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

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 S1P 1 small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P 1 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 S1P 1 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.Sphingosine 1-phosphate (S1P) 3 is a naturally occurring bioactive sphingolipid that elicits multiple cellular responses such as differentiation, proliferation, survival, and angiogenesis (1-5). S1P acts as an intracellular second messenger. Extracellular S1P also activates intracellular signaling pathways through ligation to a family of G-protein-coupled S1P receptors, S1P 1-5 (previously known as endothelial differentiation gene receptors) (6). The S1P-Rs are differentially expressed in different cell types and are coupled to G i , G q , or G 12/13 (7-9). Coupling of S1P to S1P 1 via G i activates Rac and Rho (2, 10) and stimulates cell proliferation (4), cortical actin formation (11), assembly of adherens junction, and angiogenesis (2). Binding of S1P to S1P 3 induces signaling through G q or G 13 to activate Rho (2, 10, 12), promotes the formation of stress fibers and adherens junctions (2), stimulates phospholipase D (PLD) (13), and activates phospholipase C/intracellular Ca 2ϩ /protein kinase C (PKC) pathways (7). Ligation of S1P to S1P 1 also initiates cross-talk with other receptors, especially growth factor receptors including those for epidermal growth factor (EGF), platelet-derived growth factor, and vascular endothelial growth factor (14). The functional platelet-derived growth factor (PDGF)-␤/S1P 1 signaling complex was postulated to be involved in regulating migration of mouse embryonic fibroblasts in response to PDGF (15). Furthermore, S1P binding to S1P 2 inhibits cell migration via G q or G 13 (9, 12, 16) and activates adenylate cyclase (17) and mitogen-activated protein kinases (MAPKs) (18). There are few studies related to S1P signaling via S1P 4 and...

Arachidonic Acid Metabolism Regulates Escherichia coli Penetration of the Blood-Brain Barrier

“…HBMEC were isolated and characterized as described previously (37). HBMEC (ϳ70% confluence) were infected with a dialyzed adenovirus (Ad) of AdECFP-cPLA 2 -wt (where ECFP is enhanced cyan fluorescent protein and wt is wild type), AdECFP-cPLA 2 -S505A, AdECFP-cPLA 2 -S515A (29), or Ad5CA dominant negative PKC␣ or a vector control (8). Strain RS218 grown overnight in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, MI) was resuspended in experimental medium (M199-HamF12 [1:1], containing 5% heat-inactivated fetal bovine serum, 2 mM glutamine, and 1 mM pyruvate) and added in a multiplicity of infection of 100:1 to HBMEC grown in collagen-coated 24-well plates, which were incubated at 37°C in a 5% CO 2 incubator for 90 min for binding assays.…”

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…In addition to PKCd, PKC« was also shown to regulate PLD2 activation after sphingosine 1-phosphate (S1P) stimulation in lung epithelial cells (Gorshkova et al, 2008). A direct protein-protein interaction was not measured and an intermediate protein is likely mediating the effects of PKC« on PLD2.…”

“…Cancer cells can secrete chemoattractants to recruit immune cells into tumors where they produce MMP and other factors that promote invasion (Condeelis and Pollard, 2006). In addition, PLD activity is required for fibroblast migration toward LPA (Pilquil et al, 2006) and endothelial cell migration toward S1P (Gorshkova et al, 2008). Like nontransformed cells, cancer cells, such as MDA-MB-231, also require PLD activity for migration Scott et al, 2009).…”

“…In endothelial cells, Rac activation appears to require PLD2 activity because RNAi silencing of PLD2 decreases Rac activation and cell migration in response to S1P (Gorshkova et al, 2008). The PH domain of PLD2 encodes a putative Cdc42/Rac interactive binding domain that directly interacts with Rac proteins, located between amino acids 255 and 269 Peng et al, 2011).…”

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“…PKCα and PKCβ1 stimulate PLD [129,130], but PKCδ and PKCε can act as both positive and negative regulators of PLD1 and PLD2 in vivo. PKCδ can displace PLD1 from the complex with PKCα [131,132]. Results of in vitro studies have shown that human and rat PLD1 are activated under the influence of PKCα, PKCβ1, and PKCβ2 independently of their kinase activity [26,47,130].…”