Nicolas Ancellin

Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate.

Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

Akt-mediated phosphorylation of the G protein-coupled receptor EDG-1 is required for endothelial cell chemotaxis.

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.

Differential Pharmacological Properties and Signal Transduction of the Sphingosine 1-Phosphate Receptors EDG-1, EDG-3, and EDG-5

Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In this report, pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-1, -3, and -5 are characterized using aXenopus oocyte expression system, which lacks endogenous SPP receptors. Microinjection of the EDG-3 and EDG-5 but not EDG-1 mRNA conferred SPP-responsive intracellular calcium transients; however, the EDG-5 response was quantitatively much less. Co-expression of EDG-1 receptor with the chimeric Gαqi protein conferred SPP responsiveness. Gαqi or Gαqco-injection also potentiated the EDG-5 and EDG-3 mediated responses to SPP. These data suggest that SPP receptors couple differentially to the Gq and Gi pathway. All three GPCRs were also activated by sphingosylphosphorylcholine, albeit at higher concentrations. None of the other related sphingolipids tested stimulated or blocked SPP-induced calcium responses. However, suramin, a polycyclic anionic compound, selectively antagonized SPP-activated calcium transients in EDG-3 expressing oocytes with an IC50of 22 μm, suggesting that it is an antagonist selective for the EDG-3 GPCR isotype. We conclude that the three SPP receptors signal differentially by coupling to different G-proteins. Furthermore, because only EDG-3 was antagonized by suramin, variations in receptor structure may determine differences in antagonist selectivity. This property may be exploited to synthesize receptor subtype-specific antagonists.

Sphingosine-1-phosphate: extracellular mediator or intracellular second messenger?

Sphingosine-1-phosphate (SPP), a polar sphingolipid metabolite, has received much attention recently as an extracellular mediator and an intracellular second messenger. It regulates a wide range of biological responses such as cell growth, death, differentiation, and migration. Recent identification of plasma membrane receptors and the cloning of SPP metabolizing enzymes have increased our understanding of the biology of SPP synthesis and action. However, controversy exists regarding the mode of action of this molecule. EDG-1 and related G-protein-coupled receptors were identified recently as plasma membrane receptors for SPP. In light of this recent discovery, many of the functions of SPP previously thought to be due to intracellular second messenger action should be reevaluated. In addition, signaling properties and functions of the three known receptors for SPP need to be fully delineated. The structures and the evolutionary conservation of SPP metabolizing enzymes from yeast to mammals support the hypothesis that SPP also plays a role as an intracellular second messenger. However, definitive assignment of the intracellular role of SPP awaits purification/molecular cloning of elusive intracellular receptors. Better knowledge of the molecular basis of SPP action is needed to assess the physiological and pathophysiological significance of this bioactive lipid mediator.

Sphingosine-1-phosphate signaling via the EDG-1 family of G-protein-coupled receptors.

Abstract: The bioactive lipid sphingosine‐1‐phosphate (SPP) is abundantly formed and released during the activation of platelets by thrombotic stimuli. Once exported, SPP interacts with the G‐protein‐coupled receptors (GPCR) of the EDG‐1 family. SPP binds to EDG‐1 with the dissociation constant of ∼8 nM and induces signal transduction events such as mitogen‐activated protein kinase (MAP kinase) activation, decrease of cAMP levels, remodeling of the actin cytoskeleton, among others. EDG‐1 is a prototypical member of a large family of GPCRs that interact with glycero‐ and sphingolysolipid phosphates, namely, SPP and lysophosphatidic acid (LPA). Three other GPCRs, trivially termed EDG‐3, EDG‐5, and EDG‐8, are also high‐affinity receptors for SPP. The four SPP receptor subtypes regulate different intracellular signal transduction pathways. In vascular endothelial cells, cooperative signaling between EDG‐1 and EDG‐3 subtypes of SPP receptors results in adherens junction assembly, cell survival, morphogenesis into capillary‐like networks, and angiogenesis. SPP acts distinctly, albeit cooperatively, with polypeptide angiogenic factors, resulting in the formation of mature neovessels. Thus SPP signaling as an extracellular mediator via the EDG‐1 family of GPCRs may be a heretofore unrecognized mechanism for the regulation of angiogenesis and vascular endothelial cell function.

Switching Intracellular Signaling Pathways to Study Sphingosine 1‐Phosphate Receptors

Sphingosine-1-phosphate (SPP) is a potent bioactive lipid mediator generated from ceramide by the sphingosine kinase pathway.1 SPP, released by activated platelets, triggers activation of multiple intracellular signaling pathways, including phospholipase C and D, mitogen-activated protein kinase, protein kinase C, as well as small GTP proteins Rho and Rac.2,3 How this lipid plays a role as an intracellular second messenger or an extracellular mediator is controversial.4 However, three G-proteincoupled receptors (GPCR), namely EDG-1, -3, and AGR16/H218/EDG-5, are activated by SPP. A cDNA encoding EDG-1 was originally cloned as a phorbol esterinducible immediate early gene from vascular endothelial cells.5 Subsequently, several related orphan receptors, including EDG-36 and EDG-5,7 were cloned. As these proteins are widely expressed in a variety of tissues, it is important to address how these three receptors for the same ligand trigger distinct signaling pathways linking to specific cellular functions. The presence of EDG receptors (detection of the transcript and/or characterization of SPP-induced intracellular signals) has been detected in many cell lines (HEK293, HEL60, HepG2, CHO, vascular smooth muscle cells, mouse NIH 3T3, monkey COS-1 cells, human bladder carcinoma J82 cells). Thus, characterization of a truly negative cell system is an important first step in investigating the signal transduction and pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-1, -3, and -5. Calcium monitoring by the photoprotein aequorin is a highly sensitive method to study GPCR coupling to Gq-linked signaling pathways in the Xenopus oocyte.8 We decided to use this sensitive assay to monitor intracellular calcium changes. Furthermore we used chimeric G proteins9 to switch the intracellular signaling pathway of any GPCR to the phospholipase Cβ/calcium pathway. In the first set of experiments, wild-type Gαq or chimeric Gα protein Gαqs and Gαqi were expressed in the oocyte. Protein expression was monitored by Western blot analysis. SPP (100 nM) did not induce intracellular calcium mobilization in uninjected, Gαq, chimeric Gαqs, or Gαqi-expressing oocytes. Therefore, Xenopus oocytes do not appear to express any detectable calcium-coupled SPP receptors linked to the Gαq, Gαqi, or Gαqs pathways. In contrast, lysophosphatidic acid stimulated calcium transients efficiently in uninjected oocytes. Next, oocytes were injected with capped complementary RNA to allow expression of the EDG-1, -3, and -5 proteins at the plasma membrane. When stimulated