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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 160-169, 2002
© 2002 American Thoracic Society

Activation of Bone Morphogenetic Protein/Smad Signaling in Bronchial Epithelial Cells during Airway Inflammation

Alexander Rosendahl, Evangelia Pardali, Matthaios Speletas, Peter ten Dijke, Carl-Henrik Heldin and Paschalis Sideras

Astrazeneca R&D Lund, Department of Molecular & Biosciences, Lund, Sweden; Department of Immunology, BioMedical Center, Lund University, Lund, Sweden; Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and Ludwig Institute for Cancer Research, Uppsala, Sweden

Address correspondence to: Alexander Rosendahl, Ph.D., AstraZeneca R&D Lund, Department of Biosciences, Scheelev 2, S-221 87 Lund, Sweden. E-mail: alexander.rosendahl{at}astrazeneca.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone morphogenetic proteins (BMPs) are pleiotropic secreted proteins, structurally related to transforming growth factor (TGF)-ß and activins. BMPs play pivotal roles in the regulation of embryonic lung development and branching of airways and have recently been considered to influence inflammatory processes in adults due to their chemotactic activity on fibroblasts, myocytes, and inflammatory cells. In this study, we have investigated the possible involvement of BMPs in a model of experimental allergic–airway inflammation in situ using antibodies that detect activated Smad proteins, and have monitored the modulation of BMP ligands during the inflammatory response. Inflamed bronchial epithelial cells and a few scattered alveolar cells expressed levels of phosphorylated Smad1 (pSmad1/5), indicative of active BMP/Smad signaling. This was in contrast to healthy epithelium, which was devoid of immunoreactivity. A mechanistic explanation for increased pSmad1/5 staining during inflammation was provided by the upregulated expression of all the BMP type I receptors, i.e., activin receptor–like kinase (ALK)2, ALK3, and ALK6, in the inflamed bronchial epithelial cells. Furthermore, the mRNA and protein profiles for BMP ligands were significantly altered during airway inflammation with induction of BMP2, BMP4, and BMP6, and downregulation of BMP5 and BMP7. Collectively, our data demonstrate for the first time active BMP/Smad signaling during airway inflammation in bronchial epithelial cells and thus raise the possibility that BMPs could play a determining role in respiratory pathophysiology.

Abbreviations: activin receptor–like kinase, ALK • bone morphogenetic proteins, BMP • BMP type II receptor, BMPR-II • diaminobenzidine, DAB • extracellular matrix, ECM • epidermal growth factor, EGF • immunoglobulin, Ig • interleukin, IL • ovalbumin, OVA • phosphate-buffered saline, PBS • phosphorylated Smad1 protein, pSmad1/5 • transforming growth factor-ß, TGF-ß


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Members of the transforming growth factor (TGF)-ß family play central roles in controlling cellular proliferation, differentiation, migration, and survival (1). These cytokines constitute a highly conserved set of signaling proteins whose analogs are present in organisms from Caenorhabditis elegans to mammals indicating essential roles in development and maintenance of tissue homeostasis. The TGF-ß family can be divided into three subgroups: the TGF-ßs, the activins/inhibins, and the bone morphogenetic proteins (BMPs), of which the latter constitutes the largest subfamily. BMPs are 30–35 kD hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation when injected subdermally in rats (2). Several studies have demonstrated that these proteins play essential roles during embryonic development (3, 4). For example, BMP2 knockout mice die in utero, BMP7 knockout mice exhibit neonatal lethality, BMP5-deficient mice are viable but exhibit several gross malformations, and BMP6 knockout mice are viable and fertile with only moderate malformations (4–7). These phenotypes become evident in tissue undergoing morphogenesis, suggesting that BMP signaling is particularly relevant when the cells are being directed toward specific differentiation pathways.

So far more than 20 mammalian BMPs have been identified, but only three type I and three type II receptors capable of binding BMPs have been cloned in mammals (8). BMPs induce the heteromeric complex formation between type II and type I receptors. The constitutively active type II kinase activates the type I receptor, which subsequently propagates the signal downstream by phosphorylating specific BMP receptor-regulated Smads, i.e., Smad1, Smad5, and Smad8 (8–10). Phosphorylated receptor regulated Smads (R-Smads) form heterocomplexes with the common partner Smad4 (Co-Smad) and translocate to the nucleus, where they participate in the regulation of transcription of target genes (11).

The prevalence of asthma has increased dramatically in the last 20 years, affecting today as many as 10% of the population in industrial countries. Allergic asthma is a chronic disease representing the final stage of repeated airway antigen challenges. The disease is associated with widespread narrowing of the bronchial airways, pulmonary eosinophilia, mast cell infiltration, mucus hypersecretion (12, 13), excessive IL-4 and IgE production (14, 15), and finally remodeling of the airways (16–18). The term airway remodeling refers to structural changes resulting in alteration of the airway epithelium, lamina propria, and submucosa, resulting thus in the thickening of the airway wall (16–18). In vitro experiments have shown that TGF-ßs and activins act as chemotactic agents for fibroblasts and stimulate secretion of extracellular matrix (ECM) components that contribute to fibrosis and scar formation, such as collagen, fibronectin, and proteoglycans (19, 20). Aberrant activation of the TGF-ß and activins, in bleomycin-induced airway inflammation, has indirectly been linked to excessive fibrosis and formation of scar (21). Even though BMPs are involved in lung development, their role during inflammatory responses in adult lung has not been investigated so far.

In the present study, we have evaluated if the composition of BMPs is modulated and examined the state of BMP/Smad activation during airway inflammation. We demonstrate that inflamed bronchial epithelial cells transduce signals via pSmad1/5. These results raise the possibility that inflamed epithelium could be under the influence of BMPs during the development of allergic asthma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Balb/C (H-2b, Mls1b) mice were obtained from Bomholtgård, Ry, Denmark, and were routinely used at the age of 8–12 wk. The experiments were conducted with approval of the Swedish ethical committee.

Allergen-Induced Airway Inflammation and Tissue Collection
The ovalbumin (OVA)-induced airway inflammation model used is described elsewhere (22). Briefly, mice were sensitized by an intraperitoneal injection of 7.5 µg OVA (Sigma, St. Louis, MO), absorbed to 1.5 mg aluminum hydroxide F-2200 (Reheis, Dublin, Ireland), dissolved in 0.3 ml phosphate-buffered saline (PBS) (Life Technologies Ltd., Paisley, UK), and boosted with the same administration at Day 7. On Days 14, 21, and 22 mice were challenged with 10 mg/ml aerosolized OVA for 1 h each time. Control animals were sensitized with intraperitoneal OVA and challenged with aerosolized PBS alone. Animals were terminated by an intraperitoneal injection of 0.15 ml (60 mg/ml) sodium pentobarbital (Apoteksbolaget, Umeå, Sweden) 24 h after the third aerosolic challenge. At this time the OVA-challenged mice had high levels of interleukin (IL)-4, immunoglobulin (Ig)E, and a massive eosinophilia infiltration. Before resection, lungs were inflated with Tissue-Tek O.C.T. (Sakura Fintek, Torrance, CA) to preserve morphology. The specimens were stored at -70°C until used.

Antibodies and Reagents
Polyclonal antisera were raised in rabbits against synthetic peptides as described elsewhere (23, 24). Briefly, for antibodies against BMP type I receptors and the type II receptor, peptides that corresponded to the divergent intracellular juxtamembrane domains were used. Antibodies against Smad1 corresponded to the variable proline-rich linker region; this antibody crossreacts with Smad8 but not Smad5 (25). The pSmad1/5 antibody was raised by a peptide KKK-NPISpSVpS (where pS stands for phosphorylated serine residue) (26); this antibody recognizes terminally phosphorylated pSmad1 and pSmad5 and is herein referred to as pSmad1/5. All the antisera were tested for specificity by immunoprecipitation and Western blotting on African green monkey COS-7 cells transfected with different receptors and Smads and have successfully been used in tissue sections (27–31). The antisera were purified using ImmunoPure IgG (Protein A) purification Kit according to the manufacturer's instructions (Pierce, Rockford, IL). Polyclonal antibodies against normal rabbit IgG (DAKO, Älvsjö, Sweden) were used as negative controls in the experiments. Antibodies against BMP2–7 ligands were purchased from Santa Cruz BioTechnologies, Inc. (Santa Cruz, CA) and recombinant BMP ligand proteins were purchased from R&D Systems (Minneapolis, MN).

Immunohistochemistry
Expression of receptors and Smad proteins in tissue was visualized using the standard avidin/biotin system according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA) and is described in detail in (22). Briefly, endogenous peroxidase activity in cryo-preserved tissues fixed in 2% paraformaldehyde (Sigma, Steinheim, Germany) was quenched using 1% H2O2/methanol. After blocking and 0.1% saponin (ICN Biomedicals, Inc., Aurora, OH) permeabilization, sections were incubated with primary antibody followed by biotinylated secondary antibody (Vector Laboratories). Slides were developed for 5 min in diaminobenzidine (DAB) (Vector Laboratories) and counterstained with Mayer's hematoxylin (Histolab, Gothenburg, Sweden). Images were analyzed using the Leica IM2000 and the Leica Qwin computer program (Leica Microsystems, Heerbrugg, Switzerland). Four to five mice were analyzed for expression of the investigated markers. In each animal at least five intermediate airways/vessels were analyzed and the number of positively stained epithelial and endothelial cells were counted by the Leica Qwin computer program and SEM was determined.

Western Blotting
Lung tissue from mice challenged three times with either PBS or OVA was homogenized in liquid nitrogen using mortal and pestle. The proteins were then extracted in 1% TritonX-100, 25 mM Tris-HCl (pH 7.6), 0.1 M NaCl, 1 mM EDTA, 5 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, and complete protease inhibitor cocktail (Boehringer Mannheim GmbH, Mannheim, Germany) diluted 1:25. An equal amount of lung protein was run under reduced conditions on a 10% NuPage Bis/Tris gel (NOVEX/Invitrogen, Groningen, The Netherlands) and thereafter transferred to PVDF membranes (NOVEX/Invitrogen) by electroblotting using transfer buffer supplemented with 20% methanol (NOVEX/Invitrogen). Blots were blocked overnight at 4°C in PBS-0.1% Tween20–1% BSA and then incubated with 1 µg/ml of the primary antibody for 1 h with or without the cognate peptide present at 30-fold excess, at room temperature, using the DecaProbe system (Hoefer; Amersham Pharmacia Biotech, Uppsala, Sweden). Thereafter, the membranes were washed 6 x 10 min with blocking buffer, incubated for 1 h with the secondary HRP-linked donkey-anti-rabbit/goat antibodies at 1:5,000 (Santa Cruz Biotechnology), washed 6 x 10 min with blocking buffer, incubated with ECL-substrate (Amersham Pharmacia Biotech), and finally exposed to radiographic films. The films were scanned and the relative intensity of the obtained products was measured using the QuantityOne software (Bio-Rad, Hercules, CA).

RNA Isolation and Reverse Transcription/Polymerase Chain Reaction
cDNA was synthesized from total lung RNA isolated with RNAqueous (Ambion, Austin, TX), using the First-Strand cDNA synthesis kit (Amersham Pharmacia, Piscataway, NJ). In each experiment, lung tissue from two healthy and two OVA-challenged animals was homogenized separately, and semiquantitative reverse transcription/polymerase chain reaction (RT-PCR) amplifications were performed using different concentrations of cDNA template (1:1, 1:5, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1,000, and 1:2,500). Amplification of the cDNA template was performed using 30 cycles each consisting of 60 s at 95°C, 90 s at 55°C, 60 s at 72°C, and finally followed by 10 min at 72°C at the DNA Engine/Tetrad (MJ Research Inc., Watertown, MS). The gels were scanned using the Fluor-S MultiImager (Bio-Rad) and the relative intensity of the obtained products was measured using the QuantityOne software (Bio-Rad). Titration curves, from the serial diluted templates, were drawn and the bestfit equation was obtained with the Excel software (Microsoft, Redmond, WA). The fold modulation, between PBS versus 3x OVA-treated sample for each marker, was calculated using the obtained formula. The Student's t test determined statistical power. The PCR primers used were: (ALK2 forward): 5'-GGCGGGGTCTTACACG TAA-3'; (ALK2 reverse): 5'-CTGGACCAGAGGAACAAAGG-3'; (ALK3 forward): 5'-TCATGACGCATTAAC-3'; (ALK3 reverse) 5'-CTACACTGCCCCCTG-3'; (ALK6 forward): 5'-TTTC TGGGTTCCTCT-3'; (ALK6 reverse); 5'-ACCACCTTAGACG CA-3'; (BMPR-II forward): 5'-AATTTCCGCAGAATCAA GAAC-3'; (BMPR-II reverse): 5'-TGAATGAGGTG GACT-3'; (Smad1 forward): 5'-TGCCCTGGACAGCCG-3'; (Smad1 reverse): 5'-TCTGAGCTGGTTGGG-3'; (Smad5 forward): 5'-CT TGAGCAGCCCAGG-3'; (Smad5 reverse): 5'-GCGTTGTTG GGTTGG-3'; (BMP2 forward): 5'-GAGACCCACCCCCAG-3'; (BMP2 reverse): 5'-TCCACCCCACATCAC-3'; (BMP4 forward): 5'-AGCCGAGCCAACACT-3'; (BMP4 reverse): 5'-CTTCTTCT TGGACCG-3'; (BMP5 forward): 5'-TGGGCTGGCTTGTCT-3'; (BMP5 reverse): 5'-TTCCCCGTCACAATA-3'; (BMP6 forward): 5'-AGTCCTCTTCTTCGG-3'; (BMP6 reverse): 5'-CCGTCAC CGCCTCAC-3': (BMP7 forward): 5'-GGCTTCTCCTACCCC-3'; (BMP7 reverse): 5'-TCTTGGTTCTTTGGC-3': (GAPDH forward): 5'- GAAGGGTGGAGCCA-3'; (GAPDH reverse): 5'-TG CCAGCCCCGGCA-3'. The primers were purchased from DNA Technology, Aarhus, Denmark.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Smad1 Activation in Bronchial Epithelial Cells during Airway Inflammation
We investigated the involvement of the BMP/Smad family-signaling pathway in airway inflammation using mice sensitized by intraperitoneal injection of OVA and challenged by repeated inhalation of aerosolized OVA as a model system.

The hallmark of ongoing BMP signaling is the phosphorylation and nuclear translocation of BMP receptor–activated pSmads. To monitor signaling through the BMP type I specific receptors during the development of allergic airway inflammation, the occurrence of phosphorylated Smad1 and/or Smad5 in protein extracts from PBS- and OVA-challenged lungs was analyzed by Western blotting using the pSmad1/5 antiserum, which recognizes pSmad1 and pSmad5 (Figure 1) . Whereas barely detectable levels were observed in lysates from PBS-treated lungs, a strong band in the 55-kD region corresponding to pSmads (referred to herein as pSmad1/5), was detected in cell lysates from OVA-challenged lungs. The pSmad1/5 signal was competed efficiently by a 30-fold excess of pSmad1/5 peptide and the Smad1 signal was competed by the Smad1 peptide, thus further verifying the specificity of these reagents (Figure 1). This prompted us to further define the cell types in which active BMP/Smad signaling was ongoing by immunohisto- chemistry.



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  		Figure 1. Expression of Smad1 and pSmad1/5 in total lung homogenate. Mice were treated with three aerosolic inhalations of PBS or OVA. Cell lysates were subjected to immunoblotting with Smad1 or pSmad1/5 antibodies in the presence or absence of 30-fold access of the cognate peptide to verify the specificity of the antibodies. Results from one out of three similar experiments are shown (n = 2 for each group).

 
Bronchial epithelial cells in PBS-treated animals did not show any pSmad1/5 immunostaining (Figures 2A and 2B) . A few scattered cells in the vascular endothelium, the subepithelial fibroblast layer, and among the alveolar cells expressed the activated pSmad1/5 protein (Figures 2A–2D). In sharp contrast, the majority of the intermediate airway bronchial epithelial cells from OVA-challenged animals (88 ± 5%) exhibited strong immunoreactivity (Figures 2E and 2F). Detailed analysis of these epithelial cells demonstrated that a fraction of them (20 ± 2%) showed nuclear immunostaining for pSmad1/5, indicating active BMP-mediated signaling in these cells. The remaining cells had a strong cytoplasmic expression (Figures 2E and 2F). All vascular endothelial cells had a weak expression of pSmad1/5 (Figures 2G and 2H). As in healthy animals, scattered alveolar cells (8 ± 1%) and fibroblasts (15 ± 2%) were pSmad1/5-positive in the inflamed tissue. Infiltrating inflammatory cells, which express high levels of pSmad2 (22), were negative for pSmad1/5 (Figures 2E–2H). Similar levels of total Smad1 were detected in PBS- and OVA-challenged lungs by Western blotting (Figure 1). To address the cellular expression of these Smad1 molecules in the tissue before and after OVA challenges, we analyzed their distribution in the lung using immunohistochemistry. Healthy animals showed a moderate to strong expression of Smad1 in all bronchial epithelial cells (Figures 3A and 3B) . A weaker expression was noted in all other cell types, including endothelial, alveolar, and fibroblast, demonstrating that all cells have an intrinsic capacity to signal via the BMP/Smad pathway if the right stimulus is present (Figures 3A–3D). Similar level and pattern of expression were found in the antigen-challenged animals (Figures 3E–3H), suggesting that the increase in phosphorylation of BMP receptor–activated Smads in the epithelium is a result of BMP signaling.



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  		Figure 2. Increased staining by pSmad1/5 antibodies in inflamed bronchial epithelium in tissue. Lungs from mice challenged with three aerosolic inhalations of PBS (A–D) or OVA (F–H) were dissected 24 h after the last inhalation. Lung sections were stained with pSmad1/5 antibody (4 µg/ml) and visualized as brown color by diaminobenzidine (DAB) and counterstained with hematoxylin. Expression in an intermediate airway –(AW) (A, B, E, and F) and around a small vessel (V) (C, D, G, and H) is shown in x40 (A, C, E, and G) or x100 (B, D, F, and H) magnification. Four lungs were analyzed with similar results. Arrows indicate positively stained (green) or negative (red) cells.

 


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  		Figure 3. Ubiquitous expression of Smad1 in lung in tissue. Lung from mice challenged with three aerosolic inhalations of PBS (A–D) or OVA (F–H) were stained with Smad1 antibody (1 µg/ml), which recognizes both C-terminally phosphorylated Smad1 and nonphosphorylated Smad1. Expression in an intermediate airway (AW) (A, B, E, and F) and around a small vessel (V) (C, D, G, and H) is shown in x40 (A, C, E, and G) or x100 (B, D, F, and H) magnification. One representative of four lungs is shown.

 
Our results demonstrate that antigen challenge of lungs induces activation of BMP receptor–activated Smads and that this occurs mainly in the bronchial epithelial cells.

BMP Type I Receptor Expression Is Induced in Bronchial Epithelial Cells during Inflammation
To determine which of the potential BMP receptors activated the Smads, we analyzed the expression patterns of all the known BMP type I and BMP type II receptors in inflamed and healthy lungs by Western blotting. Specificity of the recognized proteins by antibodies was determined by performing a blocking experiment with cognate peptides. Analysis of whole-lung protein extracts revealed a moderate upregulation of ALK2 (2-fold) and ALK6 (4-fold) from inflamed animals compared with healthy controls (Figure 4) . Anti-ALK3 antibodies detected three bands in lysates from healthy or inflamed lungs with molecular masses of 40, 45, and 60 kD (Figure 4). The dominant immunoreactivity in healthy lungs was obtained at 45 kD, a size ~ 5–10 kD smaller than the mouse full-length receptor. In sharp contrast, the 40–45 kD bands were significantly downregulated in the inflamed lungs and the predominantly expressed form of ALK3 was of 60 kD. Competition with the peptide used to raise the antibody completely (> 90%) inhibited detection of the 60-kD band, but partially affected (~ 30%) the lower 40–45 kD bands (Figure 4). This suggests that the 60-kD band is ALK3, and raises the possibility that the 40–45 kD bands are antigenically-related proteins. Similar expression of the BMPR-II was noted irrespective of the treatment (Figure 4). To examine if the increased receptor protein expression during inflammation was accompanied by an induction of mRNA transcripts, total lung mRNA preparations were analyzed by RT-PCR. A 5-fold induction of ALK2 mRNA transcripts and a weak induction of ALK6 were evident in the OVA-challenged mice, whereas the other receptors remained unchanged at the mRNA level (Figure 6A).



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  		Figure 4. Expression of BMP receptor protein level in total lung homogenates. Mice were treated with three aerosolic inhalation of PBS or OVA. Lung tissue cell lysates were analyzed for presence of BMP receptors by Western blots with antibodies (1 µg/ml) against ALK2, ALK3, ALK6, or BMPR-II. Specificity was determined by incubation of the antibody with a 30-fold excess of the cognate peptide for 1 h before the incubation with the blotted membrane. Results from one representative experiment out of three is shown (n = 2 mice in each group).

 


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  		Figure 6. Modulation of BMP mRNA transcripts during airway inflammation. Lung cDNA from mice treated with three aerosolic inhalations of PBS or OVA was analyzed by semiquantitative RT-PCR. The presence of transcripts for BMP receptors, Smads, and ligands was analyzed after sequential dilution steps of the cDNA template in H2O. (A) Fold-modulation in mRNA expression of BMP transcripts. (B) RT-PCR products obtained using cDNA template at 1:10 dilution. Results from one out of two similar experiments are shown (n = 2 mice in each group). Statistical analyses were performed with Student's t test. *Indicates 0.05 < P > 0.01, **0.01 < P > 0.001. SD was below 15%.

 
To identify the cell types induced to express the receptor complexes, we analyzed healthy and inflamed lungs by immunohistochemistry. ALK2 expression in healthy lung was restricted to bronchial epithelial and scattered alveolar cells (Figure 5A) . A moderate increase was noted on the epithelial cells after antigen challenge but no additional cell type began to express the receptor (Figure 5B). Healthy animals weakly expressed ALK3 on epithelial, alveolar, and fibroblast cells, whereas the vascular endothelium was highly immunoreactive (Figure 5C). Antigen challenge resulted in an increased expression of ALK3 on endothelial and epithelial cells, and fibroblasts (Figure 5D). In addition, a few infiltrating (18 ± 3%) and alveolar cells (15 ± 5%) expressed high ALK3 levels (Figure 5D). The bronchial epithelium expressed ALK6 in healthy animals and an induction was evident on these cell types after antigen challenge, whereas other cell types remained negative (Figures 5E and 5F). The BMPR-II was found only on the bronchial epithelium, in the vascular endothelium, and on a few scattered alveolar cells (Figures 5G and 5H). The level of expression and the distribution was not significantly modulated after OVA treatment (Figures 5G and 5H). Collectively, our results demonstrate a similar upregulation of BMP type I receptors at the mRNA and protein levels during the inflammatory response and that the induction is mainly restricted to inflamed bronchial epithelial and endothelial cells in the tissue.



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  		Figure 5. Dynamic changes of BMP receptors during airway inflammation in tissue. Lungs from mice challenged with three aerosolic inhalations of PBS (A, C, E, and G) or OVA (B, D, F, and H) were stained with antibodies against ALK2 (A and B), ALK3 (C and D), ALK6 (E and F) or BMPR-II (G and H) and shown at x20 magnification. All reagents were used at a concentration of 1 µg/ml. Five lungs were analyzed with similar results. Arrows indicate positively stained (green) or negative (red) cells. AW, airway; V, vessel.

 
Modulated Composition of BMP Ligands after Antigen-Induced Inflammation
To define the potential ligands responsible for the induced BMP activity noted in lung after antigen challenge, we compared expression levels of BMP ligands in PBS- and OVA-challenged lungs at the RNA and protein levels. Semiquantitative RT-PCR analyses show several of the BMP ligands to be markedly induced during the inflammatory response (Figure 6A) . A marked increase of BMP2 (4-fold) and BMP6 (7.5-fold) mRNA transcripts were noted in the OVA-challenged lung tissue (Figure 6A). In addition, neither BMP8a nor BMP9 were expressed in healthy lung, but were moderately induced in inflamed tissue (data not shown). In sharp contrast, a significant downregulation of BMP7 (9-fold) and a moderate reduction of BMP5 (2-fold) mRNA transcripts were detected in the OVA-challenged tissue (Figure 6A). BMP4 (1.3-fold) levels remained unchanged (Figure 6A). Several studies have demonstrated presence of BMP ligand mRNA without detecting the proteins in the tissue. Therefore, we analyzed the presence of some of the BMP ligands at the protein level. The antibodies used cannot discriminate between preproproteins and mature BMP ligands. Therefore, recombinant BMP proteins were included as controls. Total protein extracts from lung tissues were analyzed by Western blotting and the quantity of BMP preproprotein present inside the cell before cleavage, as well as the mature secreted polypeptides, could be visualized and compared (32). The healthy lung contained high levels of mature BMP2 and BMP5, moderate levels of BMP4, BMP6, and BMP7 (Figure 7) . A marked induction of mature BMP2 (2.5-fold) and BMP6 (8-fold) forms was recorded after antigen challenge (Figure 7). In contrast, a reduction of mature BMP4 (2-fold), BMP5 (7-fold), and BMP7 (10-fold) was noted during inflammation (Figure 7). Importantly, the preproprotein of BMP4 and BMP5 were present in healthy lung but were both upregulated 2-fold after OVA challenge (Figure 7). Multiple bands were observed for several of the ligands irrespective of this treatment that most likely correspond to various cleavage products or glycosylated forms of the preproproteins. Specificity of the reagents was demonstrated with peptide inhibition experiments in which both the prepro- and the mature forms were blocked (data not shown). Collectively our results showed that, during antigen-driven inflammation in lung, the composition of BMP ligands is modulated both at the mRNA and protein levels.



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  		Figure 7. Expression of BMP ligands at protein level in inflamed lung tissue. Total lung homogenate from mice treated with three aerosolic inhalations of PBS or OVA was analyzed for expression of BMP ligands. Cell lysates were subjected to immunoblotting with 1 µg/ml antibodies against BMP2, BMP4, BMP5, BMP6, or 3 µg/µl BMP7 antibody. Recombinant ligand protein (1 µg/ml) was included as a control and the expected size of the major preproprotein is indicated by an asterisk for each BMP ligand. One representative experiment out of three similar experiments is shown (n = 2 mice in each group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies using human biopsies and experimental models have indicated that TGF-ßs and activins can play determinative roles in the inflammatory and fibroblast proliferative response consequent to antigen-induced pulmonary inflammation and thus constitute prime candidates for regulating tissue remodeling that accompanies asthma. Using the OVA inhalation experimental model, which resembles the inflammatory response that occurs in humans exposed to short-time encounter with high-dose antigens like pollen, and antibodies recognizing the phosphorylated Smad2 protein, we recently demonstrated that the TGF-ß/activin signaling pathway is indeed strongly induced in the lung during the course of an allergen-induced airway inflammation (22).

The present study was initiated to investigate if another subgroup of the TGF-ß family ligands, the BMPs, are involved in the processes induced by allergen-challenge in the lung. To visualize active BMP-mediated signaling in the tissue we monitored the phosphorylation and subsequent nuclear translocation of BMP receptor–activated Smads, a hallmark of activated BMP signaling, during the inflammatory phase of allergen-induced airway in mice. In contrast to the healthy epithelium, which was devoid of immunoreactivity, almost all epithelial cells in the inflamed airways were stained with pSmad1/5 antiserum. Furthermore, approximately one out of five epithelial cells displayed nuclear pSmad1/5 staining, which implies active BMP/Smad signaling in these cells (11, 33). A mechanistic explanation for the increased pSmad1/5 staining in the inflamed lungs was provided by the observation that all known type I BMP receptors were upregulated on bronchial epithelial cells concurrent with a significantly altered expression profile of BMP ligands in the lung tissue. Moreover, some cells had both a cell membrane expression and a cytoplasmic expression of the BMP type I receptors. Zwaagstra and coworkers (34), recently demonstrated that engagement of the type I TGF-ß receptors resulted in a rapid internalization of the active receptor complex followed by either degradation or re-expression of the receptors (34, 35). The intracellularly localized receptors, which were observed mainly in the OVA-challenged mice, could reflect a similar process occurring in vivo in response to the allergen challenge.

Dynamic interactions between the epithelial cell layer and the underlying mesenchymal components play pivotal roles during lung development and repair. Local production of growth factors such as epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and cytokines that include members of the TGF-ß family mediate a bidirectional growth control system, effectively creating an "epithelial–mesenchymal throphic unit" that directs lung morphogenesis as well as proximo-distal epithelial lineage differentiation (18, 36). In general, signaling through tyrosine kinase receptors such as the receptors for EGF, FGF, PDGF, and IGF stimulate lung development, whereas signaling via serine/threonine kinase receptors, such as the TGF-ß receptor family, are inhibitory. In the adult lung, the bronchial epithelium acts as a physical barrier separating and protecting the internal milieu in the lung from the external environment. The integrity of the bronchial epithelium is a key feature in airway defense and thus any damage or injury to it is rapidly repaired. During the repair phase, the epithelial–mesenchymal throphic unit is reactivated in the affected areas and the normal epithelial structure is re-established in a process in which the antagonistic effects of EGF and TGF-ß play a central role (18, 37).

The pathopysiologic changes associated with asthma include an altered expression of several growth factors and cytokine receptors, implicated to contribute to the repair of the epithelial–mesenchymal throphic unit. However, the distribution and expression levels are severely changed compared with normal repair condition (38–40). Furthermore, the presence of inflammatory cells interacting with the epithelium during the progression of the disease results in a generalized activation of the epithelial–mesenchymal throphic unit. This results in a failure to reconstitute normal lung architecture, and a number of structural changes (including extensive epithelial damage, deposition of ECM, goblet cell metaplasia, smooth muscle hypertrophy, increase in nerves and blood vessels) contribute to the tissue remodeling that is associated with asthma. A mechanistic explanation for the failure of EGF signaling to mediate proper repair has been suggested to be abnormally high signaling from the antagonistic pathways stimulated by members of the TGF-ß family (41). Our previous findings demonstrating active TGF-ß/activin–mediated pSmad2 signaling (22), and the present study demonstrating active BMP-stimu- lated signaling in allergen-challenged airway epithelium, strongly support this hypothesis and raise the possibility that the BMP signaling system can potentially play a determinative role in respiratory pathophysiology.

Even though BMPs were originally identified and consequently named for their ability to induce cartilage and bone formation (42), later studies demonstrated the importance of properly balanced BMP signaling for normal lung development (4–7, 43, 44). Overexpression of BMP4 in transgenic animals under the surfactant protein C promoter/enhancer revealed a phenotype with abnormal morphogenesis associated with cystic terminal sacs and markedly inhibited overall proliferation (43). In addition, the epithelium was dominated by type I epithelial cells and the number of type II cells was markedly reduced (43). The type II cells are responsible for the ion transport as well as homeostasis, being the progenitor cell that repopulates damaged epithelium (45). The importance of controlled BMP signaling during lung development was further delineated in transgeneic animals expressing dominant-negative ALK6 or XNoggin (endogenous BMP secreted antagonist) in the lung (44). Epithelial cell proliferation was significantly inhibited in these animals. This was accompanied by severely reduced distal epithelial cell formation with a concurrent increase in proximal cell phenotype, manifested by upregulation of hepatocyte nuclear factor/forkhead homolog 4 (Hfh4) and expression of ciliated epithelium (44). Interestingly, this phenotype resembles the situation in patients with asthma, where the inflamed epithelium often shows a reduced proliferative response and a ciliated phenotype is sometimes noted in the bronchioles in the lower airways (46). The phenotypic similarities obtained in the above-mentioned animal models and asthma are consistent with the notion that the signaling cascades that operate during early embryonic development are re-utilized to repair the tissue when it is damaged by injury or prolonged inflammatory activities (18, 36). Thus, these BMP mechanisms may contribute to the disease pathophysiology when they are allowed to proceed without adequate control, and further support the notion that some aspects of asthma pathophysiology could be due to BMP-mediated signaling.

BMPs directly modulate the pathophysiology of other in- flammatory/fibrotic conditions. In injured skin both BMP2 and BMP6 are induced several fold, especially in the epithelium and the underlying fibroblast sheet where they colocalize with other members of the TGF-ß family and contribute to deposit of ECM components like collagen V and fibronectin (47–49). Moreover, skin injury during fetal development heals without scar formation, but exogenous implanted BMP2 in fetal sheep promotes scar formation and is associated with cellular growth, maturation, and fibroplasia (50). In sharp contrast, BMP7 has been shown to prevent tubulointerstitial fibrogenesis in unilateral ureteral obstruction, which in part mediated via an anti-inflammatory process downregulating the intercellular adhesion molecules, thereby reducing the number of infiltrating mononuclear cells (51). Interestingly, the composition of BMP ligands produced in OVA-challenged animals is markedly altered during the progression of airway inflammation. BMPs previously shown to be induced during fibrotic responses such as BMP2 and BMP6 (47–49) were produced in elevated levels during the airway inflammation in our study, whereas production of BMP7 that can potentially regulate resolution of the inflammatory response (51) was reduced in the OVA-challenged inflamed lung tissue. Thus, an altered intercellular communication with temporal and spatial altered composition of growth factors/cytokine networks and expression of active receptors may contribute to the pathogenesis of airway inflammation.

Collectively, our results show that inflamed bronchial epithelium in airways is induced to transmit BMP signals activation and phosphorylation of Smads. The local inflammatory response involves upregulation of BMP type I receptors and production of an altered BMP ligand profile. Adenoviral-mediated gene transfer approaches to locally administer constitutive active or inactive receptors and to locally express BMP ligands during the inflammatory response will directly address whether the BMPs contribute to the pathophysiology or are induced to counteract/protect the epithelium during the antigen challenge. Furthermore, all findings presented herein are derived from an experimental system that recapitulates parts of the asthma disease pathology spectrum like immigration of inflammatory cells to the lung tissue, but lacks the irreversibility of changes in the tissue such as thickening of basement membranes, which are characteristics of chronic asthma. Therefore, future approaches to further investigate the possibility that dysregulated growth factor signaling is an important feature during lung pathophysiology will include studies using human biopsies from patients suffering from chronic asthma, cystic fibrosis, and chronic obstructive pulmonary disease.


   Acknowledgments
 
The authors thank Mr. A. Smailagic for excellent technical assistance with the airway immunization experiments, and Mr. H. Lindberg and Dr. G. Marko-Varga for advice with protein extraction. This work was supported by ASTRAZENECA AB., Cancerfonden (2819-B99-11XAA), and the Dutch Scientific Research Council (ALW 809.67.021).

Received in original form November 19, 2001

Received in final form February 5, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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