Lipid <em>phosphate</em> phosphatases (LPPs) regulate cell signaling by modifying the concentrations of lipid <em>phosphates</em> versus their dephosphorylated products. The ecto-activity regulates the availability of extracellular lysophosphatidate (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and thereby signaling by their respective receptors. LPP products (monoacylglycerol or <em>sphingosine</em>) are taken up by cells and rephosphorylated to produce LPA and S<em>1</em>P, respectively, which activate intracellular signaling cascades. The proposed integrin binding domain on the external surface of LPP3 modifies cell/cell interactions. Expression of LPPs on internal membranes controls signaling depending on the access of lipid <em>phosphates</em> to their active sites. Different LPPs perform distinct functions, probably based on integrin binding, their locations, and their abilities to metabolize different lipid <em>phosphates</em> in vivo.

During angiogenesis, nascent vascular sprouts fuse to form vascular networks, enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S<em>1</em>P receptor-<em>1</em> (S<em>1</em>P<em>1</em>) stabilizes the primary vascular network. A gradient of S<em>1</em>P<em>1</em> expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S<em>1</em>P<em>1</em>, adherens junctions are destabilized, barrier function is breached, and flow is perturbed, resulting in abnormal vascular hypersprouting. Interestingly, S<em>1</em>P<em>1</em> responds to S<em>1</em>P as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in vitro and in vivo. These data demonstrate that blood flow and circulating S<em>1</em>P activate endothelial S<em>1</em>P<em>1</em> to stabilize blood vessels in development and homeostasis.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), an evolutionarily conserved bioactive lipid mediator, is now recognized as a potent modulator of cell regulation. In vertebrates, S<em>1</em>P interacts with cell surface G protein-coupled receptors of the EDG family and induces profound effects in a variety of organ systems. Indeed, an S<em>1</em>P receptor agonist is undergoing clinical trials to combat immune-mediated transplant rejection. Recent information on S<em>1</em>P receptor biology suggests potential utility in the control of cardiovascular processes, including angiogenesis, vascular permeability, arteriogenesis, and vasospasm. However, studies from diverse invertebrates, such as yeast, Dictyostelium, Drosophila, and Caenorhabditis elegans have shown that S<em>1</em>P is involved in important regulatory functions in the apparent absence of EDG S<em>1</em>P receptor homologues. Metabolic pathways of S<em>1</em>P synthesis, degradation, and release have recently been described at the molecular level. Genetic and biochemical studies of these enzymes have illuminated the importance of S<em>1</em>P signaling systems both inside and outside of cells. The revelation of receptor-dependent pathways, as well as novel metabolic/intracellular pathways has provided new biological insights and may ultimately pave the way for the development of novel therapeutic approaches for cardiovascular diseases.

Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T<em>1</em>α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC<em>1</em>B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

The lysophospholipid (LPL) mediators lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are generated by enzymatic cleavage of stores of glycerophospholipids and sphingomyelin, respectively, in membranes of stimulated cells. LPLs are albumin bound, distributed widely in mammalian tissues, and increased in concentration by physiological activation of platelets and some other cells, tissue injury, inflammation, and neoplasia. The principal effects of LPA and S<em>1</em>P are growth related, including induction of cellular proliferation, alterations in differentiation and survival, and suppression of apoptosis. LPA and S<em>1</em>P also evoke cellular effector functions, which are dependent on cytoskeletal responses such as contraction, secretion, adhesion, and chemotaxis. The extracellular mediator activities of LPLs are transduced by subfamilies of G-protein-coupled receptors (GPCRs), of which the most completely characterized are those encoded by the endothelial differentiation genes (edgs). One homology cluster composed of Edg-<em>1</em>, -3, and -5 recognizes and responds to S<em>1</em>P, and the other cluster of Edg-2 and -4 is dedicated to LPA. Edg proteins are developmentally regulated and differ in tissue distribution, but couple similarly to multiple types of G-proteins to signal through ras and mitogen-activated protein kinase, rho, phospholipase C, and several protein tyrosine kinases. Numerous interactions between glycerophospholipids and sphingolipids are observed in their biosynthetic and signaling pathways. Many of the cellular effects of LPA and S<em>1</em>P are attributable to modifications in the content and/or activity of a major functional protein. Examples are increases in nuclear levels of transcription factors that regulate the serum response element, suppression of death caspase activities in apoptosis, and elevation of membrane content of heparin binding-epidermal growth factor-like growth factor, which serves as an autocrine and juxtacrine stimulus of proliferation. These ubiquitous LPL mediators of cellular growth, differentiation, and activities thus act directly through complex subfamilies of GPCRs and by regulating expression of biologically critical proteins.

Regulation of intracellular cyclic adenosine 3 ',5 '-mono<em>phosphate</em> (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-di<em>phosphate</em> and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is novel. In these cells, S<em>1</em>P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S<em>1</em>P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S<em>1</em>P is mediated by the S<em>1</em>P(2) receptor and the heterotrimeric G(<em>1</em>3) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.

The G protein-coupled receptors S<em>1</em>P2/Edg5 and S<em>1</em>P3/Edg3 both mediate <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) stimulation of Rho, yet S<em>1</em>P2 but not S<em>1</em>P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Galpha<em>1</em>2 and Galpha<em>1</em>3, but not of Galphaq, by expression of respective C-terminal peptides abolished S<em>1</em>P2-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S<em>1</em>P2 and either Galpha<em>1</em>2 or Galpha<em>1</em>3, but not Galphaq, mediated S<em>1</em>P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Galphai, by contrast, specifically antagonized S<em>1</em>P2-mediated inhibition of Rac and migration. The S<em>1</em>P2 actions were mimicked by expression of V<em>1</em>4Rho and were abolished by C3 toxin and N<em>1</em>9Rho, but not Rho kinase inhibitors. In contrast to S<em>1</em>P2, S<em>1</em>P3 mediated S<em>1</em>P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G<em>1</em>2/<em>1</em>3. Upon inactivation of Gi by pertussis toxin, S<em>1</em>P3 mediated inhibition of Rac and migration just like S<em>1</em>P2. These results indicate that integration of counteracting signals from the Gi- and the G<em>1</em>2/<em>1</em>3-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S<em>1</em>P receptor isoform.

Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast- and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their <em>sphingosine</em> kinase <em>1</em> (Sphk<em>1</em>) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S<em>1</em>P<em>1</em>,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S<em>1</em>P.

OBJECTIVE

To determine whether lysophosphatidic acid (LPA) and other lysophospholipids (LPL) are useful markers for diagnosis and/or prognosis of ovarian cancer in a controlled setting.

METHODS

Plasma samples were collected from ovarian cancer patients and healthy control women in Hillsborough and Pinellas counties, Florida, and processed at the University of South Florida H. Lee Moffitt Cancer Center and Research Institute (Moffitt). Case patients with epithelial ovarian cancer (n = <em>1</em><em>1</em>7) and healthy control subjects (n = 27) participated in the study. Blinded LPL analysis, including 23 individual LPL species, was performed at the Cleveland Clinic Foundation using an electrospray ionization mass spectrometry-based method. LPL levels were transmitted to Moffitt, where clinical data were reviewed and statistical analyses were performed.

RESULTS

There were statistically significant differences between preoperative case samples (n = 45) and control samples (n = 27) in the mean levels of total LPA, total lysophosphatidylinositol (LPI), <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), and individual LPA species as well as the combination of several LPL species. The combination of <em>1</em>6:0-LPA and 20:4-LPA yielded the best discrimination between preoperative case samples and control samples, with 93.<em>1</em>% correct classification, 9<em>1</em>.<em>1</em>% sensitivity, and 96.3% specificity. In 22 cases with both preoperative and postoperative samples, the postoperative levels of several LPL, including S<em>1</em>P, total LPA, and lysophosphatidylcholine (LPC) levels and some individual species of LPA and LPC, were significantly different from preoperative levels.

CONCLUSIONS

LPA, LPI, LPC, and S<em>1</em>P appear useful as diagnostic and prognostic biomarkers of ovarian cancer.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid metabolite abundantly stored in platelets and released upon platelet activation. Recently, S<em>1</em>P has been postulated for its potential roles in angiogenesis. In this study, we provided several lines of evidence showing that S<em>1</em>P has angiogenic activity. In vitro, S<em>1</em>P stimulated DNA synthesis and chemotactic motility of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, reaching a near maximum at <em>1</em> microM. S<em>1</em>P also significantly induced tube formation of HUVECs on Matrigel. Matrigel plug assay in mice revealed that S<em>1</em>P promotes angiogenesis in vivo. In addition, exposure of HUVECs to S<em>1</em>P led to rapid activation of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in a pertussis toxin (PTX)-sensitive manner. Notably, HUVEC migration and tube formation in response to S<em>1</em>P were completely blocked by pretreatment with PTX. Further, the MEK inhibitor U0<em>1</em>26 markedly inhibited S<em>1</em>P-induced tube formation but S<em>1</em>P-induced migration was not affected by inhibition of ERK and p38 MAPK. Taken together, these results indicate that S<em>1</em>P induces angiogenesis predominantly via G(i) protein-coupled receptors in endothelial cells and suggest that S<em>1</em>P may act as an important modulator of platelet-induced angiogenesis.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are serum-borne lysophospholipids that signal through their cognate G protein-coupled receptors to evoke a great variety of responses in numerous cell types. In addition to stimulating cell proliferation and survival, LPA and S<em>1</em>P induce profound cytoskeletal changes through Rho-mediated signaling pathways, leading to such diverse responses as cell rounding, neurite retraction, and modulation of tumor cell invasiveness (transcellular migration). A major recent advance is the identification of a subfamily of heptahelical receptors for LPA and S<em>1</em>P.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a multifunctional lipid mediator that signals via the S<em>1</em>P family of G protein-coupled receptors (S<em>1</em>PR), regulates vascular maturation, permeability, and angiogenesis. In this study, we explored the role of S<em>1</em>P 2 receptor (S<em>1</em>P2R) in normal vascularization and hypoxia-triggered pathological angiogenesis of the mouse retina. S<em>1</em>P2R is strongly induced in ECs during hypoxic stress. When neonatal mice were subjected to ischemia-driven retinopathy, pathologic neovascularization in the vitreous chamber was suppressed in S<em>1</em>p2-/- mice concomitant with reduction in endothelial gaps and inflammatory cell infiltration. In addition, EC patterning and normal revascularization into the avascular zones of the retina were augmented. Reduced expression of the proinflammatory enzyme cyclooxygenase-2 (COX-2) and increased expression of eNOS were observed in the S<em>1</em>p2-/- mouse retina. S<em>1</em>P2R activation in ECs induced COX-2 expression and suppressed the expression of eNOS. These data identify the S<em>1</em>P2R-driven inflammatory process as an important molecular event in pathological retinal angiogenesis. We propose that antagonism of the S<em>1</em>P2R may be a novel therapeutic approach for the prevention and/or treatment of pathologic ocular neovascularization.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), produced by <em>sphingosine</em> kinase (SPHK), acts both by intracellular and extracellular modes. We evaluated the role of SPHK<em>1</em> and S<em>1</em>P in osteoclastogenesis using bone marrow-derived macrophage (BMM) single and BMM/osteoblast coculture systems. In BMM single cultures, the osteoclastogenic factor receptor activator of NF-kappaB ligand (RANKL) upregulated SPHK<em>1</em> and increased S<em>1</em>P production and secretion. SPHK<em>1</em> siRNA enhanced and SPHK<em>1</em> overexpression attenuated osteoclastogenesis via modulation of p38 and ERK activities, and NFATc<em>1</em> and c-Fos levels. Extracellular S<em>1</em>P had no effect in these cultures. These data suggest that intracellular S<em>1</em>P produced in response to RANKL forms a negative feedback loop in BMM single cultures. In contrast, S<em>1</em>P addition to BMM/osteoblast cocultures greatly increased osteoclastogenesis by increasing RANKL in osteoblasts via cyclooxygenase-2 and PGE(2) regulation. S<em>1</em>P also stimulated osteoblast migration and survival. The RANKL elevation and chemotactic effects were also observed with T cells. These results indicate that secreted S<em>1</em>P attracts and acts on osteoblasts and T cells to augment osteoclastogenesis. Taken together, S<em>1</em>P plays an important role in osteoclastogenesis regulation and in communication between osteoclasts and osteoblasts or T cells.

Stomata form pores on leaf surfaces that regulate the uptake of CO2 for photosynthesis and the loss of water vapour during transpiration. An increase in the cytosolic concentration of free calcium ions ([Ca2+]cyt) is a common intermediate in many of the pathways leading to either opening or closure of the stomatal pore. This observation has prompted investigations into how specificity is controlled in calcium-based signalling systems in plants. One possible explanation is that each stimulus generates a unique increase in [Ca2+]cyt, or 'calcium signature', that dictates the outcome of the final response. It has been suggested that the key to generating a calcium signature, and hence to understanding how specificity is controlled, is the ability to access differentially the cellular machinery controlling calcium influx and release from internal stores. Here we report that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> is a new calcium-mobilizing molecule in plants. We show that after drought treatment <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> levels increase, and we present evidence that this molecule is involved in the signal-transduction pathway linking the perception of abscisic acid to reductions in guard cell turgor.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a potent lysolipid involved in a variety of biological responses important for cancer progression. Therefore, we investigated the role of <em>sphingosine</em> kinase type <em>1</em> (SphK<em>1</em>), the enzyme that makes S<em>1</em>P, in the motility, growth, and chemoresistance of MCF-7 breast cancer cells. Epidermal growth factor (EGF), an important growth factor for breast cancer progression, activated and translocated SphK<em>1</em> to plasma membrane. SphK<em>1</em> was required for EGF-directed motility. Downregulation of SphK<em>1</em> in MCF-7 cells reduced EGF- and serum-stimulated growth and enhanced sensitivity to doxorubicin, a potent chemotherapeutic agent. These results suggest that SphK<em>1</em> may be critical for growth, metastasis and chemoresistance of human breast cancers.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S<em>1</em>P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S<em>1</em>P receptor subtypes were deleted. Mice lacking either S<em>1</em>P(2) or S<em>1</em>P(3) receptors and subjected to <em>1</em>-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S<em>1</em>P(2,3) receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S<em>1</em>P(2,3) receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S<em>1</em>P(2,3) receptor knockout mouse hearts. Neither S<em>1</em>P(2) nor S<em>1</em>P(3) receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S<em>1</em>P(2) and S<em>1</em>P(3) receptor mediated activation of Akt was tested by using cells from WT and S<em>1</em>P receptor knockout hearts. Akt was activated by S<em>1</em>P, and this was modestly diminished in cardiomyocytes from S<em>1</em>P(2) or S<em>1</em>P(3) receptor knockout mice and completely abolished in the S<em>1</em>P(2,3) receptor double-knockout myocytes. Our data demonstrate that activation of S<em>1</em>P(2) and S<em>1</em>P(3) receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S<em>1</em>P and receptor-mediated Akt activation in this process.

Receptors for the serine protease thrombin and for lysophospholipids are coupled to G proteins and control a wide range of cellular functions, including mitogenesis. Activators of these receptors are present in blood, and can enter the brain during central nervous system (CNS) injury. Reactive astrogliosis, a prominent component of CNS injury with potentially harmful consequences, may involve proliferation of astrocytes. In this study, we have examined the expression and activation of protease activated receptors (PARs), lysophosphatidic acid (LPA) receptors, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors on murine astrocytes. We show that activation of these three receptor classes can lead to astrogliosis in vivo and proliferation of astrocytes in vitro. Cultured murine cortical astrocytes express mRNA for multiple receptor subtypes of PAR (PAR-<em>1</em>-4), LPA (LPA-<em>1</em>-3) and S<em>1</em>P (S<em>1</em>P-<em>1</em>, -3, -4, and -5) receptors. Comparison of the intracellular signaling pathways of glial PAR-<em>1</em>, LPA, and S<em>1</em>P receptors indicates that each receptor class activates multiple downstream signaling pathways, including Gq/<em>1</em><em>1</em>-directed inositol lipid/Ca2+ signaling, Gi/o activation of mitogen-activated protein kinases (MAPK) (extracellular signal-regulated kinase <em>1</em>/2 and stress activated protein kinase/c-jun N-terminal kinase, but not p38), and activation of Rho pathways. Furthermore, activation of these different receptor classes can differentially regulate two transcription factor pathways, serum response element and nuclear factor of activated T cells. Blockade of Gi/o signaling with pertussis toxin, MAPK activation with <em>1</em>,4-diamino-2,3-dicyano-<em>1</em>,4-bis(2-aminophynyltio)butadiene (U0<em>1</em>26), or Rho kinase signaling with R-(+)-trans-N-(4-pyridyl)-4-(<em>1</em>-aminoethyl)-cyclohexane carboxamide (Y27632) can markedly reduce the proliferative response of glial cells to PAR-<em>1</em>, LPA, or S<em>1</em>P receptor activation, suggesting that each of these pathways is important in coupling of receptor activation to glial proliferation.

The bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is emerging as an important mediator of immune and inflammatory responses. S<em>1</em>P formation is catalyzed by <em>sphingosine</em> kinase (SK), of which the SK<em>1</em> isoenzyme is activated by tumor necrosis alpha (TNF-alpha). SK<em>1</em> has been shown to be required for mediating TNF-alpha inflammatory responses in cells, including induction of cyclooxygenase 2 (COX-2). Because TNF-alpha and COX-2 are increased in patients with inflammatory bowel disease (IBD), we investigated the role of SK<em>1</em> in a murine model of colitis. SK<em>1</em>(-/-) mice treated with dextran sulfate sodium (DSS) had significantly less blood loss, weight loss, colon shortening, colon histological damage, and splenomegaly than did wild-type (WT) mice. In addition, SK<em>1</em>(-/-) mice had no systemic inflammatory response. Moreover, WT but not SK<em>1</em>(-/-) mice treated with dextran sulfate sodium had significant increases in blood S<em>1</em>P levels, colon SK<em>1</em> message and activity, and colon neutrophilic infiltrate. Unlike WT mice, SK<em>1</em>(-/-) mice failed to show colonic COX-2 induction despite an exaggerated TNF-alpha response; thus implicating for the first time SK<em>1</em> in TNF-alpha-mediated COX-2 induction in vivo. Inhibition of SK<em>1</em> may prove to be a valuable therapeutic target by inhibiting systemic and local inflammation in IBD.

Lipid <em>phosphates</em> initiate key signaling cascades in cell activation. Lysophosphatidate (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are produced by activated platelets. LPA is also formed from circulating lysophosphatidylcholine by autotaxin, a protein involved tumor progression and metastasis. Extracellular LPA and S<em>1</em>P stimulate families of G-protein coupled receptors that elicit diverse responses. LPA is involved in wound repair and tumor growth. Exogenous S<em>1</em>P is a potent stimulator of angiogenesis, a process vital in development, tissue repair and the growth of aggressive tumors. Inside the cell, phosphatidate (PA), ceramide <em>1</em>-<em>phosphate</em> (C<em>1</em>P), LPA, and S<em>1</em>P act as signaling molecules with distinct functions including the stimulation of cell division, cytoskeletal rearrangement, Ca(2+) transients, and membrane movement. These observations imply that phosphatases that degrade lipid <em>phosphates</em> on the cell surface, or inside the cell, regulate cell signaling under physiological and pathological conditions. This occurs through attenuation of signaling by the lipid <em>phosphates</em> and by the production of bioactive products (diacylglycerol, ceramide, and <em>sphingosine</em>). Three lipid <em>phosphate</em> phosphatases (LPPs) and a splice variant dephosphorylate LPA, PA, CIP, and S<em>1</em>P. Two S<em>1</em>P phosphatases (SPPs) act specifically on S<em>1</em>P. In addition, there is family of four LPP-related proteins (LPRs, or plasticity-related genes, PRGs). PRG-<em>1</em> expression in neurons has been reported to increase extracellular LPA breakdown and attenuate LPA-induced axonal retraction. It is unclear whether the LRPs dephosphorylate LPA directly, stimulate LPP activity, or bind LPA and S<em>1</em>P. Also, the importance of extra- versus intra-cellular actions of the LPPs and SPPs, and the individual roles of different isoforms is not firmly established. Understanding the functions and regulation of the LPPs, SPPs and related proteins will hopefully contribute to interventions to correct dysfunctions in conditions such as wound repair, inflammation, angiogenesis, tumor growth, and metastasis.

The transactivation of enhanced growth factor receptor (EGFR) by G protein-coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase <em>sphingosine</em> kinase-<em>1</em> (SphK<em>1</em>). We show that E2 stimulates SphK<em>1</em> activation and the release of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), by which E2 is capable of activating the S<em>1</em>P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease-dependent manner. Thus, these findings reveal a key role for SphK<em>1</em> in the coupling of the signals between three membrane-spanning events induced by E2, S<em>1</em>P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., "criss-cross" transactivation.

Angiogenesis is a complex morphogenetic process whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a unique organotypic model of angiogenic sprouting and neovessel formation that originates from preformed artificial vessels fully encapsulated within a 3D extracellular matrix. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells, following stalk cells exhibiting apical-basal polarity, and lumens and branches connecting back to the parent vessels. Ultimately, sprouts bridged between preformed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis. Interestingly, the ability of VEGF receptor 2 inhibition to antagonize filopodia formation in tip cells was context-dependent, suggesting a mechanism by which vessels might be able to toggle between VEGF-dependent and VEGF-independent modes of angiogenesis. Like VEGF, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> also seemed to exert its proangiogenic effects by stimulating directional filopodial extension, whereas matrix metalloproteinase inhibitors prevented sprout extension but had no impact on filopodial formation. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting and highlight the potential utility of the model to elucidate the molecular mechanisms that coordinate the complex series of events involved in neovascularization.

G protein-coupled receptors (GPCRs) of endothelial cells transmit diverse intracellular signals that regulate adherens junction (AJ) permeability. Increased endothelial permeability contributes to pathological processes such as inflammation, atherogenesis, and acute lung injury. Thrombin, a serine protease, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive lipid, regulate endothelial barrier function by activating their respective GPCRs-the protease-activated receptor PAR(<em>1</em>) and the S<em>1</em>P receptor S<em>1</em>P(<em>1</em>)-which initiate intracellular signals that regulate AJ integrity and cytoskeleton organization. The distinct patterns of PAR(<em>1</em>) and S<em>1</em>P(<em>1</em>) signal transduction underlie the functional antagonism between thrombin and S<em>1</em>P. Evidence points to a role for activation of the S<em>1</em>P(<em>1</em>) receptor that is induced by PAR(<em>1</em>)-mediated signaling in the mechanism of AJ reannealing and endothelial barrier repair. Understanding the molecular basis of AJ integrity in the context of inflammation is important in developing novel anti-inflammatory therapeutics. This Review provides a working model for molecular mechanisms for the dual regulation of endothelial barrier function.

Vascular endothelial growth factor (VEGF) signaling is critical to the processes of angiogenesis and tumor growth. Here, evidence is presented for VEGF stimulation of <em>sphingosine</em> kinase (SPK) that affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors. VEGF or phorbol <em>1</em>2-myristate <em>1</em>3-acetate treatment of the T24 bladder tumor cell line resulted in a time- and dose-dependent stimulation of SPK activity. In T24 cells, VEGF treatment reduced cellular <em>sphingosine</em> levels while raising that of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. VEGF stimulation of T24 cells caused a slow and sustained accumulation of Ras-GTP and phosphorylated extracellular signal-regulated kinase (phospho-ERK) compared with that after EGF treatment. Small interfering RNA (siRNA) that targets SPK<em>1</em>, but not SPK2, blocks VEGF-induced accumulation of Ras-GTP and phospho-ERK in T24 cells. In contrast to EGF stimulation, VEGF stimulation of ERK<em>1</em>/2 phosphorylation was unaffected by dominant-negative Ras-N<em>1</em>7. Raf kinase inhibition blocked both VEGF- and EGF-stimulated accumulation of phospho-ERK<em>1</em>/2. Inhibition of SPK by pharmacological inhibitors, a dominant-negative SPK mutant, or siRNA that targets SPK blocked VEGF, but not EGF, induction of phospho-ERK<em>1</em>/2. We conclude that VEGF induces DNA synthesis in a pathway which sequentially involves protein kinase C (PKC), SPK, Ras, Raf, and ERK<em>1</em>/2. These data highlight a novel mechanism by which SPK mediates signaling from PKC to Ras in a manner independent of Ras-guanine nucleotide exchange factor.

The sphingolipid metabolites ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> contribute to controlling cell proliferation and apoptosis. Ceramide and its catabolite <em>sphingosine</em> act as negative regulators of cell proliferation and promote apoptosis. Conversely, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, formed by phosphorylation of <em>sphingosine</em> by a <em>sphingosine</em> kinase, has been involved in stimulating cell growth and inhibiting apoptosis. As the phosphorylation of <em>sphingosine</em> diminishes apoptosis, while dephosphorylation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> potentiates it, the role of <em>sphingosine</em> as a messenger of apoptosis is of importance. Herein, the effects of <em>sphingosine</em> on diverse signaling pathways implicated in the apoptotic process are reviewed.