OBJECTIVE

<em>Sphingosine</em> kinase <em>1</em> (SphK<em>1</em>), which phosphorylates <em>sphingosine</em> to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), is overexpressed in various types of cancers, and may act as an oncogene in tumorigenesis. However, little is known about the precise role of the SphK<em>1</em>/S<em>1</em>P pathway in human liver cancer, especially regarding the metastasis of hepatocellular carcinoma (HCC).

METHODS

The expression of SphK<em>1</em> was detected by quantitative reverse-transcription PCR. In addition, transwell cell migration and invasion assay were carried out for functional analysis. Furthermore, the level of S<em>1</em>P was quantified by ELISA and Rac<em>1</em>/Cdc42 GTPase activation was assessed by western blot analysis.

RESULTS

The levels of SphK<em>1</em> mRNA are commonly up-regulated in HCC patients and human liver cancer cell migration and invasion can be promoted by the overexpression of SphK<em>1</em>. In addition, inhibition of SphK<em>1</em> with either a SphK<em>1</em> inhibitor or siRNA reduced human liver cancer cell migration and invasion. Furthermore, overexpression of SphK<em>1</em> increased S<em>1</em>P levels, and the exogenous addition of S<em>1</em>P increased liver cell migration and invasion through the EDG<em>1</em> receptor.

CONCLUSIONS

The results from this study provide strong evidence of a role for the SphK<em>1</em>/S<em>1</em>P/EDG<em>1</em> pathway in liver metastasis, thus making it an attractive therapeutic target for the development of new anti-HCC drugs.

<em>Sphingosine</em> kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating <em>sphingosine</em> into <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). SphK/S<em>1</em>P play a critical role in angiogenesis, inflammation, and various pathologic conditions. Recently, S<em>1</em>P(<em>1</em>) receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S<em>1</em>P signaling via S<em>1</em>P(<em>1</em>) enhances synoviocyte proliferation, COX-2 expression, and prostaglandin E(2) production. Here, we examined the role of SphK/S<em>1</em>P in RA using a potent SphK inhibitor, N,N-dimethyl<em>sphingosine</em> (DMS), and a molecular approach against one of its isoenzymes, SphK<em>1</em>. We observed that levels of S<em>1</em>P in the synovial fluid of RA patients were significantly higher than those of osteoarthritis patients. Additionally, DMS significantly reduced the levels of TNF-alpha, IL-6, IL-<em>1</em>beta, MCP-<em>1</em>, and MMP-9 in cell-contact assays using both Jurkat-U937 cells and RA PBMCs. In a murine collagen-induced arthritis model, i.p. administration of DMS significantly inhibited disease severity and reduced articular inflammation and joint destruction. Treatment of DMS also down-regulated serum levels IL-6, TNF-alpha, IFN-gamma, S<em>1</em>P, and IgG<em>1</em> and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Moreover, similar reduction in incidence and disease activity was observed in mice treated with SphK<em>1</em> knock-down via small interfering RNA approach. Together, these results demonstrate SphK modulation may provide a novel approach in treating chronic autoimmune conditions such as RA by inhibiting the release of pro-inflammatory cytokines.

Legionella pneumophila infects alveolar macrophages and protozoa through establishment of an intracellular replication niche. This process is mediated by bacterial effectors translocated into the host cell via the Icm/Dot type IV secretion system. Most of the effectors identified so far are unique to L. pneumophila; however, some of the effectors are homologous to eukaryotic proteins. We performed a distribution analysis of many known L. pneumophila effectors and found that several of them, mostly eukaryotic homologous proteins, are present in different Legionella species. In-depth analysis of LegS2, a L. pneumophila homologue of the highly conserved eukaryotic enzyme <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase (SPL), revealed that it was most likely acquired from a protozoan organism early during Legionella evolution. The LegS2 protein was found to translocate into host cells using a C-terminal translocation domain absent in its eukaryotic homologues. LegS2 was found to complement the <em>sphingosine</em>-sensitive phenotype of a Saccharomyces serevisia SPL-null mutant and this complementation depended on evolutionary conserved residues in the LegS2 catalytic domain. Interestingly, unlike the eukaryotic SPL that localizes to the endoplasmic reticulum, LegS2 was found to be targeted mainly to host cell mitochondria. Collectively, our results demonstrate the remarkable adaptations of a eukaryotic protein to the L. pneumophila pathogenesis system.

Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) via ligation of the S<em>1</em>P<em>1</em> receptor. The S<em>1</em>P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S<em>1</em>P<em>1</em> receptor ligation. We examined the role of the S<em>1</em>P<em>1</em> receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S<em>1</em>P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S<em>1</em>P response. Reduction of S<em>1</em>P<em>1</em>R expression (via siRNA) attenuated S<em>1</em>P-induced TER elevations whereas the TER response to FTY was unaffected. Both S<em>1</em>P and FTY rapidly (within 5 min) induced S<em>1</em>P<em>1</em>R accumulation in membrane lipid rafts, but only S<em>1</em>P stimulated S<em>1</em>P<em>1</em>R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S<em>1</em>P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S<em>1</em>P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac<em>1</em> and cortactin (via siRNA), attenuated S<em>1</em>P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.

High-density lipoproteins (HDL) are the major plasma carriers for <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in healthy individuals, but their S<em>1</em>P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S<em>1</em>P levels in plasma and HDL are altered in coronary artery disease. S<em>1</em>P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S<em>1</em>P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S<em>1</em>P) revealed higher normalized plasma S<em>1</em>P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S<em>1</em>P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S<em>1</em>P per protein in HDL: <em>1</em>32 vs. <em>1</em>53 pmol/mg). The amount of total plasma S<em>1</em>P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S<em>1</em>P was, accordingly, higher (sCAD: 84, MI: 8<em>1</em>, controls: <em>1</em>0 pmol/mL). HDL-bound plasma S<em>1</em>P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S<em>1</em>P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S<em>1</em>P to HDL-C-normalized HDL-bound S<em>1</em>P was also higher in both sCAD (0.<em>1</em>8 mg/mL) and MI (0.<em>1</em>5 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S<em>1</em>P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S<em>1</em>P and the S<em>1</em>P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than <em>1</em>2 h had the highest levels of total and normalized plasma S<em>1</em>P, as well as the highest levels of S<em>1</em>P in isolated HDL. The HDL-C-normalized plasma level of S<em>1</em>P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S<em>1</em>P in CAD, and may represent a novel marker of HDL dysfunction.

The signaling lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) stabilizes the vasculature, directs lymphocyte egress from lymphoid organs, and shapes inflammatory responses. However, little is known about how S<em>1</em>P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. We have found that lipid <em>phosphate</em> phosphatase 3 (LPP3) enables efficient export of mature T cells from the thymus into circulation, and several lines of evidence suggest that LPP3 promotes exit by destroying thymic S<em>1</em>P. Although five additional S<em>1</em>P-degrading enzymes are expressed in the thymus, they cannot compensate for the loss of LPP3. Moreover, conditional deletion of LPP3 in either epithelial cells or endothelial cells is sufficient to inhibit egress. These results suggest that S<em>1</em>P generation and destruction are tightly regulated and that LPP3 is essential to establish the balance.

Although most of pharmacological therapies for cancer utilize the apoptotic machinery of the cells, the available anti-cancer drugs are limited due to the ability of prostate cancer cells to escape from the anti-cancer drug-induced apoptosis. A human prostate cancer cell line PC3 is resistant to camptothecin (CPT). To elucidate the mechanism of this resistance, we have examined the involvement of <em>sphingosine</em> kinase (SPHK) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK<em>1</em>, and also elevated expression of S<em>1</em>P receptors, S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3), as compared with those of LNCaP cells. The knockdown of SPHK<em>1</em> by small interfering RNA and inhibition of S<em>1</em>P receptor signaling by pertussis toxin in PC3 cells induced significant inhibition of cell growth, suggesting implication of SPHK<em>1</em> and S<em>1</em>P receptors in cell proliferation in PC3 cells. Furthermore, the treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK<em>1</em>/S<em>1</em>P signaling by induction of both SPHK<em>1</em> enzyme and S<em>1</em>P(<em>1</em>)/S<em>1</em>P(3) receptors. These findings strongly suggest that high expression and up-regulation of SPHK<em>1</em> and S<em>1</em>P receptors protect PC3 cells from the apoptosis induced by CPT.

Members of a subfamily of G protein-coupled receptors (GPCRs), encoded by five different endothelial differentiation genes (edgs), specifically mediate effects of lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) on cellular proliferation and differentiation. Mechanisms of suppression of apoptosis by LPA and S<em>1</em>P were studied in the Tsup-<em>1</em> cultured line of human T lymphoblastoma cells, which express Edg-2 and Edg-4 GPCRs for LPA and Edg-3 and Edg-5 GPCRs for S<em>1</em>P. At <em>1</em>0-<em>1</em>0 M to <em>1</em>0-7 M, both LPA and S<em>1</em>P protected Tsup-<em>1</em> cells from apoptosis induced by Abs to Fas, CD2, and CD3 plus CD28 in combination. Apoptosis elicited by C6 ceramide was inhibited by S<em>1</em>P, but not by LPA, in part because ceramide suppressed expression of Edg-2 and Edg-4 surface receptors for LPA without affecting Edg-3 surface receptors for S<em>1</em>P. At <em>1</em>0-9 M to <em>1</em>0-7 M, LPA and S<em>1</em>P significantly suppressed cellular levels of the apoptosis-promoting protein Bax, without altering the levels of Bcl-xL or Bcl-2 assessed by Western blots and immunoassays. Transfections of pairs of antisense plasmids for Edg-2 plus Edg-4 and Edg-3 plus Edg-5, and hygromycin selection of transfectants with reduced expression of the respective Edg R proteins in Western blots, inhibited both protection from apoptosis and reduction in cellular levels of Bax by LPA and S<em>1</em>P. Thus, LPA and S<em>1</em>P protection from apoptosis is mediated by distinct Edg GPCRs and may involve novel effects on Bax regulatory protein.

Hypoxia and signaling via hypoxia-inducible factor-<em>1</em> (HIF-<em>1</em>) is a key feature of solid tumors and is related to tumor progression as well as treatment failure. Although it is generally accepted that HIF-<em>1</em> provokes tumor cell survival and induces chemoresistance under hypoxia, HIF-<em>1</em>-independent mechanisms operate as well. We present evidence that conditioned medium obtained from A549 cells, incubated for 24 h under hypoxia, protected naive A549 cells from etoposide-induced cell death. Lipid extracts generated from hypoxia-conditioned medium still rescued cells from apoptosis induced by etoposide. Specifically, the bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) not only was essential for cell viability of A549 cells but also protected cells from apoptosis. We noticed an increase in <em>sphingosine</em> kinase 2 (SphK2) protein level and enzymatic activity under hypoxia, which correlated with the release of S<em>1</em>P into the medium. Knockdown of SphK2 using specific small interfering RNA relieved chemoresistance of A549 cells under hypoxia and conditioned medium obtained from SphK2 knockdown cells was only partially protective. Coincubations of conditioned medium with VPC230<em>1</em>9, a S<em>1</em>P(<em>1</em>)/S<em>1</em>P(3) antagonist, reduced protection of conditioned medium, with the further notion that p42/44 mitogen-activated protein kinase transmits autocrine or paracrine survival signaling downstream of S<em>1</em>P(<em>1</em>)/S<em>1</em>P(3) receptors. Our data suggest that hypoxia activates SphK2 to promote the synthesis and release of S<em>1</em>P, which in turn binds to S<em>1</em>P(<em>1</em>)/S<em>1</em>P(3) receptors, thus activating p42/44 mitogen-activated protein kinase to convey autocrine or paracrine protection of A549 cells.

Fingolimod (FTY720) and its phosphorylated form FTY720P are modulators of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors, which are G-protein coupled receptors linked to cell migration and vascular maturation. The efficacy of FTY720 in autoimmune diseases such as multiple sclerosis and its animal models has been attributed to its inhibition of lymphocyte trafficking to target organs. In this study, we examined the role of S<em>1</em>P receptors in cultured rat oligodendrocytes (OLGs) and OLG progenitor cells (OPCs) using the active phosphorylated form of FTY720. We found that (<em>1</em>) FTY720P improves the survival of neonatal rat OLGs during serum withdrawal, which is associated with the phosphorylation of extracellular signal regulated kinases (ERK<em>1</em>/2) and Akt; (2) FTY720P regulates OPC differentiation into OLGs in a concentration-dependent manner; and (3) S<em>1</em>P receptors are differentially modulated by platelet-derived growth factor (PDGF) resulting in downregulation of S<em>1</em>P5 and upregulation of S<em>1</em>P<em>1</em> in OPCs. In addition, siRNA studies revealed that S<em>1</em>P<em>1</em> participates in PDGF-induced OPC mitogenesis. We conclude that S<em>1</em>P<em>1</em> and S<em>1</em>P5 serve different functions during oligodendroglial development, and possibly during remyelination.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a platelet-derived sphingolipid that activates G protein-coupled S<em>1</em>P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac<em>1</em> is implicated in diverse S<em>1</em>P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S<em>1</em>P-mediated Rac<em>1</em> activation are incompletely understood. We studied the pathways involved in S<em>1</em>P-mediated Rac<em>1</em> activation in bovine aortic endothelial cells (BAEC) and found that S<em>1</em>P-induced Rac<em>1</em> activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S<em>1</em>P-mediated Rac<em>1</em> activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac<em>1</em> activation while completely blocking S<em>1</em>P-induced Akt phosphorylation. We used Rac<em>1</em>-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac<em>1</em> expression and found that siRNA-mediated Rac<em>1</em> knockdown significantly impaired basal as well as S<em>1</em>P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S<em>1</em>P-induced phosphorylation of the mitogen-activated protein kinases ERK<em>1</em>/2 was unperturbed by siRNA-mediated Rac<em>1</em> knockdown. We found that overexpression of the Rac<em>1</em> guanine nucleotide exchange factor (GEF) Tiam<em>1</em> markedly enhanced Rac<em>1</em> activity, whereas a dominant negative Tiam<em>1</em> mutant significantly attenuated S<em>1</em>P-mediated Rac<em>1</em> activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam<em>1</em> as upstream modulators of S<em>1</em>P-mediated Rac<em>1</em> activation, and establish a central role for Rac<em>1</em> in S<em>1</em>P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid mediator that promotes cell survival, proliferation, migration, angiogenesis, lymphangiogenesis, and immune response; all are critical processes of cancer progression. Although some important roles of intracellular S<em>1</em>P have recently been uncovered, the majority of its biological effects are known to be mediated via activation of five specific G protein-coupled receptors [S<em>1</em>P receptor (S<em>1</em>PR)<em>1</em>-S<em>1</em>PR5] located on the cell surface. Secretion of S<em>1</em>P produced inside cells by <em>sphingosine</em> kinases can then signal through these receptors in autocrine, paracrine, and/or endocrine manners, coined "inside-out" signaling of S<em>1</em>P. Numerous studies suggest that secreted S<em>1</em>P plays important roles in cancer progression; thus, understanding the mechanism by which S<em>1</em>P is exported out of cells, particularly cancer cells, is both interesting and important. Here we will review the current understanding of the transport of S<em>1</em>P out of cancer cells and its potential roles in the tumor microenvironment.

Dendritic cells (DCs) and lymphocytes are known to show a migratory response to the phospholipid mediator, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). However, it is unclear whether the same S<em>1</em>P receptor subtype mediates the migration of lymphocytes and DCs toward S<em>1</em>P. In this study, we investigated the involvement of S<em>1</em>P receptor subtypes in S<em>1</em>P-induced migration of CD4 T cells and bone marrow-derived DCs in mice. A potent S<em>1</em>P receptor agonist, the (S)-enantiomer of FTY720-<em>phosphate</em> [(S)-FTY720-P], at 0.<em>1</em> nM or higher and a selective S<em>1</em>P receptor type <em>1</em> (S<em>1</em>P(<em>1</em>)) agonist, SEW287<em>1</em>, at 0.<em>1</em> muM or higher induced a dose-dependent down-regulation of S<em>1</em>P(<em>1</em>). The pretreatment with these compounds resulted in a significant inhibition of mouse CD4 T cell migration toward S<em>1</em>P. Thus, it is revealed that CD4 T cell migration toward S<em>1</em>P is highly dependent on S<em>1</em>P(<em>1</em>). Mature DCs, when compared with CD4 T cells or immature DCs, expressed a relatively higher level of S<em>1</em>P(3) mRNA. S<em>1</em>P at <em>1</em>0-<em>1</em>000 nM induced a marked migration and significantly enhanced the endocytosis of FITC-dextran in mature but not immature DCs. Pretreatment with (S)-FTY720-P at 0.<em>1</em> microM or higher resulted in a significant inhibition of S<em>1</em>P-induced migration and endocytosis in mature DCs, whereas SEW287<em>1</em> up to <em>1</em>00 microM did not show any clear effect. Moreover, we found that S<em>1</em>P-induced migration and endocytosis were at an extremely low level in mature DCs prepared from S<em>1</em>P(3)-knockout mice. These results indicate that S<em>1</em>P regulates migration and endocytosis of murine mature DCs via S<em>1</em>P(3) but not S<em>1</em>P(<em>1</em>).

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a blood-borne lysosphingolipid that acts to promote endothelial cell (EC) barrier function. In plasma, S<em>1</em>P is associated with both high density lipoproteins (HDL) and albumin, but it is not known whether the carriers impart different effects on S<em>1</em>P signaling. Here we establish that HDL-S<em>1</em>P sustains EC barrier longer than albumin-S<em>1</em>P. We showed that the sustained barrier effects of HDL-S<em>1</em>P are dependent on signaling by the S<em>1</em>P receptor, S<em>1</em>P<em>1</em>, and involve persistent activation of Akt and endothelial NOS (eNOS), as well as activity of the downstream NO target, soluble guanylate cyclase (sGC). Total S<em>1</em>P<em>1</em> protein levels were found to be higher in response to HDL-S<em>1</em>P treatment as compared with albumin-S<em>1</em>P, and this effect was not associated with increased S<em>1</em>P<em>1</em> mRNA or dependent on de novo protein synthesis. Several pieces of evidence indicate that long term EC barrier enhancement activity of HDL-S<em>1</em>P is due to specific effects on S<em>1</em>P<em>1</em> trafficking. First, the rate of S<em>1</em>P<em>1</em> degradation, which is proteasome-mediated, was slower in HDL-S<em>1</em>P-treated cells as compared with cells treated with albumin-S<em>1</em>P. Second, the long term barrier-promoting effects of HDL-S<em>1</em>P were abrogated by treatment with the recycling blocker, monensin. Finally, cell surface levels of S<em>1</em>P<em>1</em> and levels of S<em>1</em>P<em>1</em> in caveolin-enriched microdomains were higher after treatment with HDL-S<em>1</em>P as compared with albumin-S<em>1</em>P. Together, the findings reveal S<em>1</em>P carrier-specific effects on S<em>1</em>P<em>1</em> and point to HDL as the physiological mediator of sustained S<em>1</em>P<em>1</em>-PI3K-Akt-eNOS-sGC-dependent EC barrier function.

The sphingolipid metabolites ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have recently been implicated in autophagy. In this study, we report that depletion of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> phosphohydrolase-<em>1</em> (SPP<em>1</em>), an endoplasmic reticulum (ER)-resident enzyme that specifically dephosphorylates S<em>1</em>P, induced autophagy. Although the mammalian target of rapamycin and class III phosphoinositide 3-kinase/Beclin-<em>1</em> pathways were not involved and this autophagy was p53 independent, C/EBP homologous protein, BiP, and phospho-eucaryotic translation initiation factor-2α, and cleavage of procaspases 2 and 4, downstream targets of ER stress, were increased after SPP<em>1</em> depletion. Autophagy was suppressed by depletion of protein kinase regulated by RNA-like ER kinase (PERK), inositol-requiring transmembrane kinase/endonuclease-<em>1</em>α, or activating transcription factor 6, three sensors of the unfolded protein response (UPR) to ER stress. Autophagy triggered by downregulation of SPP<em>1</em> did not lead to apoptosis but rather stimulated, in a PERK dependent manner, the survival signal Akt, whose inhibition then sensitized cells to apoptosis. Although depletion of SPP<em>1</em> increased intracellular levels of S<em>1</em>P and its secretion, activation of cell surface S<em>1</em>P receptors did not induce autophagy. Nevertheless, increases in intracellular pools of S<em>1</em>P, but not dihydro-S<em>1</em>P, induced autophagy and ER stress. Thus, SPP<em>1</em>, by regulating intracellular S<em>1</em>P homeostasis, can control the UPR and ER stress-induced autophagy.

Phospholipase Cepsilon (PLCepsilon) has been suggested to transduce signals from small GTPases, but its biological function has not yet been clarified. Using astrocytes from PLCepsilon-deficient mice, we demonstrate that endogenous G protein-coupled receptors (GPCRs) for lysophosphatidic acid, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, and thrombin regulate phosphoinositide hydrolysis primarily through PLCepsilon. Stimulation by lysophospholipids occurs through G(i), whereas thrombin activates PLC through Rho. Further studies reveal that PLCepsilon is required for thrombin- but not LPA-induced sustained ERK activation and DNA synthesis, providing a novel mechanism for GPCR and Rho signaling to cell proliferation. The requirement for PLCepsilon in this pathway can be explained by its role as a guanine nucleotide exchange factor for Rap<em>1</em>. Thus, PLCepsilon serves to transduce mitogenic signals through a mechanism distinct from its role in generation of PLC-derived second messengers.

The oral <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor (S<em>1</em>PR) modulator fingolimod has been shown to be effective in the treatment of patients with relapsing multiple sclerosis (MS). The drug binds with high affinity to 4 of the 5 G-protein-coupled S<em>1</em>P receptors (S<em>1</em>P(<em>1</em>-5)). After binding, the receptors are internalized, degraded, and thus functionally antagonized by fingolimod. Under physiologic conditions, S<em>1</em>P(<em>1</em>) mediates the egress of lymphocytes from secondary lymphoid organs to the peripheral circulation. Functional antagonism of S<em>1</em>P(<em>1</em>) by fingolimod results in a reduction in peripheral lymphocyte counts by inhibiting egress of lymphocytes, including potentially encephalitogenic T cells and their naïve progenitors that would otherwise be present within the circulation. Despite the fingolimod-mediated reduction of lymphocyte counts, fingolimod-treated patients with MS have been shown to have few infections and related complications and were able to mount antigen-specific immune responses in vaccination studies.

Mammalian LPPs (lipid <em>phosphate</em> phosphatases) are integral membrane proteins that belong to a superfamily of lipid phosphatases/phosphotransferases. They have broad substrate specificity in vitro, dephosphorylating PA (phosphatidic acid), S<em>1</em>P (<em>sphingosine</em> <em>1</em>-<em>phosphate</em>), LPA (lysophosphatidic acid) etc. Their physiological role may include the attenuation of S<em>1</em>P- and LPA-stimulated signalling by virtue of an ecto-activity (i.e. dephosphorylation of extracellular S<em>1</em>P and LPA), thereby limiting the activation of LPA- and S<em>1</em>P-specific G-protein-coupled receptors at the cell surface. However, our recent work suggests that an intracellular action of LPP2 and LPP3 may account for the reduced agonist-stimulated p42/p44 mitogen-activated protein kinase activation of HEK-293 (human embryonic kidney 293) cells. This may involve a reduction in the basal levels of PA and S<em>1</em>P respectively and the presence of an early apoptotic phenotype under conditions of stress (serum deprivation). Additionally, we describe a model whereby LPP2, but not LPP3, may be functionally linked to the phospholipase D<em>1</em>-derived PA-dependent recruitment of <em>sphingosine</em> kinase <em>1</em> to the perinuclear compartment. We also consider the potential regulatory mechanisms for LPPs, which may involve oligomerization. Lastly, we highlight many aspects of the LPP biology that remain to be fully defined.

Angiogenesis, the formation of new blood vessels, is an important component of restoration of hematopoiesis after BMT, but the mediators involved in hematopoietic angiogenesis have not been identified. We examined the influence of the lipid growth factors, phosphatidic acid (PA), lysophosphatidic acid (LPA), and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), on several angiogenic properties of endothelial cells, including migration and stabilization of vascular barrier integrity. In a previous study, PA was found to disrupt the permeability of established endothelial monolayers, an early event in the angiogenic response that liberates cells for subsequent mobilization. In the present study, both PA and LPA weakly induced the chemotactic migration of endothelial cells from an established monolayer. The chemotactic response induced by PA and LPA was similar in intensity to that observed with optimal levels of the known protein endothelial cell chemoattractants, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). A markedly greater chemotactic response was effected by nanomolar concentrations of S<em>1</em>P, indicating that this platelet-derived factor plays an important role in a key aspect of angiogenesis, chemotactic migration of endothelial cells. The chemotactic response to S<em>1</em>P was completely inhibited by preincubation of endothelial cells with antisense oligonucleotides to the high-affinity S<em>1</em>P receptor, Edg-<em>1</em>. In addition, chemotaxis of endothelial cells to S<em>1</em>P was inhibited by preincubation of cells with specific inhibitors of tyrosine kinases, but inhibitors of phosphatidylinositol 3' kinase had little effect. Finally, LPA effectively stabilized endothelial monolayer barrier function, a late event in angiogenesis. Thus, the phospholipid growth factors, PA, S<em>1</em>P, and LPA, display divergent and potent effects on angiogenic properties of endothelial cells and angiogenic differentiation of endothelial cells potentially act in tandem to effectively induce neovascularization. These mediators may thus exert important roles in restoration of hematopoiesis, as they facilitate blood vessel formation at sites of transplanted stem cells, allowing the progeny of engrafted progenitors to move from marrow sinusoids to the peripheral vasculature.

Agonists of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor (S<em>1</em>PR) attenuate kidney ischemia-reperfusion injury (IRI). Previous studies suggested that S<em>1</em>P<em>1</em>R-induced lymphopenia mediates this protective effect, but lymphocyte-independent mechanisms could also contribute. Here, we investigated the effects of S<em>1</em>PR agonists on kidney IRI in mice that lack T and B lymphocytes (Rag-<em>1</em> knockout mice). Administration of the nonselective S<em>1</em>PR agonist FTY720 or the selective S<em>1</em>P<em>1</em>R agonist SEW287<em>1</em> reduced injury in both Rag-<em>1</em> knockout and wild-type mice. In vitro, SEW287<em>1</em> significantly attenuated LPS- or hypoxia/reoxygenation-induced apoptosis in cultured mouse proximal tubule epithelial cells, supporting a direct protective effect of S<em>1</em>P<em>1</em>R agonists via mitogen-activated protein kinase and/or Akt pathways. S<em>1</em>P<em>1</em>Rs in the proximal tubule mediated IRI in vivo as well: Mice deficient in proximal tubule S<em>1</em>P<em>1</em>Rs experienced a greater decline in renal function after IRI than control mice and their kidneys were no longer protected by SEW287<em>1</em> administration. In summary, S<em>1</em>PRs in the proximal tubule are necessary for stress-induced cell survival, and S<em>1</em>P<em>1</em>R agonists are renoprotective via direct effects on the tubule cells. Selective agonists of S<em>1</em>P<em>1</em>Rs may hold therapeutic potential for the prevention and treatment of acute kidney injury.

OBJECTIVE

High-density lipoproteins (HDL) are endowed with cardiovascular protective activities. In addition to their role in reverse cholesterol transport, HDL exert several beneficial effects on endothelial cells, including the induction of endothelial nitric oxide synthase and prostacyclin release, and the control of the immune and inflammatory response.

RESULTS

To identify possible mechanisms involved in these effects we investigated the modulation of the expression of acute phase proteins of the pentraxin superfamily, such as C-reactive protein (CRP), serum amyloid P component protein (SAP), and the long pentraxin 3 (PTX3) by HDL in human endothelial cells. HDL induced PTX3 mRNA expression and protein release, whereas no effect was observed on CRP and SAP expression. This effect was mainly dependent on the activation of the lysosphingolipids receptors-PI3K/Akt axis and was mimicked by <em>sphingosine</em> <em>1</em> <em>phosphate</em> and other S<em>1</em>P mimetics. This observation was confirmed in vivo; indeed an increased expression of PTX3 mRNA was detected in the aorta of transgenic mice overexpressing human apoA-I, compared to apoA-I knock-out mice. Furthermore, plasma levels of PTX3 significantly increased in C57BL/6 mice injected with HDL.

CONCLUSIONS

These data suggest that part of the atheroprotective effects of HDL could result from the modulation of molecules that act as sensors of the immunoinflammatory balance in the vascular wall.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S<em>1</em>P is catalyzed by S<em>1</em>P lyase (SPL). The human SGPL<em>1</em> gene that encodes SPL maps to a region often mutated in cancers. To investigate the effect of SPL deficiency on cell survival and transformation, the susceptibility to anticancer drugs of fibroblasts generated from SPL-deficient mouse embryos (Sgpl<em>1</em>(-/-)) was compared with that of cells from heterozygous (Sgpl<em>1</em>(+/-)) or wild-type (Sgpl<em>1</em>(+/+)) embryos. First, loss of SPL caused resistance to the toxic effects of etoposide and doxorubicin. Interestingly, heterozygosity for the Sgpl<em>1</em> gene resulted in partial resistance to apoptosis. Secondly, doxorubicin-induced apoptotic signaling was strongly inhibited in Sgpl<em>1</em>(-/-) cells (phosphatidylserine externalization, caspase activation, and cytochrome c release). This was accompanied by a strong increase in Bcl-2 and Bcl-xL protein content. Whereas correction of SPL deficiency in Sgpl<em>1</em>(-/-) cells led to downregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL small interfering RNA-mediated knockdown in SPL-deficient cells resulted in increased sensitivity to doxorubicin, suggesting that Bcl-2 upregulation mediates SPL protective effects. Moreover, SPL deficiency led to increased cell proliferation, anchorage-independent cell growth, and formation of tumors in nude mice. Finally, transcriptomic studies showed that SPL expression is downregulated in human melanoma cell lines. Thus, by affecting S<em>1</em>P metabolism and the expression of Bcl-2 members, the loss of SPL enhances cell resistance to anticancer regimens and results in an increased ability of cells to acquire a transformed phenotype and become malignant.

An increasing number of studies show how changes in intracellular metabolic pathways alter tumor and immune cell function. However, little information about metabolic changes in other cell types, including synovial fibroblasts, is available. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) are the most common cell type at the pannus-cartilage junction and contribute to joint destruction through their production of cytokines, chemokines, and matrix-degrading molecules and by migrating and invading joint cartilage. In this review, we show that these cells differ from healthy synovial fibroblasts, not only in their marker expression, proto-oncogene expression, or their epigenetic changes, but also in their intracellular metabolism. These metabolic changes must occur due to the stressful microenvironment of inflamed tissues, where concentrations of crucial nutrients such as glucose, glutamine, and oxygen are spatially and temporally heterogeneous. In addition, these metabolic changes will increase metabolite exchange between fibroblast and other synovial cells, which can potentially be activated. Glucose and phospholipid metabolism as well as bioactive lipids, including <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and lysophosphatidic acid, among others, are involved in FLS activation. These metabolic changes likely contribute to FLS involvement in aspects of immune response initiation or abnormal immune responses and strongly contribute to joint destruction.

Strong evidence exists for interactions of zwitterionic <em>phosphate</em> and amine groups in <em>sphingosine</em>-<em>1</em> <em>phosphate</em> (S<em>1</em>P) to conserved Arg and Glu residues present at the extracellular face of the third transmembrane domain of S<em>1</em>P receptors. The contribution of Arg(<em>1</em>20) and Glu(<em>1</em>2<em>1</em>) for high-affinity ligand-receptor interactions is essential, because single-point R(<em>1</em>20)A or E(<em>1</em>2<em>1</em>)A S<em>1</em>P(<em>1</em>) mutants neither bind S<em>1</em>P nor transduce S<em>1</em>P function. Because S<em>1</em>P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance. Here we describe a modestly water-soluble highly selective S<em>1</em>P(<em>1</em>) agonist [2-(4-(5-(3,4-diethoxyphenyl)-<em>1</em>,2,4-oxadiazol-3-yl)-2,3-dihydro-<em>1</em>H-inden-<em>1</em>-yl amino) ethanol (CYM-5442)] that does not require Arg(<em>1</em>20) or Glu(<em>1</em>2<em>1</em>) residues for activating S<em>1</em>P(<em>1</em>)-dependent p42/p44 mitogen-activated protein kinase phosphorylation, which defines a new hydrophobic pocket in S<em>1</em>P(<em>1</em>). CYM-5442 is a full agonist in vitro for S<em>1</em>P(<em>1</em>) internalization, phosphorylation, and ubiquitination. It is noteworthy that CYM-5442 was a full agonist for induction and maintenance of S<em>1</em>P(<em>1</em>)-dependent blood lymphopenia, decreasing B lymphocytes by 65% and T lymphocytes by 85% of vehicle. Induction of CYM-5442 lymphopenia was dose- and time-dependent, requiring serum concentrations in the 50 nM range. In vitro measures of S<em>1</em>P(<em>1</em>) activation by CYM-5442 were noncompetitively inhibited by a specific S<em>1</em>P(<em>1</em>) antagonist [(R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W<em>1</em>46)], competitive for S<em>1</em>P, 2-amino-2-(4-octylphenethyl)propane-<em>1</em>,3-diol (FTY720-P), and 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-<em>1</em>,2, 4-oxadiazole (SEW287<em>1</em>). In addition, lymphopenia induced by CYM-5442 was reversed by W<em>1</em>46 administration or upon pharmacokinetic agonist clearance. Pharmacokinetics in mice also indicated that CYM-5442 partitions significantly in central nervous tissue. These data show that CYM-5442 activates S<em>1</em>P(<em>1</em>)-dependent pathways in vitro and to levels of full efficacy in vivo through a hydrophobic pocket separate from the orthosteric site of S<em>1</em>P binding that is headgroup-dependent.