High density lipoprotein-associated sphingosine 1-phosphate promotes endothelial barrier function

High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here we show that HDL increases endothelial barrier integrity as measured by electric cell substrate impedance sensing. S1P was implicated as the mediator in this process through findings showing that pertussis toxin, an inhibitor of G(i)-coupled S1P receptors, as well as antagonists of the S1P receptor, S1P1, inhibited barrier enhancement by HDL. Additional findings show that HDL stimulates endothelial cell activation of Erk1/2 and Akt, signaling pathway intermediates that have been implicated in S1P-dependent endothelial barrier activity. HDL was also found to promote endothelial cell motility, a process that may also relate to endothelial barrier function in the context of a vascular injury response. The effects of HDL on endothelial cell Erk1/2 and Akt activation and motility were suppressed by pertussis toxin and S1P1 antagonists. However, both HDL-induced barrier enhancement and HDL-induced motility showed a greater dependence on Akt activation as compared with Erk1/2 activation. Together, the findings indicate that HDL has endothelial barrier promoting activities, which are attributable to its S1P component and signaling through the S1P1/Akt pathway.     

Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.     

Intracellular generation of sphingosine 1-phosphate in human lung endothelial cells: role of lipid phosphate phosphatase-1 and sphingosine kinase 1

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 (32)P-labeled HPAECs to S1P or sphingosine (Sph) increased the intracellular accumulation of [(32)P]S1P in a dose- and time-dependent manner. The S1P formed in the cells was not released into the medium. The exogenously added S1P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S1P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-14, an inhibitor of LPPs. HPAECs expressed LPP1-3, and overexpression of LPP-1 enhanced the hydrolysis of exogenous [(3)H]S1P to [(3)H]Sph 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.     

Characterization of a novel sphingosine 1-phosphate receptor, Edg-8

Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1- phosphate-dependent increases in [(35)S]guanosine 5'-(3-O-thio)triphosphate binding with an EC(50) of 90 nm. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC(50) 0.7 nm). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G(q/i)alpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 2 nm) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G(i/o)alpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.     

Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1

The transactivation of enhanced growth factor receptor (EGFR) by G protein-coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase sphingosine kinase-1 (SphK1). We show that E2 stimulates SphK1 activation and the release of sphingosine 1-phosphate (S1P), by which E2 is capable of activating the S1P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease-dependent manner. Thus, these findings reveal a key role for SphK1 in the coupling of the signals between three membrane-spanning events induced by E2, S1P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., "criss-cross" transactivation.     

Functional characterization of sphingosine 1-phosphate receptor agonist in human endothelial cells

Sphingosine 1-phosphate (S1P) is a pleiotropic lysophospholipid mediator involved in many cellular responses, including transient calcium mobilization, activation of MAP kinase signaling, inhibition of adenylyl cyclase and increased cell migration. S1P has been shown to be an effective activator of vascular endothelial cells via the interaction with cell surface G protein-coupled receptors (GPCRs), namely S1P-R (formerly EDG-R). The potent immunomodulator, FTY720, is phosphorylated by sphingosine kinase (SK) to FTY720-P. Recently it was shown that FTY720-P, not FTY720, can bind to four out of five of the S1P-R. In the present study, we evaluated the effects of FTY720, FTY720-P, and analogues of FTY720-P: an active (R)-enantiomer [AFD(R)] and an inactive (S)-enantiomer [AFD(S)], on endothelial cell functions. Treatment of HUVEC with FTY720-P, but not FTY720, lead to a robust transient increase in calcium mobilization, detected using the fluorometric imaging plate reader (FLIPR) assay. Additionally, only the phosphorylated derivative (FTY720-P) stimulated MAPK activation. We also observed complementary activities of S1P and FTY720-P in an established in vitro endothelial morphogenesis (Matrigel tube formation) assay and an in vitro endothelial cell migration assay. Using a potent inhibitor of sphingosine kinase, N,N-dimethylsphingosine (DMS), FTY720's effects were inhibited in the migration assay, suggesting that FTY720-P is the active mediator. The effects of FTY720-P in these assays were inhibited by pre-treatment with PTx (pertussis toxin), indicating the requirement of a Gi-coupled S1P receptor. These findings suggest that agonist of S1P-R are able to regulate important endothelial cell properties, which may lead to a greater insight into vascular functions.     

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P₁) phosphorylation was studied. Activation of S1P₁ receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P₁ phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P₁ phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P₁ phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P₁ immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P₁ phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P₁.     

The role of sphingosine-1-phosphate in endothelial barrier function

Loss of endothelial barrier function is implicated in the etiology of metastasis, atherosclerosis, sepsis and many other diseases. Studies suggest that sphingosine-1-phosphate (S1P), particularly HDL-bound S1P (HDL–S1P) is essential for endothelial barrier homeostasis and that HDL–S1P may be protective against the loss of endothelial barrier function in disease. This review summarizes evidence providing mechanistic insights into how S1P maintains endothelial barrier function, highlighting the recent findings that implicate the major S1P carrier, HDL, in the maintenance of the persistent S1P-signaling needed to maintain endothelial barrier function. We review the mechanisms proposed for HDL maintenance of persistent S1P-signaling, the evidence supporting these mechanisms and the remaining fundamental questions.     

Involvement of released sphingosine 1-phosphate/sphingosine 1-phosphate receptor axis in skeletal muscle atrophy

Skeletal muscle (SkM) atrophy is caused by several and heterogeneous conditions, such as cancer, neuromuscular disorders and aging. In most types of SkM atrophy overall rates of protein synthesis are suppressed, protein degradation is consistently elevated and atrogenes, such as the ubiquitin ligase Atrogin-1/MAFbx, are up-regulated. The molecular regulators of SkM waste are multiple and only in part known.Sphingolipids represent a class of bioactive molecules capable of modulating the destiny of many cell types, including SkM cells. In particular, we and others have shown that sphingosine 1phosphate (S1P), formed by sphingosine kinase (SphK), is able to act as trophic and morphogenic factor in myoblasts.Here, we report the first evidence that the atrophic phenotype observed in both muscle obtained from mice bearing the C26 adenocarcinoma and C2C12 myotubes treated with dexamethasone was characterized by reduced levels of active phospho-SphK1. The importance of SphK1 activity is also confirmed by the specific pharmacological inhibition of SphK1 able to increase Atrogin-1/MAFbx expression and reduce myotube size and myonuclei number. Furthermore, we found that SkM atrophy was accomplished by significant increase of S1P transporter Spns2 and in changes in the pattern of S1P receptor (S1PRs) subtype expression paralleled by increased Atrogin-1/MAFbx expression, suggesting a role for the released S1P and of specific S1PR-mediated signaling pathways in the control of the ubiquitin ligase. Altogether, these findings provide the first evidence that SphK1/released S1P/S1PR axis acts as a molecular regulator of SkM atrophy, thereby representing a new possible target for therapy in many patho-physiological conditions.     

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.     

The sphingosine 1-phosphate receptor 5 and sphingosine kinases 1 and 2 are localised in centrosomes: Possible role in regulating cell division

We show here that the endogenous sphingosine 1-phosphate 5 receptor (S1P₅, a G protein coupled receptor (GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 and SK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where they may participate in regulating mitosis. The centrosome is a site for active GTP–GDP cycling involving the G-protein, G_i and tubulin, which are required for spindle pole organization and force generation during cell division. Therefore, the presence of S1P₅ (which normally functions as a plasma membrane guanine nucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P₅ may function as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. The addition of S1P to cells inhibits trafficking of S1P₅ to the centrosome, suggesting a dynamic shuttling endocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that the centrosomal S1P₅ receptor might function as an intracellular target of S1P linked to regulation of mitosis.     

Transactivation of sphingosine 1-phosphate receptors is essential for vascular barrier regulation. Novel role for hyaluronan and CD44 receptor family

The role for hyaluronan (HA) and CD44 in vascular barrier regulation is unknown. We examined high and low molecular weight HA (HMW-HA, approximately 1,000 kDa; LMW-HA, approximately 2.5 kDa) effects on human transendothelial monolayer electrical resistance (TER). HMW-HA increased TER, whereas LMW-HA induced biphasic TER changes ultimately resulting in EC barrier disruption. HMW-HA induced the association of the CD44s isoform with, and AKT-mediated phosphorylation of, the barrier-promoting sphingosine 1-phosphate receptor (S1P1) within caveolin-enriched lipid raft microdomains, whereas LMW-HA induced brief CD44s association with S1P1 followed by sustained association of the CD44v10 isoform with, and Src and ROCK 1/2-mediated phosphorylation of, the barrier-disrupting S1P3 receptor. HA-induced EC cytoskeletal reorganization and TER alterations were abolished by either disruption of lipid raft formation, CD44 blocking antibody or siRNA-mediated reductions in expression of CD44 isoforms. Silencing S1P1, AKT1, or Rac1 blocked the barrier enhancing effects of HA whereas silencing S1P3, Src, ROCK1/2, or RhoA blocked the barrier disruption induced by LMW-HA. In summary, HA regulates EC barrier function through novel differential CD44 isoform interaction with S1P receptors, S1P receptor transactivation, and RhoA/Rac1 signaling to the EC cytoskeleton.     

Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.     

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.     

Differential effects of sphingosine 1-phosphate and lysophosphatidic acid on endothelial cells

This review discusses multiple effects of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) on endothelial cells and proposes that S1P and LPA are important regulators of the vascular system. Two physiologic sources of S1P and LPA are platelets and lipoproteins. S1P is an inducer of angiogenesis in vivo whereas LPA is not. S1P and LPA act through endothelial cell surface Edg receptors. S1P stimulates endothelial cell migration, but inhibits migration of most nonendothelial cells. Edg1 and Edg3 receptors, working through G(i), play an important role in regulation of S1P-stimulated endothelial cell migration. LPA effects on endothelial cells are more restricted than the effects of S1P on endothelial cells. LPA stimulates migration of certain endothelial cells on certain extracellular matrix proteins. However, LPA acts like S1P in its effects on the endothelial cell cytoskeleton, proliferation, cell-cell adhesion molecule expression, and vascular permeability. LPA receptors on endothelial cells are likely Edg2 and Edg4. Future studies should better delineate the roles of Edg receptors and downstream pathways on effects of extracellular S1P and LPA and the contributions of intracellularly generated S1P and nitric oxide (NO).     

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.     

Differential involvement of ezrin/radixin/moesin proteins in sphingosine 1-phosphate-induced human pulmonary endothelial cell barrier enhancement

Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.     

Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors.

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphingosine 1-phosphate have been cloned and characterized. We found human and mouse sequences closely related to rat S1P(5) (97% identical amino acids) and report now the characterization of the human and mouse S1P(5) gene products as encoding sphingosine 1-phosphate receptors. When HEK293T cells were cotransfected with S1P(5) and G protein DNAs, prepared membranes showed sphingosine 1-phosphate concentration-dependent increases in [gamma-(35)S]GTP binding (EC(50) = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5) DNAs into rat hepatoma RH7777 cells (IC(50) = 0.22 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Northern blot analysis showed high expression of human S1P(5) mRNA in spleen, corpus collosum, peripheral blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one enantiomer of a sphingosine 1-phosphate analogue wherein the 3-hydroxyl and 4,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).     

Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1−/− mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.     

A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes.

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D-erythro-sphingosyl-phosphorylcholine; SPC) and sphingosine 1-phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D-erythro-(2S,3R)-SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure-activity relationship: it requires an unsubstituted amino group because N-acetyl-SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein-coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.