Differential regulation of sphingosine-1-phosphate and VEGF-induced endothelial cell chemotaxis - Involvement of G(i alpha 2)-linked rho kinase activity

We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by sphingosin e-l-phosphate (S1P), a potent bioactive lipid released from activated plate lets, and by vascular endothelial growth factor (VEGF), a well-recognized a ngiogenic factor. S1P-induced endothelial cell migration was maximum at 1 m uM (similar to 8-fold increase with PA endothelium) and surpassed the maxim al 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). Migratio n induced by S1P, but not by VEGF, was significantly inhibited by treatment with antisense oligonucleotides directed to Edg-1 and Edg-3 (endothelial d ifferentiation gene) S1P receptors and by G protein modification. These str ategies included pretreatment with pertussis toxin, or transfection with mi ni-genes encoding a py subunit inhibitory peptide of the beta -adrenergic r eceptor kinase, or an 11-amino-acid peptide that inhibits G(1 alpha2) signa ling. Various strategies to interrupt Rho family signaling, including C-3 e xotoxin, dominant/negative Rho, or the addition of Y27632, a cell-permeable Rho kinase inhibitor, significantly attenuated S1P- but not VEGF-induced m igration. Conversely, pharmacologic Inhibition of either myosin light chain kinase, src family tyrosine kinases, or phosphatidylinositol-3' kinase red uced basal endothelial cell migration and abolished VEGF-induced endothelia l 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 ki nase inhibition significantly reduced VEGF- and S1P-stimulated migration. T hese data confirm S1P as a potent endothelial cell chemoattractant through G(1 alpha2)-coupled Edg receptors linked to Rho-associated kinase and p38 M AP kinase activation. The divergence in signaling pathways evoked by S1P an d VEGF suggests complex and agonist-specific regulation of endothelial cell angiogenic responses.