Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway

doi:10.1016/j.bbrc.2007.05.104

Biochemical and Biophysical Research Communications

Volume 359, Issue 2, 27 July 2007, Pages 341-347

https://doi.org/10.1016/j.bbrc.2007.05.104 Get rights and content

Abstract

We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway.

Section snippets

Materials and methods

MDPC isolation. MDPC isolation was performed as previously described [6]. Briefly, the primary hindlimb muscle cells were isolated from 8-week-old C57BL/6J mice (Shimizu Laboratories Supplies) using 470 U/ml collagenase type II (Worthington) for digestion. Cells were suspended in the isolation medium, which was DMEM/F12 (Invitrogen) supplemented with B27 (Invitrogen), 20 ng/ml EGF (SIGMA), and 40 ng/ml recombinant bFGF (Promega). Cell suspensions were then cultured onto a non-coated dish at 20

Identification of osteopontin

To explore the potential molecules regulating MDPC proliferation, we performed SAGE and SST using RNA isolated from MDPCs. SAGE provides a comprehensive approach for elucidation of quantitative gene expression patterns that does not depend on prior availability of transcript information [12], and SST is an efficient strategy to identify secreted and cell-surface molecules [11]. By using the two distinct methods, we identified OPN, a secretory molecule, as one of the most abundant molecules

Discussion

OPN has been known to be involved in many physiological and pathological processes including cell adhesion, angiogenesis, apoptosis, inflammatory responses and tumor metastasis [16]. Here, we identify OPN as a myosphere-derived secretory molecule, and provide a novel role of OPN in regulating the proliferative growth of MDPCs through the PI3K/Akt signaling pathway.

bFGF and EGF have been revealed to affect the rates of proliferation for neural stem and progenitor cells [17]. Studies from single

Acknowledgments

We are grateful to Ms. A. Kosugi, Ms. M. Nishikawa, and Mr. M. Kuramoto for their skillful technical assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Grants-in-Aid from the Ministry of Health, Labor, and Welfare of Japan, Japan Association for the Advancement of Medical Equipment, Takeda Science Foundation, Novartis Research Award on Molecular and Cellular Cardiology, Kanae Foundation for Life &

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The role of the inflammatory secretory protein TNF-LIGHT (LIGHT) in the molecular mechanisms underlying persistent airflow limitation (PAL) in asthma remains unclear. We hypothesized that high airway LIGHT expression may be a feature of asthma with PAL associated with specific expression patterns of inflammatory molecules.
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Increased levels of osteopontin in sputum supernatant of smoking asthmatics
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These effects have been associated with reduced efficacy of currently available treatments, especially inhaled corticosteroids (ICS), in smoking asthmatics [7,8]. Osteopontin (OPN) is a phosphorylated acidic glycoprotein that can function as both an extracellular matrix (ECM) molecule and a cytokine [9]. Its origin is not well clarified but epithelial cells, macrophages, T cells and fibroblasts have been shown to express OPN [10–12].
Smoking may modify the inflammatory pattern of the asthmatic airways. Osteopontin (OPN) has been associated with inflammation and fibrosis. In asthma, sputum levels of OPN are elevated and have been related to the underlying severity and to mediators expressing remodeling and inflammation.
To evaluate the levels of OPN in sputum supernatants of asthmatic patients and to investigate the possible role of smoking as well as associations with mediators and cells involved in the inflammatory and remodeling process.
We studied 103 asthma patients (49 smokers) and 40 healthy subjects (20 smokers) who underwent lung function tests, bronchial hyperresponsiveness to methacholine, and sputum induction for cell count identification and measurement of OPN, TGF-β1, IL-8, IL-13 and ECP in sputum supernatants. The concentrations of all mediators were measured using enzyme immunoassays.
OPN levels (pg/ml) were significantly higher in smoking asthmatics compared to non-smoking asthmatics, and both non-smoking and smoking controls [median (interquartile ranges) 1120 (651, 1817) vs. 197 (118, 341) vs. 50 (42, 70) vs. 102 (77, 110) pg/ml, respectively; p < 0.001]. Regression analysis provided significant associations between OPN and sputum neutrophils, IL-8 and TGF-β1, the most significant being the one with TGF-β1. These associations were present only in smoking asthmatics.
Smoking habit significantly affects sputum OPN levels in asthma. The associations of OPN with sputum neutrophils, TGF-β1 and IL-8 in smoking asthmatics suggest a possible role for OPN in the neutrophilic inflammation and remodeling process in this phenotype of asthma.
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Our recent studies have shown that osteopontin does not increase the proliferation of neural progenitor cells in the absence of FGF2 (Yan et al., 2009). Activation of the IGF-1 mediated Akt pathway in the presence of FGF2 increases the proliferation of neural progenitor cells (Kalluri et al., 2007) and osteopontin stimulates Akt pathway (Ogata et al., 2007; Fong et al., 2009) to increase the proliferation of cells (Ogata et al., 2007). Likewise, activation of Akt alone does not increase the proliferation of neural progenitor cells (Kalluri et al., 2007).
We examined the role of osteopontin in the proliferation of neural progenitor cells in vitro. Osteopontin increased the proliferation of neural progenitor cells in the presence of FGF2 as measured by cell proliferation assay and bromodeoxy uridine incorporation studies. In addition, immunoblot analysis demonstrated an increase in the phosphorylation of retinoblastoma protein with a concurrent increase in the content of phospho-Akt and cyclin D1. These results indicate that osteopontin can upregulate the content of phospho-Akt, cyclin D1 and phospho-Rb to subsequently enhance the proliferation of neural progenitor cells in the presence of FGF2.
Sonic hedgehog induces angiogenesis via Rho kinase-dependent signaling in endothelial cells
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Several of these effects (e.g., increased migration, MMP-9 expression, and OPN expression) were induced by a Shh concentration of 10 ng/mL, which is likely similar to physiological Shh concentrations. It is reasonable to consider whether these Shh-induced, ROCK-mediated effects support the function of other nearby cells, such as EPCs, smooth muscle cells [45,46], and muscle-derived progenitor cells [47], and whether Shh activation of the PI3K/Akt pathway, reported in ECs [16], EPCs [48], and fibroblasts [30]in vitro, may also induce favorable effects. These data cumulatively indicate that Shh has an important and complex role in neovascularization, and the diverse nature of Shh activity could provide many promising approaches to therapeutic revascularization.
The morphogen Sonic hedgehog (Shh) promotes neovascularization in adults by inducing pro-angiogenic cytokine expression in fibroblasts; however, the direct effects of Shh on endothelial cell (EC) function during angiogenesis are unknown. Our findings indicate that Shh promotes capillary morphogenesis (tube length on Matrigel™ increased to 271 ± 50% of the length in untreated cells, p = 0.00003), induces EC migration (modified Boyden chamber assay, 191 ± 35% of migration in untreated cells, p = 0.00009), and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN) mRNA (real-time RT-PCR), which are essential for Shh-induced angiogenesis both in vitro and in vivo. Shh activity in ECs is mediated by Rho, rather than through the “classic” Shh signaling pathway, which involves the Gli transcription factors. The Rho dependence of Shh-induced EC angiogenic activity was documented both in vitro, with dominant-negative RhoA and Rho kinase (ROCK) constructs, and in vivo, with the ROCK inhibitor Y27632 in the mouse corneal angiogenesis model. Finally, experiments performed in MMP-9- and OPN-knockout mice confirmed the roles of the ROCK downstream targets MMP-9 and OPN in Shh-induced angiogenesis. Collectively, our results identify a “nonclassical” pathway by which Shh directly modulates EC phenotype and angiogenic activity.
A short-hairpin RNA targeting osteopontin downregulates MMP-2 and MMP-9 expressions in prostate cancer PC-3 cells
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Thus, our in vitro and in vivo data showed that OPN can regulate the activities of MMP-2 and MMP-9 in PC-3 cells. Cumulative evidence also suggests that there are a number of OPN-dependent signaling pathways correlating with these processes [43–46]. Das et al. [44] recently reported that OPN induction of urokinase plasminogen activator (uPA) secretion is mediated by the NF-κB/IκBα/IKK pathway and dependent on phosphatidylinositol 3-kinase/IKK/Akt signaling pathways.
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Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway

Abstract

Section snippets

Materials and methods

Identification of osteopontin

Discussion

Acknowledgments

Mol. Ther.

Biochem. Biophys. Res. Commun.

Neuron

J. Biol. Chem.

J. Biol. Chem.

Trends Genet.

J. Biol. Chem.

Trends Cell Biol.

Biochem. Biophys. Res. Commun.

Curr. Opin. Neurobiol.

Dev. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Invest. Dermatol.

Am. J. Pathol.

Blood