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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The experimental evidence that Adhesion G Protein-Coupled Receptors (aGPCRs) functionally couple to heterotrimeric G proteins has been emerging in incremental steps, but attributing biological significance to their G protein signalling function still presents a major challenge. Here, utilising activated truncated forms of the receptors, we show that ADGRE2/EMR2 and ADGRE5/CD97 are G protein-coupled in a variety of recombinant systems. In a yeast-based assay, where heterologous GPCRs are coupled to chimeric G proteins, EMR2 showed broad G protein-coupling, whereas CD97 coupled more specifically to Gα12, Gα13, Gα14 and Gαz chimeras. Both receptors induced pertussis-toxin (PTX) insensitive inhibition of cyclic AMP (cAMP) levels in mammalian cells, suggesting coupling to Gαz. EMR2 was shown to signal via Gα16, and via a Gα16/Gαz chimera, to stimulate IP1 accumulation. Finally, using an NFAT reporter assay, we identified a polyclonal antibody that activates EMR2 G protein signalling in vitro. Our results highlight the potential for the development of soluble agonists to understand further the biological effects and therapeutic opportunities for ADGRE receptor-mediated G protein signalling.
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Anticuerpos/inmunología , Antígenos CD/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Western Blotting , Células HEK293/metabolismo , Humanos , Inmunoprecipitación , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
In order to optimize the potency of the first serum-stable peptide agonist of CD47 (PKHB1) in triggering regulated cell death of cancer cells, we designed a maturation process aimed to mimic the trimeric structure of the thrombospondin-1/CD47 binding epitope. For that purpose, an N-methylation scan of the PKHB1 sequence was realized to prevent peptide aggregation. Structural and pharmacological analyses were conducted in order to assess the conformational impact of these chemical modifications on the backbone structure and the biological activity. This structure-activity relationship study led to the discovery of a highly soluble N-methylated peptide that we termed PKT16. Afterward, this monomer was used for the design of a homotrimeric peptide mimic that we termed [PKT16]3, which proved to be 10-fold more potent than its monomeric counterpart. A pharmacological evaluation of [PKT16]3 in inducing cell death of adherent (A549) and nonadherent (MEC-1) cancer cell lines was also performed.
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Diseño de Fármacos , Péptidos/química , Péptidos/farmacología , Trombospondina 1/química , Células A549 , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Péptidos/síntesis química , Conformación Proteica , Estabilidad Proteica , Relación Estructura-Actividad , Trombospondina 1/farmacologíaRESUMEN
Recently, we reported a novel role for KMO in the pathogenesis of acute pancreatitis (AP). A number of inhibitors of kynurenine 3-monooxygenase (KMO) have previously been described as potential treatments for neurodegenerative conditions and particularly for Huntington's disease. However, the inhibitors reported to date have insufficient aqueous solubility relative to their cellular potency to be compatible with the intravenous (iv) dosing route required in AP. We have identified and optimized a novel series of high affinity KMO inhibitors with favorable physicochemical properties. The leading example is exquisitely selective, has low clearance in two species, prevents lung and kidney damage in a rat model of acute pancreatitis, and is progressing into preclinical development.
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Inhibidores Enzimáticos/farmacología , Quinurenina 3-Monooxigenasa/antagonistas & inhibidores , Pancreatitis/tratamiento farmacológico , Enfermedad Aguda , Animales , Inhibidores Enzimáticos/uso terapéutico , Humanos , RatasRESUMEN
To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-(5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched chain amino acids as a consequence of BCATm inhibition.
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Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.
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Mitocondrias/enzimología , Transaminasas/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Cristalografía por Rayos X , Ensayos Analíticos de Alto Rendimiento , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.
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Benzoxazoles/farmacología , Quinurenina 3-Monooxigenasa/antagonistas & inhibidores , Insuficiencia Multiorgánica/genética , Oxazolidinonas/farmacología , Pancreatitis/genética , Propionatos/farmacología , ARN Mensajero/metabolismo , Enfermedad Aguda , Animales , Cromatografía Liquida , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Células HEK293 , Hepatocitos/metabolismo , Humanos , Técnicas In Vitro , Riñón/metabolismo , Riñón/patología , Quinurenina 3-Monooxigenasa/genética , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Noqueados , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/patología , Páncreas/metabolismo , Páncreas/patología , Pancreatitis/complicaciones , Pancreatitis/patología , Ratas , Espectrometría de Masas en Tándem , Triptófano/metabolismoRESUMEN
As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-N-methyl-2'-(methylsulfonamido)-[1,1'-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki-Miyaura cross-coupling showed BCATm inhibitory activity with IC50 = 2.0 µM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors.
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The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.
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Proteínas Mitocondriales/antagonistas & inhibidores , Pirazoles/química , Pirimidinonas/química , Transaminasas/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Animales , Cristalografía por Rayos X , Humanos , Isoleucina/sangre , Leucina/sangre , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Pirazoles/síntesis química , Pirazoles/farmacología , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Relación Estructura-Actividad , Transaminasas/química , Valina/sangreRESUMEN
Identification of indazole derivatives acting as dual angiotensin II type 1 (AT1) receptor antagonists and partial peroxisome proliferator-activated receptor-γ (PPARγ) agonists is described. Starting from Telmisartan, we previously described that indole derivatives were very potent partial PPARγ agonists with loss of AT1 receptor antagonist activity. Design, synthesis and evaluation of new central scaffolds led us to the discovery of pyrrazolopyridine then indazole derivatives provided novel series possessing the desired dual activity. Among the new compounds, 38 was identified as a potent AT1 receptor antagonist (IC50=0.006 µM) and partial PPARγ agonist (EC50=0.25 µM, 40% max) with good oral bioavailability in rat. The dual pharmacology of compound 38 was demonstrated in two preclinical models of hypertension (SHR) and insulin resistance (Zucker fa/fa rat).
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Angiotensina II/metabolismo , Descubrimiento de Drogas , Indazoles/farmacología , PPAR gamma/agonistas , Animales , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/síntesis química , Indazoles/química , Ratones , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.
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Starting from the structure of Telmisartan, a new series of potent and selective PPARgamma modulators was identified. The synthesis, in vitro and in vivo evaluation of the most potent compounds are reported and the X-ray structure of compound 7b bound to the PPARgamma ligand binding domain is described.
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Diseño de Fármacos , Indoles/síntesis química , PPAR gamma/efectos de los fármacos , Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Bencimidazoles/química , Bencimidazoles/farmacología , Benzoatos/química , Benzoatos/farmacología , Cristalografía por Rayos X , Humanos , Indoles/química , Indoles/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Ratas , Relación Estructura-Actividad , TelmisartánRESUMEN
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the LDL receptor (LDLr) in hepatocytes, and its expression in mouse liver has been shown to decrease with fenofibrate treatment. METHODS: We developed a sandwich ELISA using recombinant human PCSK9 protein and 2 affinity-purified polyclonal antibodies directed against human PCSK9. We measured circulating PCSK9 concentrations in 115 diabetic patients from the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study before and after fenofibrate treatment. RESULTS: We found that plasma PCSK9 concentrations correlate with total (r = 0.45, P = 0.006) and LDL (r = 0.54, P = 0.001) cholesterol but not with triglycerides or HDL cholesterol concentrations in that cohort. After 6 weeks of treatment with comicronized fenofibrate (200 mg/day), plasma PCSK9 concentrations decreased by 8.5% (P = 0.041 vs pretreatment). This decrease correlated with the efficacy of fenofibrate, as judged by a parallel reduction in plasma triglycerides (r = 0.31, P = 0.015) and LDL cholesterol concentrations (r = 0.27, P = 0.048). CONCLUSIONS: We conclude that this decrease in PCSK9 explains at least in part the LDL cholesterol-lowering effects of fenofibrate. Fenofibrate might be of interest to further reduce cardiovascular risk in patients already treated with a statin.
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Colesterol/sangre , Diabetes Mellitus/tratamiento farmacológico , Fenofibrato/uso terapéutico , Hipolipemiantes/uso terapéutico , Serina Endopeptidasas/sangre , Biomarcadores/sangre , Línea Celular Tumoral , LDL-Colesterol/sangre , Diabetes Mellitus/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Fenofibrato/farmacología , Humanos , Hipolipemiantes/farmacología , Masculino , Persona de Mediana Edad , Plasma , Proproteína Convertasa 9 , Proproteína ConvertasasRESUMEN
OBJECTIVE: Exercise increases fatty acid oxidation (FAO), improves serum high density lipoprotein cholesterol (HDLc) and triglycerides (TG), and upregulates skeletal muscle peroxisome proliferator activated receptor (PPAR)delta expression. In parallel, PPARdelta agonist-upregulated FAO would induce fatty-acid uptake (via peripheral lipolysis), and influence HDLc and TG-rich lipoprotein particle metabolism, as suggested in preclinical models. METHODS AND RESULTS: Healthy volunteers were allocated placebo (n=6) or PPARdelta agonist (GW501516) at 2.5 mg (n=9) or 10 mg (n=9), orally, once-daily for 2 weeks while hospitalized and sedentary. Standard lipid/lipoproteins were measured and in vivo fat feeding studies were conducted. Human skeletal muscle cells were treated with GW501516 in vitro and evaluated for lipid-related gene expression and FAO. Serum TG trended downwards (P=0.08, 10 mg), whereas TG clearance post fat-feeding improved with drug (P=0.02). HDLc was enhanced in both treatment groups (2.5 mg P=0.004, 10 mg P<0.001) when compared with the decrease in the placebo group (-11.5+/-1.6%, P=0.002). These findings complimented in vitro cell culture results whereby GW501516 induced FAO and upregulated CPT1 and CD36 expression, in addition to a 2-fold increase in ABCA1 (P=0.002). However, LpL expression remained unchanged. CONCLUSIONS: This is the first report of a PPARdelta agonist administered to man. In this small study, GW501516 significantly influenced HDLc and TGs in healthy volunteers. Enhanced in vivo serum fat clearance, and the first demonstrated in vitro upregulation in human skeletal muscle fat utilization and ABCA1 expression, suggests peripheral fat utilization and lipidation as potential mechanisms toward these HDL:TG effects.
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Lipoproteínas HDL/metabolismo , PPAR delta/agonistas , Tiazoles/farmacología , Triglicéridos/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adulto , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células Cultivadas , Ácidos Grasos/metabolismo , Humanos , Masculino , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Oxidación-Reducción/efectos de los fármacos , PPAR delta/genética , PPAR delta/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Protein kinase C (PKC)-mediated signal transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the role of various PKC isoforms in sphingosine 1-phosphate (S1P)-stimulated endothelial cells is not well understood. PKCalpha and PKCepsilon activity are increased in endothelial cell cultures, and S1P receptor transfection studies indicate S1P(3) stimulates PKCalpha and S1P1 leads to PKCepsilon activity. Infection of endothelial cells with dominant negative (DN)PKCalpha adenovirus reduces cell migration and greatly inhibits morphogenesis in cells stimulated with S1P. This effect is specific to PKCalpha, as infection with DN PKCepsilon does not alter either migration or morphogenesis. The PKC-specific chemical inhibitor GF109203X also inhibits these two responses. Infection of endothelial cells with dominant negative PKCalpha reduces S1P-induced calcium rise. This maximal rise requires calcium uptake, but it does not require enzymatic activity of the kinase. Pretreatment of these cells with the PKC-specific inhibitor GF109203X does not inhibit S1P-induced calcium rise. S1P-induced morphogenesis but not cell migration is critically dependent on extracellular calcium. Pretreatment of endothelial cells with phorbol 12-myristate 13-acetate for 5min abolishes S1P-stimulated rise in calcium but had little or no effect on migration. The PMA-inhibited calcium rise can be prevented by PKC inhibitor or infection with dominant negative PKCalpha.
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Endotelio Vascular/fisiología , Lisofosfolípidos/fisiología , Proteína Quinasa C-alfa/fisiología , Esfingosina/análogos & derivados , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Humanos , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-epsilon/fisiología , Transducción de Señal/fisiología , Esfingosina/fisiología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The alkaloid drug berberine (BBR) was recently described to decrease plasma cholesterol and triglycerides (TGs) in hypercholesterolemic patients by increasing expression of the hepatic low density lipoprotein receptor (LDLR). Using HepG2 human hepatoma cells, we found that BBR inhibits cholesterol and TG synthesis in a similar manner to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Significant increases in AMPK phosphorylation and AMPK activity were observed when the cells were incubated with BBR. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK, correlated with a subsequent increase in fatty acid oxidation. All of these effects were abolished by the mitogen-activated protein kinase kinase inhibitor PD98059. Treatment of hyperlipidemic hamsters with BBR decreased plasma LDL cholesterol and strongly reduced fat storage in the liver. These findings indicate that BBR, in addition to upregulating the LDLR, inhibits lipid synthesis in human hepatocytes through the activation of AMPK. These effects could account for the strong reduction of plasma TGs observed with this drug in clinical trials.
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Adenilato Quinasa/metabolismo , Berberina/farmacología , Hipolipemiantes/farmacología , Lípidos/biosíntesis , Acetil-CoA Carboxilasa/metabolismo , Aminoimidazol Carboxamida/farmacología , Animales , Línea Celular Tumoral , Cricetinae , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/metabolismo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Fosforilación/efectos de los fármacos , Receptores de LDL/metabolismoRESUMEN
AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.
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Hígado Graso/enzimología , Hepatocitos/fisiología , Hipoglucemia/enzimología , Hígado/enzimología , Complejos Multienzimáticos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Activadas por AMP , Animales , Secuencia de Bases , Glucemia/metabolismo , Clonación Molecular , Cartilla de ADN , Activación Enzimática , Ácidos Grasos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
The enzyme sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that acts extracellularly on G protein-coupled receptors of the S1P(1)/EDG-1 subfamily. Although S1P is formed in the cytosol of various cells, S1P release is not understood and is controversial because this lipid mediator is also regarded as a second messenger. In this report, we describe the existence of an extracellular S1P-generating system in vascular endothelial cells. Endothelial cells release SK constitutively and form S1P in the range of receptor stimulation. Levels of sphingosine but not ATP in the extracellular environment are rate-limiting. Treatment of endothelial cells with small interfering RNA for SK-1 transcript specifically inhibited SK export, and SK-1-transfected human embryonic kidney 293 cells exhibited enhanced release of SK-1. The export of SK-1 is constitutive and is inhibited by cytochalasin D and treatment at 4 degrees C but not by brefeldin A or nocodazole, suggesting that a nonclassical secretory pathway that requires the actin cytoskeleton dynamics is involved. Because S1P regulates angiogenesis and vascular maturation, we overexpressed SK-1 using an adenoviral vector in vivo in the Matrigel system of angiogenesis. Overexpression of SK-1 resulted in enhanced release of SK activity and induced angiogenesis and vascular maturation. These findings suggest that S1P is made in the extracellular milieu and that extracellular export of SK contributes to the action of S1P in the vascular system.