Osteopontin Is Induced by TGF-β2 and Regulates Metabolic Cell Activity in Cultured Human Optic Nerve Head Astrocytes

<div><p>The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (<i>sq</i>) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by <i>sq</i>RT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by <i>sq</i>RT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.</p></div

<i>Semiquantitative</i> RT-PCR analyses of most prevalent OPN receptors in cultivated ONH astrocytes and effect of TGF-β2.

<p>(A) Representative RT-PCR results of OPN receptor gene expression in untreated (co) and TGF-β2 (1 ng/ml, 72 h) treated ONH astrocytes. (B) Densitometric analysis of <i>sq</i> RT-PCR results does not demonstrate any regulation of OPN receptors in TGF-β2 treated cells compared to controls. OPN signal is normalized to GAPDH. Values represent mean ± SD of 11 independent experiments (n = 11).</p

TGF-β2 and OPN effects on metabolic activity in cultured ONH astrocytes.

<p>Cell viability was assessed by MTS assays. Quantification of the metabolic cell activity of ONH astrocytes treated with TGF-β2 (1 ng/ml) and different OPN concentrations (0, 250, 1000, 2000 ng/ml) for 48, 96 and 144 hours. Values represent mean ± SD of 11 independent experiments (n = 11). Statistical significance was calculated by student’s t-test (*p<0.05; **p<0.01; ***p<0.001).</p

<i>Semiquantitative</i> RT-PCR analysis of key regulator of ECM synthesis in ONH astrocytes after treatment with different OPN concentrations.

<p>Increasing OPN concentration has no effects to the gene expression of key regulator of ECM synthesis (CTGF) or degeneration (PAI-1) in cultivated ONH astrocytes. (A) Representative RT-PCR results of connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1) in untreated (co) and OPN treated ONH astrocytes. (B) Densitometric analysis of <i>sq</i> RT-PCR results reveal no gene induction of CTGF and PAI-1 after treatment with 250, 1000 or 2000 ng/ml OPN compared to untreated cells (co). OPN signal is normalized to GAPDH. Values represent mean ± SD of 11 independent experiments (n = 11).</p

Quantification of OPN induction in TGF-β2 treated ONH astrocytes.

<p>Analysis of OPN expression in ONH astrocytes after treatment with 1/ml TGF-β2 for 72 hours. (A) S<i>emiquantitative (sq)</i> RT-PCR analysis indicates an up-regulation of OPN transcript in TGF-β2 treated ONH astrocytes compared to control cells (co). (B) Western blot analysis demonstrates an increase in secreted OPN protein (MMP-cleaved) in TGF-β2 treated ONH astrocytes compared to controls. (C) Densitometric quantification of <i>sq</i> RT-PCR reveals significant induction of OPN mRNA (2.3-fold, p = 0.008). OPN signals are normalized to GAPDH. (D) Quantification of western blot results reveals significantly increased OPN secretion into supernatant (2.5-fold, p = 0.0054). OPN western blots are normalized within the β-actin signal on the same nitrocellulose membrane. (E) Statistical analysis of real time RT-PCR shows significant up-regulation of OPN transcript (6-fold, p = 0.0073). Values represent mean ± SD of 11 independent experiments (n = 11).</p

<i>Semiquantitative</i> RT-PCR analysis of glaucoma-associated ECM components in ONH astrocytes after treatment with OPN.

<p>Treatment with 250/ml OPN for 72 hours has no effect on gene expression of glaucoma-associated ECM components (A, B), genes of the ECM degradation system (C–F), as well as stress response genes (G, H) in cultivated ONH astrocytes compared to controls (0 OPN). (A, C, E and G) Representative RT-PCR results upon treatment with 250 ng/ml OPN () and controls (0 OPN). (B, D, F and H) Densitometric analysis of <i>sq</i> RT-PCR results normalized to GAPDH. Values represent mean ± SD of 11 independent experiments (n = 11).</p

Effects of selective OPN receptor blocking on metabolic activity in cultured ONH astrocytes.

<p>Metabolic cell activity of ONH astrocytes incubated with an anti-CD44 antibody or a synthetic RGD peptide for 36 and 72 hours compared to untreated cells (co). Values represent mean ± SD of 11 independent experiments (n = 11). Statistical significance is calculated by student’s t-test (*p<0.05; **p<0.01; ***p<0.001).</p

3D reconstruction by digitized and computer based processed histological sections of the pig lacrimal gland.

<p>A) Visualization of seven excretory lacrimal ducts (different colors) within the paraffin embedded lacrimal gland (grey). B) Separate exposure of each lacrimal duct in different colors with surrounding gland tissue reconstruction. Only the yellow-marked duct system is displayed alone, without gland tissue reconstruction.</p

Excretory ducts of the left lacrimal system in pig.

<p>Conjunctival view at the level of the eye angle, left scale bar: 2 mm. EB: eyeball; LEA: left eye angle; 1–6: excretory ducts. Right bottom square: higher magnification of an excretory duct, scale bar: 200 µm.</p

Comparison of lipids reported in the current study to those reported for HMGECs [4] and epithelial cells [37] in previous reports.

<p>phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM), diacylglycerol (DAG), ceramide (Cer), cholesterol ester (CE), free cholesterol (Chol), and wax ester (WE)</p><p>Comparison of lipids reported in the current study to those reported for HMGECs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128096#pone.0128096.ref004" target="_blank">4</a>] and epithelial cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128096#pone.0128096.ref037" target="_blank">37</a>] in previous reports.</p