Protein Kinase C-ϵ Regulates Sphingosine 1-Phosphate-mediated Migration of Human Lung Endothelial Cells through Activation of Phospholipase D2, Protein Kinase C-ζ, and Rac1

• Published in: The Journal of biological chemistry 2008-04, Vol.283 (17), p.11794-11806
• Main Authors: Gorshkova, Irina, He, Donghong, Berdyshev, Evgeny, Usatuyk, Peter, Burns, Michael, Kalari, Satish, Zhao, Yutong, Pendyala, Srikanth, Garcia, Joe G.N., Pyne, Nigel J., Brindley, David N., Natarajan, Viswanathan
• Format: Article
• Language: English
• Subjects: Cell Movement; Endothelium, Vascular - cytology; Gene Expression Regulation, Enzymologic; Humans; Lysophospholipids - chemistry; Models, Biological; Neovascularization, Pathologic; Phospholipase D - metabolism; Protein Isoforms; Protein Kinase C - metabolism; Protein Kinase C-epsilon - chemistry; Protein Kinase C-epsilon - physiology; Pulmonary Artery - cytology; rac1 GTP-Binding Protein - metabolism; RNA, Small Interfering - metabolism; Signal Transduction; Sphingosine - analogs & derivatives; Sphingosine - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M800250200

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 S1P1 small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P1 to Gi. Overexpression of dominant negative (dn) PKC-ϵ or -ζ, but not PKC-α or -δ, 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-ϵ, but not PKC-ζ, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-ϵ, but not PKC-ζ, 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-ζ, or treatment with myristoylated PKC-ζ peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P1 and Gi to activate PKC-ϵ and, subsequently, a PLD2-PKC-ζ-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.