Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells.
Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.
Deo
Dayanand D
DD
Department of Biochemistry and Molecular Biology, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Bazan
Nicolas G
NG
Hunt
Jay D
JD
D000262:Adenylyl Cyclases / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D008954:Models, Biological; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013997:Time Factors; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology*
D004268:DNA-Binding Proteins; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; C039351:platelet activating factor receptor; D019716:Tissue Inhibitor of Metalloproteinase-2; D014443:Tyrosine; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000262:Adenylyl Cyclases