¤ Current address: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America
AP, KG, SY, VR, MS, and NK designed the study. KG, JC, TJR, YY, CB, IH, VR, and NK performed experiments. AP, KG, JC, YY, SY, IH, VR, MS, and NK analyzed the data. AP, KG, MS, and NK enrolled patients. AP, KG, MS, and NK contributed to writing the paper.
The authors have declared that no competing interests exist.
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung.
Using oligonucleotide arrays, we identified
Our results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease.
Osteopontin may have a critical role in the pathogenesis of idiopathic pulmonary fibrosis, and be a target for therapeutic intervention in this disease.
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 [
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 [
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 [
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 [
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
Patients from the National Institute of Respiratory Diseases, Mexico City, México (
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).
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 [
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
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).
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 [
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).
Primary human normal lung fibroblasts were obtained as previously described [
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 [
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.
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 [
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
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 [
Serum-free conditioned media was centrifuged at 300
The complete microarray dataset is available at the Gene Expression Omnibus database with GEO serial accession number GSE2052 (
Total RNA was used to generate double-stranded cDNA and biotin-labeled cRNA. Fragmented cRNA was hybridized to Codelink Uniset I slides and stained and scanned as described in Materials and Methods.
(A) A log scale scatter plot of the average of intensity of all the genes on the arrays in controls (x-axis) and IPF (y-axis). Colored points indicate 178 genes that were significantly changed (
(B) Osteopontin levels in individual samples are shown. The y-axis is expression level in arbitrary fluorescence levels (log scale). The blue quadrangles are osteopontin levels in individual samples. Heat map shows sample osteopontin levels normalized to the geometric mean of osteopontin in controls and log base 2-transformed.
Osteopontin protein was quantified in BAL fluids from 18 IPF patients and 10 healthy controls. As shown in
Quantification of osteopontin by ELISA was performed in BAL fluid samples from 18 IPF patients and 10 healthy individual controls. An increased concentration of soluble osteopontin was found in the BAL fluid obtained from IPF patients compared with healthy controls. The data represent the mean ± standard deviation (SD). *
To examine the cellular source of osteopontin we analyzed IPF and control lungs by immunohistochemistry. As illustrated in
Immunoreactive protein was revealed with AEC, and samples were counterstained with hematoxylin. Two representative IPF lung samples exhibited strong epithelial staining (original magnification, 40×) (A,B). Control lung showed no staining (C). Negative control section from IPF lung in which the primary antibody was replaced with nonimmune serum also showed no staining (40×) (D).
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 (
Osteopontin-induced fibroblast proliferation (2 μg/ml) was significantly suppressed by GRGDS-pentapeptide, which interrupts binding of RGD-containing proteins to cell surface integrins (
Human normal lung fibroblasts (A) and A549 epithelial cells (B) were grown in Ham's F-12 medium with 0.1% FBS and stimulated with 2 μg/ml osteopontin. In parallel, osteopontin-stimulated cells were treated with anti-αvβ3, anti-CD44, and GRGDS. Each bar represents the mean ± SD of three experiments performed in triplicate; *
OPN, osteopontin
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
Fibroblasts (A) and A549 epithelial cells (B) were placed in the upper compartment of a Boyden-type chamber, and Ham's F-12 medium containing 5% BSA alone or with 10 μg/ml of osteopontin was added to the lower compartment. After 8 h of incubation, the migrating cells were stained, and the absorbance of the stained solution was measured by ELISA. In parallel experiments, osteopontin-stimulated cells were treated with anti-αvβ3, anti-CD44, and GRGDS. Each bar represents the mean ± SD of three experiments; *
OPN, osteopontin
A549 lung cells also showed a significant increase in cell migration in response to osteopontin (
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 (
(A) Representative Northern blot of 20 μg total cellular RNA per lane extracted from control cells and fibroblasts stimulated with 0.4 μg/ml and 1 μg/ml osteopontin. Both concentrations of osteopontin induced a down-regulation in the expression of MMP-1.
(B) Osteopontin also reduced overexpression of MMP-1 in APMA-stimulated cells.
(C) The expression level of MMP-1 by real-time PCR was determined as described in Materials and Methods and normalized to the level of 18S ribosomal RNA. In parallel experiments, osteopontin-stimulated cells were treated with anti-αvβ3 and anti-CD44. Bars represent mean ± SD (*
(D) Representative Western blot demonstrating a decrease of immunoreactive MMP-1 in conditioned media from fibroblasts stimulated with osteopontin. Fibroblasts treated with APMA and FGF-1 plus heparin used as positive controls strongly induced MMP-1 expression.
C, control; FGF1, FGF-1 plus heparin; OPN, osteopontin; PMA, APMA-stimulated.
The effect of osteopontin on
(A) Northern blot of 20 μg total cellular RNA per lane extracted from control and fibroblasts stimulated with 0.4 μg/ml and 1 μg/ml osteopontin. Both concentrations of osteopontin induced an increase of TIMP-1 expression.
(B) The expression level of TIMP-1 by real-time PCR normalized to the level of 18S ribosomal RNA corroborates TIMP-1 up-regulation by osteopontin (*
(C) Western blot demonstrating an increase of immunoreactive TIMP-1 in conditioned media from fibroblasts stimulated with osteopontin.
C, control; OPN, osteopontin.
The effect of osteopontin on collagen gene expression is depicted in
(A) Northern blot of 20 μg total cellular RNA per lane extracted from control and fibroblasts stimulated with 0.4 μg/ml and 1 μg/ml osteopontin. Both concentrations of osteopontin induced an up-regulation in the expression of α1 type I collagen.
(B) Western blot showing no effect of osteopontin on immunoreactive α smooth muscle actin. Recombinant TGF-β1 was used as a positive control.
C, control; OPN, osteopontin.
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
(A) Northern blot of 20 μg total cellular RNA per lane extracted from control and A549 cells stimulated with 0.4, 1, and 2 μg/ml osteopontin.
(B) Densitometry of Northern blot and normalization of MMP-7 to 18S demonstrates a 3- to 4-fold increase in MMP-7 over control.
(C) Real-time PCR showing up-regulation of MMP-7 expression by osteopontin (*
(D) Western blot demonstrating an increase of immunoreactive MMP-7 in conditioned medium from A549 epithelial cells stimulated with osteopontin. Activation of pro-MMP-7 by APMA is shown in leftmost lane.
(E) Zymography of conditioned media in 12.5% SDS gels containing bovine CM-transferrin (0.3 mg/ml) and heparin as substrate.
C, control; OPN, osteopontin.
Since we have previously demonstrated that IPF lungs strongly express MMP-7 in alveolar epithelial cells [
(A−C) MMP-7 staining is shown in green (A), osteopontin is shown as red staining (B), and overlap of staining is shown in yellow (C), suggest colocalization of MMP-7 and osteopontin in alveolar epithelial cells in IPF lungs (60×).
(D) A lower-magnification image (20×) of the same region (A–C) with the same color coding. The white rectangle depicts area shown in (A−C).
To determine whether MMP-7 and osteopontin expression levels jointly distinguish IPF and control samples, we applied weakest link statistical models [
IPF samples are depicted in solid dots and controls in open dots; the y-axis is MMP-7 expression and x-axis is osteopontin expression. Black solid line is the curve of optimal use showing that the expression levels for MMP-7 and osteopontin jointly interact to determine the IPF phenotype. The probability contour plot is shown in terms of the observed expression data (scale is log base 2 for gene expression data) (A); and probability contour plot is shown in terms of percentiles of the data (scale is percentiles) (B).
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 [
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 [
Positive feedback mechanisms have been previously proposed for osteopontin and MMP-2 [
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. [
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 [
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 [
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.
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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.
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.
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.
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.
Medline Plus has links to many pages with information on the disease:
The Coalition for Pulmonary Fibrosis is a nonprofit organization that has information for patients as well as physicians:
The Dorothy P. and Richard P. Simmons Center for Interstitial Lung Diseases contains information about IPF for patients and their families:
The authors wish to acknowledge Inna Loutaev and Lara Chensny for their technical help and Mary Williams for her help in administering the collaborative efforts that underlie this manuscript. The authors also thank Dr. A. Choi, Dr. J. Dauber, and Dr. N. Friedman for their critical review and insightful comments. This work was supported National Institutes of Health grant 1R01 HL073745–01 and by a generous donation from the Simmons family. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
aminophenylmercuric acetate
bronchoalveolar lavage
epidermal growth factor (receptor)
idiopathic pulmonary fibrosis
matrix metalloprotease
standard deviation
transforming growth factor-beta 1
tissue inhibitor of metalloprotease