Abstract
BackgroundEarlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.Methodology/Principal FindingsInhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1Pint, and attenuated S1Pext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1Pint and potentiated motility of HPAECs to S1Pext or serum. S1Pext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1Pext or 4-deoxypyridoxine-dependent endothelial cell motility.Conclusions/SignificanceThese results suggest S1Pext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.
Highlights
Sphingolipid metabolites such as ceramides and sphingoid bases are important modulators of cell survival, cell proliferation, angiogenesis, and vascular integrity
These results demonstrate that SphK1, but not SphK2, regulates S1Pext mediated endothelial cell migration
Having established that S1P lyase (S1PL) siRNA or 4-DP treatment increases S1P levels in cells and stimulates endothelial cell motility, we evaluated whether these effects were mediated by an autocrine pathway of S1P secretion and signaling via S1P1 and/or 3 by using a highly specific anti-S1P monoclonal antibody [33,34]
Summary
Sphingolipid metabolites such as ceramides and sphingoid bases are important modulators of cell survival, cell proliferation, angiogenesis, and vascular integrity. Sphingosine-1-phosphate (S1P), elicits a plethora of cellular responses such as proliferation, survival, chemotaxis and endothelial barrier regulation. We have recently shown that S1P signals through S1P1 and Gi to activate PKC-e and subsequently, a PLD2-PKC-f-Rac cascade to induce migration of human lung ECs [12]. These studies strongly suggest a role for extracellular action of S1P through S1P1 and other S1PRs in stimulating migration of ECs. Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P1 receptor, PKCe, and PLD2-PKCf-Rac signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility
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