Protein kinase C-epsilon regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-zeta, and Rac1.
The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P(1) small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P(1) to G(i). Overexpression of dominant negative (dn) PKC-epsilon or -zeta, but not PKC-alpha or -delta, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-epsilon, but not PKC-zeta, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-epsilon, but not PKC-zeta, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-zeta, or treatment with myristoylated PKC-zeta peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P(1) and G(i) to activate PKC-epsilon and, subsequently, a PLD2-PKC-zeta-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.
Gorshkova
Irina
I
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
He
Donghong
D
Berdyshev
Evgeny
E
Usatuyk
Peter
P
Burns
Michael
M
Kalari
Satish
S
Zhao
Yutong
Y
Pendyala
Srikanth
S
Garcia
Joe G N
JG
Pyne
Nigel J
NJ
Brindley
David N
DN
Natarajan
Viswanathan
V
D002465:Cell Movement; D004730:Endothelium, Vascular / Q000166:cytology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008246:Lysophospholipids / Q000737:chemistry*; D008954:Models, Biological; D009389:Neovascularization, Pathologic; D010739:Phospholipase D / Q000378:metabolism*; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000737:chemistry / Q000502:physiology*; D011651:Pulmonary Artery / Q000166:cytology; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D000099221:Protein Kinase C zeta
D008246:Lysophospholipids; D010739:Phospholipase D; D020033:Protein Isoforms; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D034741:RNA, Small Interfering; D013110:Sphingosine; D020830:rac1 GTP-Binding Protein; D000099221:Protein Kinase C zeta; C060506:sphingosine 1-phosphate; C105606:phospholipase D2