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

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.