<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differentiation gene) family G protein-coupled receptors EDG<em>1</em>, EDG3, and EDG5 (AGR<em>1</em>6). Among prominent activities of S<em>1</em>P is the regulation of cell motility; S<em>1</em>P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG<em>1</em> or EDG3 (EDG<em>1</em> cells or EDG3 cells, respectively) S<em>1</em>P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase- and Rac-dependent manners. Both S<em>1</em>P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG<em>1</em> and EDG3 cells, S<em>1</em>P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S<em>1</em>P. In EDG5 cells, S<em>1</em>P stimulated PI 3-kinase activity as it did in EDG<em>1</em> cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S<em>1</em>P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S<em>1</em>P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N(<em>1</em>7)-Cdc42, but not N(<em>1</em>9)-RhoA, suppressed S<em>1</em>P- and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.

A significant and sustained increase in vascular permeability is a hallmark of acute inflammatory diseases such as acute lung injury (ALI) and sepsis and is an essential component of tumor metastasis, angiogenesis, and atherosclerosis. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), an endogenous bioactive lipid produced in many cell types, regulates endothelial barrier function by activation of its G-protein coupled receptor S<em>1</em>P(<em>1</em>). S<em>1</em>P enhances vascular barrier function through a series of profound events initiated by S<em>1</em>P(<em>1</em>) ligation with subsequent downstream activation of the Rho family of small GTPases, cytoskeletal reorganization, adherens junction and tight junction assembly, and focal adhesion formation. Furthermore, recent studies have identified transactivation of S<em>1</em>P(<em>1</em>) signaling by other barrier-enhancing agents as a common mechanism for promoting endothelial barrier function. This review summarizes the state of our current knowledge about the mechanisms through which the S<em>1</em>P/S<em>1</em>P(<em>1</em>) axis reduces vascular permeability, which remains an area of active investigation that will hopefully produce novel therapeutic agents in the near future.

The sphingolipid metabolite, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), has emerged as a critical player in a number of fundamental biological processes and is important in cancer, angiogenesis, wound healing, cardiovascular function, atherosclerosis, immunity and asthma, among others. Activation of <em>sphingosine</em> kinases, enzymes that catalyze the phosphorylation of <em>sphingosine</em> to S<em>1</em>P, by a variety of agonists, including growth factors, cytokines, hormones, and antigen, increases intracellular S<em>1</em>P. Many of the biological effects of S<em>1</em>P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface in an autocrine and/or paracrine manner. Therefore, understanding the mechanism by which intracellularly generated S<em>1</em>P is released out of cells is both interesting and important. In this review, we will discuss how S<em>1</em>P is formed and released. We will focus particularly on the current knowledge of how the S<em>1</em>P gradient between tissues and blood is maintained, and the role of ABC transporters in S<em>1</em>P release.

OBJECTIVE

FTY720 is a known <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor agonist. In the present study, we investigated the neuroprotective effect of postischemic administration of FTY720 in rats with 2 hours transient middle cerebral artery occlusion (MCAO).

METHODS

One hundred eleven male rats were randomly assigned to sham-operated and MCAO treated with vehicle, 0.25 mg/kg and <em>1</em> mg/kg of FTY720, another selective <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-<em>1</em> agonist SEW287<em>1</em> (5 mg/kg), or 0.25 mg/kg of FTY720 plus a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> antagonist, VPC230<em>1</em>9 (0.5 mg/kg). Drugs were injected intraperitoneally immediately after reperfusion. Neurological score and infarct volume were assessed at 24 and 72 hours after MCAO. Western blotting, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated uridine 5'-tri<em>phosphate</em>-biotin nick end-labeling were conducted at 24 hours after MCAO.

RESULTS

FTY720 significantly reduced infarct volume and improved neurological score at 24 and 72 hours after MCAO compared with the vehicle group. SEW287<em>1</em> showed similar neuroprotective effects to FTY720, whereas VPC 203<em>1</em>9 abolished the neuroprotective effects of FTY720. FTY720 significantly retained Akt and extracellular signal-regulated kinase phosphorylation and Bcl-2 expression and decreased cleaved caspase-3 expression and terminal deoxynucleotidyl transferase-mediated uridine 5'-tri<em>phosphate</em>-biotin nick end-labeling-positive neurons at 24 hours after MCAO. VPC230<em>1</em>9 blocked the antiapoptotic effects of FTY720.

CONCLUSIONS

These data suggest that activation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>-<em>1</em> by FTY720 reduces neuronal death after transient MCAO.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a product of sphingomyelin (SM) metabolism. It is present in most eukaryotic organisms; however, in higher vertebrates (bony fishes to mammals), it is utilized as a regulator of cell function by binding to extracellular receptors. Its role as a second messenger function was originally proposed; however, the unequivocal role of S<em>1</em>P as a second messenger in higher eukaryotes is lacking at present. As an extracellular mediator, S<em>1</em>P binds to a family of G-protein-coupled receptors named S<em>1</em>P(<em>1</em>-5), originally referred to as (EDG-<em>1</em>, 3, 5, 6 and 8). These receptors couple to multiple G-proteins and regulate intracellular signaling pathways. Most tissues express one or more S<em>1</em>P receptor subtypes; therefore, S<em>1</em>P is known as a multifunctional physiologic mediator. Its precise physiologic roles in multiple organ systems are just beginning to be elucidated; however, at present, regulation of cardiogenesis, vascular system formation, oocyte survival and immune cell trafficking has been recently reported. Currently, a S<em>1</em>P receptor agonist, is undergoing clinical trials as an agent to fight organ transplant rejection. Since S<em>1</em>P is a fundamental multifunctional mediator, better understanding of the biology of S<em>1</em>P holds great promise to develop novel tools to control various diseases.

Recent studies have identified factors responsible for angiogenesis within developing tumors, but mediators of vessel formation at sites of trauma, injury, and wound healing are not clearly established. Here we show that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) released by platelets during blood clotting is a potent, specific, and selective endothelial cell chemoattractant that accounts for most of the strong endothelial cell chemotactic activity of blood serum, an activity that is markedly diminished in plasma. Preincubation of endothelial cells with pertussis toxin inhibited this effect of S<em>1</em>P, demonstrating the involvement of a Galphai-coupled receptor. After S<em>1</em>P-induced migration, endothelial cells proliferated avidly and differentiated forming multicellular structures suggestive of early blood vessel formation. S<em>1</em>P was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Our results show that blood coagulation initiates endothelial cell angiogenic responses through the release of S<em>1</em>P, a potent endothelial cell chemoattractant that exerts its effects by activating a receptor-dependent process.

The sphingolipid metabolite, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), formed by phosphorylation of <em>sphingosine</em>, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S<em>1</em>P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of <em>sphingosine</em> kinase type <em>1</em> (SPHK<em>1</em>) increased S<em>1</em>P levels and blocked MCF-7 cell death induced by anti-cancer drugs, <em>sphingosine</em>, and TNF-alpha. SPHK<em>1</em> also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK<em>1</em> stimulated ERK<em>1</em>/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S<em>1</em>P or dihydro-S<em>1</em>P, ligands for the S<em>1</em>P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK<em>1</em> cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK<em>1</em> may play an important role in breast cancer progression by regulating tumor cell growth and survival.

XBP-<em>1</em>, a transcription factor that drives the unfolded protein response (UPR), is activated in B cells when they differentiate to plasma cells. Here, we show that in the B cells, whose capacity to secrete IgM has been eliminated, XBP-<em>1</em> is induced normally on induction of differentiation, suggesting that activation of XBP-<em>1</em> in B cells is a differentiation-dependent event, but not the result of a UPR caused by the abundant synthesis of secreted IgM. Without XBP-<em>1</em>, B cells fail to signal effectively through the B-cell receptor. The signalling defects lead to aberrant expression of the plasma cell transcription factors IRF4 and Blimp-<em>1</em>, and altered levels of activation-induced cytidine deaminase and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor. Using XBP-<em>1</em>-deficient/Blimp-<em>1</em>-GFP transgenic mice, we find that XBP-<em>1</em>-deficient B cells form antibody-secreting plasmablasts in response to initial immunization; however, these plasmablasts respond ineffectively to CXCL<em>1</em>2. They fail to colonize the bone marrow and do not sustain antibody production. These findings define the role of XBP-<em>1</em> in normal plasma cell development and have implications for management of B-cell malignancies.

The S<em>1</em>P(2) receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite <em>sphingosine</em> <em>1</em>-<em>phosphate</em> with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S<em>1</em>P(2) receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S<em>1</em>P(2) receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S<em>1</em>P(2) receptor-null mice showed markedly disturbed structures. JTE0<em>1</em>3, an S<em>1</em>P(2) receptor-specific antagonist, blocked the S<em>1</em>P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S<em>1</em>P(2) receptor-null mice.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) is an enzyme that catalyses the phosphorylation of <em>sphingosine</em> to produce the bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). We demonstrate here that FTY720 (Fingolimod) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK<em>1</em> catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK<em>1</em> in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK<em>1</em> is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called 'inside-out' signalling by S<em>1</em>P, as LNCaP-AI cells exhibit increased expression of S<em>1</em>P(2/3) receptors and reduced lipid <em>phosphate</em> phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S<em>1</em>P at S<em>1</em>P(2/3) receptors.

OBJECTIVE

Pronounced inflammatory reactions occurring shortly after intracerebral hemorrhage (ICH) contribute to the formation and progression of perihematomal edema (PHE) and secondary brain injury. We hypothesized that modulation of brain inflammation reduces edema, thus improving clinical outcomes in patients with ICH.

OBJECTIVE

To investigate whether oral administration of fingolimod, a Food and Drug Administration-approved <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor modulator for multiple sclerosis, is safe and effective in alleviating PHE and neurologic deficits in patients with ICH.

METHODS

In this 2-arm, evaluator-blinded study, we included 23 patients with primary supratentorial ICH with hematomal volume of 5 to 30 mL. Clinical and neuroimaging feature-matched patients were treated with standard care with or without oral fingolimod. The study was conducted in Tianjin Medical University General Hospital, Tianjin, China.

METHODS

All patients received standard management alone (control participants) or combined with fingolimod (FTY720, Gilenya), 0.5 mg, orally for 3 consecutive days. Treatment was initiated within <em>1</em> hour after the baseline computed tomographic scan and no later than 72 hours after the onset of symptoms.

METHODS

Neurologic status and hematomal and PHE volumes (Ev) and relative PHE, defined as Ev divided by hematomal volume, were monitored by clinical assessment and magnetic resonance imaging, respectively, for 3 months.

RESULTS

Patients treated with fingolimod exhibited a reduction of neurologic impairment compared with control individuals, regained a Glasgow Coma Scale score of <em>1</em>5 by day 7 (<em>1</em>00% vs 50%, P = .0<em>1</em>), and had a National Institutes of Health Stroke Scale score reduction of 7.5 vs 0.5 (P < .00<em>1</em>). Neurologic functions improved in these patients in the first week coincident with a reduction of circulating lymphocyte counts. At 3 months, a greater proportion of patients receiving fingolimod achieved full recovery of neurologic functions (modified Barthel Index score range, 95-<em>1</em>00; 63% vs 0%; P = .00<em>1</em>; modified Rankin Scale score range, 0-<em>1</em>; 63% vs 0%; P = .00<em>1</em>), and fewer reported ICH-related lung infections. Perihematomal edema volume and rPHE were significantly smaller in fingolimod-treated patients than in control individuals (Ev at day 7, 47 mL vs <em>1</em>08 mL, P = .04; Ev at day <em>1</em>4, 55 mL vs <em>1</em>24 mL, P = .07; rPHE at day 7, 2.5 vs 6.4, P < .00<em>1</em>; rPHE at day <em>1</em>4, 2.6 vs 7.7, P = .003, respectively). We recorded no differences between groups in the occurrence of adverse events.

CONCLUSIONS

In patients with small- to moderate-sized deep primary supratentorial ICH, administration of oral fingolimod within 72 hours of disease onset was safe, reduced PHE, attenuated neurologic deficits, and promoted recovery. The efficacy of fingolimod in preventing secondary brain injury in patients with ICH warrants further investigation in late-phase trials.

BACKGROUND

clinicaltrials.gov Identifier:NCT02002390.

Lipid metabolism is regulated by multiple signaling pathways, and generates a variety of bioactive lipid molecules. These bioactive lipid molecules known as signaling molecules, such as fatty acid, eicosanoids, diacylglycerol, phosphatidic acid, lysophophatidic acid, ceramide, <em>sphingosine</em>, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, phosphatidylinositol-3 <em>phosphate</em>, and cholesterol, are involved in the activation or regulation of different signaling pathways. Lipid metabolism participates in the regulation of many cellular processes such as cell growth, proliferation, differentiation, survival, apoptosis, inflammation, motility, membrane homeostasis, chemotherapy response, and drug resistance. Bioactive lipid molecules promote apoptosis via the intrinsic pathway by modulating mitochondrial membrane permeability and activating different enzymes including caspases. In this review, we discuss recent data in the fields of lipid metabolism, lipid-mediated apoptosis, and cancer therapy. In conclusion, understanding the underlying molecular mechanism of lipid metabolism and the function of different lipid molecules could provide the basis for cancer cell death rationale, discover novel and potential targets, and develop new anticancer drugs for cancer therapy.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) is a key enzyme critical to the sphingolipid metabolic pathway responsible for catalyzing the formation of the bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. SK<em>1</em>-mediated production of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> has been shown to stimulate such biological processes as cell growth, differentiation, migration, angiogenesis, and inhibition of apoptosis. In this study, cell type-specific immunolocalization of SK<em>1</em> was examined in the bronchus/terminal bronchiole of the lung. Strong immunopositive staining was evident at the apical surface of pseudostratified epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, immature chondrocytes, type II alveolar cells, foamy macrophages, endothelial cells of blood vessels, and neural bundles. Immunohistochemical screening for SK<em>1</em> expression was performed in 25 samples of normal/tumor patient matched non-small-cell lung cancer tissue and found that 25 of 25 tumor samples (carcinoid [5 samples], squamous [<em>1</em>0 samples], and adenocarcinoma tumors [<em>1</em>0 samples]), exhibited overwhelmingly positive immunostaining for SK<em>1</em> as compared with patient-matched normal tissue. In addition, an approximately 2-fold elevation of SK<em>1</em> mRNA expression was observed in lung cancer tissue versus normal tissue, as well as in several other solid tumors. Taken together, these findings define the localization of SK<em>1</em> in lung and provide clues as to how SK<em>1</em> may play a role in normal lung physiology and the pathophysiology of lung cancer.

Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential effect of camptothecin and docetaxel on human PC-3 and LNCaP prostate cancer cells to determine their effect on <em>sphingosine</em> kinase-<em>1</em> (SphK<em>1</em>) activity and subsequent ceramide/<em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) balance in relation with cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK<em>1</em> and elevation of the ceramide/S<em>1</em>P ratio only in cell lines sensitive to these drugs. SphK<em>1</em> overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing the ceramide/S<em>1</em>P ratio. Alternatively, silencing SphK<em>1</em> by RNA interference or pharmacologic inhibition induced apoptosis coupled with ceramide elevation and loss of S<em>1</em>P. The differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/green fluorescent protein model established in nude mice. Docetaxel induced a stronger SphK<em>1</em> inhibition and ceramide/S<em>1</em>P ratio elevation than camptothecin. This was accompanied by a smaller tumor volume and the reduced occurrence and number of metastases. SphK<em>1</em>-overexpressing PC-3 cells implanted in animals developed remarkably larger tumors and resistance to docetaxel treatment. These results provide the first in vivo demonstration of SphK<em>1</em> as a sensor of chemotherapy.

SphK (<em>sphingosine</em> kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S<em>1</em>P (<em>sphingosine</em> <em>1</em>-<em>phosphate</em>). S<em>1</em>P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S<em>1</em>P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK<em>1</em>. PF-543 inhibits SphK<em>1</em> with a K(i) of 3.6 nM, is <em>sphingosine</em>-competitive and is more than <em>1</em>00-fold selective for SphK<em>1</em> over the SphK2 isoform. In <em>1</em>483 head and neck carcinoma cells, which are characterized by high levels of SphK<em>1</em> expression and an unusually high rate of S<em>1</em>P production, PF-543 decreased the level of endogenous S<em>1</em>P <em>1</em>0-fold with a proportional increase in the level of <em>sphingosine</em>. In contrast with past reports that show that the growth of many cancer cell lines is SphK<em>1</em>-dependent, specific inhibition of SphK<em>1</em> had no effect on the proliferation and survival of <em>1</em>483 cells, despite a dramatic change in the cellular S<em>1</em>P/<em>sphingosine</em> ratio. PF-543 was effective as a potent inhibitor of S<em>1</em>P formation in whole blood, indicating that the SphK<em>1</em> isoform of <em>sphingosine</em> kinase is the major source of S<em>1</em>P in human blood. PF-543 is the most potent inhibitor of SphK<em>1</em> described to date and it will be useful for dissecting specific roles of SphK<em>1</em>-driven S<em>1</em>P signalling.

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S<em>1</em>P receptor <em>1</em> (S<em>1</em>P₁) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S<em>1</em>P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

OBJECTIVE

To report the results of a 24-month extension of a phase II trial assessing the efficacy, safety, and tolerability of the once-daily oral <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor modulator, fingolimod (FTY720), in relapsing multiple sclerosis (MS).

METHODS

In the randomized, double-blind, placebo-controlled core study, 28<em>1</em> patients received placebo or FTY720, <em>1</em>.25 or 5.0 mg/day, for 6 months. During the subsequent dose-blinded extension, patients assigned to placebo were re-randomized to either dose of FTY720; those originally assigned to FTY720 continued at the same dose. Patients receiving FTY720 5.0 mg were switched to <em>1</em>.25 mg during the month <em>1</em>5 to month 24 study visits.

RESULTS

Of 28<em>1</em> patients randomized in the core study, 250 (89%) entered the extension phase, and <em>1</em>89 (75.6%) received treatment for 24 months. During the core study, FTY720 significantly reduced gadolinium-enhanced (Gd(+)) lesions and annualized relapse rate (ARR) compared with placebo, with no differences between doses. During the extension phase, patients who switched from placebo to FTY720 showed clear reductions in ARR and lesion counts compared with the placebo phase; ARR and lesion counts remained low in patients who continued FTY720 treatment. After 24 months, 79 to 9<em>1</em>% of patients were free from Gd(+) lesions and up to 77% of patients remained relapse free. FTY720 was well tolerated; no new safety concerns emerged during months 7 to 24 compared with the 6-month core study.

CONCLUSIONS

Once-daily oral treatment with FTY720, <em>1</em>.25 or 5.0 mg, for up to 2 years, was well tolerated and was associated with low relapse rates and lesion activity.

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), <em>sphingosine</em> (Sph), and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>P) and this has necessitated the development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base <em>1</em>-<em>phosphates</em> (SB-<em>1</em>Ps), ceramides (Cers), ceramide <em>1</em>-<em>phosphates</em> (Cer-<em>1</em>P), glucosyl/galactosyl-ceramides (Glu-Cers), and sphingomyelins (SMs) is performed on a Thermo Fisher Scientific triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues, or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a LC/MS/MS system. Qualitative analysis (identification) of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target analyte, synthetic standards, and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration, using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit of subpicomole level. This methodology constitutes a "Lipidomic" approach to study the SPLs metabolism, defining a function of distinct subspecies of individual bioactive SPL classes.

Mice deficient in <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor type 2 (S<em>1</em>P(2)) develop diffuse large B cell lymphoma. However, the role of S<em>1</em>P(2) in normal germinal center (GC) physiology is unknown. Here we show that S<em>1</em>P(2)-deficient GC B cells outgrew their wild-type counterparts in chronically established GCs. We found that antagonism of the kinase Akt mediated by S<em>1</em>P(2) and its downstream mediators Gα(<em>1</em>2), Gα(<em>1</em>3) and p<em>1</em><em>1</em>5RhoGEF regulated cell viability and was required for growth control in chronically proliferating GCs. Moreover, S<em>1</em>P(2) inhibited GC B cell responses to follicular chemoattractants and helped confine cells to the GC. In addition, S<em>1</em>P(2) overexpression promoted the centering of activated B cells in the follicle. We suggest that by inhibiting Akt activation and migration, S<em>1</em>P(2) helps restrict GC B cell survival and localization to an S<em>1</em>P-low niche at the follicle center.

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -<em>1</em> cells, which expressed approximately 2.3- and <em>1</em>9-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C<em>1</em>8-ceramide, which is generated by the human longevity assurance gene <em>1</em> (hLASS<em>1</em>), in sensitive, but not in resistant cells. Inhibition of hLASS<em>1</em> by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS<em>1</em>, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C<em>1</em>8-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS<em>1</em> for ceramide generation in K562/IMA-<em>1</em> cells. However, expression levels of <em>sphingosine</em> kinase-<em>1</em> (SK<em>1</em>) and generation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) were increased significantly in K562/IMA-<em>1</em> cells, channeling sphingoid bases to the <em>sphingosine</em> kinase pathway. The partial inhibition of SK<em>1</em> expression by small interference RNA modulated S<em>1</em>P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK<em>1</em> in K562 cells increased the ratio between total S<em>1</em>P/C<em>1</em>8-ceramide levels approximately 6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK<em>1</em>/S<em>1</em>P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C<em>1</em>8-ceramide synthesis mainly via hLASS<em>1</em> in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S<em>1</em>P by overexpression of SK<em>1</em> result in resistance to imatinib-induced apoptosis.

Sphingolipids are formed via the metabolism of sphingomyelin, a constituent of the plasma membrane, or by de novo synthesis. Enzymatic pathways result in the formation of several different lipid mediators, which are known to have important roles in many cellular processes, including proliferation, apoptosis and migration. Several studies now suggest that these sphingolipid mediators, including ceramide, ceramide <em>1</em>-<em>phosphate</em> and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), are likely to have an integral role in inflammation. This can involve, for example, activation of pro-inflammatory transcription factors in different cell types and induction of cyclooxygenase-2, leading to production of pro-inflammatory prostaglandins. The mode of action of each sphingolipid is different. Increased ceramide production leads to the formation of ceramide-rich areas of the membrane, which may assemble signalling complexes, whereas S<em>1</em>P acts via high-affinity G-protein-coupled S<em>1</em>P receptors on the plasma membrane. Recent studies have demonstrated that in vitro effects of sphingolipids on inflammation can translate into in vivo models. This review will highlight the areas of research where sphingolipids are involved in inflammation and the mechanisms of action of each mediator. In addition, the therapeutic potential of drugs that alter sphingolipid actions will be examined with reference to disease states, such as asthma and inflammatory bowel disease, which involve important inflammatory components. A significant body of research now indicates that sphingolipids are intimately involved in the inflammatory process and recent studies have demonstrated that these lipids, together with associated enzymes and receptors, can provide effective drug targets for the treatment of pathological inflammation.

Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of alphav beta3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional beta3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-<em>1</em> hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of alphav beta3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to alphav beta3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin <em>1</em> (Ang-<em>1</em>) and the platelet-derived lipid mediator <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-<em>1</em>, S<em>1</em>P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of beta3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering beta3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism for vascular leakage after infection by pathogenic hantaviruses and the means to inhibit hantavirus-directed endothelial cell permeability that may be applicable to additional vascular leak syndromes.

Angiogenesis, or new blood vessel formation, is critical for the growth and spread of tumors. Multiple phases of this process, namely, migration, proliferation, morphogenesis, and vascular stabilization, are needed for optimal tumor growth beyond a diffusion-limited size. The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor-<em>1</em> (S<em>1</em>P(<em>1</em>)) is required for stabilization of nascent blood vessels during embryonic development. Here we show that S<em>1</em>P(<em>1</em>) expression is strongly induced in tumor vessels. We developed a multiplex RNA interference technique to downregulate S<em>1</em>P(<em>1</em>) in mice. The small interfering RNA (siRNA) for S<em>1</em>P(<em>1</em>) specifically silenced the cognate transcript in endothelial cells and inhibited endothelial cell migration in vitro and the growth of neovessels into subcutaneous implants of Matrigel in vivo. Local injection of S<em>1</em>P(<em>1</em>) siRNA, but not a negative control siRNA, into established tumors inhibited the expression of S<em>1</em>P(<em>1</em>) polypeptide on neovessels while concomitantly suppressing vascular stabilization and angiogenesis, which resulted in dramatic suppression of tumor growth in vivo. These data suggest that S<em>1</em>P(<em>1</em>) is a critical component of the tumor angiogenic response and argue for the utility of siRNA technology in antiangiogenic therapeutics.

Three G protein-coupled receptors (Edg-<em>1</em>, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator <em>sphingosine</em> <em>1</em>-<em>phosphate</em> have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed <em>sphingosine</em> <em>1</em>- <em>phosphate</em>-dependent increases in [(35)S]guanosine 5'-(3-O-thio)tri<em>phosphate</em> binding with an EC(50) of 90 nm. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC(50) 0.7 nm). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G(q/i)alpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 2 nm) binding for radiolabeled <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor that couples to G(i/o)alpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.