Effect of Osteopontin on MMP-7 Gene and Protein Expression and Activity in A549 Epithelial Cells

<div><p>(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.</p> <p>(B) Densitometry of Northern blot and normalization of MMP-7 to 18S demonstrates a 3- to 4-fold increase in MMP-7 over control.</p> <p>(C) Real-time PCR showing up-regulation of MMP-7 expression by osteopontin (*<i>p</i> < 0.01) and inhibition by anti-α_vβ₃, anti-CD44, anti-EGFR, and GRGDS.</p> <p>(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.</p> <p>(E) Zymography of conditioned media in 12.5% SDS gels containing bovine CM-transferrin (0.3 mg/ml) and heparin as substrate.</p> <p>C, control; OPN, osteopontin.</p></div

Localization of Osteopontin in IPF Lungs

<p>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).</p

Osteopontin Expression Levels by Microarray Analysis from Controls and IPF Lungs

<div><p>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.</p> <p>(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 (<i>p</i> < 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.</p> <p>(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.</p></div

Osteopontin and MMP-7 Colocalization in IPF Lungs

<div><p>(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×).</p> <p>(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).</p></div

Effect of Osteopontin on TIMP-1 Gene and Protein Expression by Fibroblasts

<div><p>(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.</p> <p>(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 (*<i>p</i> < 0.01). In parallel experiments, osteopontin-stimulated cells were treated with anti-α_vβ₃ and anti-CD44.</p> <p>(C) Western blot demonstrating an increase of immunoreactive TIMP-1 in conditioned media from fibroblasts stimulated with osteopontin.</p> <p>C, control; OPN, osteopontin.</p></div

Weakest Link Models of Osteopontin and MMP-7

<p>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).</p

Osteopontin Levels in BAL Fluid

<p>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). * <i>p</i> < 0.01.</p

Effect of Osteopontin on Fibroblasts and Epithelial Cell Migration

<div><p>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-α_vβ₃, anti-CD44, and GRGDS. Each bar represents the mean ± SD of three experiments; *<i>p</i> < 0.01; **<i>p</i> < 0.05.</p> <p>OPN, osteopontin</p></div

Effects of Osteopontin on Collagen Gene Expression and Smooth Muscle Alpha Actin Protein in Human Lung Fibroblasts

<div><p>(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.</p> <p>(B) Western blot showing no effect of osteopontin on immunoreactive α smooth muscle actin. Recombinant TGF-β1 was used as a positive control.</p> <p>C, control; OPN, osteopontin.</p></div

Effect of Osteopontin on Fibroblasts and Epithelial Cell Proliferation

<div><p>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-α_vβ₃, anti-CD44, and GRGDS. Each bar represents the mean ± SD of three experiments performed in triplicate; *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p> <p>OPN, osteopontin</p></div