Differential regulation of sphingosine-1-phosphate- and VEGF-induced endothelial cell chemotaxis. Involvement of G(ialpha2)-linked Rho kinase activity.
We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by sphingosine-1-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 microM (approximately 8-fold increase with PA endothelium) and surpassed the maximal response evoked by either VEGF (10 ng/ml) (approximately 2.5-fold increase) or hepatocyte growth factor (HGF) (approximately 2.5-fold 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 betagamma subunit inhibitory peptide of the beta-adrenergic receptor kinase, or an 11-amino-acid peptide that inhibits G(1alpha2) 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(1alpha2)-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.
Liu
F
F
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Verin
A D
AD
Wang
P
P
Day
R
R
Wersto
R P
RP
Chrest
F J
FJ
English
D K
DK
Garcia
J G
JG
D000818:Animals; D001808:Blood Vessels / Q000187:drug effects; D002417:Cattle; D002633:Chemotaxis / Q000187:drug effects*; D003599:Cytoskeleton / Q000187:drug effects; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects*; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008168:Lung / Q000098:blood supply; D008222:Lymphokines / Q000494:pharmacology*; D008246:Lysophospholipids; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D018919:Neovascularization, Physiologic; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases
D016228:Endothelial Growth Factors; D047908:Intracellular Signaling Peptides and Proteins; D008222:Lymphokines; D008246:Lysophospholipids; D018994:Myosin Light Chains; D011518:Proto-Oncogene Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C060506:sphingosine 1-phosphate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020928:Mitogen-Activated Protein Kinases; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020741:rho GTP-Binding Proteins; D013110:Sphingosine