G protein-coupled sphingosine-1-phosphate receptors: potential molecular targets for angiogenic and anti-angiogenic therapies

Sphingosine-1-phosphate (S1P) is a plasma lipid mediator with pleiotropic activities; it is constitutively produced in red blood cells and vascular endothelial cells through phosphorylation of sphingosine by one of two S1P synthesizing enzymes, sphingosine kinase 1 and 2 (SphK 1, 2), and exported into plasma to bind to high density lipoprotein and albumin. Sphingosine-1-phosphate acts through five members of the G protein-coupled S1P receptors (S1PR1-S1PR5) to exert diverse actions, which include vascular maturation in embryonic stage and postnatal angiogenesis, maintenance of functional integrity of vascular endothelium, regulation of vascular tonus, and lymphocyte trafficking. Sphingosine-1-phosphate is unique in its ability to regulate cell migration either positively or negatively by acting through different receptor subtypes. S1PR1 and S1PR3 mediate chemotactic cell migration toward S1P via Gi/Rac pathway, whereas S1PR2 mediates S1P inhibition of chemotaxis via G12/13/Rho-dependent inhibition of Rac. Sphingosine-1-phosphate positively or negatively regulates tumor cell migration, invasion in Matrigel, and hematogenous metastasis in manners strictly dependent on S1P receptor subtypes expressed in tumor cells. S1PR1 (and S1PR3) also mediates activation of Gi/phosphatidylinositol 3-kinase (PI3K)/Akt and stimulation of cell proliferation/survival, whereas S1PR2 could mediate suppression of cell proliferation/survival through G12/13/Rho/Rho kinase/PTEN-dependent Akt inhibition. S1PR1 (and S1PR3) expressed in endothelial cells mediates angiogenic action of S1P by stimulating endothelial cell migration, proliferation and tube formation. In a mouse model of hindlimb ischemia after femoral artery resection, repeated local administration or sustained delivery of S1P, or transgenic overexpression of SphK1, accelerates post-ischemic angiogenesis, through the S1P actions on both endothelial cells and bone marrow-derived myeloid cells (BMDCs). In tumor cells, SphK1 is upregulated especially in advanced stages, through mechanisms involving both activating Ras mutation and hypoxia, which leads to increased S1P production and also decreased cellular content of pro-apoptotic sphingolipid ceramide, a metabolic precursor of S1P. Apoptotic tumor cells also produce S1P through SphK2 activation, thus implicated in tumor angiogenesis by acting on endothelial cells through S1PR1/S1PR3, as well as tumor-infiltrating macrophages and BMDCs. Inhibition of S1PR1 function by either an anti-S1P antibody or FTY720 inhibits tumor angiogenesis and tumor growth. Differently from S1PR1, S1PR2 expressed in host cells mediates inhibition of tumor angiogenesis and tumor growth, through mechanisms involving the suppression of endothelial cell migration, proliferation and tube formation, and inhibition of BMDC recruitment to tumor stroma with suppressed expression of pro-angiogenic factor and matrix metalloprotease 9. These findings provide the molecular basis for S1P receptor subtype-selective targeting strategies aiming at angiogenic therapy for occlusive peripheral arterial diseases, and anti-angiogenic and anti-tumor therapies against cancer.Biomedical Reviews 2011; 22: 15-29

Calcium-dependent regulation of Rho and myosin phosphatase in vascular smooth muscle

Phosphorylation of 20 kD myosin light chain (MLC) is a critical process in eliciting smooth muscle contraction. Excitatory receptor agonists increase the extent of MLC phosphorylation by both activating myosin light chain kinase (MLCK) and inhibiting myosin phosphatase (MP). Activation of MLCK is dependent on Ca2+ and calmodulin, while inhibition of MP is dependent on the small guanosine triphosphatase Rho and Rho kinase. Receptor agonists were previously shown to induce Rho activation via the heterotrimeric G12/13 protein, largely in non-muscle cells. We recently discovered the novel Ca2+-dependent activation of Rho in vascular smooth muscle. This Ca2+-dependent Rho activation mechanism operates upon stimulation of vascular smooth muscle by either membrane depolarization or Gq-coupled vasoconstrictor receptors. Thus, Ca2+ induces MLC phosphorylation through both MLCK stimulation and MP inhibition. We found that phosphoinositide 3-kinase class II . isoform (PI3K-C2.) is involved in the Ca2+-dependent Rho activation and MP inhibition. PI3K-C2. appears to participate in regulation of vascular Rho activity and tone in vivo. These observations also indicate that PI3Ks exert isoform-specificeffectsonvasculartonethrough mechanisms involving regulation of endothelial nitric oxide production and smooth muscle MP activity.Biomedical Reviews 2005; 16: 13-21

Sphingosine-1-phosphate signaling and biological activities in the cardiovascular system

金沢大学医薬保健研究域医学系The plasma lysophospholipid mediator sphingosine-1-phosphate (S1P) is produced exclusively by sphingosine kinase (SPHK) 1 and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S1P receptors, S1P1, S1P2 and S1P3. S1P1 expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S1P2, is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G12/13-and Rho-dependent mechanism. In rat neointimal VSMCs, S1P1 is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S1P3 expressed on endothelial cells also mediates chemotaxis toward S1P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S1P3 expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S1P3, together with S1P2 and SPHK1, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK1 is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S1P activation of the S1P3 signaling. We also demonstrated that exogenously administered S1P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S1P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer. © 2008 Elsevier B.V. All rights reserved

Sphingosine-1-phosphate signaling in physiology and diseases

Sphingosine-1-phosphate (S1P), which acts as both the extracellular and intracellular messenger, exerts pleiotropic biological activities including regulation of formation of the vasculature, vascular barrier integrity, and lymphocyte trafficking. Many of these S1P actions are mediated by five members of the G protein-coupled S1P receptors (S1P 1-S1P 5) with overlapping but distinct coupling to heterotrimeric G proteins. The biological activities of S1P are based largely on the cellular actions of S1P on migration, adhesion, and proliferation. Notably, S1P often exhibits receptor subtype-specific, bimodal effects in these cellular actions. For example, S1P 1 mediates cell migration toward S1P, that is, chemotaxis, via G i/Rac pathway whereas S1P 2 mediates inhibition of migration toward a chemoattractant, that is, chemorepulsion, via G 12/13/Rho pathway, which induces Rac inhibition. In addition, S1P 1 mediates stimulation of cell proliferation through the G i-mediated signaling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and ERK whereas S1P 2 mediates inhibition of cell proliferation through mechanisms involving G 12/13/Rho/Rho kinase/PTEN-dependent Akt inhibition. These differential effects of S1P receptor subtypes on migration and proliferation lead to bimodal regulation of various biological responses. An observed biological response is likely determined by an integrated outcome of the counteracting signals input by S1P receptor subtypes. More recent studies identified the new intracellular targets of S1P including the inflammatory signaling molecule TRAF2 and histone deacetylases HDAC1 and HDAC2. These interactions of S1P regulate NF-κB activity and gene expression, respectively. Development of S1P receptor agonists and antagonists with improved receptor subtype-selectivity, inhibitors, or modulators of sphingolipid-metabolizing enzymes, and their optimal drug delivery system provide novel therapeutic tactics. © 2012 International Union of Biochemistry and Molecular Biology, Inc

Signaling mechanisms for positive and negative regulation of cell motility by sphingosine-1-phosphate receptors

Sphingosine-1-phosphate (S1P) exerts positive and negative effects on cell migration apparently in a cell-type-dependent manner. Our data suggest that the bimodal actions of S1P on cell migration is due to receptor subtype-specific positive and negative regulation of Rho family GTPase, Rac; S1P1 and S1P3 mediate Rac stimulation and chemotaxis whereas S1P2 mediates Rac inhibition and chemorepulsion. The stimulatory effects of S1P 1 and S1P3 on Rac and, subsequently on migration, are mediated by Gi. The inhibitory effect of SlP2 acts on G12/13 and Rho. S1P exerts inhibitory effects on some tumor cell migration and invasion via S1P2. S1P2 also mediates the inhibition of hematogenous metastasis. In contrast, exogenously expressed S1P1 has the reverse effect, it stimulates invasion and metastasis. S1P also exerts a similar bimodal action on vascular endothelial cells and, thereby, angiogenesis. The examples suggest that control of S1P receptor activity using a receptor subtype-specific agonist and antagonist may have beneficial effects on disorders, including cancer, and vascular diseases. © Springer-Verlag Tokyo 2006. All rights reserved.[Book Chapter] Y. Hirabayashi, Y. Igarashi, A.H. Merrill, Jr. (eds.), Sphingolipid biology, Springer-Verlag, c200

Phosphatidylinositol 3-kinase class II α-isoform PI3K-C2α is required for transforming growth factor β-induced smad signaling in endothelial cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P1 and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc

Sphingosine-1-phosphate as a mediator involved in development of fibrotic diseases

Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-β (TGFβ), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFβ signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFβ, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research. © 2012 Elsevier B.V

Sphingosine-1-phosphate receptor 2 protects against anaphylactic shock through suppression of endothelial nitric oxide synthase in mice

Background: Sphingosine-1-phosphate receptor 2 (S1P2) is expressed in vascular endothelial cells (ECs). However, the role of S1P2 in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P2 inhibits Akt, an activating kinase of eNOS. Objective: We tested the hypothesis that endothelial S1P2 might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. Methods: Mice deficient in S1P2 and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. Results: S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of β-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. Conclusion: S1P2 diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P2 agonists as novel therapeutic agents for anaphylaxis. © 2013 American Academy of Allergy, Asthma & Immunology

S1P2, the G protein-coupled receptor for sphingosine-1- phosphate, negatively regulates tumor angiogenesis and tumor growth in vivo in mice

金沢大学医薬保健研究域医学系Sphingosine-1-phosphate (S1P) has been implicated in tumor angiogenesis by acting through the Gi-coupled chemotactic receptor S1P1. Here, we report that the distinct receptor S1P2 is responsible for mediating the G12/13/Rho-dependent inhibitory effects of S1P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S1P2LacZ/+ mice, we found that S1P2 was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD11b-positive bone marrow-derived cells (BMDC). Lewis lung carcinoma or B16 melanoma cells implanted in S1P2-deficient (S1P2-/-) mice displayed accelerated tumor growth and angiogenesis with enhanced association of vascular smooth muscle cells and pericytes. S1P2-/- ECs exhibited enhanced Rac activity, Akt phosphorylation, cell migration, proliferation, and tube formation in vitro. Coinjection of S1P2-/- ECs and tumor cells into wild-type mice also produced a relative enhancement of tumor growth and angiogenesis in vivo. S1P2-/- mice were also more efficient at recruiting CD11b-positive BMDCs into tumors compared with wild-type siblings. Bone marrow chimera experiments revealed that S1P2 acted in BMDCs to promote tumor growth and angiogenesis. Our results indicate that, in contrast to endothelial S1P1, which stimulates tumor angiogenesis, S1P 2 on ECs and BMDCs mediates a potent inhibition of tumor angiogenesis, suggesting a novel therapeutic tactic for anticancer treatment. ©2010 AACR

G12/13 and Gq mediate S1P2-induced inhibition of Rac and migration in vascular smooth muscle in a manner dependent on Rho but not Rho kinase

金沢大学医薬保健研究域医学系Aims: The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs. Methods and results: S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P2 receptor antagonist JTE-013. The C-terminal peptides of Gα subunits (Gα-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Gα12-CT, Gα13-CT, and Gα q-CT, but not that of Gαs-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G12/13 and Gq classes are necessary for the S1P inhibition. The expression of Gαq-CT as well as Gα12-CT and Gα13-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT1, which robustly couples to Gq, did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of Gq alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT1 receptor fused to Gα12 was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway. Conclusion: These observations together suggest that S1P2 mediates inhibition of Rac and migration through the coordinated action of G 12/13 and Gq for Rho activation in VSMCs. © The Author 2008.