Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation
Cytoskeleton organization, cortactin and vinculin expression, and cell proliferation in BM-MSCs cultured under hypoxic conditions. (a, A–F) Representative immunofluorescence confocal images of BM-MSCs cultured on glass coverslips for 48 h under hypoxic conditions in the absence (vehicle) or in the presence of 2 μM S1PR1 receptor antagonist, W146, or 2 μM S1PR1 receptor agonist, SEW2871, fixed and stained (A–C) with Alexa 568-phalloidin to detect actin filaments (red) and immunostained with antibodies against cortactin (green) and (D–F) with antibodies against vinculin (cyan). Scale bar 50 μm. The images are representative of at least three independent experiments with similar results. (a, G and H) Densitometric analyses of the intensity of the (G) cortactin and (H) vinculin fluorescence signals performed on digitized images. Data are mean ± S.E.M. (b) Cell proliferation analysis by cell counting. Synchronized BM-MSCs were incubated in growth medium for 48 h in normoxia and hypoxia (histogram on left) or in hypoxia in the absence (vehicle) or in presence of 2 μM S1PR1 receptor antagonist, W146, or 2 μM S1PR1 receptor agonist, SEW2871 (histogram on the right). Cells were collected and counted by TALI Cytometer as reported in Section 2. Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. Significance of differences in (a) (G and H, Student’s t-test), versus vehicle; in (b) (Student’s t-test), versus normoxia or vehicle.