Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells

https://doi.org/10.1016/j.bbrc.2006.06.110Get rights and content

Abstract

The roles of various soluble factors in promoting the osteogenic differentiation of adult mesenchymal stem cells (MSCs) have been widely studied, but little is known about how the extracellular matrix (ECM) instructs the phenotypic transition between growth and differentiation. To investigate this question, we cultured MSCs on purified vitronectin or type-I collagen, motivated by our earlier tissue engineering work demonstrating that MSC adhesion to polymer scaffolds is primarily mediated by the passive adsorption of these two ECM ligands from serum. Using alkaline phosphatase activity and matrix mineralization as indicators of the early and late stages of osteogenesis, respectively, we report here that both substrates supported differentiation, but the mechanism was substrate dependent. Specifically, osteogenesis on vitronectin correlated with enhanced focal adhesion formation, the activation of focal adhesion kinase (FAK) and paxillin, and the diminished activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) pathways. By contrast, MSCs on type-I collagen exhibited reduced focal adhesion formation, reduced activation of FAK and paxillin, and increased activation of ERK and PI3K. Inhibition of ERK and FAK blocked mineral deposition on both substrates, suggesting that the observed differences in signaling pathways ultimately converge to the same cell fate. Understanding these mechanistic differences is essential to predictably control the osteogenic differentiation of MSCs and widen their use in regenerative medicine.

Section snippets

Materials and methods

Materials. Purified type-I collagen and human vitronectin were purchased from Cohesion (Palo Alto, CA) and Chemicon International (Temecula, CA), respectively. Phospho-p44/42 MAPK antibody (anti-phospho-ERK1/2, monoclonal) was purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies to ERK1, paxillin, and p-FAK (Y397) were purchased from BD Transduction Laboratories (Lexington, KY). Anti-pser473-Akt (rabbit polyclonal) and anti-pY31-paxillin (rabbit polyclonal) antibodies were

Integrin expression and utilization in MSCs

Based in part on our recent report that MSCs undergo osteogenesis to differing degrees when cultured on thin polymer films due to the differential deposition of serum-derived ECM proteins [32], we hypothesized that MSC differentiation must be influenced by integrins. Our first goal in testing this hypothesis was to use flow cytometry to quantify the integrin expression repertoire and adhesion blocking to determine which integrins are actually used to adhere to Col I and VN. Flow cytometric

Discussion

It is widely recognized that cell–ECM interactions play an important role in tissue development [37], [38], and that signaling pathways initiated by integrin binding are involved in regulating nearly every aspect of cell function, including cell cycle progression, migration, apoptosis, and differentiation [16], [39], [40]. In this study, we investigated the impact of integrin-mediated adhesion to vitronectin and type-I collagen on the osteogenic differentiation of MSCs. Our exclusive focus on

Acknowledgments

This work was partially supported by a grant from the NIH/NIDCR (1R03-DE016117) awarded to A.J.P. and by internal set-up funds provided by UC Irvine. In addition, we acknowledge Aswathi Sreedharan for cell culture assistance, Cyrus Ghajar for critically reading the manuscript, and the UC Irvine Center for Immunology for assistance with flow cytometry.

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