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      Combined inhibition of TGFβ and PDGF signaling attenuates radiation-induced pulmonary fibrosis

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          ABSTRACT

          Background : Radiotherapy (RT) is a mainstay for the treatment of lung cancer, but the effective dose is often limited by the development of radiation-induced pneumonitis and pulmonary fibrosis. Transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) play crucial roles in the development of these diseases, but the effects of dual growth factor inhibition on pulmonary fibrosis development remain unclear. Methods : C57BL/6 mice were treated with 20 Gy to the thorax to induce pulmonary fibrosis. PDGF receptor inhibitors SU9518 and SU14816 (imatinib) and TGFβ receptor inhibitor galunisertib were applied individually or in combinations after RT. Lung density and septal fibrosis were measured by high-resolution CT and MRI. Lung histology and gene expression analyses were performed and Osteopontin levels were studied. Results : Treatment with SU9518, SU14816 or galunisertib individually attenuated radiation-induced pulmonary inflammation and fibrosis and decreased radiological and histological signs of lung damage. Combining PDGF and TGFβ inhibitors showed to be feasible and safe in a mouse model, and dual inhibition significantly attenuated radiation-induced lung damage and extended mouse survival compared to blockage of either pathway alone. Gene expression analysis of irradiated lung tissue showed upregulation of PDGF and TGFβ-dependent signaling components by thoracic irradiation, and upregulation patterns show crosstalk between downstream mediators of the PDGF and TGFβ pathways. Conclusion : Combined small-molecule inhibition of PDGF and TGFβ signaling is a safe and effective treatment for radiation-induced pulmonary inflammation and fibrosis in mice and may offer a novel approach for treatment of fibrotic lung diseases in humans. Translational statement : RT is an effective treatment modality for cancer with limitations due to acute and chronic toxicities, where TGFβ and PDGF play a key role. Here, we show that a combined inhibition of TGFβ and PDGF signaling is more effective in attenuating radiation-induced lung damage compared to blocking either pathway alone. We used the TGFβ-receptor I inhibitor galunisertib, an effective anticancer compound in preclinical models and the PDGFR inhibitors imatinib and SU9518, a sunitinib analog. Our signaling data suggest that the reduction of TGFβ and PDGF signaling and the attenuation of SPP1 (Osteopontin) expression may be responsible for the observed benefits. With the clinical availability of similar compounds currently in phase-I/II trials as cancer therapeutics or already approved for certain cancers or idiopathic lung fibrosis (IPF), our study suggests that the combined application of small molecule inhibitors of TGFβ and PDGF signaling may offer a promising approach to treat radiation-associated toxicity in RT of lung cancer.

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          Signal transduction by the TGF-beta superfamily.

          Transforming growth factor-beta (TGF-beta) superfamily members regulate a plethora of developmental processes, and disruption of their activity has been implicated in a variety of human diseases ranging from cancer to chondrodysplasias and pulmonary hypertension. Intense investigations have revealed that SMAD proteins constitute the basic components of the core intracellular signaling cascade and that SMADs function by carrying signals from the cell surface directly to the nucleus. Recent insights have revealed how SMAD proteins themselves are regulated and how appropriate subcellular localization of SMADs and TGF-beta transmembrane receptors is controlled. Current research efforts investigating the contribution of SMAD-independent pathways promise to reveal advances to enhance our understanding of the signaling cascade.
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            Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.

            IL-1beta is one of a family of proinflammatory cytokines thought to be involved in many acute and chronic diseases. Although it is considered to participate in wound repair, no major role has been attributed to IL-1beta in tissue fibrosis. We used adenoviral gene transfer to transiently overexpress IL-1beta in rat lungs after intratracheal administration. The high expression of IL-1beta in the first week after injection was accompanied by local increase of the proinflammatory cytokines IL-6 and TNF-alpha and a vigorous acute inflammatory tissue response with evidence of tissue injury. The profibrotic cytokines PDGF and TGF-beta1 were increased in lung fluid samples 1 week after peak expression of IL-1beta. Although PDGF returned to baseline in the third week, TGF-beta1 showed increased concentrations in bronchoalveolar lavage fluid for up to 60 days. This was associated with severe progressive tissue fibrosis in the lung, as shown by the presence of myofibroblasts, fibroblast foci, and significant extracellular accumulations of collagen and fibronectin. These data directly demonstrate how acute tissue injury in the lung, initiated by a highly proinflammatory cytokine, IL-1beta, converts to progressive fibrotic changes. IL-1beta should be considered a valid target for therapeutic intervention in diseases associated with fibrosis and tissue remodeling.
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              Up-Regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

              Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia of unknown etiology characterized by alveolar epithelial cell injury/activation, fibroblast proliferation, and exaggerated accumulation of extracellular matrix in the lung parenchyma [1,2]. The disease is usually progressive and largely unresponsive to corticosteroid and immunosuppressive therapy [2,3]. A distinctive morphological feature of IPF is the development of fibroblastic/myofibroblastic foci, represented by widely scattered, small aggregates of subepithelial mesenchymal cells immersed within a myxoid-appearing extracellular matrix [4]. These fibroblastic/myofibroblastic foci represent areas of active fibrogenesis and play a crucial role in the progressive fibrotic response [1,5]. These sites are characterized by inappropriate re-epithelialization and impaired extracellular matrix remodeling. Although significant advances have been made in the characterization of the clinical and morphological features of this disease, the molecular mechanisms that underlie IPF pathogenesis in humans are still largely unknown [1,4–7]. Osteopontin (also termed secreted phosphoprotein 1) is a phosphorylated acidic glycoprotein that contains an Arg-Gly-Asp motif that binds to the integrin family of adhesion molecules [8]. It functions as a cell adhesion and migration molecule that can bind to several ligands, including αvβ3 integrin, some CD44 isoforms, and fibronectin [9,10]. Osteopontin has been implicated in a number of physiological and pathological processes including bone resorption, malignant transformation, and metastasis [11,12]. It is also considered a key cytokine regulating inflammation, cellular immune response, and tissue repair, with a unique effect on T cell function [11,12]. Using oligonucleotide microarrays we previously demonstrated that osteopontin is highly up-regulated in bleomycin-induced lung fibrosis in mice, and we reported similar results in a preliminary report involving five IPF lungs and four control samples [13,14]. Interestingly, osteopontin seems to have a profibrotic effect in the development of bleomycin-induced pulmonary fibrosis [15]. After bleomycin instillation, osteopontin-null mice developed reduced lung fibrosis characterized by dilated distal air spaces with decreased active transforming growth factor-beta 1 (TGF-β1) and reduced type I collagen expression compared with wild-type controls [16]. The mechanisms by which osteopontin, a cytokine with primarily TH1 effects (i.e., antifibrotic) on T lymphocytes [11,17,18], may cause profibrotic effects are not fully understood. In this study, we applied microarrays to analyze gene expression patterns in a larger cohort of IPF lungs (13 IPF samples and 11 controls), and we analyzed the direct effects of osteopontin on human lung fibroblasts, alveolar epithelial cell migration and proliferation, and matrix metalloprotease (MMP) gene expression in vitro. Methods Study Population Patients from the National Institute of Respiratory Diseases, Mexico City, México (Protocol S1), and the University of Pittsburgh, Pittsburgh, Pennsylvania, United States (Protocol S2), were included in this study. The protocol was approved by both Institutions, and written informed consent was obtained where required. Samples for oligonucleotide microarray were obtained from the tissue bank of the Department of Pathology at the University of Pittsburgh. The use of archived tissue has been approved by the local Institutional review board. Diagnosis of IPF was supported by history, physical examination, pulmonary function studies, chest HRCT, and bronchoalveolar lavage (BAL) findings, and was corroborated by open lung biopsy. The morphologic diagnosis of IPF was based on typical microscopic findings consistent with usual interstitial pneumonia [4]. The patients fulfilled the criteria of the American Thoracic Society and European Respiratory Society [7]. BAL samples were obtained at first consult as part of the initial diagnostic work-up. None of the patients had been treated with corticosteroids or immunosuppressive drugs at the time of BAL. Demographic data, pulmonary function data, and BAL differential cell counts are provide in Table 1. Levels of osteopontin in BAL fluids were evaluated in ten healthy individuals (two current smokers, two former smokers, and six that had never smoked). All had normal chest X-rays and spirometries. Likewise, histologically normal lung tissues obtained at necropsy from six nonsmoking adult individuals who had died of causes unrelated to lung diseases were utilized for immunohistochemistry. For oligonucleotide microarrays, control samples included normal histology lung samples resected from patients with lung cancer obtained from the Pittsburgh Tissue Bank (Pittsburgh, Pennsylvania, United States). Oligonucleotide Microarrays Surgical remnants of biopsies or lungs explanted from patients with IPF that underwent pulmonary transplant were the sources of 13 IPF samples. Lung samples resected from patients with lung cancer, obtained from the tissue bank of the Department of Pathology at the University of Pittsburgh, were the sources of 11 normal samples. None of these samples had been included in our previous study. Total RNA was extracted and used as a template to generate double-stranded cDNA and biotin-labeled cRNA, as recommended by the manufacturer of the arrays and previously described [11]. Fragmented cRNA was hybridized to Codelink Uniset I slides. After hybridization, arrays were washed and stained with streptavidin-AlexaFluor 647. The arrays were scanned using a Genepix 4000B microarray scanner. Images were analyzed using Codelink expression II analysis suite. They were visually inspected for defects and quality control parameters as recommended by the manufacturer. Data files were imported into a microarray database and linked with updated gene annotations using SOURCE (http://genome-www5.stanford.edu/cgi-bin/SMD/source/sourceSearch) and then median scaled. Based on our previous experience, all expression levels below 0.01 were brought to 0.01. Statistical analysis was performed using Scoregene gene expression package (http://www.cs.huji.ac.il/labs/compbio/scoregenes, and data visualization was performed using Genexpress (http://genexpress.stanford.edu) and Spotfire Decision Site 8.0 (Spotfire, Göteborg, Sweden). The complete set of gene array data has been deposited in the Gene Expression Omnibus database with GEO serial accession number GSE2052 (http://www.ncbi.nlm.nih.gov/geo) according to MIAME guidelines. The general approach to analysis was previously described by us [19]. Weakest link models [20,21] were fitted using the Weaklink package for the R statistical software system (http://www.r-project.org). The p-value was obtained from fitting a logistic regression model with a single independent variable that is the minimum of the percentiles for the expression levels for the two genes. A Bonferroni correction was applied for multiple testing. Bronchoalveolar Lavage BAL was performed through flexible fiberoptic bronchoscopy under local anesthesia. Briefly, 300 ml of normal saline was instilled in 50-ml aliquots, with an average recovery of 60%–70%. The recovered BAL fluid was centrifuged at 250 g for 10 min at 4 °C. The cell pellet was resuspended in 1 ml of PBS and an aliquot was used to evaluate the total number of cells. Other aliquots were fixed in carbowax, stained with hematoxylin and eosin, and used for differential cell count. Supernatants were kept at −70 °C until use. ELISA Quantification of osteopontin was performed in BAL fluid samples from 18 IPF patients and 10 healthy individual controls, by using a commercial sensitive and specific ELISA following the instructions of the manufacturer (Calbiochem, La Jolla, California, United States). Immunohistochemistry Tissue sections were deparaffinized, rehydrated, and then blocked with 3% H2O2 in methanol for 30 min, then antigen was retrieved with citrate buffer (10 mM, pH 6.0) for 5 min in a microwave. Rabbit polyclonal antibody to human osteopontin (2 ng/ml; Calbiochem) was applied and samples were incubated at 4 °C overnight. A secondary biotinylated anti-immunoglobulin followed by horseradish peroxidase-conjugated streptavidin (BioGenex, San Ramon, California, United States) was used according to manufacturer's instructions. AEC (BioGenex) in acetate buffer containing 0.05% H2O2 was used as substrate [15,19]. The sections were counterstained with hematoxylin. The primary antibody was replaced by non-immune serum for negative control slides. Two-Color Immunofluorescence Analysis A standard two-stage double-immunofluorescence labeling technique was used. Briefly, frozen sections were washed in PBS (0.01 M [pH 7.4]) for 5 min and then fixed in cold acetone for 10 min, twice. Tissues were incubated in blocking buffer (1% BSA, 5% normal serum, 0.05% NP-40, in PBS) for 30 min. The slides were then incubated with a rabbit antibody to osteopontin (1:100; Abcam, Cambridge, Massachusetts, United States) for 1 h at room temperature and washed in PBST (0.05% Tween-20 in 0.01 M PBS [pH 7.4]) for 10 min, three times. A mouse anti-MMP-7 monoclonal antibody (1:1,000; Chemicon International, Temecula, California, United States) was added and the slides were incubated for an additional 1 h at room temperature. After three 10-min washes in PBST, slides were incubated with secondary antibodies (sheep anti-rabbit IgG-Cy3, 1:1,000; and goat anti-mouse IgG-FITC, 1:1,000; Sigma-Aldrich, St. Louis, Missouri, United States) for 30 min. Slides were then washed in the same buffer and mounted with antifade medium (containing DAPI to stain cell nuclei). Cell Culture Primary human normal lung fibroblasts were obtained as previously described [22], and the A549 cell line was obtained from ATCC (Rockville, Maryland, United States). Growth Rate Assay Lung fibroblasts or A549 cells were seeded in 96-well culture plates at a cell density of 7.5 × 103 and 5 × 103 cells/well respectively, and incubated in Ham's F-12 and DMEM media, respectively (GIBCO-BRL, Grand Island, New York, United States), supplemented with 10% FBS at 37 °C in 5% CO2 and 95% air. After 12 h, the medium was replaced by medium with 0.1% FBS alone or 0.1% FBS plus increasing concentrations of osteopontin (0.4, 1, and 2 μg/ml) and the cells were maintained in culture for another 48 h. Cell growth was determined using the cell proliferation reagent WST-1 (Boehringer Mannheim, Mannheim, Germany) as previously described [19]. All assays were performed in triplicate. In parallel experiments, fibroblasts and A549 cells were pretreated for 25 min with antibody to human αvβ3 integrin (10 μg/ml; Chemicon), the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS; Calbiochem), or antibody to human CD44 (NeoMarkers, Fremont, California, United States), and then osteopontin was added (2 μg/ml). Additionally, A549 cells were pretreated with antibody to human epidermal growth factor receptor (EGFR; 10 μg/ml; Chemicon). Cell Migration Assay Migration of fibroblasts and A549 cells was assayed using commercially available 24-well collagen-coated Boyden chambers (Chemicon) with an 8-μm pore size. Briefly, a semi confluent (∼80%) monolayer of lung fibroblasts or A549 cells was harvested with trypsin-EDTA, centrifuged, and resuspended in Ham's F-12 medium containing 5% BSA. The cell suspensions (3 × 105 cells/well) were added to the upper chamber. The lower chamber contained 0.3 ml of medium with 5% BSA alone or with 10 μg/ml of recombinant human osteopontin (Calbiochem). PDGF (8 ng/ml) and epidermal growth factor (EGF; 50 ng/ml) were used as positive controls for fibroblasts and for A549 cells, respectively. Additional BSA-coated chambers were used as blanks for each sample. After incubation for 8 h at 37 °C in a humidified incubator with 5% CO2 and 95% air, the nonmigrating cells on the top of Boyden chamber were scraped and washed. The migrating cells were quantitated according to manufacturer's instructions. Briefly, the cells were stained and the color eluted with 300 μl of extraction buffer, and 150-μl aliquots were measured in an ELISA plate reader at 545 nm. All assays were performed in duplicate. In parallel experiments, A549 cells and fibroblasts were pretreated as described above for growth rate. RNA Isolation and Northern Blot Analysis Total RNA was extracted from lung fibroblasts and A549 epithelial cells using the RNeasy Mini Kit (Qiagen GmbH, Germany). Cells were lysed and homogenized in the presence of a highly denaturing guanidine isothiocyanate-containing buffer. The samples were then applied to an RNeasy minicolumn, and RNA was eluted in 30 μl of water. Total RNA (20 μg/lane) was fractionated on a 1% agarose gel containing 0.66 M formaldehyde [21]. Ribosomal RNA was visualized with ethidium bromide, and the fractionated RNA was transferred onto a Nytran transfer membrane (NEN Life Science Products, Boston, Massachusetts, United States) by capillary blotting overnight. RNA was immobilized by baking at 80 °C for 2 h, and then prehybridized at 42 °C for 18 h in 5× SSC, 50% formamide, 5× Denhardt's solution, and 0.5% SDS, containing 100 μg/ml of denatured salmon sperm DNA. Hybridization was carried out at 42 °C for 18 h in hybridization buffer composed of formamide, 0.5% SDS, and heat-denatured 32P-labeled cDNA probes. After washing, membranes were dried and exposed to Kodak BIOMAX MS film at −70 °C with an intensifying screen. The cDNA clones for human MMP-1, TIMP-1, α1 type I collagen, and RNA ribosomal 18S were obtained from ATCC. Human MMP-7 cDNA was a kind gift of Lynn Matrisian, Vanderbilt University, Nashville, Tennessee, United States. The cDNA probes were radiolabeled with 32P-dCTP to a specific activity of 200 × 106 dpm/μg using a Random Primer Labeling Kit (Stratagene, La Jolla, California, United States). All experiments were repeated twice. Quantitative Real-Time RT-PCR 1 μg of RNA was treated with 1 unit of DNAase (Life Technologies, Grand Island, New York, United States). First-strand cDNA was synthesized by reverse transcription with random primers and Moloney-murine leukemia virus reverse transcriptase according to manufacturer's protocol (Advantage RT-for-PCR Kit; Clontech, Palo Alto, California, United States). Real-time PCR amplification was performed using i-Cycler iQ Detection System (BioRad, Hercules, California, United States), using TAQMAN probes (PE Applied Biosystems, Wellesley, California, United States) labeled with FAM and TET. PCR was performed with the cDNA working mixture in a 25-μl reaction volume containing 3 μl of cDNA, 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 μM dNTPs, 0.2 μM specific 5′ and 3′ primers for 18S rRNA, 0.6 μM specific 5′ and 3′ primers for gene target, 0.2 μM of each probe TAQMAN (18S rRNA and gene target), and 1.25 units of AmpliTaq GOLD DNA polymerase (PE Applied Biosystems). A dynamical range was built with each product of PCR on copy number serial dilutions from 1 × 108 to 1 × 101; all PCRs were performed in triplicate. Standard curves were calculated referring the threshold cycle (Ct) to the log of each cDNA dilution step. Results were expressed as the number of copies of the target gene normalized to 18S rRNA. Some primers used in PCR reactions were designed using Beacon Designer software 2.1 (BioRad) and checked for homology in BLAST. The cycling conditions for PCR amplification were performed using the following protocol: Initial activation of AmpliTaq Gold DNA polymerase at 95 °C for 7 min; and 40 cycles of denaturation at 95 °C for 30s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The sequences of the PCR primer pairs and probes for each gene are shown in Table 2. All PCR experiments were done in duplicate. PAGE Zymography For MMP-7 analysis, conditioned media was electrophoresed in 12.5% SDS gels containing as substrate bovine CM-transferrin (0.3 mg/ml) and heparin [22]. After electrophoresis, gels were washed in a solution of 2.5% Triton X-100, and incubated overnight at 37 °C in 100 mM glycine (pH 7.6), plus 10 mM CaCl2 and 50 nM ZnCl2. Identical gels were incubated but in the presence of 20 mM EDTA. Gels were stained with Coomassie Brilliant Blue R250 and destained in a solution of 7.5% acetic acid and 5% methanol. Recombinant human MMP-7 (Chemicon) was used as positive control. Western Blot Analysis Serum-free conditioned media was centrifuged at 300 g at 4 °C for 30 min to remove cell debris, then concentrated by lyophilization. Samples were solubilized in water, and aliquots containing 5 μg of protein were mixed with Laemmli sample buffer and electrophoresed on 10% or 12.5% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose filters at 15 V for 20 min using semi-dry transfer cell (BioRad). Nonspecific sites were blocked overnight with 4% (w/v) nonfat dried milk in PBS, and membranes were incubated with rabbit antibody to human interstitial collagenase IgG (1:250 in PBS with 1% BSA; Santa Cruz Biotechnology, California, United States), antibody to human TIMP-1 (Chemicon), antibody to human MMP-7 (Oncogene, San Diego, California, United States), or monoclonal antibody to human α-smooth muscle actin (2 μg/ml; Cymbus Biotechnology, Chandlers Ford, Hants, United Kingdom) for 2 h at room temperature. Filters were incubated with secondary antibody conjugated to peroxidase for 1 h at room temperature. Finally, the filters were developed with the Enhanced Chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, New Jersey, United States) using radiograph film (Hyperfilm, Amersham Pharmacia Biotech) according to the instructions of the manufacturer. Western blots were repeated twice. Results Osteopontin Gene Expression Is Highly Up-Regulated in IPF Lungs The complete microarray dataset is available at the Gene Expression Omnibus database with GEO serial accession number GSE2052 (http://www.ncbi.nlm.nih.gov/geo). Gene expression patterns clearly distinguished IPF lungs from normal lungs. Osteopontin was the most up-regulated gene among the genes that distinguished IPF lungs (Figure 1A). Osteopontin levels were increased more than 20-fold (mean expression 0.62 in control lungs, 16.8 in IPF lungs; Figure 1B). This increase was statistically significant whether we applied threshold number of misclassifications (TNoM) score (p = 0.000221), or Student's t-test (p = 0.0000035). We corrected for multiple testing by controlling the false discovery rate at the 5% level [23]. Osteopontin expression levels were higher in most IPF lungs than in any control lung (Figure 1B). Osteopontin Is Increased in BAL Fluid from IPF Patients Osteopontin protein was quantified in BAL fluids from 18 IPF patients and 10 healthy controls. As shown in Figure 2, ELISA measurement revealed a significant increase in the immunoreactive protein in the fluids derived from IPF lungs (148.8 ± 83 ng/ml in IPF lungs versus 3.8 ± 2.0 ng/ml in healthy controls, p < 0.01). Immunoreactive Osteopontin Is Localized Primarily in Epithelial Cells To examine the cellular source of osteopontin we analyzed IPF and control lungs by immunohistochemistry. As illustrated in Figure 3A–3C, osteopontin was localized primarily in alveolar epithelial cells that exhibited an intense cytoplasmic staining in IPF lungs. Occasionally, clusters of alveolar macrophages were also positive (Figure 3C). Immunohistochemical staining for osteopontin was negative in normal lungs as well as in lung tissue samples incubated with nonimmune sera (Figure 3D). Osteopontin Induces Fibroblast and Epithelial Cell Growth Rate To determine the effect of osteopontin on the growth rates of fibroblasts and epithelial cells, cells were stimulated with increasing concentrations of osteopontin, and the cell number was determined after 48 h using the cell proliferation reagent WST-1. Significant dose-dependent increases in cell proliferation were observed with 1 μg/ml and 2 μg/ml. Two different fibroblast lines reached 220% and 380% over controls (p < 0.01), while in two different experiments A549 cell lines exhibited 60% and 80% increases of growth rate over control (p < 0.01). Osteopontin-induced fibroblast proliferation (2 μg/ml) was significantly suppressed by GRGDS-pentapeptide, which interrupts binding of RGD-containing proteins to cell surface integrins (p < 0.01) and by antibody to αvβ3 integrin (p < 0.05), suggesting that the effect of osteopontin on growth rate was mediated by the interaction of the GRGDS domain of osteopontin with αvβ3 integrin (Figure 4A). In contrast, epithelial cell growth was partially inhibited by antibody to CD44 (p < 0.05) (Figure 4B). Epithelial cell growth was also significantly suppressed by GRGDS but not by antibodies to αvβ3 (Figure 4B). Osteopontin Induces Fibroblast and Epithelial Cell Migration To examine the effect of osteopontin on cell migration, we used collagen-coated Boyden chambers, a well-established in vitro assay system. The number of cells that migrated in absence of osteopontin was used as control (0% migration). As revealed in Figure 5A, fibroblasts significantly moved toward osteopontin compared with cells exposed to medium plus 5% BSA alone in the lower chamber. Osteopontin (10 μg) enhanced fibroblast migration by 120% ± 11% (p < 0.01), an enhancement similar to that obtained with the potent fibroblast mitogen platelet-derived growth factor, used as a positive control (157% ± 12%, unpublished data). To analyze possible mechanisms by which osteopontin stimulates migration, different blockers were used. Fibroblast migration was significantly reduced by GRGDS and antibody to αvβ3 integrin (p < 0.01), and by antibody to CD44 (p < 0.05). The inhibition of cell migration was specific, since incubation with IgG had no effect (unpublished data). A549 lung cells also showed a significant increase in cell migration in response to osteopontin (Figure 5B). After 8 h of incubation, A549 cells passing through the membrane increased by 114% ± 25% compared to control cells (p < 0.01). Epidermal growth factor, used as a positive control, induced a 168% ± 14% increase in cell migration (unpublished data). Osteopontin-induced migration was abolished when the epithelial cells were pretreated individually with GRGDS, anti-αvβ3 integrin, or anti-CD44 (p < 0.01; Figure 5B). Osteopontin Induces an Environment That Favors Extracellular Matrix Deposition in Fibroblasts MMP-1 expression by fibroblasts Under basal conditions, some primary human lung fibroblast cell lines may express MMP-1, while others express the enzyme only when stimulated, for example, by aminophenylmercuric acetate (APMA). In this context, the effect of osteopontin on MMP-1 expression was examined by Northern blot analysis in a cell line producing MMP-1 under basal conditions, and in a cell line that did not produce MMP-1 but was stimulated by APMA (Figure 6A and 6B). Osteopontin down-regulated both the basal and the APMA-induced MMP-1 transcript level. When the signal of MMP-1 mRNA was normalized to the level of 18S RNA and quantified by densitometry, a reduction of ∼50% was noticed. This result was confirmed by real-time PCR that showed ∼40% inhibition (p < 0.05; Figure 6C). Inhibition of MMP-1 expression was partially blocked by antibody to CD44, while it was not affected by antibody to αvβ3 integrin. The inhibitory effect of osteopontin on MMP-1 was also observed at the protein level by Western blot analysis. As shown in Figure 6D, the level of immunoreactive MMP-1 present in the conditioned medium was decreased in the fibroblasts stimulated with osteopontin as compared with control cells. Osteopontin increases TIMP-1 expression by fibroblasts The effect of osteopontin on TIMP-1 gene expression in fibroblasts is illustrated in Figure 7. Northern blot analysis (Figure 7A) revealed an increase in TIMP-1 gene expression at 0.4 μg/ml and 1 μg/ml osteopontin when compared to control. This result was confirmed by real-time PCR (Figure 7B). Osteopontin increased the TIMP-1/18S ribosomal RNA from 55.7 ± 14.2 copies to 121.2 ± 13.1 copies (p < 0.01). Both anti-CD44 and anti-αvβ3 integrin abolished this increase. Osteopontin also increased TIMP-1 protein expression in conditioned medium as illustrated in Figure 7C. Osteopontin induces collagen gene expression in fibroblasts The effect of osteopontin on collagen gene expression is depicted in Figure 8. Northern blot analysis (Figure 8A) revealed a 2-fold increase in α1 type I collagen gene expression. Expression of α-smooth muscle actin in fibroblasts was induced by TGF-β1 but not by osteopontin (Figure 8B). Osteopontin Increases MMP-7 Expression by Epithelial Cells Stimulation of A549 cells with osteopontin (0.4 μg/ml and 2.0 μg/ml) induced an up-regulation of MMP-7 gene expression as illustrated by Northern blot in Figure 9A. Real-time PCR revealed a 6-fold increase at 6 h after osteopontin stimulation, which was abolished by treatment with anti-αvβ3, anti-CD44, anti-EGFR, or GRGDS as shown in Figure 9B. Pro-MMP-7 overexpression was confirmed at the protein level by Western blot analysis of the epithelial cells conditioned medium (Figure 9C). As recombinant MMP-7 used as control showed a lower molecular weight band to that of conditioned medium, this one was treated with APMA to activate pro-MMP-7. Activated proenzyme showed a similar molecular weight band to the recombinant protein. Zymography using CM-transferrin as substrate showed that treatment of A549 epithelial cells with 0.4 μg/ml and 1 μg/ml osteopontin induced an increase of both the pro-MMP-7 and MMP-7 activity bands (Figure 9D). Osteopontin Colocalizes with MMP-7 in Alveolar Epithelial Cells from IPF Lungs Since we have previously demonstrated that IPF lungs strongly express MMP-7 in alveolar epithelial cells [14], we evaluated whether the staining pattern of this enzyme is associated with that of osteopontin. Figure 10 illustrates confocal microscopy images showing that MMP-7 (Figure 10A) is partially overlapped with that of osteopontin (Figure 10B), giving substantial double labeling (Figure 10C and 10D). Normal lungs did not exhibit any significant staining for MMP-7 or osteopontin (unpublished data). Weakest Link Models Identify a Statistically Significant Interaction between Osteopontin and MMP-7 To determine whether MMP-7 and osteopontin expression levels jointly distinguish IPF and control samples, we applied weakest link statistical models [20]. Weakest link models assume that two genes jointly impact the probability that a randomly selected sample belongs to a certain phenotype (IPF in this case) only if expression levels of the two genes lie on a low-dimensional curve whose form is specified by the model and estimated using the observed quantiles of the data. Our analysis detected a statistically significant joint effect of MMP-7 and osteopontin on the IPF phenotype (p < 0.001). This joint relationship is illustrated in Figure 11 for both the gene expression values (Figure 11A) and the sample percentiles (Figure 11B). To assess the relevance of this association, we chose both substantively meaningful genes and a more general set of genes to evaluate the contribution of the weakest link model relating osteopontin and MMP-7 (Figure 11). Among 14 matrix metalloproteases and their inhibitors represented on the Human Uniset I chip, osteopontin and MMP-7 exhibited the most significant biological interaction. Osteopontin also had significant interactions (weakest link models with p < 0.05) with MMP-1, MMP-2, and MMP-11 but none was as statistically significant as MMP-7. We also ran weakest link models combining osteopontin with each of 400 genes that passed the false discovery rate of less than 0.05 for differential expression between IPF and control samples. The interaction of osteopontin with MMP-7 in a weakest link model was more significant than 371 of these 400 genes. Discussion In the present study we focused on the profibrotic effects of osteopontin, a multifunctional cytokine that mediates diverse biological functions, including cell adhesion, chemotaxis, and signaling, as well as tissue reparative processes [8,11,12]. We demonstrated that osteopontin was the most up-regulated gene in lungs of IPF patients, and that it was mainly expressed by alveolar epithelial cells. To better understand the potential local profibrotic effects of osteopontin, we studied its effects on lung fibroblasts and epithelial cells. Functionally, osteopontin induced fibroblast and epithelial cell proliferation and migration. The effect on fibroblast migration and proliferation was dependent mainly on integrins, while in epithelial cells proliferation was mainly dependent on CD44 and migration was dependent on CD44 and integrin signaling. Osteopontin exhibited profibrotic effects on molecules involved in extracellular matrix remodeling. Thus, in fibroblasts, osteopontin increased TIMP-1 and type I collagen and inhibited MMP-1 expression, while in alveolar epithelial cells it induced MMP-7. The effects on TIMP-1 and MMP-1 expression appeared to be mostly dependent on CD44, while the effect on MMP-7 expression was dependent on CD44 and integrin signaling. Interestingly, osteopontin was colocalized with MMP-7 in alveolar epithelial cells of IPF lungs, and application of the weakest link models to microarray data suggested that the genes of both interacted to affect the IPF phenotype. Our results provide a potential mechanism by which osteopontin secreted from epithelial cells exerts its profibrotic effects through direct signaling on fibroblasts and epithelial cells. Several studies in experimental tissue fibrosis have suggested a possible profibrotic role of osteopontin. In kidney fibrosis, osteopontin enhances macrophage recruitment and stimulates the development of renal scarring after an acute ischemic insult [24]. Also, osteopontin expression is increased in the myocardium after myocardial infarction, and the lack of this mediator is associated with decreased collagen accumulation [25]. Similarly, osteopontin appears to be an important mediator of the cardiac profibrotic effects of angiotensin II by promoting collagen synthesis and remodeling in the interstitial myocardium [26]. In experimental lung fibrosis it has been suggested that osteopontin produced by alveolar macrophages functions as a fibrogenic cytokine [15,16]. In granulomatous lung diseases, osteopontin is also up-regulated and its main sources are macrophages and T lymphocytes [27,28]. We observed that in human IPF lungs, hyperplastic alveolar epithelial cells seemed to be a source of osteopontin, which is consistent with the findings of Berman et al. [15,16]. These observations support the key role of alveolar epithelial cells as regulators of the lung profibrotic microenvironment in IPF, thus emphasizing the critical difference between the mechanisms of fibrosis in IPF compared with animal models or with lung fibrosis associated with inflammatory disorders. Positive feedback mechanisms have been previously proposed for osteopontin and MMP-2 [29], where osteopontin induces MMP-2 expression [30] and is cleaved and activated by MMP-2 [29]. The same mechanism has been proposed for osteopontin and MMP-3, where osteopontin binds and activates MMP-3, which in turn can cleave and activate it [31]. Similarly, osteopontin is cleaved and activated by MMP-7 [31], and we observe that MMP-7 is induced by osteopontin in epithelial cells, suggesting that this positive feedback mechanism is also applicable to osteopontin and MMP-7. This is also supported by the colocalization of MMP-7 and osteopontin in IPF epithelial cells, and by the computational relationship of the expression levels of osteopontin and MMP-7. Interestingly, MMP-7 and osteopontin are β-catenin target genes [32,33]. Recently, Chilosi et al. reported impressive activation of WNT/β-catenin in IPF lungs [34]. They have mainly observed β-catenin nuclear localization in proliferative bronchiolar lesions, where it colocalized with MMP-7 [34]. Collectively, these results could indicate a mechanism by which osteopontin and MMP-7 are induced by aberrant activation of the WNT/β-catenin pathway, and each affects the function and expression of the other gene, thus representing a local positive feedback mechanism that facilitates a chronic, relentless lung disease. Although intriguing, this proposed positive, self-perpetuating loop in itself cannot explain increased collagen deposition, the critical hallmark of fibrosis. In this context, our results suggest that osteopontin affects the critical balance between MMPs and their inhibitors through its cell-specific effects. In agreement with the inhibition of interleukin 1β-stimulated increases in MMP-2 and MMP-9 observed by Xie et al. [35], we observed that in human lung fibroblasts, osteopontin caused a significant reduction of baseline as well as APMA-stimulated MMP-1, an MMP responsible for fibrillar collagen degradation. Additionally, we observed a concomitant increase in TIMP-1, the main inhibitor of MMP-1 (as well as MMP-2 and −9), and in type I collagen gene expression, suggesting that osteopontin may facilitate a profibrotic environment not only by its effect on epithelial cells but also by inducing a nondegradative microenvironment similar to the one observed in IPF and experimental lung fibrosis [36,37]. Interestingly, osteopontin did not induce α-smooth muscle actin in lung fibroblasts in vitro, suggesting that although it had a role in facilitating the profibrotic environment in IPF, it had weaker role in the formation of myofibroblast foci. However, further experiments will be needed to determine this point. Migration and proliferation of fibroblasts are essential for the expansion of their populations and the formation of the fibroblastic foci that seem to represent the “leading edge” of the progressive fibrotic process [5]. The observation that osteopontin induced both migration and proliferation of primary human lung fibroblasts suggests that osteopontin may be involved in this process and is supported by previous observations in murine and human fibroblast cell lines [15]. Osteopontin promotes fibroblast collagen gel contraction and rat cardiac fibroblast proliferation, and it has been suggested that it is an important mediator of angiotensin II regulation of fibroblast behavior in the cardiac remodeling process [38]. It also induced proliferation of vessel smooth muscle cells, suggesting that it is involved in vascular remodeling during the development of atherosclerosis [39,40]. These results highlight the sufficiency of a cytokine secreted from alveolar epithelial cell to induce many of the phenotypic changes associated with lung fibrosis. The mechanisms by which osteopontin influences epithelial and fibroblast cells are not fully understood. In general it has been proposed that osteopontin affects cells by binding to CD44 isoforms, certain integrins, and EGFR [41,42]. It is unknown whether the expression of these receptors is changed in IPF; CD44 is expressed on lung fibroblasts and epithelial cells [43], is induced in radiation- and bleomycin-induced pulmonary fibrosis [44] and in acute alveolar fibrosis [45], and is critical for resolution of noninfectious lung inflammation [46]. The integrin receptors for osteopontin are widely expressed in lung epithelial cells and fibroblasts [47], but a change in their repertoire has not been reported yet with IPF. In this paper we did not seek to fully dissect these mechanisms; however, we explored some of the possible receptors that may be important to osteopontin effect on fibroblasts and epithelial cells. Our results support some of the previous observations regarding the importance of integrin-mediated signaling in fibroblast migration. Interestingly, we observed a differential effect in epithelial and fibroblasts cells. Inhibition of CD44 significantly reduced the effects of osteopontin on cell proliferation in epithelial cells, while αvβ3 inhibition seemed to affect mostly fibroblasts. This integrin is a receptor for a wide variety of extracellular matrix ligands with an exposed RGD sequence, including vitronectin, fibronectin, fibrinogen, thrombospondin, proteolyzed collagen, von Willebrand factor, as well as osteopontin, and appears to play a critical role in cell migration [48]. The hyaluronic acid receptor CD44 is also a receptor for osteopontin, and it has been implicated in chemotaxis mediated by this mediator [10,49,50]. In our experiments, the pan-integrin antagonist GRGDS-pentapeptide added to the culture medium abolished the effects in fibroblasts and epithelial cells, suggesting that different integrins may be involved in both cell types, and that for some effects, both integrins and CD44 are required. Detailed studies of the effects of variant osteopontin isoforms as well as cells that express different receptors will be needed to elucidate these mechanisms. Our study focused on human tissues and human cell lines because of the unique features of IPF that are not readily mimicked by any animal models. We insisted on using primary human lung fibroblasts in our experiments; therefore, we are confident that these results represent a mechanism that may actually occur in the human lung. Unfortunately, it is nearly impossible to work with primary alveolar epithelial cells, and we had to resort to the epithelial cell line A549. However, we present evidence obtained from human lungs that suggest that the mechanisms that we proposed in vitro do exist in the human IPF lung. In summary, in this study we highlight the role of osteopontin in human IPF. Although previous studies have suggested that osteopontin has a potential profibrotic effect in animal models of lung fibrosis, its role in human IPF was unclear. We demonstrated that osteopontin is highly expressed in IPF lungs, and that it is primarily expressed by hyperplastic alveolar epithelial cells. We demonstrated that osteopontin affected fibroblast and epithelial cell proliferation and migration, and that it had fibrosis-relevant effects on MMP and TIMP expressions. Our results suggest a mechanism explaining most of the profibrotic effects of osteopontin by its direct effects on fibroblasts and epithelial cells in the lungs. Furthermore, our results suggest that in IPF the interaction between MMP-7 and osteopontin may be involved in the relentlessly progressive nature of the disease, and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease. Supporting Information Protocol S1 Sample Mexico (746 KB ZIP). Click here for additional data file. Protocol S2 Microarrays (2.1 MB ZIP). Click here for additional data file. Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers of the genes discussed in this paper are R18S (X03205), MMP-1 (NM_002421), MMP-7 (XM_017384), and TIMP-1 (X03124). Patient summary Background Idiopathic pulmonary fibrosis is a chronic progressive disease of the lung that leads to increasing amounts of scar tissue with subsequent destruction of the lung. There is no specific cure at present. Patients may be treated with corticosteroids or drugs to suppress their immune system, although these drugs are usually not effective. Some patients receive lung transplants. Why Was This Study Done? Previous work has suggested that a protein called osteopontin is increased in mice that have lung fibrosis and that mice that do not have the gene for osteopontin are protected from lung fibrosis. The researchers wanted to investigate if osteopontin was also involved in the human disease. What Did the Researchers Do and Find? They looked at samples taken from the lungs of people with idiopathic pulmonary fibrosis and other diseases and measured many genes that are expressed there. They found that osteopontin was increased in the lungs of people with idiopathic pulmonary fibrosis. They then looked at cultures of lung cells and found that osteopontin caused an increase in the number and movement of cells that are involved in lung fibrosis. Its presence also affected other proteins that seem to be involved in fibrosis. What Do These Findings Mean? Osteopontin may have a key role in the pathway that causes fibrosis to occur in the lungs of people with idiopathic pulmonary fibrosis. Further work will need to be done to confirm these results, but in the future drugs directed against osteopontin or one of the related proteins might be a possible treatment for the disease. Currently there are no such drugs. Additionally osteopontin may be useful in the diagnosis and early detection of the disease, but further studies are required. Where Can I Get More Information Online? Medline Plus has links to many pages with information on the disease: http://www.nlm.nih.gov/medlineplus/pulmonaryfibrosis.html The Coalition for Pulmonary Fibrosis is a nonprofit organization that has information for patients as well as physicians: http://www.coalitionforpf.org/ The Dorothy P. and Richard P. Simmons Center for Interstitial Lung Diseases contains information about IPF for patients and their families: http://simmonscenterild.upmc.com/
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                Author and article information

                Journal
                Oncoimmunology
                Oncoimmunology
                KONI
                Oncoimmunology
                Taylor & Francis
                2162-4011
                2162-402X
                May 2016
                29 December 2015
                29 December 2015
                : 5
                : 5
                : e1123366
                Affiliations
                [a ]Department of Molecular & Radiation Oncology, German Cancer Research Center (DKFZ), 280 INF , Heidelberg, Germany
                [b ]Department of Radiology, University Hospital Center, 400 INF , Heidelberg, Germany
                [c ]Department of Radiation Oncology, University Hospital Center, 400 INF , Heidelberg, Germany
                [d ]Department of Radiation Oncology, Ludwig-Maximilians University Munich , Munich, Germany
                [e ]Oncology Early Clinical Investigation, Lilly Research Laboratories, Indianapolis , IN, USA
                Author notes

                Supplemental data for this article can be accessed on the publisher's website.

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                Shared first authorship.

                Article
                1123366
                10.1080/2162402X.2015.1123366
                4910723
                27467922
                33716f50-5d15-4c7c-8c10-4938fc0bee97
                © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License http://creativecommons.org/licenses/by-nc/3.0/, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

                History
                : 6 July 2015
                : 11 November 2015
                : 18 November 2015
                Page count
                Figures: 5, Tables: 0, References: 56, Pages: 12
                Categories
                Original Research

                Immunology
                lung fibrosis,pdgf,radiotherapy,small molecule inhibitors,tgfβ
                Immunology
                lung fibrosis, pdgf, radiotherapy, small molecule inhibitors, tgfβ

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