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Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.Sphingosine 1-phosphate (S1P) 2 is a bioactive lipid mediator that plays an important role in regulating intracellular mobilization of Ca 2ϩ , cytoskeletal reorganization, cell growth, differentiation, motility, angiogenesis, and survival (1-5). In biological fluids such as plasma, S1P is present at 0.2-0.5 M, whereas higher concentrations (1-5 M) in serum are attributed to enhanced release from activated platelets (1, 5). S1P is generated by phosphorylation of free sphingosine (Sph) by two sphingosine kinases (SphKs) 1 and 2, which are highly conserved enzymes present in most of the mammalian cells and tissues (6 -9). Cellular levels of S1P are regulated through its formation via SphKs and by its degradation by S1P lyase (SPL) (10 -12), S1P phosphatases (SPPs) (13-15), and intracellular lipid phosphate phosphatases (LPPs) (16 -18). Platelets lack S1P lyase (19), but in most cells the balance between S1P formation and degradation translates to low basal levels of intracellular S1P. S1P exerts dual actions in cells; it acts as an intracellular second messenger and functions extracellularly as a ligand for a family of five G-protein-coupled receptors formerly known as endothelial differentiation gene (Edg) receptors. To date, five G-protein-coupled receptors, S1P-1 (Edg-1), S1P-2 (Edg-5), S1P-3 (Edg-3), S1P-4 (Edg-6), and S1P-5 (Edg-8), have been identified. All these receptors bind to and are activated by extracellular S1P and dihydro-S1P (1, 5, 20 -22). In the vessel wall extracellular S1P is a potent stimulator of angiogenesis (23, 24) and is a major chemotactic factor for endothelial cells (ECs). Recently, circulating S1P and the immunosuppressive drug FTY720, which is also phosphorylated by SphKs, have been implicated in lymphocyte homing and immunoregulation (25,26). In addition to its extracellular action, S1P functions as an intr...

Sphingosine kinase 2 (SK2) catalyzes the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). We report here, the stereospecific synthesis of an analogue of FTY720 called (R)-FTY720-OMe, which we show is a competitive inhibitor of SK2. (R)-FTY720-OMe failed to inhibit sphingosine kinase 1 activity, thereby demonstrating specificity for SK2. Prolonged treatment of HEK 293 cells with (R)-FTY720-OMe also induced a reduction in SK2 expression. In addition, (R)-FTY720-OMe inhibited DNA synthesis and prevented S1P-stimulated rearrangement of actin in MCF-7 breast cancer cells. These findings demonstrate that SK2 functions as a pro-survival protein and is involved in promoting actin rearrangement into membrane ruffles/lamellipodia in response to S1P in MCF-7 breast cancer cells.

Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)-and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)-and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 M) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G i -coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.Sustained vascular barrier leak, a marked characteristic of acute inflammatory diseases, such as acute lung injury (ALI) and sepsis, contributes to the high mortality of these conditions. Disruption of the pulmonary vascular endothelial cell (EC) monolayer in the lung microcirculation results in flooding of interstitial and alveolar compartments with fluid, protein, and inflammatory cells, resulting in respiratory failure (Dudek and Garcia, 2001). Specific therapies that prevent or reverse inflammation-mediated vascular barrier leak are lacking (Wheeler and Bernard, 2007). We have previously demonstrated the potent barrier-enhancing properties of sphingosine 1-phosphate (S1P), a platelet-derived sphingolipid that rapidly induces EC cytoskeletal rearrangements, leading to augmented EC monolayer integrity . Through the ligation of the G i -coupled S1P 1 receptor (S1P 1 R), S1P initiates a series of downstream events, including Rac activation, cortactin translocation, peripheral myosin light chain (MLC) phosphorylation, and focal adhesion rearrangement, culminating in enhancement of the EC cortical

FTY720 (Fingolimod™), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P 1 receptor internalization and targeting to the proteasomal degradative pathway, and thus acts as a functional antagonist of S1P 1 by depleting the functional S1P 1 receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)-and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P 1 (EC 50 20 ± 3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P 1,3,4 (K i 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P 2 , and S1P 5 . Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with K i values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)-and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P 2 transcripts and traces of S1P 5 . (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The biological effects of the (R)-and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets. NIH Public Access