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

The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery

Autophagosome formation involves the sequential actions of conserved ATG proteins to coordinate the lipidation of the ubiquitin-like modifier Atg8-family proteins at the nascent phagophore membrane. Although the molecular steps driving this process are well understood, the source of membranes for the expanding phagophore and their mode of delivery are only now beginning to be revealed. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12–ATG5 conjugate, a crucial player during Atg8-family protein lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins alters autophagic flux in cell-based starvation assays. To determine whether the ATG12–ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. We report changes in the relative proportions of individual cell surface proteins and show that cell surface levels of the SLC7A5-SLC3A2 amino acid transporter are influenced by autophagy capability. Our data provide evidence for direct regulatory coupling between the ATG12–ATG5 conjugate and the clathrin membrane trafficking system and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery. Abbreviations: ATG, autophagy related; BafA1, bafilomycin A(1); GFP, green fluorescent protein; HIP1R, huntingtin interacting protein 1 related; MEF, mouse embryo fibroblast; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha; SILAC, stable isotope labelling with amino acids in culture; SQSTM1, sequestosome 1; STRING, search tool for the retrieval of interacting genes/protein

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

モルフォーゲン活性を有する新規生理活性脂質S1Pの生理機能の分子生物学的解析

金沢大学医学系研究科Edg5ノックアウト(KO)マウス、S1P合成酵素Sphキナーゼトランスジェニック(Tg)マウス、S1P分解酵素S1PリアーゼTgマウスを作出した。また、これと平行してS1P・Edg受容体システムによる二方向性細胞運動制御の分子機構の解析を進めた。Edg5 KOマウスはメンデル則にしたがって出生した後、離乳までに半数近くが死亡するほか、成長後も死亡率が高い。Edg5KOマウスに10mmHg程度の血圧低下、Edg5KO雌マウスに子宮形成不全、少仔傾向を認める他は明らかな形態形成の異常は見られない。現在死因を解析中である。Sphキナーゼ、S1PリアーゼTgマウスでは、創傷治癒ならびに血管傷害後内皮肥厚のそれぞれ促進、遅延を認めている。Edg5による化学遊走及びRacの抑制は、三量体G蛋白質G_と、そのエフェクターRhoを介していることを初めて明らかにした。Edg3はEdg5と同様にG_に共役してRhoを活性化するが、Edg5とは異なりRacを活性化して遊走を促進した。Edg3のこの作用は百日咳毒素前処理により消失し、逆にEdg5同様Rac活性ならびに遊走の抑制に転じたことから、Edg3では優勢なGi共役がG_を介した抑制作用をマスクしている結果、Rac活性及び遊走が促進されることが判明した。またさらに、マウスB16メラノーマ細胞を用いたin vivo浸潤、尾静脈注射後肺転移のモデルシステムを活用し、癌の浸潤・転移を典型例とする生体内細胞運動が、発現するEdg受容体アイソフォーム依存性に特異的に制御されていることを初めて明らかにした。We tried to determine physiological and pathological roles of the sphingosine-1-phosphate (S1P)/Edg receptor axis, by using gene targeting and transgenic techniques and cell biological analysis.We generated Edg5 knockout mice, by the standard method. We found that mice homozygous for null mutation of Edg5 receptor gene are born, accordingly to the Mendelian law. Gross abnormality in the appearance and behavior of Edg5-/- mice is not observed. After birth, approximately 40 % of Edg5-/- mice die within 1 month. The reason of this increased mortality in the early stage is not known at present. Adult Edg5-/- mice display slightly lower blood pressure. Sphingosine kinase is a rate-limiting enzyme for the production of S1P. In sphingosine kinase-transgenic mice, the wound-healing process is promoted compared to wild type mice.Edg5 has unique activities to suppress cell migration and cellular Rac, different from Edg1 or Edg3, which both inhibit Rac activity cell migration. We found for the first time that Edg5 couples to inhibition of Rac via G12/13 and the small GTPase Rho. We observed that Edg5 exerts inhibition of cell motility and invasion of tumor cells, resulting in suppression of metastasis in vivo.研究課題/領域番号:13470008, 研究期間(年度):2001 – 2002出典：「モルフォーゲン活性を有する新規生理活性脂質S1Pの生理機能の分子生物学的解析」研究成果報告書　課題番号13470008（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-13470008/134700082002kenkyu_seika_hokoku_gaiyo/)を加工して作

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

遺伝子改変マウスを用いた生理活性脂質S1P・Edg受容体システムの統合的研究

スフィンゴシン-1-リン酸(S1P)/Edg受容体システムの心血管における生理機能および心血管系および腫瘍における病態生理的役割を様々な遺伝子改変マウスを用いて解析し、さらにヒト疾患モデルを作成して治療法開発のための基礎研究を行った。S1P受容体のひとつであるEdg5は、血管壁では主に平滑筋に発現している。Edg5ノックアウト(KO)マウスは野生型マウスに比較して低血圧を示した。アンギオテンシンII・ACTH負荷、NO合成酵素阻害薬負荷に対する昇圧反応には差異が見られなかった。現在、マウス血管のRho経路の活性を検討中である。マウス後肢虚血モデルにおいて野生型マウス虚血局所へのS1Pの投与は虚血後血管新生を促進して血流回復を早めた。また、S1P産生が亢進しているスフィンゴシンキナーゼ(SphK)トランスジェニック(Tg)マウスでは野生型マウスに比して虚血後血管新生が促進していた。また、インビトロ管腔形成アッセイでEdg5受容体遮断はS1Pの血管新生作用を増強した。Edg1を過剰発現するEdg1-Tgマウスが生後3カ月より進行性の心肥大を呈することを見い出した。心筋細胞ではEdg1発現が亢進しており、ERKリン酸化も増加していた。また、心肥大はアンギオテンシンIIシグナルに依存していた。Edg5を介した癌(B16)細胞遊走抑制はRhoに依存していたがRhoキナーゼには依存せず、またPTENを必要としなかった。Edg5は同細胞のフィブロネクチンへの接着を強く抑制した。宿主Edg1受容体の活性化が癌細胞の尾静脈注入後肺転移を促進することを見いだした。以上の結果は、S1P/Edgシステムが血圧調節などの心血管ホメオスターシスや心肥大・血管新生・腫瘍転移などの病態生理において重要な役割を有していることを示して+おり、S1P/Edgシステムを標的とした治療が有効である可能性を示唆する。We investigated physiological and pathophysiological roles of sphingosine-l-phosphate (S1P) and its receptors in the cardiovascular system and tumor progression. Slp exerts its pleiotropic effects through acting on the Edg family receptors. The mice that is null for the S1P receptor Edg5 exhibits hypotension compared with wild type littermates. The Edg5-knockout (Edg5-KO) mice showed similar extents of pressor responses to angiotensin II, ACTH, and the nitric oxide synthase inhibitor L-NAME, as the wild type mice. In the murine hindlimb ischemic model due to femoral artery ligation, daily local intramuscular injection into the ischemic limb promoted the recovery of the blood flow with a concomitant increase in the capillary density in the ischemic limb. The PLGA-based slow-releasing S1P that we invented was also effective in stimulating the blood flow recovery. We also found that the mice transgenic for the S1P-synthesizing enzyme sphingosine kinase-1 showed stimulated recovery in the post-ischemic blood flow. In the in vitro capillary-like tube formation assay, the anti-migratory receptor Edg5 inhibited tube formation, and the blockade of Edg5 stimulated tube formation. Thus, Edg5 is likely anti-angiogenic receptor. Edgl is expressed in cardiomyocytes, however, its role is not known. We found that the mice transgenic for Edgl exhibited cardiac hypertrophy, which was prevented by an AT1 receptor antagonist. In the murine tail vein injection model, an Edgl agonist stimulated lung metastasis of B16 melanoma cells probably through acting on Edgl in the host tissues. These results collectively indicate that S1P-Edg system plays important roles in the cardiovascular homeostasis and the cardiovascular and tumor pathophysiology, and suggest potential usefulness of novel therapeutic strategy to target the S1P-Edg system.研究課題/領域番号:17390054, 研究期間(年度):2005-2006出典：「遺伝子改変マウスを用いた生理活性脂質S1P・Edg受容体システムの統合的研究」研究成果報告書　課題番号17390054 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作