Matrix metalloproteinase 2-integrin alpha(v)beta3 binding is required for mesenchymal cell invasive activity but not epithelial locomotion: a computational time-lapse study - PubMed (original) (raw)

Matrix metalloproteinase 2-integrin alpha(v)beta3 binding is required for mesenchymal cell invasive activity but not epithelial locomotion: a computational time-lapse study

Paul A Rupp et al. Mol Biol Cell. 2008 Dec.

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 epithelial-to-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 alpha(v)beta3 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 alpha(v)beta3 binding. Inhibitors of MMP enzymatic activity and molecules that prevent integrin alpha(v)beta3 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.

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Figures

Figure 1.

Figure 1.

Protein extracts from early, intermediate, and late stage AV cushions were subjected to immunoblot analysis and gel substrate zymography. (a) A Western immunoblot prepared using the IgG fraction of a polyclonal MMP2 antiserum. The antibodies detect a dominant band, _M_r 72,000, at each stage. Three other bands were detected at greatly reduced levels. (b) Gelatin zymography using affinity purified MMP2 as a positive control enzyme. Significant gelatinolytic activity is present in a prominent band, _M_r 72,000, with two faint bands of substrate clearance corresponding to lower molecular mass forms of MMP. Note that the bands present at each AV cushion stage were also present in the authentic immunoaffinity purified MMP2. A duplicate gel incubated in the presence of 1,10-phenanthroline, a general inhibitor of MMP activity, exhibits no corresponding bands of MMP-specific gelatinolytic activity.

Figure 2.

Figure 2.

MMP2 tissue immunoreactivity at early, intermediate, and late stages of AV cushion morphogenesis. Immunofluorescence microscopy detects MMP2 associated with endocardial cushion mesenchymal cells/tissues. (a) A transverse section through an early stage heart shows mesenchymal cells surrounded by wispy MMP2 immunolabeling (mes). MMP2 antigen is also associated with endocardial cells (arrows), but no immunostaining is detected in the myocardium (myo). The lumen of the AV canal is collapsed (L) in this specimen. A sagittal section through the AV canal of an intermediate stage heart in b shows strong MMP2 immunofluorescence associated with the superior (s) and inferior (i) endocardial cushion tissue. (c) An intermediate stage heart at higher magnification; the MMP2 antigen shows up as fluorescent foci near cells embedded in the cardiac jelly, i.e., cushion mesenchyme. (d) A transverse section through the atrioventricular canal of a late developmental stage heart. Bright punctate MMP2 immunofluorescence is associated with the cushion tissue. Occasionally, there are isolated sites of immunostaining associated with the myocardium (bracket). Insets in a, b, and d provide the anatomical frame of reference for the histological sections.

Figure 3.

Figure 3.

Simultaneous MMP2 and αvβ3 integrin double immunofluorescence microscopy. (a–c) Intermediate stage endocardial cushions. (d–f) Single whole-mounted mesenchymal cell within a 3D collagen gel. At the tissue level, αvβ3 (a) and MMP2 (b) antigens show considerable overlap (c). The images in a and b were compiled from z-axis projections of 12 1-μm optical sections by using a laser scanning confocal microscope (LSCM). (d) αvβ3 immunoreactivity outlining the surface of a cushion mesenchymal cell fixed while moving through a 3D collagen gel. (e) MMP2 immunoreactivity. (f) Superimposition of the two images. Note the abundant yellow fluorescence that indicates a considerable degree of MMP2-αvβ3 colocalization (f), including the distal tips of filapodia (arrows). d–f images were acquired near the resolution limit of light optics using a 100× objective lens (LSCM). To detect cryptic αvβ3 antibody binding sites, in this whole-mounted specimen, it was necessary to unmask the epitopes using detergent (Triton X-100) and mild trypsin treatment (see Materials and Methods for details).

Figure 4.

Figure 4.

Time-lapse DIC microscopy of motile cells within and on collagen gels (see Supplemental Movie 1). Each image is an individual frame from time-lapse movies of cultures either treated with rhPEX (a–f) or left untreated (a′–f′). In the absence of a perturbing reagent, spindle-shaped cells move through the 3D collagen gels by extending slender filopodia (e.g., e′). Normally, their motility is highly persistent with infrequent changes of direction (a′–f′). Conversely, mesenchymal cell motility is significantly reduced in cultures exposed to rhPEX domain; in addition to impaired motility, the rhPEX-treated cells are amorphous and poorly extended (a–f). Contrary to expectations, cushion-derived endocardial-like cells, moving across the surface of the 3D collagen gels, are not affected by rhPEX (g). The data strongly suggest that PEX-mediated MMP2-integrin αvβ3 binding is required for persistent mesenchymal cell motility but not for endocardial-like cell locomotion across a free collagen gel surface.

Figure 5.

Figure 5.

Trajectories of cells emigrating from AV cushion explants. Colored lines represent cell trajectories. The red color designates the beginning of the path, which changes to blue at the end of the path. A′ and b′ show cultures incubated with a biologically active hemopexin-domain mimetic compound TSRI265, whereas a and b depict cultures treated with TSRI359, a control organic compound. a and a′ are images taken at the surface of the collagen gel, whereas b and b′ depict a focal plane 100 μm below the collagen gel surface. Cells in control cultures (a and b) display normal motility, as do TSRI265-treated cells moving across the collagen gel surface (a′). In distinct contrast, the active TSRI265 compound inhibits mesenchymal cell locomotion through the collagen gel (b′). Note the significantly shorter, more tortuous, trajectory plots. These data are entirely consistent with the rhPEX polypeptide data. For clarity, the explanted cushion tissue is shown, but the surrounding outgrowth of motile cells was erased using Adobe PhotoShop software.

Figure 6.

Figure 6.

Statistical analysis of digital time-lapse data. (a) Average displacement (micrometers) versus elapsed time (hours) in a double logarithmic plot, which was calculated for the two cells shown in Figure 4 and Supplemental Movie 1. In the case of a control cell that moves persistently (green symbols), the displacement is proportional to the elapsed time (green line). For a PEX-treated cell, which seems to move randomly, the displacement is proportional to the square root of time (red line). Such analysis of displacement versus time curves permits the classification of cell motility as persistent or diffusive as described in Materials and Methods. (b) The graph depicts the proportion of mesenchymal cells that display highly persistent motility through 3D collagen gels under various treatment conditions. Agents designed to interfere with MMP2-αvβ3 association reduce the percentage of persistently moving cells. (c) Displacements of control and inhibitor-treated mesenchymal cells moving within the 3D collagen gel. Compared with control cultures, the PEX domain and its mimetic analog inhibited the motility of mesenchymal cells moving through the substance of the collagen gels. In contrast, d shows the displacements of endocardial-like (epithelial) cells moving across the scaffold at the gel/medium interface. Unlike mesenchymal cells, the motility of the endocardial-like cells was unaffected by the two inhibitors; indeed, endocardial cell motility was statistically undistinguishable from control cultures (d). PEX, a recombinant human hemopexin polypeptide; TSR1265, a synthetic organic inhibitor of MMP2-αvβ3 integrin binding; TSR1359, a control compound for the active organic inhibitor TSRI265.

Figure 7.

Figure 7.

Endocardial cushion cell outgrowth in the presence and absence of MMP proteolysis inhibitors. General inhibitors of MMP enzyme activity decrease cell outgrowth from explanted endocardial cushions. The divalent cation chelator, EDTA (10 μM), decreased outgrowth by 39%, whereas a zinc chelator (44463; 25/50 μg/ml) diminished up to 35%. Specific inhibition of MMP2 activity also decreased outgrowth of endocardial cushion cells. Antibodies to MMP2 decreased outgrowth up to 33% (20/50 μg/ml), whereas TIMP2 (10 μg/ml) reduced diameter of outgrowth by 44%. These data connect MMP2-mediated proteolysis to cell motility. All values were statistically different (p < 0.001) based on Student's t test.

Figure 8.

Figure 8.

Endocardial cushion explants grown on collagen gels mixed with DQ-gelatin, a reagent that displays induced fluorescence when enzymatically cleaved. (a) An untreated (control) endocardial cushion incubated for 24 h. The colors have been inversed for better contrast such that fluorescence signal is represented by dark color. The strongest signal is associated with the cells (black punctate dots). Fainter tracks of fluorescence can be observed in the collagen. Treatment with the control organic reagent (b) displays similar results. Explants incubated in the presence of 1,10-phenanthroline (c), a general metalloproteinase inhibitor, display minimal cell migration, with the majority having migrated before exposure to the reagent. (d) An endocardial cushion exposed to the TSRI265 organic reagent. Migrating cells are present but in fewer numbers. Additionally, fluorescence tracks are apparent but are lighter in intensity. The insets in a, b, and d are single sections from a z-series through the collagen gels collected using a laser scanning confocal microscope with a 10× objective. The images in a–d were collected with a 5× objective. The fields of view are 1700 μm × 1360 μm, and the inserts are 165 × 165 μm.

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