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J. Biol. Chem., Vol. 283, Issue 17, 11794-11806, April 25, 2008

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

Irina Gorshkova, Donghong He, Evgeny Berdyshev, Peter Usatuyk, Michael Burns, Satish Kalari, Yutong Zhao, Srikanth Pendyala, Joe G. N. Garcia, Nigel J. Pyne, David N. Brindley^¶1, and Viswanathan Natarajan²

From the Department of Medicine, University of Chicago, Chicago, Illinois 60637, Department of Physiology and Pharmacology, University of Strathclyde, Glasgow G4 ONR, United Kingdom, and ^¶Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

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

Received for publication, January 10, 2008 , and in revised form, February 21, 2008.

^* This work was supported by National Institutes of Health Grant HL RO1 79396 (to V. N.) and Canadian Institute of Health Research Grant MOP 81137 (to D. N. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4 and Table 1.

¹ Recipient of a Medical Scientist Award from the Alberta Heritage Foundation for Medical Research.

² To whom correspondence should be addressed: Dept. of Medicine, The University of Chicago, CIS Bldg., Rm. W408B, 929 East 57th St., Chicago, IL 60637. Tel.: 773-834-2638; Fax: 773-834-2687; E-mail: vnataraj{at}medicine.bsd.uchicago.edu.

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