( 0.01 by two-way ANOVA. Effect of Rho and Rho Kinase in Mediating S1P Augmentation of HFL-1 Cell Chemotaxis Since Icotinib S1P has been reported to activate Rho and Rho kinase in a variety of cell Icotinib types (22, 23), their role in mediating S1P-stimulated HFL-1 chemotaxis was evaluated. was blocked by the Rho-kinase inhibitor Y-27632 in a concentration-dependent manner. Inhibition of FAK with siRNA reduced basal chemotaxis toward fibronectin slightly but significantly, and almost completely blocked S1P augmented chemotaxis. These results suggest that S1P-augmented fibroblast chemotaxis toward fibronectin depends on the S1P2 receptor and requires Rho and Rho-kinase, and FAK phosphorylation. By augmenting fibroblast recruitment, S1P has the potential to modulate tissue repair after injury. The pathways by which S1P mediates this effect, therefore, represent a potential therapeutic target VGR1 to affect tissue repair and remodeling. 0.05. Data are expressed as means SEM. RESULTS Effect of S1P on Chemotactic Response of Human Fibroblasts to Fibronectin S1P stimulated HFL-1 cell migration toward human fibronectin in a concentration-dependent manner (Physique 1A). S1P at concentrations less than 1 M had no effect on fibronectin-induced chemotaxis, whereas S1P concentrations greater than 1 M markedly augmented the chemotaxis. S1P had a similar effect on human foreskin fibroblast chemotaxis toward fibronectin (data not shown). At concentrations of S1P above 20 M, chemotaxis was inhibited. To determine whether the stimulatory effect was due to only S1P or the combination of S1P and fibronectin, varying concentrations of fibronectin were placed in the bottom wells of the chemotactic chamber. The number of migrated cells increased as the concentration of fibronectin increased (Physique 1B). In the absence of chemoattractant (fibronectin), S1P had little effect on HFL-1 cell migration (data not shown), indicating that S1P augmented fibronectin-directed chemotactic activity rather than enhancing chemokinetic activity. The S1P metabolites sphingosine and ceramide did not affect the HFL-1 cell chemotaxis (data not shown). Open in a separate window Physique 1. S1P stimulates human lung fibroblast migration toward human fibronectin (HFn). (= 3). S1P (10 M, 0.05 compared with 0 M S1P ( 0.01 compared with or without S1P. Each panel represents one experiment performed in triplicate that was repeated three times. Expression of S1P Receptors in HFL-1 Cells To determine the expression of S1P receptors by HFL-1 cells, two methods were used. First, mRNA expression for S1P1, S1P2, S1P3, S1P4, and S1P5 was examined by RT-PCR. HFL-1 cells expressed S1P1, S1P2, S1P3, and S1P5, but not S1P4 (Physique 2A). A similar expression pattern Icotinib was observed in human bronchial fibroblasts and human airway smooth muscle cells (data not shown). In contrast, Jurkat cells expressed S1P1, S1P2, S1P3, and S1P4, but not S1P5 (Physique 2A). Second, to confirm protein expression, immunoblots were performed, and immunoreactive protein was detected for S1P1, S1P2, and S1P5 in HFL-1 cell lysates (Physique 2B). Open in a separate window Physique 2. Expression of S1P receptors in human lung fibroblasts. ( 0.01 by two-way ANOVA. Effect of Rho and Rho Kinase in Mediating S1P Augmentation of HFL-1 Cell Chemotaxis Since S1P has been reported to activate Rho and Rho kinase in a variety of cell types (22, 23), their role in mediating S1P-stimulated HFL-1 chemotaxis was evaluated. S1P (2 M) significantly stimulated Rho activation, as evidenced by GTP binding to Rho, and this was completely blocked by the S1P2 antagonist (JTE-013; Physique 4A). LPA (2 M) slightly increased Rho activation Icotinib and this was not affected by JTE-013 (Physique 4A), indicating that S1P activates Rho through the S1P2 receptor, but LPA was activated through other mechanisms. Activation of Rho through the S1P2 receptor was further confirmed by showing that suppression of the S1P2 receptor with siRNA blocked S1P-induced Rho activation (Physique 4B). Open in a separate window Open in a separate window Physique 4. Role of the S1P2 receptor in mediating S1P-stimulated Rho activation. ( 0.05) inhibition of chemotaxis toward fibronectin in the absence of S1P. In contrast, Y-27632 resulted in a concentration-dependent inhibition of S1P-augmented chemotaxis ( 0.05, Figure 5), with essentially complete inhibition of the S1P augmentation by 10 M Y-27632. Open in a separate window Physique 5. Effect of Rho kinase inhibitor on S1P-stimulated cell migration. Fibroblast chemotaxis was performed to fibronectin (20 g/ml) with varying concentrations of Y27632 added to the cells in the top wells. 0.01 by two-way ANOVA. Other Potential Signaling Pathways for S1P-Augmented HFL-1 Cell Chemotaxis To further investigate the mechanism by which S1P stimulates HFL-1 chemotaxis, pharmacologic inhibitors of other potential signal transduction pathways were used. To examine.