ICAM-1-activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration.
Polymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)-dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)-mediated transendothelial migration. An important unanswered question is whether ICAM-1-activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1-dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1(-/-) and eNOS(-/-) mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr(518)Phe ICAM-1 mutant, induced SHP-2-dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr(518)Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho-ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti-PECAM-1 mAb-binding activity. These results collectively show that ICAM-1-activated Src and eNOS signaling sequentially induce PECAM-1-mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation.
Department of Pharmacology, University of Illinois, Chicago 60612, USA. rminsh@uic.edu
Guoquan
G
Liu
Aaron T
AT
Place
Zhenlong
Z
Chen
Viktor M
VM
Brovkovych
Stephen M
SM
Vogel
William A
WA
Muller
Randal A
RA
Skidgel
Asrar B
AB
Malik
Richard D
RD
Minshall
D000818:Animals; D015153:Blotting, Western; D002351:Carrageenan / Q000494:pharmacology; D002448:Cell Adhesion; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020556:Neutrophil Infiltration / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction; D057705:Transendothelial and Transepithelial Migration / Q000187:drug effects; D019061:src-Family Kinases / Q000378:metabolism*
D019408:Platelet Endothelial Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D009569:Nitric Oxide; D002351:Carrageenan; D052250:Nitric Oxide Synthase Type III; D019061:src-Family Kinases