The bioactive sphingolipids including, ceramide, <em>sphingosine</em>, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have important roles in several types of signaling and regulation of many cellular processes including cell proliferation, apoptosis, senescence, angiogenesis, and transformation. Recent accumulating evidence suggests that ceramide- and S<em>1</em>P-mediated pathways have been implicated in cancer development, progression, and chemotherapy. Ceramide mediates numerous cell-stress responses, such as induction of apoptosis and cell senescence, whereas S<em>1</em>P plays pivotal roles in cell survival, migration, and inflammation. These sphingolipids with opposing roles can be interconverted within cells, suggesting that the balance between them is related to cell fate. Importantly, these sphingolipids are metabolically related through actions of enzymes including ceramidases, ceramide synthases, <em>sphingosine</em> kinases, and S<em>1</em>P phosphatases thereby forming a network of metabolically interrelated bioactive lipid mediators whose importance in normal cellular function and diseases is gaining appreciation. In this review, we summarize involvement of sphingolipids and their related enzymes in pathogenesis and therapy of cancer and discuss future directions of sphingolipid field in cancer research.

BACKGROUND

Metabolomics is a powerful tool that is increasingly used in clinical research. Although excellent sample quality is essential, it can easily be compromised by undetected preanalytical errors. We set out to identify critical preanalytical steps and biomarkers that reflect preanalytical inaccuracies.

METHODS

We systematically investigated the effects of preanalytical variables (blood collection tubes, hemolysis, temperature and time before further processing, and number of freeze-thaw cycles) on metabolomics studies of clinical blood and plasma samples using a nontargeted LC-MS approach.

RESULTS

Serum and heparinate blood collection tubes led to chemical noise in the mass spectra. Distinct, significant changes of 64 features in the EDTA-plasma metabolome were detected when blood was exposed to room temperature for 2, 4, 8, and 24 h. The resulting pattern was characterized by increases in hypoxanthine and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (800% and 380%, respectively, at 2 h). In contrast, the plasma metabolome was stable for up to 4 h when EDTA blood samples were immediately placed in iced water. Hemolysis also caused numerous changes in the metabolic profile. Unexpectedly, up to 4 freeze-thaw cycles only slightly changed the EDTA-plasma metabolome, but increased the individual variability.

CONCLUSIONS

Nontargeted metabolomics investigations led to the following recommendations for the preanalytical phase: test the blood collection tubes, avoid hemolysis, place whole blood immediately in ice water, use EDTA plasma, and preferably use nonrefrozen biobank samples. To exclude outliers due to preanalytical errors, inspect the biomarker signal intensities reflecting systematic as well as accidental and preanalytical inaccuracies before processing the bioinformatics data.

Endothelial differentiation gene (edg)-encoded G protein-coupled receptors (Edg Rs)-<em>1</em>, -3, and -5 bind <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), and Edg-2 and -4 bind lysophosphatidic acid (LPA). Edg Rs transduce signals from LPA and S<em>1</em>P that stimulate ras- and rho-dependent cellular proliferation, enhance cellular survival, and suppress apoptosis. That high levels of LPA in plasma and ascitic fluid of patients with ovarian cancer correlate with widespread invasion suggested the importance of investigating expression and functions of Edg Rs in ovarian cancer cells (OCCs) as compared with nonmalignant ovarian surface epithelial cells (OSEs). Analyses of Edg Rs by semiquantitative reverse transcription-PCR, a radioactively quantified variant of PCR, and Western blots developed with monoclonal antibodies showed prominent expression of Edg-4 R in primary cultures and established lines of OCCs but none in OSEs. In contrast, levels of Edg-2, -3, and -5 were higher in OSEs than OCCs. LPA stimulated proliferation and signaled a serum response element-luciferase reporter of immediate-early gene activation in OCCs but not OSEs, whereas S<em>1</em>P evoked similar responses in both OSEs and OCCs. Pharmacological inhibitors of Edg R signaling suppressed OCC responses to LPA. A combination of monoclonal anti-Edg-4 R antibody and phorbol myristate acetate, which were inactive separately, evoked proliferative and serum response element-luciferase responses of OCCs but not OSEs. Thus the Edg-4 R may represent a distinctive marker of OCC that transduces growth-promoting signals from the high local concentrations of LPA characteristic of aggressive ovarian cancer.

Soluble mediators such as thrombin and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac<em>1</em> activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac<em>1</em> is inhibited. In contrast, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> addition leads to comparatively weak and delayed activation of RhoA and it activates Rac<em>1</em>. In addition, we show here that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.

FTY720 is an immunomodulator with demonstrated efficacy in a phase II trial of relapsing multiple sclerosis. FTY720-<em>phosphate</em>, the active metabolite generated upon phosphorylation in vivo, acts as a potent agonist on four of the five known <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P(<em>1</em>)) receptors. AUY954, an aminocarboxylate analog of FTY720, is a low nanomolar, monoselective agonist of the S<em>1</em>P(<em>1</em>) receptor. Due to its selectivity and pharmacokinetic profile, AUY954 is an excellent pharmacological probe of S<em>1</em>P(<em>1</em>)-dependent phenomena. Oral administration of AUY954 induces a profound and reversible reduction of circulating lymphocytes and, in combination with RAD00<em>1</em> (Certican/Everolimus, an mTOR inhibitor), is capable of prolonging the survival of cardiac allografts in a stringent rat transplantation model. This demonstrates that a selective agonist of the S<em>1</em>P(<em>1</em>) receptor is sufficient to achieve efficacy in an animal model of transplantation.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> that determines the allergic responsiveness of mast cells. High intracellular concentrations of <em>sphingosine</em> act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> are dominant over <em>sphingosine</em> as they counteract its inhibitory potential. Therefore, it might be inferred that <em>sphingosine</em>-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.

Transforming growth factor beta (TGF-beta) contributes to the progression of pulmonary fibrosis through up-regulation of alpha-smooth muscle actin (alpha-SMA) as lung myofibroblast differentiation. Bioactive <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) has been shown to mimic TGF-beta signals; however, the function of S<em>1</em>P in lung fibrotic process has not been well documented. We found, in a mouse model of bleomycin lung fibrosis, that SPHK<em>1</em> and alpha-SMA were colocalized within lung fibrotic foci and that these expressions were significantly increased in primary cultured fibroblasts. Using human lung fibroblasts WI-38, we explored the rationale of <em>sphingosine</em> kinase (SPHK) with TGF-beta<em>1</em> stimulation. SPHK inhibitors and small interference RNA (siRNA) targeted SPHK<em>1</em> decreased alpha-SMA and fibronectin expression up-regulated by TGF-beta<em>1</em>. In the meantime, SPHK<em>1</em> inhibition did not affect smad2 phosphorylation in response to TGF-beta<em>1</em>. Then we examined whether S<em>1</em>P receptors transactivation may affect TGF-beta signals. siRNA against S<em>1</em>P(2) and S<em>1</em>P(3), but not S<em>1</em>P(<em>1</em>), reduced alpha-SMA expression as well as Y-27632, Rho kinase inhibitor. We also detected activation of Rho GTPase upon stimulation of TGF-beta<em>1</em> on the cell membrane where S<em>1</em>P(2) or S<em>1</em>P(3) was overexpressed. These data suggested that SPHK<em>1</em> activation by TGF-beta<em>1</em> leads to Rho-associated myofibroblasts differentiation mediated by transactivated S<em>1</em>P receptors in the lung fibrogenic process.

Recent studies demonstrated a role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) in the protection against the stress of ischemia/reperfusion (I/R) injury. In experiments reported here, we have investigated the signaling through the S<em>1</em>P cascade by FTY720, a sphingolipid drug candidate displaying structural similarity to S<em>1</em>P, underlying the S<em>1</em>P cardioprotective effect. In ex vivo rat heart and isolated sinoatrial node models, FTY720 significantly prevented arrhythmic events associated with I/R injury including premature ventricular beats, VT, and sinus bradycardia as well as A-V conduction block. Real-time PCR and Western blot analysis demonstrated the expression of the S<em>1</em>P receptor transcript pools and corresponding proteins including S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3 in tissues dissected from sinoatrial node, atrium and ventricle. FTY720 (25 nM) significantly blunted the depression of the levels of phospho-Pak<em>1</em> and phospho-Akt with ischemia and with reperfusion. There was a significant increase in phospho-Pak<em>1</em> levels by 35%, <em>1</em>99%, and 205% after 5, <em>1</em>0, and <em>1</em>5 min of treatment with 25 nM FTY720 compared with control nontreated myocytes. However, there was no significant difference in the levels of total Pak<em>1</em> expression between nontreated and FTY720 treated. Phospho-Akt levels were increased by 44%, 63%, and 6<em>1</em>% after 5, <em>1</em>0, and <em>1</em>5 min of treatment with 25 nM FTY720, respectively. Our data provide the first evidence that FTY720 prevents I/R injury-associated arrhythmias and indicate its potential significance as an important and new agent protecting against I/R injury. Our data also indicate, for the first time, that the cardioprotective effect of FTY720 is likely to involve activation of signaling through the Pak<em>1</em>.

Although IgM serves as a first barrier to Ag spreading, the cellular and molecular mechanisms following B lymphocyte activation that lead to IgM secretion are not fully understood. By virtue of their anatomical location, marginal zone (MZ) B cells rapidly generate Ag-specific IgM in response to blood-borne pathogens and play an important role in the protection against these potentially harmful Ags. In this study, we have explored the contribution of TLR agonists to MZ B cell activation and mobilization as well as their ability to promote primary IgM responses in a mouse model. We demonstrate that diverse TLR agonists stimulate MZ B cells to become activated and leave the MZ through pathways that are differentially dependent on MyD88 and IFN-alphabeta receptor signaling. Furthermore, in vivo stimulation of MZ B cells with TLR agonists led to a reduction in the expression of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors expressed by MZ B cells and/or increased CD69 cell surface levels. Importantly, as adjuvants for a T cell-dependent protein Ag, TLR agonists were found to accelerate the kinetics but not magnitude of the Ag-specific IgM response. Together, these data demonstrate that in vivo TLR agonist treatment enhances the early production of Ag-specific IgM and activates MZ B cells to promote their relocation.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP) is an important sphingolipid-derived second messenger in mammalian cells that acts to promote proliferation and to inhibit apoptosis. Various growth factors increase the intracellular concentration of SPP by activating <em>sphingosine</em> kinase, the molecular cloning of which has revealed that it defines a new type of lipid kinase. Cell fate is influenced by the balance between the intracellular concentration of SPP and that of ceramide, a pro-apoptotic sphingolipid metabolite. The observation that a similar "rheostat" is a determinant of cell survival in yeast cells exposed to heat shock indicates that it is an evolutionarily conserved mechanism of stress regulation. SPP also acts extracellularly to inhibit cell motility and to influence cell morphology, effects that appear to be mediated by the G protein-coupled receptor EDG<em>1</em>. These observations indicate that SPP is the prototype of a new class of lipid mediators that exert both intracellular and extracellular actions.

We examined the actions of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) on signaling pathways in Chinese hamster ovary cells transfected with putative S<em>1</em>P receptor subtypes, i.e. Edg-<em>1</em>, AGR<em>1</em>6/H2<em>1</em>8 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S<em>1</em>P receptors and their affinities to S<em>1</em>P, which were estimated by [(3)H]S<em>1</em>P binding to the cells. In vector-transfected cells, S<em>1</em>P slightly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in association with inositol <em>phosphate</em> production, reflecting phospholipase C activation; the S<em>1</em>P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR<em>1</em>6-transfected cells. In comparison with vector-transfected cells, the S<em>1</em>P-induced [Ca(2+)](i) increase was also slightly enhanced in the Edg-<em>1</em>-transfected cells. In all cases, the inositol <em>phosphate</em> and Ca(2+) responses to S<em>1</em>P were partially inhibited by pertussis toxin (PTX). S<em>1</em>P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S<em>1</em>P receptor subtypes was AGR<em>1</em>6>> Edg-3>> Edg-<em>1</em>. In the presence of forskolin, however, S<em>1</em>P significantly inhibited cAMP accumulation at a lower concentration (<em>1</em>-<em>1</em>00 nM) of S<em>1</em>P in a manner sensitive to PTX in the Edg-<em>1</em>-transfected cells but not in either the Edg-3 or AGR<em>1</em>6-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-<em>1</em> and Edg-3 were equally potent, but AGR<em>1</em>6 was ineffective. Thus, S<em>1</em>P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca(2+) system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulates cell proliferation, apoptosis, motility, and neurite retraction. Contradictory reports propose that S<em>1</em>P acts as either an intracellular second messenger or an extracellular ligand for cell-surface receptors. Hence, the precise signaling mechanisms mediating the diverse cellular effects of S<em>1</em>P remain to be determined. Here, we investigate whether S<em>1</em>P stimulation of cell proliferation, survival, and related signaling events can be mediated by the recently cloned Edg family members of G protein-coupled receptors. We observed that S<em>1</em>P treatment significantly increased proliferation of HTC4 hepatoma cells stably transfected with human S<em>1</em>P receptor Edg3 or Edg5, which was attributable to stimulation of cell growth and inhibition of apoptosis caused by serum starvation. Edg3 and Edg5 transduced S<em>1</em>P-evoked signaling events relevant to cell proliferation and survival, including activation of the ERK/MAP kinases, and immediate-early induction of c-Jun and c-Fos. Trancriptional activation of reporter genes for the c-fos promoter and the serum response element by Edg3 and Edg5 transfected in Jurkat cells was inhibited by pertussis toxin and C3 exoenzyme, implicating G(i/o)- and Rho-dependent pathways. Our data also indicated that Edg3 and Edg5 mediated the serum response element activation through transcriptional factors Elk-<em>1</em> and serum response factor. Thus, specific G protein-coupled receptors Edg3 and Edg5 account for, at least in part, S<em>1</em>P-induced cell proliferation, survival, and related signaling events.

Adult skeletal muscle is able to repeatedly regenerate because of the presence of satellite cells, a population of stem cells resident beneath the basal lamina that surrounds each myofiber. Little is known, however, of the signaling pathways involved in the activation of satellite cells from quiescence to proliferation, a crucial step in muscle regeneration. We show that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> induces satellite cells to enter the cell cycle. Indeed, inhibiting the sphingolipid-signaling cascade that generates <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> significantly reduces the number of satellite cells able to proliferate in response to mitogen stimulation in vitro and perturbs muscle regeneration in vivo. In addition, metabolism of sphingomyelin located in the inner leaflet of the plasma membrane is probably the main source of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> used to mediate the mitogenic signal. Together, our observations show that sphingolipid signaling is involved in the induction of proliferation in an adult stem cell and a key component of muscle regeneration.

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with <em>sphingosine</em> <em>1</em>-<em>phosphate</em> recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF27<em>1</em> and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S<em>1</em>P(<em>1</em>-5). S<em>1</em>P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S<em>1</em>P, <em>sphingosine</em> kinase-<em>1</em>, correlate with short survival of glioma patients. In this study we examined the mechanism of S<em>1</em>P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S<em>1</em>P receptor expression in glioma cell lines. S<em>1</em>P(<em>1</em>), S<em>1</em>P(2) and S<em>1</em>P(3) all contribute positively to S<em>1</em>P-stimulated glioma cell proliferation, with S<em>1</em>P(<em>1</em>) being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S<em>1</em>P(5) blocks glioma cell proliferation, and inhibits ERK activation. S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) enhance glioma cell migration and invasion. S<em>1</em>P(2) inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S<em>1</em>P(2) also potently enhances expression of the matricellular protein CCN<em>1</em>/Cyr6<em>1</em>, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN<em>1</em> blocked S<em>1</em>P(2)-stimulated glioma invasion. Thus, while S<em>1</em>P(2) decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix.

Resistance to chemotherapeutic drugs often limits their clinical efficacy. Previous studies have implicated the bioactive sphingolipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S-<em>1</em>-P) in regulating sensitivity to cisplatin [cis-diamminedichloroplatinum(II)] and showed that modulating the S-<em>1</em>-P lyase can alter cisplatin sensitivity. Here, we show that the members of the <em>sphingosine</em> kinase (SphK<em>1</em> and SphK2) and dihydroceramide synthase (LASS<em>1</em>/CerS<em>1</em>, LASS4/CerS4, and LASS5/CerS5) enzyme families each have a unique role in regulating sensitivity to cisplatin and other drugs. Thus, expression of SphK<em>1</em> decreases sensitivity to cisplatin, carboplatin, doxorubicin, and vincristine, whereas expression of SphK2 increases sensitivity. Expression of LASS<em>1</em>/CerS<em>1</em> increases the sensitivity to all the drugs tested, whereas LASS5/CerS5 only increases sensitivity to doxorubicin and vincristine. LASS4/CerS4 expression has no effect on the sensitivity to any drug tested. Reflecting this, we show that the activation of the p38 mitogen-activated protein (MAP) kinase is increased only by LASS<em>1</em>/CerS<em>1</em>, and not by LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to cause a specific translocation of LASS<em>1</em>/CerS<em>1</em>, but not LASS4/CerS4 or LASS5/CerS5, from the endoplasmic reticulum (ER) to the Golgi apparatus. Supporting the hypothesis that this translocation is mechanistically involved in the response to cisplatin, we showed that expression of SphK<em>1</em>, but not SphK2, abrogates both the increased cisplatin sensitivity in cells stably expressing LASS<em>1</em>/CerS and the translocation of the LASS<em>1</em>/CerS<em>1</em>. The data suggest that the enzymes of the sphingolipid metabolic pathway can be manipulated to improve sensitivity to the widely used drug cisplatin.

BACKGROUND

Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are bioactive phospholipids with mitogenic and growth factor-like activities that act via specific cell-surface receptors present in many normal and transformed cell types. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. The presence of LPA-like activity and the hypothesis that levels of this bioactivity in effusions of ovarian cancer patients are higher than those in effusions of other cancer patients was studied.

METHODS

A neurite retraction bioassay in a neuroblastoma cell line previously developed for in vitro detection of LPA activity on cell lines was employed and bioactivity was expressed in virtual LPA-equivalent levels. LPA-equivalent levels were tested in effusions of 62 patients with a range of malignancies, including <em>1</em>3 ovarian cancer patients. Biochemical and clinical parameters were evaluated for correlations with LPA-equivalent levels.

RESULTS

Average LPA-equivalent levels were 50.2 microns (range 5.4-200) for all patients, and 94.5 microns (range <em>1</em>5-200) for ovarian cancer patients (P = 0.004). There were no additional independent significant correlations between LPA-equivalent levels in effusions and a range of other biochemical and clinical characteristics.

CONCLUSIONS

These data suggest a role for LPA-like lipids in the peritoneal spread of ovarian cancer and possibly that of other predominantly intraperitoneal malignancies.

Clinical and epidemiological studies have shown that HDLs, a class of plasma lipoproteins, heterogeneous in size and density, have an atheroprotective role attributed, for years, to their capacity to promote the efflux of cholesterol from activated cholesterol-loaded arterial macrophages. Recent studies, however, have recognized that the physical heterogeneity of HDLs is associated with multiple functions that involve both the protein and the lipid components of these particles. ApoA-I, quantitatively the major protein constituent, has an amphipathic structure suited for transport of lipids. It readily interacts with the ATP-binding cassette transporter ABCA<em>1</em>, the SR-B<em>1</em> scavenger receptor; activates the enzyme lecithin-cholesterol acyl transferase (LCAT), which is critical for HDL maturation. It also has antioxidant and antiinflammatory properties, along with the HDL-associated enzymes paraoxonase, platelet activating factor acetylhydrolase (PAF), and glutathione peroxidase. Regarding the lipid moiety, an atheroprotective role has been recognized for lysosphingolipids, particularly <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). All of these atheroprotective functions are lost in the post-translational dependent dysfunctional plasma HDLs of subjects with systemic inflammation, coronary heart disease, diabetes, and chronic renal disease. The emerging notion that particle quality has more predictive power than quantity has stimulated further exploration of the HDL proteome, already revealing unsuspected pro- or antiatherogenic proteins/peptides associated with HDL.

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (Mr = 32,<em>1</em>58) and -2b (Mr = 35, <em>1</em><em>1</em>9), both of which contained six putative transmembrane domains. Both enzymes were glycosylated and cleaved by N-glycanase and endo-beta-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b shared 47% identical sequence and were judged to be the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34-39% identical to that of Drosophila Wunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyzed lysophosphatidate and ceramide-<em>1</em>-<em>phosphate</em> in addition to phosphatidate, the hydrolysis of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> was detected only for PAP-2b. PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation.

Tertiary lymphoid organs (TLOs) are accumulations of lymphoid cells in chronic inflammation that resemble LNs in their cellular content and organization, high endothelial venules, and lymphatic vessels (LVs). Although acute inflammation can result in defective LVs, TLO LVs appear to function normally in that they drain fluid and transport cells that respond to chemokines and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) gradients. Molecular regulation of TLO LVs differs from lymphangiogenesis in ontogeny with a dependence on cytokines and hematopoietic cells. Ongoing work to elucidate the function and molecular regulation of LVs in TLOs is providing insight into therapies for conditions as diverse as lymphedema, autoimmunity, and cancer.

<em>Sphingosine</em> kinases (SK) regulate the balance between proapoptotic ceramides and mitogenic <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P); however, the functions of the two isoenzymes (SK<em>1</em> and SK2) in tumor cells are not well defined. Therefore, RNA interference was used to assess the individual roles of SK<em>1</em> and SK2 in tumor cell sphingolipid metabolism, proliferation, and migration/invasion. Treatment of A498, Caki-<em>1</em>, or MDA-MB-23<em>1</em> cells with siRNAs specific for SK<em>1</em> or SK2 effectively suppressed the expression of the target mRNA and protein. Ablation of SK<em>1</em> did not affect mRNA or protein levels of SK2 and reduced intracellular levels of S<em>1</em>P while elevating ceramide levels. In contrast, ablation of SK2 elevated mRNA, protein, and activity levels of SK<em>1</em> and increased cellular S<em>1</em>P levels. Interestingly, cell proliferation and migration/invasion were suppressed more by SK2-selective ablation than by SK<em>1</em>-selective ablation, showing that the increased S<em>1</em>P does not rescue these phenotypes. Similarly, exogenous S<em>1</em>P did not rescue the cells from the antiproliferative or antimigratory effects of the siRNAs. Consistent with these results, differential effects of SK<em>1</em>- and SK2-selective siRNAs on signaling proteins, including p53, p2<em>1</em>, ERK<em>1</em>, ERK2, FAK, and VCAM<em>1</em>, indicate that SK<em>1</em> and SK2 have only partially overlapping functions in tumor cells. Overall, these data indicate that loss of SK2 has stronger anticancer effects than does suppression of SK<em>1</em>. Consequently, selective inhibitors of SK2 may provide optimal targeting of this pathway in cancer chemotherapy.

Asthma is a common debilitating inflammatory lung disease affecting over 200 million people worldwide. Here, we investigated neurogenic components involved in asthmatic-like attacks using the ovalbumin-sensitized murine model of the disease, and identified a specific population of neurons that are required for airway hyperreactivity. We show that ablating or genetically silencing these neurons abolished the hyperreactive broncho-constrictions, even in the presence of a fully developed lung inflammatory immune response. These neurons are found in the vagal ganglia and are characterized by the expression of the transient receptor potential vanilloid <em>1</em> (TRPV<em>1</em>) ion channel. However, the TRPV<em>1</em> channel itself is not required for the asthmatic-like hyperreactive airway response. We also demonstrate that optogenetic stimulation of this population of TRP-expressing cells with channelrhodopsin dramatically exacerbates airway hyperreactivity of inflamed airways. Notably, these cells express the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor 3 (S<em>1</em>PR3), and stimulation with a S<em>1</em>PR3 agonist efficiently induced broncho-constrictions, even in the absence of ovalbumin sensitization and inflammation. Our results show that the airway hyperreactivity phenotype can be physiologically dissociated from the immune component, and provide a platform for devising therapeutic approaches to asthma that target these pathways separately.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-<em>1</em>-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-<em>1</em>-P, possibly resulting from <em>sphingosine</em> (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-<em>1</em>-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-<em>1</em>-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-<em>1</em>-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-<em>1</em>-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-<em>1</em>-P, especially for plasma and serum Sph-<em>1</em>-P. Platelet-derived Sph-<em>1</em>-P seems to play an important role in vascular biology.

P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-α) and sphingolipid-based signaling. Critical to this pathway was signaling through <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>PR<em>1</em>). In brain capillaries, S<em>1</em>P acted through S<em>1</em>PR<em>1</em> to rapidly and reversibly reduce P-glycoprotein transport activity. <em>Sphingosine</em> reduced transport by a <em>sphingosine</em> kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S<em>1</em>P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S<em>1</em>P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, (3)H-verapamil (threefold increase), (3)H-loperamide (fivefold increase), and (3)H-paclitaxel (fivefold increase); blocking S<em>1</em>PR<em>1</em> abolished this effect. Tight junctional permeability, measured as brain (<em>1</em>4)C-sucrose accumulation, was not altered. Therefore, targeting signaling through S<em>1</em>PR<em>1</em> at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain.