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Comparative Study
, 2 (9), e251

Up-regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

Affiliations
Comparative Study

Up-regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

Annie Pardo et al. PLoS Med.

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung.

Methods and findings: Using oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to alphavbeta3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-alphavbeta3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7.

Conclusions: 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Osteopontin Expression Levels by Microarray Analysis from Controls and IPF Lungs
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 (p < 0.01 in TNoM and Student's t-test). Points are colored by their fold ratios; progressive shades of blue indicate increase, and progressive shades of red indicate decrease. Points colored in gray did not reach significance. Oblique solid line indicates the line of equality. Two green dashed lines are lines that depict 10-fold change. (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.
Figure 2
Figure 2. Osteopontin Levels in BAL Fluid
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). * p < 0.01.
Figure 3
Figure 3. Localization of Osteopontin in IPF Lungs
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).
Figure 4
Figure 4. Effect of Osteopontin on Fibroblasts and Epithelial Cell Proliferation
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; *p < 0.05; **p < 0.01. OPN, osteopontin
Figure 5
Figure 5. Effect of Osteopontin on Fibroblasts and Epithelial Cell Migration
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; *p < 0.01; **p < 0.05. OPN, osteopontin
Figure 6
Figure 6. Effects of Osteopontin on MMP-1 Gene and Protein Expression in Two Human Lung Fibroblast Cell Lines
(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 (*p < 0.05). (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.
Figure 7
Figure 7. Effect of Osteopontin on TIMP-1 Gene and Protein Expression by Fibroblasts
(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 (*p < 0.01). In parallel experiments, osteopontin-stimulated cells were treated with anti-αvβ3 and anti-CD44. (C) Western blot demonstrating an increase of immunoreactive TIMP-1 in conditioned media from fibroblasts stimulated with osteopontin. C, control; OPN, osteopontin.
Figure 8
Figure 8. Effects of Osteopontin on Collagen Gene Expression and Smooth Muscle Alpha Actin Protein in Human Lung Fibroblasts
(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.
Figure 9
Figure 9. Effect of Osteopontin on MMP-7 Gene and Protein Expression and Activity in A549 Epithelial Cells
(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 (*p < 0.01) and inhibition by anti-αvβ3, anti-CD44, anti-EGFR, and GRGDS. (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.
Figure 10
Figure 10. Osteopontin and MMP-7 Colocalization in IPF Lungs
(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).
Figure 11
Figure 11. Weakest Link Models of Osteopontin and MMP-7
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).

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