Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation
S1P receptor subtype expression and SphK/S1PR axis role in cell gelatinolytic activity. (a and c) Expression of S1P receptors by reverse transcription (RT) and real-time PCR analysis. mRNA were determined by RT of total RNA (1 μg) obtained from BM-MSCs at low- (L-) and high- (H-) density culture and 2 μl of cDNA (for S1PR1, S1PR2, and S1PR3 detection) or 4 μl of cDNA (for S1PR4 and S1PR5 detection) were amplified as described in Section 2. Representative agarose gels of amplified DNA are shown. GAPDH amplification was used for data normalization. (b and d) Quantification of mRNA expression by real-time PCR analysis. Data are reported as mean ± S.E.M. of the ratio between the fold of variation of S1P receptor expression obtained from high- and low-density BM-MSCs culture. (e and f) BM-MSCs seeded onto fluorescein-labeled gelatin substrate- (DQ gelatin-) coated plastic culture plates (e) or glass coverslips (f) were cultured for 48 h in absence (vehicle) or in presence of the following compounds: 5 μM sphingosine kinase inhibitor (iSK), 1 μM exogenous sphingosine-1-phosphate (exoS1P), 2 μM S1PR1 receptor antagonist, W146, and 2 μM S1PR1 receptor agonist, SEW2871. (e) Spectrophotometrical quantification of the DQ gelatin fluorescence intensity revealed after proteolytic digestion of the gelatin by MMP gelatinases. (f) Representative superimposed DIC (grey) and fluorescent confocal microscopy images (green; gelatin fluorescence intensity) of fixed cells. Scale bar 30 μm. Histogram shows the densitometric analysis of the intensity of the gelatin fluorescence signals performed on digitized images. Data reported as mean ± S.E.M. are representative of at least three independent experiments with similar results. Significance of differences in (b) and (d) (Student’s t-test), and ; in (e) and (f) (one-way ANOVA and Newman-Keuls multiple comparison test), versus vehicle, versus SEW2871.
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