Differential regulation of sphingosine-1-phosphate and VEGF-induced endothelial cell chemotaxis -: Involvement of Giα2-linked rho kinase activity

We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by sphingosine-l-phosphate (S1P), a potent bioactive lipid released from activated platelets, and by vascular endothelial growth factor (VEGF), a well-recognized angiogenic factor. S1P-induced endothelial cell migration was maximum at 1 muM (similar to 8-fold increase with PA endothelium) and surpassed the maximal response evoked by either VEGF (10 ng/ml) (similar to 2.5-fold increase) or hepatocyte growth factor (HGF) (similar to 2.5-foId increase). Migration induced by S1P, but not by VEGF, was significantly inhibited by treatment with antisense oligonucleotides directed to Edg-1 and Edg-3 (endothelial differentiation gene) S1P receptors and by G protein modification. These strategies included pretreatment with pertussis toxin, or transfection with mini-genes encoding a py subunit inhibitory peptide of the beta -adrenergic receptor kinase, or an 11-amino-acid peptide that inhibits G(1 alpha2) signaling. Various strategies to interrupt Rho family signaling, including C-3 exotoxin, dominant/negative Rho, or the addition of Y27632, a cell-permeable Rho kinase inhibitor, significantly attenuated S1P- but not VEGF-induced migration. Conversely, pharmacologic Inhibition of either myosin light chain kinase, src family tyrosine kinases, or phosphatidylinositol-3' kinase reduced basal endothelial cell migration and abolished VEGF-induced endothelial cell migration but did not inhibit the increase in S1P-induced migration. Whereas VEGF and S1P increased both p42/p44 extracellular regulated kinase and p38 mitogen-activated protein (MAP) kinase activities, only p38 MAP kinase inhibition significantly reduced VEGF- and S1P-stimulated migration. These data confirm S1P as a potent endothelial cell chemoattractant through G(1 alpha2)-coupled Edg receptors linked to Rho-associated kinase and p38 MAP kinase activation. The divergence in signaling pathways evoked by S1P and VEGF suggests complex and agonist-specific regulation of endothelial cell angiogenic responses.