Matrix Metalloproteinase 2-Integrin αvβ3 Binding Is Required for Mesenchymal Cell Invasive Activity but Not Epithelial Locomotion: A Computational Time-Lapse Study

Abstract

Cellular invasive behavior through three-dimensional collagen gels was analyzed using computational time-lapse imaging. A subpopulation of endocardial cells, derived from explanted quail cardiac cushions, undergoes an epithelialto-mesenchymal transition and invades the substance of the collagen gels when placed in culture. In contrast, other endocardial cells remain epithelial and move over the gel surface. Here, we show that integrin ␣v␤3 and matrix metalloproteinase (MMP)2 are present and active in cushion mesenchymal tissue. More importantly, functional assays show that mesenchymal invasive behavior is dependent on MMP2 activity and integrin ␣v␤3 binding. Inhibitors of MMP enzymatic activity and molecules that prevent integrin ␣v␤3 binding to MMP2, via its hemopexin domain, result in significantly reduced cellular protrusive activity and invasive behavior. Computational analyses show diminished intensity and persistence time of motility in treated invasive mesenchymal cells, but no reduction in motility of the epithelial-like cells moving over the gel surface. Thus, quantitative time-lapse data show that mesenchymal cell invasive behavior, but not epithelial cell locomotion over the gel surface, is partially regulated by the MMP2-integrin interactions. INTRODUCTIONCell motility within a three-dimensional (3D) tissue space, often termed invasion, is a dynamic process involved in a host of morphological and pathological events (Harris, 1987). Extracellular matrix (ECM) fibers provide mechanical support for tissue integrity/deformations, act as a scaffold for cell motility, and act as a repository of growth factors and latent enzymes (McCarthy and Turley, 1993;Damsky et al., 1997;Friedl and Brocker, 2000;Davis and Senger, 2005). Cell invasion is often coupled to local ECM degradation (Ellis and Murphy, 2001). In an extreme case, structures termed invadopodia have been observed on the surface of invasive tumor cells. These structures are cellular protrusions that contain surface-bound, externally facing proteases (Chen, 1989;Mueller and Chen, 1991;Monsky et al., 1993Monsky et al., , 1994Kelly et al., 1994).During normal embryonic development extracellular matrix protease activity is important for successful epithelialto-mesenchymal transformations (EMT) that accompany neural crest, sclerotome, and renal tubular cell delamination (Cheng and Lovett, 2003;Song et al., 2000;Duong and Erickson, 2004). During epithelial-to-mesenchymal transformations ECM proteolysis may not only remove a physical barrier, but also expose cell receptor sites and regulatory molecules sequestered in the ECM. Similar proteolytic events are recapitulated during angiogenesis and tubulogenesis in vitro (Bayless and Davis, 2003;Fisher et al., 2006). When deprived of specific proteolytic mechanisms, some cell types change their motility strategy and move through the ECM by means of amoeboid behavior (Wolf et al., 2003).Degradation of the ECM occurs by a number of secreted proteinases. One family, the matrix metalloproteinases (MMPs), is compose...

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