-25, which abides by National Institute of Health Guide for the Care and Use of Laboratory Animals.

Histology

Following the gross inspection, 10% formalin-fixed lung tissues were embedded in paraffin and cut into 5-μm thick sections. The sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT) stains for histological examination. The light microscope (Olympus D72, Japan) was used to examine at a magnification of 200×. In addition, the severity of pulmonary fibrosis was assessed according to the percentage of collagen measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, Maryland, USA).

Culture of A549 cells and treatments

A549 cell, a cell line of human type II alveolar epithelial carcinoma, was purchased from Xiang Ya School of Medicine, Central South University (Hunan, China). Cells was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, California, USA) containing 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 µm streptomycin in 5% carbon dioxide incubator (SANYO, Japan) at 37°C.

Cells were plated in six-well plates (total 51 plates) at a density of 5 × 10⁴cells/well. When approximately 70–80% confluent, A549 cells were maintained in 0.5% FCS-DMEM for 24 h and then treated in the absence or presence of silica at a concentration of 0, 12.5, 25, 50, 100, or 200 μg/ml to observe the dose-dependent changes for 24 h. The time-dependent profiles of A549 cell treated with the silica suspension (100 μg/ml, 2.5 ml) were investigated for 6, 12, 24, 48, or 72 h.

A549 cells were treated with the silica suspension (100 μg/ml, 2.5 ml) and different concentrations of BMP-7 (0, 100, 200, 400, 800, or 1600 ng/ml) for 24 h. The cells were harvested following laboratory standard procedures, and cellular proteins were extracted by using the total protein extraction kit for Western blot analysis according to the manufacturer’s instructions.

Western blot analysis

Western blots were performed as previously described by Yang et al.¹⁹ Briefly, 80 μg proteins from A549 cells or lung homogenates were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) transfer membrane (GE Healthcare, Atlanta, Georgia, USA), which was blocked with fat-free milk and incubated with the primary antibodies, namely vimentin and β-actin (Cell Signaling Technology, Beverly, Massachusetts, USA) and E-cadherin (Santa Cruz biotechnology, Santa Cruz, California, USA), respectively. Membrane was incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). Finally, pierce enhanced chemiluminescence Western Blotting Substrate (Thermo, Pittsburgh, Pennsylvania, USA) and Kodak film (Kodak, Rochester, New York, USA) were used for the visualization of the target protein. Western blot images were acquired and analyzed by Molecular Imager BIO-RAD Gel Doc™ XR + with Image lab™ software (BIO-RAD, Berkeley, California, USA).

Immunohistochemistry

After deparaffinization and rehydration of the paraffin-embedded lung tissue sections, the sections for antigen retrieval were heated in citrate buffer (pH 6.0) for 10 min at 100°C. Then, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Thereafter, the sections were blocked with 5% normal goat serum and incubated overnight at 4°C with phosphate-buffered saline containing rabbit polyclonal anti-E-cadherin (1:100, Santa Cruz Biotechnology) or vimentin (1:100, Cell Signaling Technology). A 30-min incubation in biotinylated secondary antibody (1:1000) was followed by 30 min in 3,3′-diaminobenzidine reagent. Slides were counterstained with hematoxylin for 3 min. At the end, immunohistochemistry images were obtained using the same exposure times and conditions with Olympus X71-F22PH microscope (Olympus, Japan) and analyzed by Image-Pro Plus 6.0 software (Media Cybernetics).The ratio of integrated optical density/pulmonary parenchyma area represented the quantitative expression of vimentin and E-cadherin.

Statistical analysis

In this study, the SPSS 18.0 software was used to evaluate statistical significance through a one-way analysis of variance followed by pairwise comparison with the Student–Newman–Keuls test. All values were means ± SD. The values of p < 0.05 were statistically significant.

Results

BMP-7 inhibits EMT in a rat model of silicosis

The previous studies have demonstrated that EMT can contribute to bleomycin-induced lung fibrosis²⁰ and several organs.^21,22 To analyze the effect of BMP-7 on EMT, the expressions of epithelial and mesenchymal markers were determined by immunohistochemistrical staining for lungs from rats. The results showed that silica induced a marked upregulation of mesenchymal marker vimentin on days 15 and 30 after instillation (Figure 1(a)), but BMP-7 treatment markedly reduced the upregulation of vimentin on days 15 and 30 (p < 0.05, Figure 1(b)). Meanwhile, downregulation of the epithelial marker E-cadherin was obvious in silica groups compared with control groups (Figure 2(a)). By contrast, downregulation of E-cadherin was significantly inhibited in BMP-7 treatment groups on day 30 (p < 0.05, Figure 2(b)).

Figure 1.

Effect of BMP-7 on vimentin level of lung tissue of silica-exposed rats. (a) Representative images of immumohistochemical staining for vimentin are presented in three groups over time. Positive staining presents a yellow brown color (magnification ×200). Scale bar: 50 μm. (b) Histogram showing the changes of mean IOD/pulmonary area of vimentin in silica-induced pulmonary fibrosis after BMP-7 treatment at different times. Values are expressed as mean ± SD (n = 8 rats/group). #p < 0.05: compared with saline control group; *p < 0.05: compared with silica group. BMP-7: bone morphogenetic protein 7; IOD: integrated optical density.

Figure 2.

Effect of BMP-7 on E-cadherin level of lung tissue of silica-exposed rats. (a). Representative images of immumohistochemical staining for E-cadherin are presented in three groups over time. Positive staining presents a yellow brown color (magnification ×200). (b). Histogram showing the changes of mean IOD/pulmonary area of E-cadherin in silica-induced pulmonary fibrosis after BMP-7 treatment at different times. Values are expressed as mean ± SD (n = 8 rats/group). #p < 0.05: compared with saline control group; *p < 0.05: compared with silica group. Scale bar: 50 μm. BMP-7: bone morphogenetic protein 7; IOD: integrated optical density.

Inhibition of EMT correlates with reduction of pulmonary fibrosis in vivo

BMP-7 can reduce asbestos-induced fibrotic alterations in the lung.²³ However, there are insufficient data to show whether it has antifibrotic effect in silica-induced lung fibrosis. In this study, we assessed the effect of BMP-7 on silica-induced lung fibrosis in rats. MT-stained sections of lungs from rats demonstrated collagen fiber areas (blue areas) was increased in silica groups compared with control groups but was decreased in BMP-7-treated group compared with silica groups (Figure 3(a)). To further confirm the effects of BMP-7 on the histopathology of silica-induced lung fibrosis, the percentage of fibrotic area (collagen area/whole pulmonary parenchyma area) was measured. Analysis of the result showed that the pulmonary fibrosis was reduced in the BMP-7-treated rats by approximately 11–21% as compared to silica alone-treated rats on days 15 and 30, respectively (p < 0.05, Figure 3(b)). H&E and Masson stains in each group also showed a similar change over time (Figure 4). At 15 days after instillation, we observed alveolar septal thickening, inflammatory infiltration, and irregular cellular nodules in the lungs of silica group; while in BMP-7-treated group, there were less cellular nodules in the lungs of rats at day 15 (Figure 4). However, we noticed more and larger cellular nodules on day 30 after instillation, compared with day 15. BMP-7-treated rats showed less number and smaller size of cellular nodules in the lungs compared with the silica group (Figure 4).

Figure 3.

BMP-7 attenuates intratracheally silica-induced pulmonary fibrosis. (a) Representative Masson-stained sections to detect collagen fiber are shown from three groups at days 15 and 30 after silica instillation. The fibrotic areas were stained blue (Magnification ×200). Scale bar: 50 μm. (b) The fibrotic areas are significantly increased in silica group compared with control rats. BMP-7 treatment significantly reduces the fibrotic areas when compared with silica group at days 15 and 30. Values are expressed as mean ± SD (n = 8 rats/group). #p < 0.05: compared with saline control group; *p < 0.05: compared with silica group. BMP-7: bone morphogenetic protein 7.

Figure 4.

H&E staining for pathological changes in rat lung (magnification ×200). Silica group: infiltration of inflammatory cells and alveolar septal thickening, and irregular cellular nodules were observed at day 15; more and larger cellular nodules at day 30; BMP-7-treated group: compared to silica group, less cellular nodules at day 15 were observed; the number and volume of nodules were fewer and smaller at day 30. Scale bar: 50 μm. H&E: hematoxylin and eosin; BMP-7: bone morphogenetic protein 7.

BMP-7 counteracts silica-induced EMT in vitro

It has been proven that BMP-7 substantially inhibited TGF-β-induced EMT in human coronary endothelial cells²⁴ and NP1 cells.¹³ We tested the effect of BMP-7 on silica-induced EMT in A549 cells exposing to various concentrations of BMP-7- and silica-treated macrophage supernatants. A549 cells in control group exhibited the typical features of epithelial cells, including a cubic, cobblestone-like formation. After the silica suspension (100 μg/ml) treatment, inverted microscope revealed that the A549 cells underwent a morphological change, turned into a spindle-like mesenchymal phenotype, and lost their cell–cell contact. A549 cells treated with BMP-7 in the supernatant recovered epithelial phenotype (Figure 5(a)). The expression of E-cadherin, a characteristic epithelial phenotypic marker, gradually reduced in a dose-dependent manner. On the contrary, as a mesenchymal marker, vimentin level gradually increased. The level of E-cadherin was significantly (p < 0.05) decreased in the presence of silica suspension as well as increases of vimentin expression (Figure 5(b)). Meanwhile, the expressions of E-cadherin and vimentin present an opposite change in a time-dependent manner. Incubation with the silica suspension (100 μg/ml) for 72 h induced an obvious decrease in E-cadherin expression (p < 0.05) and an increase in vimentin expression (p < 0.05; Figure 5(c)). After treatment with BMP-7(100–1600 ng/ml), reduction of E-cadherin level by the silica suspension (100 μg/ml) was inhibited, and vimentin expressed was downregulated in A549 cells (Figure 5(d)). These suggested that BMP-7 inhibited silica-induced EMT in A549 cells.

Figure 5.

Regulation of silica-induced vimentin and E-cadherin expression by BMP-7. (a) Changes in A549 cells morphology. (b) Effect of silica on expression of mesenchymal marker vimentin and epithelial marker E-cadherin in a dose-dependent manner. (c) Effect of silica on expression of mesenchymal marker vimentin and epithelial marker E-cadherin in a time-dependent manner. (d) Effect of BMP-7 on regulation of vimentin and E-cadherin expression by silica. Densitometric analysis values for each blot were normalized to β-actin and expressed as a ratio of basal levels. Each point represents mean ± SD (n = 3 independent experiments) *p < 0.05: compared with control group. BMP-7: bone morphogenetic protein 7.

BMP-7 inhibits TGF-β1/Smad3 in silica-induced EMT in vivo

TGF-β signaling has been well known as mediators of TGF-β-induced EMT through Smad2/3.²⁵ Therefore, we examined the changes of TGF-β1 and Smad3 proteins after BMP-7 treatment in a rat model of silicosis. We found that TGF-β1 expression in silica group was significantly higher than that in the control group at days 15 and 30 (p < 0.05; Figure 6(b)). However, TGF-β1 expression was significantly reduced in BMP-7-treated rats than the silica groups at days 15 and 30 (p < 0.05; Figure 6(b)). Similarly, Smad3 expression was significantly reduced in BMP-7-treated groups compared with silica groups on day 30 post instillation (p < 0.05; Figure 6(c)). All these data suggested BMP-7 treatment could inhibit TGF-β1/Smad3 in silica-induced EMT.

Figure 6.

Effects of BMP-7 on TGF-β1/Smad3 in rat lung. The expression of TGF-β1 and Smad3 was detected. (a) Representative Western blot results were presented; (b) relative densitometry diagram demonstrates a significant increase in TGF-β1 level in the silica group compared with the saline control group. TGF-β1 was decreased in the BMP-7-treated group; (c) relative densitometry diagram demonstrates an increase in Smad3 level in the silica group compared with the saline control group; and (d) Smad3 is decreased in the BMP-7-treated group. Values are expressed as mean ± SD (n = 5 rats/group). #p < 0.05: compared with saline control group; *p < 0.05: compared with silica group. BMP-7: bone morphogenetic protein 7; TGF-β1: transforming growth factor-β1.

Discussion

EMT, a potential source of fibroblasts, is assumed to be an important link for various fibrotic diseases such as cardiac and renal fibrosis.^26,27 Theoretically, fibroblasts can derive directly from epithelial cells via EMT,²⁰ which may contribute to the development of silicosis, a fibrotic lung disease caused by inhalation of free crystalline silica.²⁸ The recent study indicates that approximately one-third of the lung fibroblasts derive from the lung epithelium 2 weeks after bleomycin administration and argue that epithelial cells in human forms of lung fibrosis are capable of phenotypic transition to a mesenchymal phenotype.²⁰ Consistent with these results, our present studies found that there are increases of vimentin as a mesenchymal marker and decreases of E-cadherin as an epithelial marker in silica-induced pulmonary fibrosis. These results suggested that epithelial cells undergo EMT in vivo in the rat model of pulmonary fibrosis caused by silica. Similarly, in vitro studies revealed that silica induced EMT in A549 treated with silica suspension, including loss of E-cadherin and increases of vimentin expression. Here, we demonstrate that EMT has also crucial implications for silica-induced pulmonary fibrosis.

Myllärniemi and colleagues²³ observed that BMP-7 treatment inhibited asbestos-induced fibrotic changes, when the treatment was started 7 days after asbestos exposure. In this study, we established a rat model of silicosis by intratracheal instillation of silica dust. BMP-7 was administered to treat the animals on day 8 after the intratracheal instillation of silica dust. So, our research focused on the remedial treatment of BMP-7 for silica-induced pulmonary fibrosis. To elucidate the antifibrotic efficacy of BMP-7, we first analyzed lung histopathologic changes from MT and H&E stains. Masson staining for collagen fibers confirmed that BMP-7 alleviated pulmonary fibrosis in BMP-7-treated rats versus rats exposed to silica, especially at day 30 post instillation. Meanwhile, H&E histological staining showed the same pattern as Masson’s findings in the lung of BMP-7-treated groups versus positive control groups (silica). These findings suggested the potential antifibrotic function of BMP-7 in the rat model of silicosis.

Additionally, BMP-7 has also been identified as an antagonist of EMT.¹³ To explain further whether the antifibrotic effects of BMP-7 in the rat model of silicosis were involved in EMT, we analyzed the potential efficacy and molecular mechanisms of BMP-7 to attenuate silica-induced EMT. Therefore, we tested whether BMP-7 could inhibit silica-induced EMT. Animal experimental results demonstrated that significantly less vimentin and more E-cadherin was expressed in the BMP-7-treated group than in the silica group. Similarly, following the exposure of A549 cells to silica suspension for 72 h, the expression of epithelial marker E-cadherin was increased and expression of the mesenchymal marker vimentin decreased. The above data are consistent with the literature report that BMP-7 reversed TGF-β₁-induced EMT by reinduction of E-cadherin.¹³ This study demonstrated that BMP-7 inhibits silica-induced EMT in the rat pulmonary fibrosis model and in A549 cells.

It has been identified that BMP-7 counteracts EMT involving Smad signaling pathway in chronic renal injury and kidney fibrosis.^13,29 In order to elucidate the potential mechanism in the regulation of BMP-7 to inhibit EMT, we further investigated the expressions of TGF-β1 and Smad3 proteins in vivo. These two Smad proteins were considered to be involved in TGF-β-induced EMT.²⁵ Our data showed that BMP-7 treatment suppressed the expression of TGF-β1 and Smad3 in countering EMT. This is in agreement with the previously published observation that BMP-7 directly blocks Smad2/3. Therefore, our results show that the inhibition of silica-induced EMT with BMP-7 might be explained by the downregulation of TGF-β1/Smad3.

In conclusion, this article provides evidence that EMT contributes to silica-induced pulmonary fibrosis and the treatment of BMP-7 can inhibit silica-induced EMT in vitro and in vivo. Furthermore, these results indicate a potential therapeutic intervention for the treatment of silicosis.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This work was supported by grants of National Natural Science Foundation of China (No. 81273047).

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Bone morphogenetic protein 7 attenuates epithelial–mesenchymal transition induced by silica

Abstract

Keywords

Introduction

Materials and methods

Animals and reagents

Animal model of pulmonary fibrosis

Histology

Culture of A549 cells and treatments

Western blot analysis

Immunohistochemistry

Statistical analysis

Results

BMP-7 inhibits EMT in a rat model of silicosis

Inhibition of EMT correlates with reduction of pulmonary fibrosis in vivo

BMP-7 counteracts silica-induced EMT in vitro

BMP-7 inhibits TGF-β1/Smad3 in silica-induced EMT in vivo

Discussion

Footnotes

Conflict of interest

Funding

References