Coordination between the vascular system and forming organs is essential for proper embryonic development. The vasculature expands by sprouting angiogenesis, during which tip cells form filopodia that incorporate into capillary loops. Although several molecules, such as vascular endothelial growth factor A (Vegfa), are known to induce sprouting, the mechanism that terminates this process to ensure neovessel stability is still unknown. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P(<em>1</em>)) has been shown to mediate interaction between endothelial and mural cells during vascular maturation. In vitro studies have identified S<em>1</em>P(<em>1</em>) as a pro-angiogenic factor. Here, we show that S<em>1</em>P(<em>1</em>) acts as an endothelial cell (EC)-autonomous negative regulator of sprouting angiogenesis during vascular development. Severe aberrations in vessel size and excessive sprouting found in limbs of S<em>1</em>P(<em>1</em>)-null mouse embryos before vessel maturation imply a previously unknown, mural cell-independent role for S<em>1</em>P(<em>1</em>) as an anti-angiogenic factor. A similar phenotype observed when S<em>1</em>P(<em>1</em>) expression was blocked specifically in ECs indicates that the effect of S<em>1</em>P(<em>1</em>) on sprouting is EC-autonomous. Comparable vascular abnormalities in S<em>1</em>p(<em>1</em>) knockdown zebrafish embryos suggest cross-species evolutionary conservation of this mechanism. Finally, genetic interaction between S<em>1</em>P(<em>1</em>) and Vegfa suggests that these factors interplay to regulate vascular development, as Vegfa promotes sprouting whereas S<em>1</em>P(<em>1</em>) inhibits it to prevent excessive sprouting and fusion of neovessels. More broadly, because S<em>1</em>P, the ligand of S<em>1</em>P(<em>1</em>), is blood-borne, our findings suggest a new mode of regulation of angiogenesis, whereby blood flow closes a negative feedback loop that inhibits sprouting angiogenesis once the vascular bed is established and functional.

Angiogenesis is critical for development and repair, and is a prominent feature of many pathological conditions. Based on evidence that insulin-like growth factor binding protein (IGFBP)-3 enhances cell motility and activates <em>sphingosine</em> kinase (SphK) in human endothelial cells, we have investigated whether IGFBP-3 plays a role in promoting angiogenesis. IGFBP-3 potently induced network formation by human endothelial cells on Matrigel. Moreover, it up-regulated proangiogenic genes, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 and -9. IGFBP-3 even induced membrane-type <em>1</em> MMP (MT<em>1</em>-MMP), which regulates MMP-2 activation. Decreasing SphK<em>1</em> expression by small interfering RNA (siRNA), blocked IGFBP-3-induced network formation and inhibited VEGF, MT<em>1</em>-MMP but not IGF-I up-regulation. IGF-I activated SphK, leading to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) formation. The IGF-I effect on SphK activity was blocked by specific inhibitors of IGF-IR, PI3K/Akt and ERK<em>1</em>/2 phosphorylation. The disruption of IGF-I signaling prevented the IGFBP-3 effect on tube formation, SphK activity and VEGF release. Blocking ERK<em>1</em>/2 signaling caused the loss of SphK activation and VEGF and IGF-I up-regulation. Finally, IGFBP-3 dose-dependently stimulated neovessel formation into subcutaneous implants of Matrigel in vivo. Thus, IGFBP-3 positively regulates angiogenesis through involvement of IGF-IR signaling and subsequent SphK/S<em>1</em>P activation.

Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are extracellular lipid mediators that signal through distinct members of the Edg/LP subfamily of G protein-coupled receptors (GPCRs). LPA and S<em>1</em>P receptors are expressed in almost every cell type and can couple to multiple G proteins (G(i), G(q) and G(<em>1</em>2/<em>1</em>3)) to mediate a great variety of responses, ranging from rapid morphological changes to long-term stimulation of cell proliferation. LPA serves as the prototypic GPCR agonist that activates the small GTPases Ras (via G(i)) and RhoA (via G(<em>1</em>2/<em>1</em>3)), leading to activation of the mitogen-activated protein kinase (MAPK) cascade and reorganization of the actin cytoskeleton, respectively. This review focuses on our current insights into how Ras-MAPK signaling is regulated by GPCR agonists in general, and by LPA in particular.

The major sphingolipid metabolite, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), has important biological functions. S<em>1</em>P is the ligand for a family of five G-protein-coupled receptors with distinct signaling pathways that regulate angiogenesis, vascular maturation, immunity, chemotaxis, and other important biological pathways. Recently, clinical trials have targeted S<em>1</em>P receptors (S<em>1</em>PRs) for autoimmune diseases and transplantation and have generated considerable interest in developing additional, more selective compounds. This review summarizes current knowledge on the biology of S<em>1</em>P and S<em>1</em>PRs that forms the basis for future drug development and the treatment of kidney disease.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive lipid mediator, stimulates proliferation and contractility in hepatic stellate cells, the principal matrix-producing cells in the liver, and inhibits proliferation via S<em>1</em>P receptor 2 (S<em>1</em>P(2)) in hepatocytes in rats in vitro. A potential role of S<em>1</em>P and S<em>1</em>P(2) in liver regeneration and fibrosis was examined in S<em>1</em>P(2)-deficient mice. Nuclear 5-bromo-2'-deoxy-uridine labeling, proliferating cell nuclear antigen (PCNA) staining in hepatocytes, and the ratio of liver weight to body weight were enhanced at 48 h in S<em>1</em>P(2)-deficient mice after a single carbon tetrachloride (CCl(4)) injection. After dimethylnitrosamine (DMN) administration with a lethal dose, PCNA staining in hepatocytes was enhanced at 48 h and survival rate was higher in S<em>1</em>P(2)-deficient mice. Serum aminotransferase level was unaltered in those mice compared with wild-type mice in both CCl(4)- and DMN-induced liver injury, suggesting that S<em>1</em>P(2) inactivation accelerated regeneration not as a response to enhanced liver damage. After chronic CCl(4) administration, fibrosis was less apparent, with reduced expression of smooth-muscle alpha-actin-positive cells in the livers of S<em>1</em>P(2)-deficient mice, suggesting that S<em>1</em>P(2) inactivation ameliorated CCl(4)-induced fibrosis due to the decreased accumulation of hepatic stellate cells. Thus, S<em>1</em>P plays a significant role in regeneration and fibrosis after liver injury via S<em>1</em>P(2).

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of <em>sphingosine</em> kinase <em>1</em> (SK<em>1</em>). Exosomes derived from an SK<em>1</em>-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK<em>1</em> exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK<em>1</em> levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK<em>1</em> mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S<em>1</em>PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK<em>1</em>-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, <em>sphingosine</em>, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.

Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor 2 (S<em>1</em>PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S<em>1</em>PR2 on sites distinct from the pocket of the sphingolipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and signals via the G protein G<em>1</em>3, the Rho GEF LARG, and RhoA. Deleting or blocking S<em>1</em>PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S<em>1</em>PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A(-/-) mice, indicating a repressor function of the Nogo-A/S<em>1</em>PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S<em>1</em>PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S<em>1</em>PR2 signaling platforms will provide new insights into regulation of synaptic plasticity.

FTY720 is a <em>sphingosine</em>-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors. Here we demonstrate that FTY720 enhances the activity of the <em>sphingosine</em> transporter Abcb<em>1</em> (Mdr<em>1</em>) and the leukotriene C(4) transporter Abcc<em>1</em> (Mrp<em>1</em>). Both transporters must be active for FTY720-mediated T cell migration and LN homing. Migration and homing driven by FTY720, phosphorylated FTY720, or S<em>1</em>P also require 5-lipoxygenase-mediated synthesis of cysteinyl leukotrienes and their efflux from the cell. FTY720-mediated LN homing events further downstream are dependent on CCL<em>1</em>9, CCL2<em>1</em>, VLA-4alpha, and CD44. Use of T cells deficient in 5-lipoxygenase, Abcb<em>1</em>, and Abcc<em>1</em>, and comparison of the effects of FTY720 with those of S<em>1</em>P, suggest a model of sequential engagement of Abcb<em>1</em>, SP<em>1</em> receptors, 5-lipoxygenase, and Abcc<em>1</em> to enhance T cell migration and homing.

Cysteine-rich protein 6<em>1</em> (Cyr6<em>1</em>/CCN<em>1</em>) is an angiogenic factor and a member of a family of growth factor-inducible immediate-early genes with functions in cell adhesion, proliferation and differentiation. We investigated the regulatory mechanisms and signaling pathways involved in Cyr6<em>1</em>/CCN<em>1</em>gene activation in smooth muscle cells. Treatment of these cells with <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive lysolipid, increased rapidly but transiently the expression of the Cyr6<em>1</em>/CCN<em>1</em> gene at both the mRNA and protein levels. Cyr6<em>1</em>/CCN<em>1</em> mRNA stability was not altered but the transcription rate of the Cyr6<em>1</em>/CCN<em>1</em> gene was increased fivefold in isolated nuclei from S<em>1</em>P-stimulated cells indicating that the level of control is primarily transcriptional. Transfection experiments showed that a 936-bp promoter fragment of the human Cyr6<em>1</em>/CCN<em>1</em> gene is functional and induces a reporter gene activity in S<em>1</em>P-treated cells. Using a combination of cis-element mutagenesis and expression of dominant negative inhibitors of transcription factors, we showed that both a CRE and AP-<em>1</em> site and their cognate transcription factors, cAMP response element binding protein (CREB) and AP-<em>1</em>, were responsible for the promoter activity in S<em>1</em>P-stimulated cells. Furthermore, by using either pharmacological inhibitors or active forms of known signaling molecules, we showed that inducible Cyr6<em>1</em>/CCN<em>1</em> gene expression occurs through RhoA GTPase and that additional signaling through the p38 pathway is required. In particular, p38 seems to regulate Cyr6<em>1</em>/CCN<em>1</em> promoter activity through modulation of phosphorylation of CREB and the CREB kinase, MSK<em>1</em>. These findings demonstrate the transcriptional regulation of the Cyr6<em>1</em>/CCN<em>1</em> gene and provide clues to the signaling molecules and transcription factors involved in such regulation.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em>, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid <em>phosphate</em> phosphatases (<em>1</em>, <em>1</em>a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid <em>phosphate</em> phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid <em>phosphate</em> phosphatases <em>1</em>, <em>1</em>a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid <em>phosphate</em> phosphatase activity. These findings were supported by results showing that lipid <em>phosphate</em> phosphatases <em>1</em>, <em>1</em>a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid <em>phosphate</em> phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid <em>phosphate</em> phosphatases <em>1</em>, <em>1</em>a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.

The lipid mediator <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) confers survival benefits in cardiomyocytes and isolated hearts subjected to oxidative stress. High-density lipoprotein (HDL) is a major carrier of S<em>1</em>P in the serum, but whether HDL-associated S<em>1</em>P directly mediates survival in a preparation composed exclusively of cardiomyocytes has not been demonstrated. Accordingly, we tested the hypothesis that signal activation and survival during simulated ischemia-reperfusion injury in response to HDL require lipoprotein-associated S<em>1</em>P. As a model, we used adult mouse cardiomyocytes subjected to hypoxia-reoxygenation. Cells were treated or not with autologous mouse HDL, which significantly increased myocyte viability as measured by trypan blue exclusion. This survival effect was abrogated by the S<em>1</em>P(<em>1</em>) and SIP(3) receptor antagonist VPC 230<em>1</em>9. The selective S<em>1</em>P(3) antagonist CAY<em>1</em>0444, the G(i) antagonist pertussis toxin, the MEK (MAPK/ERK) kinase inhibitor PD-98059, and the phosphoinositide-3 kinase inhibitor wortmannin also inhibited the prosurvival effect of HDL. We observed that HDL activated both Akt (protein kinase B) and the MEK<em>1</em>/2-ERK<em>1</em>/2 pathway and also stimulated phosphorylation of glycogen synthase kinase-3beta. ERK<em>1</em>/2 activation was through an S<em>1</em>P(<em>1</em>) subtype receptor-G(i) protein-dependent pathway, whereas the activation of Akt was inhibited by CAY<em>1</em>0444, indicating mediation by S<em>1</em>P(3) subtype receptors. We conclude that HDL, via its cargo of S<em>1</em>P, can directly protect cardiomyocytes against simulated oxidative injury in the absence of vascular effects and that prosurvival signal activation is dependent on both S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) subtype receptors.

The anaphylatoxin C5a is produced following the activation of the complement system and is associated with a variety of pathologies, including septic shock and adult respiratory distress syndrome, and with immune complex-dependent diseases such as rheumatoid arthritis. C5a has been shown to regulate inflammatory functions by interacting with its receptor, C5aR, which belong to the rhodopsin family of seven-transmembrane GPCRs. However, the intracellular signaling pathways triggered by C5aR on immune-effector cells are not well understood. In this report we present data showing that, in human monocyte-derived macrophages, C5aR uses the intracellular signaling molecule <em>sphingosine</em> kinase (SPHK)<em>1</em> to trigger various physiological responses. Our data show that C5a rapidly stimulates the generation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, SPHK activity, and membrane translocation of SPHK<em>1</em>. Using an antisense oligonucleotide against SPHK<em>1</em>, we show that knockdown of SPHK<em>1</em> abolishes the C5a-triggered intracellular Ca(2+) signals, degranulation, cytokine generation, and chemotaxis. Our study shows for the first time that SPHK<em>1</em> not only plays a key role in the generation and release of proinflammatory mediators triggered by anaphylatoxins from human macrophages but is also involved in the process of immune cell motility, thus pointing out SPHK<em>1</em> as a potential therapeutic target for the treatment of inflammatory and autoimmune diseases.

Naive T cells continuously recirculate between secondary lymphoid tissue via the blood and lymphatic systems, a process that maximizes the chances of an encounter between a T cell and its cognate antigen. This recirculation depends on signals from chemokine receptors, integrins, and the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor. The authors of previous studies in other cell types have shown that Rac GTPases transduce signals leading to cell migration and adhesion; however, their roles in T cells are unknown. By using both 3-dimensional intravital and in vitro approaches, we show that Rac<em>1</em>- and Rac2-deficient T cells have multiple defects in this recirculation process. Rac-deficient T cells home very inefficiently to lymph nodes and the white pulp of the spleen, show reduced interstitial migration within lymph node parenchyma, and are defective in egress from lymph nodes. These mutant T cells show defective chemokine-induced chemotaxis, chemokinesis, and adhesion to integrin ligands. They have reduced lateral motility on endothelial cells and transmigrate in-efficiently. These multiple defects stem from critical roles for Rac<em>1</em> and Rac2 in transducing chemokine and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> signals leading to motility and adhesion.

Lysophospholipid (LP) research has experienced a period of renaissance with the discovery of the lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors in the late <em>1</em>990s. Vertebrate LP receptors regulate embryogenesis, vascular development, neurogenesis, uterine development, oocyte survival, immune cell trafficking and inflammatory reactions. LP signaling is important in cancer, autoimmunity and inflammatory diseases. Research on LP biology has contributed to the development of a first-generation S<em>1</em>P receptor modulator that has entered phase III clinical trials for the treatment of multiple sclerosis. Further basic research on LP signaling is anticipated to lead to novel therapeutic tools to combat various human diseases.

Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S<em>1</em>P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S<em>1</em>P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium.

Francisella tularensis, the etiological agent of the inhalation tularemia, multiplies in a variety of cultured mammalian cells. Nevertheless, evidence for its in vivo intracellular residence is less conclusive. Dendritic cells (DC) that are adapted for engulfing bacteria and migration towards lymphatic organs could serve as potential targets for bacterial residence and trafficking. Here, we focus on the in vivo interactions of F. tularensis with DC following airway infection of mice. Lethal airway infection of mice with the live vaccine strain (LVS) results in trafficking of a CD<em>1</em><em>1</em>b(high)/CD<em>1</em><em>1</em>c(med)/autofluorescence(low) DC subset from the respiratory tract to the draining mediastinal lymph node (MdLN). Simultaneously, a rapid, massive bacterial colonization of the MdLN occurs, characterized by large bacterial foci formation. Analysis of bacteria in the MdLN revealed a major population of extracellular bacteria, which co-exists with a substantial fraction of intracellular bacteria. The intracellular bacteria are viable and reside in cells sorted for DC marker expression. Moreover, in vivo vital staining experiments indicate that most of these intracellular bacteria ( approximately 75%) reside in cells that have migrated from the airways to the MdLN after infection. The correlation between DC and bacteria accumulation in the MdLN was further demonstrated by manipulating DC migration to the MdLN through two independent pathways. Impairment of DC migration to the MdLN, either by a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor agonist (FTY720) or by the D prostanoid receptor <em>1</em> agonist (BW245C), resulted in reduced bacterial colonization of MdLN. Moreover, BW245C treatment delayed the onset of morbidity and the time to death of the infected mice. Taken together, these results suggest that DC can serve as an inhabitation niche for F. tularensis in the early stages of infection, and that DC trafficking plays a role in pathogen dissemination. This underscores the therapeutic potential of DC migration impairing drugs in tularemia treatment.

The signaling pathways by which <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S<em>1</em>P-induced migration of human pulmonary artery endothelial cells (HPAECs). S<em>1</em>P-induced migration was sensitive to S<em>1</em>P(<em>1</em>) small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S<em>1</em>P(<em>1</em>) to G(i). Overexpression of dominant negative (dn) PKC-epsilon or -zeta, but not PKC-alpha or -delta, blocked S<em>1</em>P-induced migration. Although S<em>1</em>P activated both PLD<em>1</em> and PLD2, S<em>1</em>P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD<em>1</em>. Blocking PKC-epsilon, but not PKC-zeta, activity attenuated S<em>1</em>P-mediated PLD stimulation, demonstrating that PKC-epsilon, but not PKC-zeta, was upstream of PLD. Transfection of HPAECs with dnRac<em>1</em> or Rac<em>1</em> siRNA attenuated S<em>1</em>P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-zeta, or treatment with myristoylated PKC-zeta peptide inhibitor abrogated S<em>1</em>P-induced Rac<em>1</em> activation. These results establish that S<em>1</em>P signals through S<em>1</em>P(<em>1</em>) and G(i) to activate PKC-epsilon and, subsequently, a PLD2-PKC-zeta-Rac<em>1</em> cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.

We examined expression of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors and <em>sphingosine</em> kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S<em>1</em>P-induced 20-kDa myosin light chain (MLC(20)) phosphorylation and contraction. RT-PCR demonstrated expression of SPK<em>1</em> and SPK2 and S<em>1</em>P(<em>1</em>) and S<em>1</em>P(2) receptors. S<em>1</em>P activated G(q), G(<em>1</em>3), and all G(i) isoforms and stimulated PLC-beta<em>1</em>, PLC-beta3, and Rho kinase activities. PLC-beta activity was partially inhibited by pertussis toxin (PTX), Gbeta or Galpha(q) antibody, PLC-beta<em>1</em> or PLC-beta3 antibody, and by expression of Galpha(q) or Galpha(i) minigene, and was abolished by a combination of antibodies or minigenes. S<em>1</em>P-stimulated Rho kinase activity was partially inhibited by expression of Galpha(<em>1</em>3) or Galpha(q) minigene and abolished by expression of both. S<em>1</em>P stimulated Ca(2+) release that was inhibited by U-73<em>1</em>22 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC(50) <em>1</em> nM). Initial contraction and MLC(20) phosphorylation were abolished by U-73<em>1</em>22 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and Galpha(q) or Gbeta antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC(20) phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73<em>1</em>22 or ML-9. These results indicate that S<em>1</em>P induces <em>1</em>) initial contraction mediated by S<em>1</em>P(2) and S<em>1</em>P(<em>1</em>) involving concurrent activation of PLC-beta<em>1</em> and PLC-beta3 via Galpha(q) and Gbetagamma(i), respectively, resulting in inositol <em>1</em>,4,5-tris<em>phosphate</em>-dependent Ca(2+) release and MLCK-mediated MLC(20) phosphorylation, and 2) sustained contraction exclusively mediated by S<em>1</em>P(2) involving activation of RhoA via Galpha(q) and Galpha(<em>1</em>3), resulting in Rho kinase- and PKC-dependent MLC(20) phosphorylation.

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type <em>1</em> insulin-like growth factor receptor (IGFR<em>1</em>) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-<em>1</em>0A breast epithelial cells, focusing on a possible involvement of the <em>sphingosine</em> kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK<em>1</em>, but not SphK2, expression. Similarly, IGFR<em>1</em> phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK<em>1</em> silencing. SphK<em>1</em> expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>) or S<em>1</em>P3, but not S<em>1</em>P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S<em>1</em>P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S<em>1</em>P<em>1</em> and S<em>1</em>P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-<em>1</em>0A cells requires SphK<em>1</em> activity and S<em>1</em>P<em>1</em>/S<em>1</em>P3, suggesting that S<em>1</em>P, the product of SphK activity and ligand for S<em>1</em>P<em>1</em> and S<em>1</em>P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.

Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S<em>1</em>P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAQ) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S<em>1</em>P stimulation. Our results demonstrated that S<em>1</em>P mediates rapid and specific recruitment (<em>1</em> microM, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S<em>1</em>P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S<em>1</em>P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.

Plasmalemmal caveolae are membrane microdomains that are specifically enriched in sphingolipids and contain a wide array of signaling proteins, including the endothelial isoform of nitric-oxide synthase (eNOS). EDG-<em>1</em> is a G protein-coupled receptor for <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) that is expressed in endothelial cells and has been implicated in diverse vascular signal transduction pathways. We analyzed the subcellular distribution of EDG-<em>1</em> in COS-7 cells transiently transfected with cDNA constructs encoding epitope-tagged EDG-<em>1</em>. Subcellular fractionation of cell lysates resolved by ultracentrifugation in discontinuous sucrose gradients revealed that approximately 55% of the EDG-<em>1</em> protein was recovered in fractions enriched in caveolin-<em>1</em>, a resident protein of caveolae. Co-immunoprecipitation experiments showed that EDG-<em>1</em> could be specifically precipitated by antibodies directed against caveolin-<em>1</em> and vice versa. The targeting of EDG-<em>1</em> to caveolae-enriched fractions was markedly increased (from 5<em>1</em> +/- <em>1</em><em>1</em>% to 93 +/- <em>1</em>4%) by treatment of transfected cells with S<em>1</em>P (5 microm, 60 min). In co-transfection experiments expressing EDG-<em>1</em> and eNOS cDNAs in COS-7 cells, we found that S<em>1</em>P treatment significantly and specifically increased nitric-oxide synthase activity, with an EC(50) of 30 nm S<em>1</em>P. Overexpression of transfected caveolin-<em>1</em> cDNA together with EDG-<em>1</em> and eNOS markedly diminished S<em>1</em>P-mediated eNOS activation; caveolin overexpression also attenuated agonist-induced phosphorylation of EDG-<em>1</em> receptor by >90%. These results suggest that the interaction of the EDG-<em>1</em> receptor with caveolin may serve to inhibit signaling through the S<em>1</em>P pathway, even as the targeting of EDG-<em>1</em> to caveolae facilitates the interactions of this receptor with ligands and effectors that are also targeted to caveolae. The agonist-modulated targeting of EDG-<em>1</em> to caveolae and its dynamic inhibitory interactions with caveolin identify new points for regulation of sphingolipid-dependent signaling in the vascular wall.

Overexpression of vascular endothelial growth factor receptors (VEGFRs) has been reported in a variety of tumor types. Here we find that <em>1</em><em>1</em> out of the <em>1</em>4 bladder tumor cell lines examined express one or more VEGF receptors. Analysis of the T24 bladder tumor cell line reveals a functional autocrine loop involving VEGF and the Flk-<em>1</em> receptor. Blocking VEGF expression in T24 cells results in a decrease in DNA synthesis. The Flk-<em>1</em> receptor in T24 cells is phosphorylated in response to VEGF-<em>1</em>2<em>1</em> or VEGF-<em>1</em>65, and an Flk-<em>1</em> inhibitor blocks VEGF to ERK signaling. We report that VEGF stimulation of T24 cells results in activation of H- and N-Ras and this is dependent on cellular <em>sphingosine</em> kinase <em>1</em> (SPK<em>1</em>) activity. Previously, we found VEGF-induced activation of Ras appears to be independent of a Ras-guanine nucleotide exchange factors (GEFs). Here we report that <em>sphingosine</em> can stimulate Ras-GTPase activating protein (GAP) activity in vitro, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) can block the stimulatory effects of <em>sphingosine</em>. We present a model where the balance between <em>sphingosine</em> and SPP regulates Ras-GAP activity such that stimulation of SPK<em>1</em> favors downregulation of Ras-GAP and thereby the activation of Ras proteins. These data highlight a VEGF pathway that may be involved in the survival and proliferation of bladder tumor cells as well as other tumor cell types.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid that mediates a wide array of biologic effects through its interaction with a family of five G protein-coupled receptors. Cytokines and growth factors interact with this signaling pathway in a variety of ways, including both activation and regulation of the expression of the enzymes that regulate synthesis and degradation of S<em>1</em>P. Not only do many growth factors and cytokines stimulate S<em>1</em>P production, leading to transactivation of S<em>1</em>P receptors, ligation of S<em>1</em>P receptors by S<em>1</em>P can also transactivate growth factor tyrosine kinase receptors and stimulate growth factor and cytokine signaling cascades. This review discusses the mechanisms involved in cross-talk between S<em>1</em>P, cytokines, and growth factors and the impact of that cross-talk on cell signaling and cell biology.