title: Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase.
abstract: BACKGROUND
  Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) 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.
  
  
  RESULTS
  Inhibition 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 S1P(int), and attenuated S1P(ext) or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P(int) and potentiated motility of HPAECs to S1P(ext) or serum. S1P(ext) 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 S1P(ext) or 4-deoxypyridoxine-dependent endothelial cell motility.
  
  
  CONCLUSIONS
  These results suggest S1P(ext) mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.
authors.affiliation: Department of Medicine, The University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Evgeny V
authors.initials: EV
authors.lastname: Berdyshev
authors.forename: Irina
authors.initials: I
authors.lastname: Gorshkova
authors.forename: Peter
authors.initials: P
authors.lastname: Usatyuk
authors.forename: Satish
authors.initials: S
authors.lastname: Kalari
authors.forename: Yutong
authors.initials: Y
authors.lastname: Zhao
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Pyne
authors.forename: Susan
authors.initials: S
authors.lastname: Pyne
authors.forename: Roger A
authors.initials: RA
authors.lastname: Sabbadini
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D000446:Aldehyde-Lyases / Q000502:physiology*; D002465:Cell Movement; D042783:Endothelial Cells / Q000166:cytology*; D006801:Humans; D008168:Lung / Q000166:cytology*; D008246:Lysophospholipids / Q000096:biosynthesis* / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000502:physiology; D000099157:Sphingosine Kinase
chemical_list: D000446:Aldehyde-Lyases; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D034741:RNA, Small Interfering; D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate; C022649:sphingosine 1-phosphate lyase (aldolase)