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Am J Physiol Lung Cell Mol Physiol 290: L120-L126, 2006. First published August 26, 2005; doi:10.1152/ajplung.00171.2005
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BMP-7 opposes TGF-{beta}1-mediated collagen induction in mouse pulmonary myofibroblasts through Id2

Nobuhiro Izumi,1 Shinjiro Mizuguchi,1 Yutaka Inagaki,2 Shizuya Saika,3 Norifumi Kawada,4 Yuji Nakajima,5 Kiyotoshi Inoue,1 Shigefumi Suehiro,1 Scott L. Friedman,6 and Kazuo Ikeda5

Departments of 1Surgery, 4Hepatology, and 5Anatomy, Graduate School of Medicine, Osaka City University, Osaka; 2Liver Fibrosis Research Unit, Tokai University School of Medicine, Isehara; 3Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan; and 6Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York

Submitted 15 April 2005 ; accepted in final form 12 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mesenchymal cells, primarily fibroblasts and myofibroblasts, are the principal matrix-producing cells during pulmonary fibrogenesis. Transforming growth factor (TGF)-{beta} signaling plays an important role in stimulating the expression of type I collagen of these cells. Bone morphogenetic protein (BMP)-7, a member of the TGF-{beta} superfamily, has been reported to oppose the fibrogenic activity of TGF-{beta}1. Here, we have addressed the effects of BMP-7 on the fibrogenic activity of pulmonary myofibroblasts. We first established cell lines from the lungs of transgenic mice harboring the COL1A2 upstream sequence fused to luciferase. They displayed a spindle shape and expressed vimentin and {alpha}-smooth muscle actin, but not E-cadherin. COL1A2 promoter activity was dose dependently induced by TGF-{beta}1, which was further augmented by adenoviral overexpression of Smad3, but was downregulated by Smad7. Under the identical condition, adenoviral overexpression of BMP-7 attenuated the TGF-{beta}1-dependent COL1A2 promoter activity. By immunocytochemistry, the ectopic expression of BMP-7 led to the nuclear localization of phospho-Smad1/5/8 and suppressed that of Smad3. BMP-7 suppressed the expression of mRNAs for COL1A2 and tissue inhibitor of metalloproteinase-2 while increasing those of inhibitors of differentiation (Id) 2 and 3. Ectopic expression of Id2 and Id3 was found to decrease the COL1A2 promoter activity. Finally, BMP-7 and Id2 decreased TGF-{beta}1-dependent collagen protein secretion. In conclusion, these data demonstrate that BMP-7 antagonizes the TGF-{beta}1-dependent fibrogenic activity of mouse pulmonary myofibroblastic cells by inducing Id2 and Id3.

bone morphogenetic protein; transforming growth factor; inhibitors of differentiation

PULMONARY FIBROSIS is an interstitial lung disorder associated with a range of acute and chronic lung injuries, characterized by the presence of chronic inflammation and increased deposition of collagens in lung parenchyma, leading to pulmonary dysfunction (8). Several cell types, including macrophages, T cells, and neutrophils, have been implicated as potential sources of cytokines that amplify inflammatory reactions and provoke the activation and proliferation of fibroblasts (1, 6). In particular, TGF-{beta} plays a pivotal role in the process of fibrosis. TGF-{beta} stimulates matrix deposition in mesenchymal cells by promoting the expression of extracellular matrix (ECM) genes and suppressing the activity of matrix metalloproteinases (3, 9, 32). Indeed, TGF-{beta} is most potent in promoting collagen gene transcription (15, 22). We previously identified a COL1A2 upstream sequence that responds to TGF-{beta} stimulation and designated this region the TGF-{beta}-responsive element (TbRE) (16). TGF-{beta} stimulates COL1A2 transcription via the interaction of Sp1 and Smad3, both of which bind to the TbRE (14).

Bone morphogenetic protein (BMP)-7, a member of the transforming growth factor (TGF)-{beta} superfamily of cysteine knot cytokines, has been implicated in the induction of epithelial mesenchymal transition (EMT) and the prevention of fibrosis progression in experimental renal diseases (41). BMP-7 may also counteract the profibrogenic activity of TGF-{beta} (38). In the absence of BMP-7, the activation of TGF-{beta} receptors by their ligand(s) leads to the phosphorylation and activation of Smad2 and Smad3, and their heterodimerization with Smad4, followed by their translocation into the nucleus (31, 37). In contrast, BMPs utilize another series of receptor-regulated Smads, Smad1 and Smad5 (43). In BMP-7, as well as TGF-{beta} signaling, Smad4 acts as a transcriptional coactivator together with Smad1 and Smad5. However, a detailed mechanism of how BMP-7 antagonizes TGF-{beta}-dependent fibrogenesis has been lacking to date.

In the present study, we established cell lines of pulmonary myofibroblasts (PM) from lung tissues of transgenic mice harboring a construct containing the sequences from –17 kb to +54 bp of the mouse COL1A2 gene that had been cloned upstream of the firefly luciferase reporter gene. By using these cells, we show here that BMP-7 opposes the TGF-{beta}1-mediated fibrogenic activity of PM in culture via the induction of inhibitors of differentiation 2 and 3 (Id2 and Id3).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All experimental procedures were conducted in accordance with the standard guidelines for animal experiments of the Graduate School of Medicine, Osaka City University, and approved by the DNA Recombinant Experiment Safety Committee of the Graduate School of Medicine, Osaka City University.

Cell lines. We established two PM cell lines from transgenic mice, which contained a construct that includes the sequences from –17 kb to +54 bp of the mouse pro {alpha}2(I) collagen gene (4) that was cloned upstream of the firefly luciferase reporter gene. These cell lines were obtained by explant outgrowth. Briefly, lung tissues were dissected from 7-wk-old transgenic mice and washed three times with PBS. They were separated into fragments of 0.5–2.0 mm3. Explants were plated onto 35-mm collagen-coated tissue culture dishes (Iwaki, Chiba, Japan), and sterilized cover glasses were placed on them to facilitate adhesion to the dishes. Subsequently, they were cultured in DMEM (Sigma, St. Louis, MO) with 10% heat-inactivated FCS (Invitrogen), 50 mg/l penicillin G, and 100 mg/l streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The culture medium was changed twice a week. The explants were removed after 2–3 wk. The cells were allowed to reach confluence within an additional 1–2 wk. The cells were then trypsinized for 5 min, resuspended in the same medium, and seeded in 25-cm2 tissue culture flasks (Greiner, Frickenhausen, Germany). Subsequently, they were split 1:4 at confluence and used at passages 8–20 in the present studies.

The mouse embryonic fibroblast cell line NIH/3T3 (RCB1862) was provided by Riken Cell Bank (Tsukuba, Japan).

Recombinant adenoviruses. We used the Adenovirus Cre/loxP-Regulated Expression Vector Set (Takara Biomedicals, Tokyo, Japan) to generate recombinant adenovirus as previously reported (34). Cosmid pAxCALNL-mBMP7, -LacZ, -mSmad3, -mSmad7, -mId2, and -mId3 were constructed by the insertion of respective mouse cDNAs into the SwaI cloning site of cosmid pAxCALNLw (35). With the use of the cosmid terminal protein complex method (26), recombinant adenoviruses were generated by transfecting HEK-293 cells with the respective cosmid DNAs. Similarly, AxCANCre was generated by transfecting 293 cells with pAxCANCre DNA-TPC according to the manufacturer’s protocol (Takara Biomedicals). The titer of recombinant adenoviruses was measured with the 50% tissue culture infectious dose method (18). Each adenovirus vector was used at the concentration of 100 multiplicities of infection (MOI).

Cell culture. Each established cell line was seeded at a density of 1.0–1.2 x 105 cells/ml in either 12-well dishes (Nunc, Roskilde, Denmark) for COL1A2 promoter activity assay or 60-mm dishes (Nunc) for mRNA extraction and incubated for 5 days in DMEM with 10% heat-inactivated FCS, 50 mg/l penicillin G, and 100 mg/l streptomycin in a humidified atmosphere of 5% CO2 at 37°C. When the cells had become confluent, they were coinfected with AxCANCre and one of pAxCALNL-mBMP7, -LacZ, -mSmad3, -mSmad7, -mId2, and -mId3 at 100 MOI for 1 h. Sequentially, the medium containing adenoviruses was removed and replaced with 0.8 ml/well (12-well dish) or 2.5 ml/dish (60-mm dish) of serum-free DMEM, followed by the culture for 2 days. On day 7, human recombinant TGF-{beta}1 was added to the medium (final concentration 5 ng/ml). The cultures of 12-well and 60-mm dishes were incubated, respectively, for 24 or 48 h.

Western blot analysis. Samples were homogenized in a lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 0.5% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4) supplemented with a protease inhibitor cocktail from Roche (Summerville, NJ). After heat denaturation at 95°C for 5 min, the samples (10 or 20 µg protein) were subjected to SDS-PAGE and then transferred onto a nitrocellulose membrane (Bio-Rad). The membranes were subsequently treated with 5% skim milk and then with primary antibodies [vimentin (1:1,000 dilution; Dako, Trappes, France), {alpha}-smooth muscle actin ({alpha}-SMA; 1:1,000 dilution, Sigma), E-cadherin (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Smad1/5/8 (1:1,000 dilution; Cell Signaling, Beverly, MA), or Smad1/5 (1:1,000 dilution; Upstate, Charlottesville, VA)] overnight at 4°C. After vigorous washing, the membranes were incubated with secondary horseradish peroxidase-conjugated anti-immunoglobulins for 2 h at room temperature. After being washed, immunoreactive bands were visualized using ECL detection reagent (Amersham, Buckinghamshire, UK) and documented with RAS 1000 (Fuji Photo Film).

Luciferase assay. Cell extracts were prepared from adenovirus-coinfected cells by using Cell Culture Lysis Reagent (Promega, Madison, WI). Briefly, the culture medium in 12-well dishes was removed, and the cells were washed with ice-cold PBS. After PBS was removed, 100 µl of Cell Culture Lysis Reagent were added to each well. Then, the cell layer was scraped, and the cellular debris was transferred to a microcentrifuge tube. Tubes were vortexed for 15 s and centrifuged at top speed for 60 s. The supernatant was recovered for COL1A2 promoter activity assay by measuring the luciferase activity, which was measured according to the manufacturer’s protocol (Promega) using a luminometer (Wallac 1420 Victor 2 multilabel counter system) and normalized with protein concentrations measured with DC Protein Assay (Bio-Rad, Hercules, CA). Values represent the means ± SD of six independent experiments.

Quantification of BMP-7, COL1A2, tissue inhibitor of metalloproteinase-2, Id2, and Id3 mRNA in PM using real-time RT-PCR. Total RNA was prepared using the GenElute Mammalian Total RNA Miniprep kit (Sigma) according to the manufacturer’s protocol. Isolated total RNA was diluted in RNase-free water and quantified by the absorbance measurement at 260 nm. Expression of COL1A2, Smad7, and GAPDH mRNA was measured using the Taq-Man One-Step RT-PCR Master Mix Reagents (PE Applied Biosystems, Foster City, CA). Expression of BMP-7, Id2, and Id3 mRNAs was measured with the One Step SYBR RT-PCR kit (Takara Biomedicals). The analysis of mRNA expression was performed using the Applied Biosystems Prism 7700 (PE Applied Biosystems) according to the previously reported procedure (13). Primers and oligonucleotide probes were designed according to the cDNA sequences in the GenBank database using Primers Express software (PE Applied Biosystems; see Table 1). The GAPDH gene primers and probe were used to normalize the relative expression levels.


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  		Table 1. Sequences of primers and probes used for real-time RT-PCR

 
In experiments using the Taq-Man One-Step RT-PCR Master Mix Reagents (PE Applied Biosystems), the RT-PCR reaction mixture contained 10 µl TaqMan 2x Universal PCR Master Mix, 0.5 µl 40x MultiScribe and RNase Inhibitor Mix, 0.5 µl forward primer (10 µM), 0.5 µl reverse primer (10 µM), 0.5 µl probe (50 µM), 100 ng total RNA, and distilled water to a final volume of 20 µl. The following RT-PCR conditions were used: 1 cycle of 50°C for 20 min followed by 1 cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Standard curves for the expression of each gene were generated by serial dilution (200, 40, 8, and 1.6 ng) of RNA prepared from PM.

In experiments using the One Step SYBR RT-PCR kit (Takara), the RT-PCR reaction mixture contained 10 µl 2x One Step SYBR RT-PCR Buffer, 0.4 µl TaKaRa Ex Taq HS (5 U/µl), 0.2 µl Moloney murine leukemia virus reverse transcriptase (200 U/µl), 0.4 µl RNase Inhibitor (40 U/µl), 0.4 µl forward primer (10 µM), 0.4 µl reverse primer (10 µM), 0.4 µl ROX reference dye (50x), 100 ng total RNA, and distilled water to a final volume of 20 µl. The following RT-PCR conditions were used: 1 cycle of 42°C for 15 min followed by 1 cycle of 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Dissociation curve analysis was performed by 1 cycle of 95°C for 15 s, 1 cycle of 60°C for 1 min, and 1 cycle of 95°C for 15 s. Standard curves for the expression of each gene were generated by serial dilution (200, 40, 8, and 1.6 ng) of total RNA prepared from PM. Each run was completed with a dissociation curve analysis to confirm the specificity of amplification and lack of primer dimers.

Immunocytostaining for Smad3 and phospho-Smad1/5/8. PM were seeded at a density of 1.0–1.2 x 105 cells/ml on eight-well glass slides (Nalge Nunc, Rochester, NY) and 60-mm dishes (Nunc) and incubated for 5 days in DMEM with 10% heat-inactivated FCS, 50 mg/l penicillin G, and 100 mg/l streptomycin in a humidified atmosphere of 5% CO2 at 37°C. When these cells had become confluent, the medium was removed and replaced with serum-free DMEM. Cells in 60-mm dishes were coinfected with AxCANCre and either AxCALNL-mBMP7 or -LacZ, respectively, at 100 MOI for 1 h. The media containing adenoviruses were sequentially removed and replaced with 2.5 ml/dish of serum-free DMEM. On day 7, the supernatant of culture medium in 60-mm dishes was harvested. To perform immunocytostaining for Smad3, the cells in eight-well glass slides were stimulated with BMP-7-infected cell medium plus TGF-{beta}1 (5 ng/ml), LacZ-infected cell medium plus TGF-{beta}1 (5 ng/ml), or LacZ-infected cell medium in the absence of TGF-{beta}1 for 30 min. To perform immunocytostaining for phospho-Smad 1/5/8, cells in eight-well glass slides were coinfected with AxCANCre and either AxCALNL-mBMP7 or -LacZ for 1 h and incubated in serum-free DMEM. Then, the cells were washed three times in PBS, fixed in 4% paraformaldehyde for 60 min, and washed three times in PBS. After being blocked with 1% bovine serum albumin in PBS (BSA/PBS) containing 0.1% Triton X-100 for 1 h at room temperature, samples were incubated with rabbit anti-Smad3 antibody (Zymed, S. San Francisco, CA) or rabbit anti-phospho-Smad1/5/8 antibody (Cell Signaling) in BSA/PBS at a 1:100 dilution overnight at 4°C. The cells were then washed with PBS three times followed by incubation with Texas red isothiocyanate-conjugated anti-rabbit secondary antibody (Dako) in 1% BSA/PBS for 1 h at room temperature. Cells were washed three times in PBS for 5 min, mounted in PermaFluor (Shandon, Pittsburgh, PA), and observed under a confocal laser scanning microscope LSM510 (Carl Zeiss, Oberkochen, Germany).

Measurement of collagen content in culture medium. Confluent cells in 25-mm2 culture flasks were coinfected with AxCANCre and either AxCALNL-LacZ, -BMP-7, or -Id2 for 1 h. Sequentially, the medium containing adenoviruses was removed, and 2 ml of serum-free DMEM supplemented with 50 µg/ml {beta}-aminopropionitrile fumarate (a lysyl oxidase inhibitor) in the absence or presence of TGF-{beta} was induced. The medium was harvested, and the collagen concentration was determined using a Sircol Collagen Assay kit (Biocolor, Belfast, N. Ireland) as previously reported (17). In brief, the culture medium was allowed to react with Sirius red dye, and the collagen-dye complexes were precipitated by centrifugation. Then, the dye was removed from the precipitate by treatment with 0.5 N sodium hydroxide. The absorption of the solution at 540 nm was determined by spectrophotometry.

Statistical analysis. Results were compared by ANOVA, and P < 0.05 was accepted as indicating statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Phenotype of PM and effect of TGF-{beta}1 on COL1A2 promoter activity. We first investigated the phenotype of PM that was established from lung tissues of transgenic mice harboring the luciferase-COL1A2 upstream sequence. Morphologically, PM have a spindle shape as shown in Fig. 1A. According to Western blotting, PM expressed vimentin and {alpha}-SMA, which are markers of contractile mesenchymal cells. In contrast, E-cadherin, a marker of epithelial cells, was not expressed, indicating that the established cell lines represent the myofibroblastic phenotype. In addition, the PM expressed luciferase activity that was dose dependently stimulated by TGF-{beta}1 (Fig. 2A). The luciferase activity was upregulated in TGF-{beta}1-treated PM infected with recombinant adenovirus expressing Smad3, but downregulated in those infected with recombinant adenovirus expressing Smad7 (Fig. 2B).



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  		Fig. 1. Phenotype of pulmonary myofibroblasts (PM) is depicted. A: phase-contrast photomicrographs of subconfluent cultures of PM. They were cultured in DMEM with 10% FCS. B: Western blot analysis of vimentin, {alpha}-smooth muscle actin (SMA), and E-cadherin was performed on PM1, PM2, and NIH/3T3 cells as described in MATERIALS AND METHODS. Note that {alpha}-SMA was highly expressed in PM compared with NIH/3T3 cells.

 


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  		Fig. 2. Effect of transforming growth factor (TGF)-{beta} signal on COL1A2 promoter activity in PM. A: after PM had become confluent and serum-starved for 48 h, they were treated with incremental concentrations of TGF-{beta}1 in serum-free DMEM for 24 h. Analysis of COL1A2 promoter activity was performed by luciferase assay as described in MATERIALS AND METHODS. Values were normalized by protein concentrations. Data represent the mean values of 6 independent experiments. B: after PM had become confluent, they were coinfected with AxCANCre and one of AxCALNL-LacZ, -mSmad3, or -mSmad7 for 1 h and incubated in serum-free DMEM for 48 h. They were then incubated in the presence of TGF-{beta}1 (5 ng/ml) for another 24 h. Analysis of the COL1A2 promoter activity was performed by luciferase assay as described in MATERIALS AND METHODS. Values were normalized by protein concentrations. Data represent means ± SD of 6 independent experiments. *Significant difference against all other groups. Cont, noninfected control.

 
BMP-7 attenuates the promoter activity and mRNA expression of COL1A2 in PM. These PM cell lines were used to evaluate the effect of BMP-7. When the PM were infected with recombinant adenovirus expressing BMP-7, the TGF-{beta}1-stimulated COL1A2 promoter activity was significantly downregulated as shown in Fig. 3A. Furthermore, the TGF-{beta}1-stimulated expression of COL1A2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNAs was significantly inhibited by BMP-7 overexpression (Fig. 3, B–D).



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  		Fig. 3. Effect of bone morphogenetic protein (BMP)-7 overexpression on collagen promoter activity and expression of COL1A2 and tissue inhibitor of metalloproteinase (TIMP)-2 mRNA in PM. After PM had become confluent, they were coinfected with AxCANCre and either AxCALNL-LacZ or -mBMP7 for 1 h and were incubated in serum-free DMEM for 48 h. They were then incubated in the presence or absence of TGF-{beta}1 (5 ng/ml) for another 24 h. A: analysis of COL1A2 promoter activity was performed by luciferase assay as described in MATERIALS AND METHODS. Values were normalized by protein concentrations. Data represent means ± SD of 6 independent experiments. *Significant difference. BMP-7 (B), COL1A2 (C), and TIMP-2 (D) mRNA expression was analyzed using real-time RT-PCR as described in MATERIALS AND METHODS. The relative levels of mRNA were normalized by that of GAPDH. Data represent the means ± SD of 6 independent experiments. *Significant difference.

 
BMP-7 stimulates nuclear localization of Smad1/5/8 and inhibits that of Smad3. To investigate whether the treatment of recombinant adenovirus expressing BMP-7 activates the BMP-specific Smad signaling pathway, we utilized the PM and the supernatant of the cells that were coinfected with AdLNL-BMP-7 and Ad-CANCre, as described in MATERIALS AND METHODS. PM untreated or stimulated by BMP-7 were stained with an antibody against phospho-Smad1/5/8 as an indicator of the activation of BMP-Smad signaling. Figure 4, A and B, demonstrates that Smad 1/5/8 was phosphorylated and was translocalized to the nucleus by BMP-7 stimulation.



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  		Fig. 4. Effect of BMP-7 on nuclear accumulation of phospho-Smad1/5/8 and Smad3 in PM. A: cells on 8-well glass slides were coinfected with AxCANCre and either AxCALNL-mBMP7 or -LacZ for 1 h and incubated in serum-free DMEM for 48 h. Immunocytostaining for phospho-Smad1/5/8 was performed as described in MATERIALS AND METHODS. B: Western blot analysis of phospho-Smad1/5/8 and Smad1/5 was performed as described in MATERIALS AND METHODS. C: cells on 8-well glass slides were stimulated with BMP-7-infected cell medium + TGF-{beta}1 (5 ng/ml), LacZ-infected cell medium + TGF-{beta}1 (5 ng/ml), or only LacZ-infected cell medium for 30 min for immunocytostaining of Smad3. Immunocytostaining was performed as described in MATERIALS AND METHODS.

 
To further investigate whether BMP-7 antagonizes TGF-{beta} signaling, PM were stimulated by 5 ng/ml TGF-{beta}1 in the presence or absence of BMP-7. Cells were immunostained using an antibody against Smad3. Figure 4C demonstrates that the nuclear localization of Smad3 by TGF-{beta} stimulation was clearly inhibited by BMP-7.

Inhibition of COL1A2 promoter activity by Ids induced by BMP-7. A regulatory relationship between Id proteins and TGF-{beta} signaling has recently been identified (20). Id proteins act as dominant negative antagonists of basic helix-loop-helix (bHLH) family of transcription factors. Therefore, we first determined the expression of Id2 and Id3 mRNAs in PM with or without TGF-{beta}1 stimulation. As shown in Fig. 5, A and B, TGF-{beta}1 stimulation significantly suppressed the expression of Id2 and Id3 mRNAs in PM. In contrast, BMP-7 significantly upregulated their mRNA expressions (Fig. 5, C and D). Successive experiments showed that the overexpression of Id2 or Id3 decreased the COL1A2 promoter activity in TGF-{beta}1-stimulated PM (Fig. 6).



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  		Fig. 5. Effect of TGF-{beta}1 and BMP-7 on inhibitors of differentiation (Id)2 and Id3 mRNA expression in PM. A and B: after PM had become confluent, they were incubated in serum-free DMEM for 48 h. They were then incubated in the presence or absence of TGF-{beta}1 (5 ng/ml) for another 24 h. C and D: after PM had become confluent, they were coinfected with AxCANCre and either AxCALNL-LacZ or -mBMP7 for 1 h and incubated in serum-free DMEM for 48 h. They were then incubated in the presence of TGF-{beta}1 (5 ng/ml) for another 24 h. Id2 and Id3 mRNA expressions were analyzed by using real-time RT-PCR. Relative levels of mRNA were normalized by that of GAPDH. Data represent the means ± SD of 6 independent experiments. *Significant difference.

 


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  		Fig. 6. Effect of Id2 and Id3 overexpression on collagen promoter activity in PM. After PM had become confluent, they were coinfected with AxCANCre and one of AxCALNL-LacZ, -mId2, or -mId3 for 1 h and incubated in serum-free DMEM for 48 h. They were then incubated in the presence or absence of TGF-{beta}1 (5 ng/ml) for another 24 h. Analysis of COL1A2 promoter activity was performed by luciferase assay as described in MATERIALS AND METHODS. Data represent the means ± SD of 6 independent experiments. *Significant difference.

 
Effect of BMP-7 and Id2 on secretion of collagens in PM. Finally, we examined whether BMP-7 and Id2 stimulation downregulate the production of Sirius red-reactive collagens. As shown in Fig. 7, BMP-7 and Id2 overexpression decreased the concentration of soluble collagens in the culture medium of PM in the presence of TGF-{beta}1.



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  		Fig. 7. Effect of BMP-7 and Id2 overexpression on the soluble collagen levels in the culture medium of PM. Confluent PM were coinfected with AxCANCre and either AxCALNL-LacZ, -mBMP7, or -Id2 for 1 h and incubated in serum-free DMEM for 48 h in the absence or presence of TGF-{beta}1 (5 ng/ml). The medium was harvested, and the collagen concentration was measured using Sircol Collagen Assay kit as described in MATERIALS AND METHODS. Data represent the means ± SD of 6 independent experiments. *Significant difference.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We successfully established PM cell lines from transgenic mice expressing a construct containing the sequences from –17 kb to +54 bp of the mouse pro {alpha}2(I) collagen gene that was cloned into the upstream of the firefly luciferase reporter gene. These cell lines provide useful tools to study mechanisms of pulmonary fibrogenesis and to test antifibrotic molecules. By using these cells, we found that BMP-7 decreased TGF-{beta}1-stimulated COL1A2 promoter activity and the mRNA expression of COL1A2 and TIMP-2, while it increased Id2 and Id3 mRNA expression, even in the presence of TGF-{beta}1.

Lung fibrosis is a major cause of death in scleroderma lung disease, idiopathic pulmonary fibrosis, radiation- and chemotherapy-induced lung fibrosis, and in conditions caused by the occupational inhalation of dust particles (2, 7, 11, 19, 24, 28, 36). Fibrosis is generally considered to arise from the impaired regulation of the wound healing response, which is characterized by the excessive accumulation of newly formed ECM, i.e., collagens and fibronectin, generated by mesenchymal cells (21). Such ECM accumulation in pulmonary alveoli interferes with gas exchange, ultimately leading to death (21).

Lung fibrosis is largely dependent on local TGF-{beta} activity, since bleomycin-induced experimental fibrosis is attenuated by the intratracheal administration of Smad7-expressing adenovirus (30). Also, mice lacking {beta}6-integrin, which is required for TGF-{beta} activation in the lung, are relatively resistant to pulmonary fibrosis (29).

We had therefore predicted that BMP-7, which reportedly antagonizes the effects of TGF-{beta} in renal mesangial cells, might have a similar effect against lung fibroblastic cells (38, 39). Indeed, the present investigations clearly demonstrated that BMP-7 gene transfer successfully attenuated COL1A2 gene expression in TGF-{beta}1-stimulated PM cell lines. Therefore, we next explored the mechanism underlying this effect.

BMP-7 and TGF-{beta}1 bind to distinct type II receptors that successively form complexes of specific type I serine-threonine kinase receptors (27). BMP-7 binds specifically to activin-like kinase (ALK)-3 and ALK-6 receptors, whereas TGF-{beta}1 binds to the ALK-5 receptor (43). After BMP-7 or TGF-{beta}1 binds to type II and ALK type I receptors, the complex triggers the initiation of intracellular signal streams mediated by Smad proteins. BMP-7 phosphorylates Smad1, Smad5, and Smad8, whereas TGF-{beta}1 activates Smad2 and Smad3 (43). Here, we confirm these interactions also in PM. As shown in Fig. 4, exogenous BMP-7 induces the translocalization of Smad1/5/8 from the cytoplasm into the nucleus and their phosphorylation and partially blocks the nuclear accumulation of Smad3 by TGF-{beta}1 stimulation.

Inhibition of COL1A2 promoter activity by either Smad7 or BMP-7 constructs produced ~30% inhibition in each case, although COL1A2 mRNA expression was suppressed at least fourfold with increased BMP-7 (Fig. 3). This discrepancy may be explained by one or both of the following mechanisms: 1) although the –17 kb COL1A2 sequence driving the reporter luciferase gene contains the TGF-{beta}-responsive element as well as an upstream enhancer sequence, it may still lack additional regions that enhance the stimulatory effect of TGF-{beta} and/or antagonistic action of BMP-7 on COL1A2 activity; and 2) Smad7 or BMP-7 may influence the degradation or instability of COL1A2 mRNA, a possibility that needs to be established experimentally.

BMP-7 is also reported to antagonize EMT in tissue repair (42), a hallmark of which is the deprivation of E-cadherin expression. E-cadherin is a central component of cell-cell adhesion junctions and is required for the formation of epithelium (12). Currently, we have not assessed whether BMP-7 triggers EMT also in the lung. However, we observed that the expression of {alpha}-SMA in PM was reduced by exogenous BMP-7 without affecting the E-cadherin expression (data not shown), suggesting the possible involvement of BMP-7 in the EMT of PM.

Id proteins have emerged recently as critical targets of BMPs (33). Although TGF-{beta} is reported to induce Ids in some cell types (10, 33), the current study revealed that the expression of Id2 and Id3 was inhibited by TGF-{beta}1 stimulation in PM, as was reported in the other fibroblasts. Ids can act as positive regulators of cell proliferation and negative regulators of cell differentiation (40). Ids have also been identified as dominant negative regulators of bHLH transcription factors (5, 25). They lack a basic DNA binding region but possess an HLH dimerization motif, allowing them to interact with ubiquitous bHLH transcription factors. As a result, these heterodimers containing Id proteins are unable to bind DNA. Interestingly, c-myc, a member of the bHLH family, upregulates the activity of COL1A2 promoter (23), whereas Id2 and Id3 are repressors of bHLH, which could antagonize the activity of the COL1A2 promoter. Collectively, these studies, along with our findings, point to a potentially new pathway for regulating collagen gene expression through precise interactions between TGF-{beta}, BMP-7, Ids, and their downstream signal molecules.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K. Inoue and K. Ikeda).


   ACKNOWLEDGMENTS
 
We thank Dr. Benoit de Crombrugghe for providing COL1A2 transgenic reporter mice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Ikeda, Dept. of Anatomy, Graduate School of Medicine, Osaka City Univ., 1-4-3, Asahimachi, Abeno, Osaka, 545-8585, Japan (e-mail: m6488795{at}med.osaka-cu.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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