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

Irina Gorshkova, Donghong He, Evgeny Berdyshev, Peter Usatuyk, Michael Burns, Satish Kalari, Yutang Zhao, Srikanth Pendyala, Joe G.N. Garcia, N.J. Pyne, David N. Brindley, Viswanathan Natarajan

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Abstract

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.
Original languageEnglish
Pages (from-to)11794-11806
Number of pages13
JournalJournal of Biological Chemistry
Volume283
Issue number17
DOIs
Publication statusPublished - 25 Apr 2008

Keywords

  • protein Kinase C-
  • sphingosine 1-
  • phosphates-
  • lungs
  • endothelial cells
  • phospholipase D2
  • proteins
  • pharmacology

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    Gorshkova, I., He, D., Berdyshev, E., Usatuyk, P., Burns, M., Kalari, S., Zhao, Y., Pendyala, S., Garcia, J. G. N., Pyne, N. J., Brindley, D. N., & Natarajan, V. (2008). 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. Journal of Biological Chemistry, 283(17), 11794-11806. https://doi.org/10.1074/jbc.M800250200