In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that <em>sphingosine</em> (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethyl<em>sphingosine</em> (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 microM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT<em>1</em>8, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethyl<em>sphingosine</em> (TMS). Under the same conditions, in the presence of serum, neither Sph-<em>1</em>-<em>phosphate</em> nor ceramide analogues C2-, C6- or C8-ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after <em>1</em>8 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B<em>1</em>. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C2Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway.

Plasma <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulates vascular permeability, and plasma and lymph S<em>1</em>P guide lymphocyte egress from lymphoid organs. S<em>1</em>P is made intracellularly, and little is known about how S<em>1</em>P is delivered into circulatory fluids. Here, we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S<em>1</em>P, but only a minor decrease in plasma S<em>1</em>P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S<em>1</em>P and blocked egress from lymph nodes directed by lymph S<em>1</em>P. Spns2 is needed in endothelial cells to supply lymph S<em>1</em>P and support lymphocyte circulation. As a differential requirement for lymph and blood S<em>1</em>P, Spns2 may be an attractive target for immune suppressive drugs.

Instrumental conditioning allows animals to learn about the consequences of their own actions, but the underpinning molecular mechanisms remain elusive. Here we show that the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor Gpr6 is selectively expressed in the striatopallidal neurons in the striatum. Gpr6-deficient mice showed reduced striatal cyclic AMP production in vitro and selective alterations in instrumental conditioning in vivo. Thus, Gpr6 is the first striatopallidal neuron-specific genetic regulator of instrumental conditioning in a mammal.

The bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) fulfils manifold tasks in the immune system acting in auto- and/or paracrine fashion. This includes regulation of apoptosis, migration and proliferation. Upon its generation by <em>sphingosine</em> kinases from plasma membrane sphingolipids, S<em>1</em>P can either act as a second messenger within cells or can be released from cells to occupy a family of specific G-protein-coupled receptors (S<em>1</em>P<em>1</em>-5). This diversity is reflected by the impact of S<em>1</em>P on macrophage biology and function. Over the last years it became apparent that the <em>sphingosine</em> kinase/S<em>1</em>P/S<em>1</em>P-receptor signalling axis in macrophages might play a central role in the pathogenesis of inflammatory diseases such as atherosclerosis, asthma, rheumatoid arthritis and cancer. Here, we summarize the current knowledge of the function of S<em>1</em>P in macrophage biology and discuss potential implications for pathology.

Systemic sclerosis (SSc) is an often fatal disease characterized by autoimmunity and inflammation, leading to widespread vasculopathy and fibrosis. Lysophosphatidic acid (LPA), a bioactive phospholipid in serum, is generated from lysophospholipids secreted from activated platelets in part by the action of lysophospholipase D (lysoPLD). <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a member of the bioactive lysophospholipid family, is also released from activated platelets. Because activated platelets are a hallmark of SSc, we wanted to determine whether subjects with SSc have altered serum lysophospholipid levels or lysoPLD activity. Lysophospholipid levels were measured using mass spectrometric analysis. LysoPLD activity was determined by quantifying choline released from exogenous lysophosphatidylcholine (LPC). The major results were that serum levels of arachidonoyl (20:4)-LPA and S<em>1</em>P were significantly higher in SSc subjects versus controls. Furthermore, serum LPA:LPC ratios of two different polyunsaturated phospholipid molecular species, and also the ratio of all species combined, were significantly higher in SSc subjects versus controls. No significant differences were found between other lysophospholipid levels or lysoPLD activities. Elevated 20:4 LPA, S<em>1</em>P levels and polyunsaturated LPA:LPC ratios may be markers for and/or play a significant role in the etiology of SSc and may be future pharmacological targets for SSc treatment.

OBJECTIVE

The importance of bioactive lipid signaling under physiological and pathophysiological conditions is progressively becoming recognized. The disparate distribution of <em>sphingosine</em> kinase (SphK) isoform activity in normal and ischemic brain, particularly the large excess of SphK2 in cerebral microvascular endothelial cells, suggests potentially unique cell- and region-specific signaling by its product <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. The present study sought to test the isoform-specific role of SphK as a trigger of hypoxic preconditioning (HPC)-induced ischemic tolerance.

METHODS

Temporal changes in microvascular SphK activity and expression were measured after HPC. The SphK inhibitor dimethylsphingosine or sphingosine analog FTY720 was administered to adult male Swiss-Webster ND4 mice before HPC. Two days later, mice underwent a 60-minute transient middle cerebral artery occlusion and at 24 hours of reperfusion, infarct volume, neurological deficit, and hemispheric edema were measured.

RESULTS

HPC rapidly increased microvascular SphK2 protein expression (<em>1</em>.7+/-0.2-fold) and activity (2.5+/-0.6-fold), peaking at 2 hours, whereas SphK<em>1</em> was unchanged. SphK inhibition during HPC abrogated reductions in infarct volume, neurological deficit, and ipsilateral edema in HPC-treated mice. FTY720 given 48 hours before stroke also promoted ischemic tolerance; when combined with HPC, even greater (and dimethyl<em>sphingosine</em>-reversible) protection was noted.

CONCLUSIONS

These findings indicate hypoxia-sensitive increases in SphK2 activity may serve as a proximal trigger that ultimately leads to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-mediated alterations in gene expression that promote the ischemia-tolerant phenotype. Thus, components of this bioactive lipid signaling pathway may be suitable therapeutic targets for protecting the neurovascular unit in stroke.

Migration of mast cells is essential for their recruitment within target tissues where they play an important role in innate and adaptive immune responses. These processes rely on the ability of mast cells to recognize appropriate chemotactic stimuli and react to them by a chemotactic response. Another level of intercellular communication is attained by production of chemoattractants by activated mast cells, which results in accumulation of mast cells and other hematopoietic cells at the sites of inflammation. Mast cells express numerous surface receptors for various ligands with properties of potent chemoattractants. They include the stem cell factor (SCF) recognized by c-Kit, antigen, which binds to immunoglobulin E (IgE) anchored to the high affinity IgE receptor (FcεRI), highly cytokinergic (HC) IgE recognized by FcεRI, lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which binds to G protein-coupled receptors (GPCRs). Other large groups of chemoattractants are eicosanoids [prostaglandin E(2) and D(2), leukotriene (LT) B(4), LTD(4), and LTC(4), and others] and chemokines (CC, CXC, C, and CX3C), which also bind to various GPCRs. Further noteworthy chemoattractants are isoforms of transforming growth factor (TGF) β<em>1</em>-3, which are sensitively recognized by TGF-β serine/threonine type I and II β receptors, adenosine, C<em>1</em>q, C3a, and C5a components of the complement, 5-hydroxytryptamine, neuroendocrine peptide catestatin, tumor necrosis factor-α, and others. Here we discuss the major types of chemoattractants recognized by mast cells, their target receptors, as well as signaling pathways they utilize. We also briefly deal with methods used for studies of mast cell chemotaxis and with ways of how these studies profited from the results obtained in other cellular systems.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has emerged as a bioactive lipid modulator that mediates a variety of cell functions. However, the effects of S<em>1</em>P on melanogenesis are not well known. Therefore, we investigated the actions of S<em>1</em>P on melanin synthesis using a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. This study shows that S<em>1</em>P significantly inhibits melanin synthesis in a concentration-dependent manner, and also that the activity of tyrosinase was reduced in S<em>1</em>P-treated cells. In contrast, a specific extracellular signal-regulated protein kinase (ERK) pathway inhibitor, PD98059, increased tyrosinase activity and melanin production, and PD98059 also restored the S<em>1</em>P-induced reduction of tyrosinase activity and pigmentation. In addition, we found that S<em>1</em>P induces the sustained activation of ERK and the subsequent degradation of microphthalmia-associated transcription factor (MITF), which plays a key role in melanogenesis. Thus, we further studied the relationship between the ERK pathway and melanin synthesis. PD98059 was found to prevent the S<em>1</em>P-induced MITF phosphorylation and degradation and to abrogate the S<em>1</em>P-induced downregulation of tyrosinase and of tyrosinase-related protein <em>1</em> (TRP<em>1</em>) production. These results indicate that the ERK pathway is potently involved in the melanogenic signaling cascade, and that S<em>1</em>P-induced ERK activation contributes to reduced melanin synthesis via MITF degradation. Therefore, we suggest that S<em>1</em>P reduces melanin synthesis by ERK activation, MITF phosphorylation and degradation, and by the subsequent downregulation of tyrosinase and TRP-<em>1</em> production.

<em>Sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) are interconvertible sphingolipid metabolites with opposing effects on cell growth and apoptosis. Based on sequence homology with LBP<em>1</em>, a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast, we report here the cloning, identification, and characterization of a mammalian SPP phosphatase (mSPP<em>1</em>). This hydrophobic enzyme, which contains the type 2 lipid phosphohydrolase conserved sequence motif, shows substrate specificity for SPP. Partially purified Myc-tagged mSPP<em>1</em> was also highly active at dephosphorylating SPP. When expressed in yeast, mSPP<em>1</em> can partially substitute for the function of LBP<em>1</em>. Membrane fractions from human embryonic kidney HEK293 cells transfected with mSPP<em>1</em> markedly degraded SPP but not lysophosphatidic acid, phosphatidic acid, or ceramide-<em>1</em>-<em>phosphate</em>. Enforced expression of mSPP<em>1</em> in NIH 3T3 fibroblasts not only decreased SPP and enhanced ceramide levels, it also markedly diminished survival and induced the characteristic traits of apoptosis. Collectively, our results suggest that SPP phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate.

The novel immunomodulator FTY720 down-modulates <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> on lymphocytes at low nanomolar concentrations, thereby inhibiting <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em>-dependent egress of lymphocytes from lymph nodes into efferent lymphatics and blood. At high micromolar concentration, FTY720 has been shown to induce growth inhibition and/or apoptosis in human cancer cells in vitro. In this study, we investigated the biological effects of FTY720 on multiple myeloma cells. We found that FTY720 induces potent cytotoxicity against drug-sensitive and drug-resistant multiple myeloma cell lines as well as freshly isolated tumor cells from multiple myeloma patients who do not respond to conventional agents. FTY720 triggers activation of caspase-8, -9, and -3, followed by poly(ADP-ribose) polymerase cleavage. Interestingly, FTY720 induces alterations in mitochondrial membrane potential (DeltaPsim) and Bax cleavage, followed by translocation of cytochrome c and Smac/Diablo from mitochondria to the cytosol. In combination treatment studies, both dexamethasone and anti-Fas antibodies augment anti-multiple myeloma activity induced by FTY720. Neither interleukin-6 nor insulin-like growth factor-I, which both induce multiple myeloma cell growth and abrogate dexamethasone-induced apoptosis, protect against FTY720-induced growth inhibition. Importantly, growth of multiple myeloma cells adherent to bone marrow stromal cells is also significantly inhibited by FTY720. Finally, it down-regulates interleukin-6-induced phosphorylation of Akt, signal transducers and activators of transcription 3, and p42/44 mitogen-activated protein kinase; insulin-like growth factor-I-triggered Akt phosphorylation; and tumor necrosis factor alpha-induced IkappaBalpha and nuclear factor-kappaB p65 phosphorylation. These results suggest that FTY720 overcomes drug resistance in multiple myeloma cells and provide the rationale for its clinical evaluation to improve patient outcome in multiple myeloma.

The bioactive signaling molecule D-erythro-<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is irreversibly degraded by the enzyme S<em>1</em>P lyase (SPL). The reaction of SPL with C<em>1</em>8-S<em>1</em>P generates ethanolamine <em>phosphate</em> and a long-chain fatty aldehyde, trans-2-hexadecenal. Modulation of SPL expression in cells and organisms produces significant phenotypes, most of which have been attributed to corresponding changes in S<em>1</em>P-dependent signaling. However, the physiological functions of SPL products are not well understood. In the present study, we explored the biological activities of trans-2-hexadecenal in human and murine cells. We demonstrate that trans-2-hexadecenal causes cytoskeletal reorganization leading to cell rounding, detachment and eventual cell death by apoptosis in multiple cell types, including HEK293T, NIH3T3 and HeLa cells. Trans-2-hexadecenal stimulated a signaling pathway involving MLK3 and the respective phosphorylation of MKK4/7 and JNK, whereas ERK, AKT and p38 were unaffected. Trans-2-hexadecenal-induced apoptosis was accompanied by activation of downstream targets of JNK including c-Jun phosphorylation, cytochrome c release, Bax activation, Bid cleavage and increased translocation of Bim into mitochondria. The antioxidant N-acetylcysteine prevented JNK activation by trans-2-hexadecenal. Further, inhibition of JNK abrogated the cytoskeletal changes and apoptosis caused by trans-2-hexadecenal, whereas Rac<em>1</em> and RhoA were not involved. In conclusion, our studies provide a new paradigm of sphingolipid signaling by demonstrating for the first time that S<em>1</em>P metabolism generates a bioactive product that induces cellular effects through oxidant stress-dependent MAP kinase cell signaling.

Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S<em>1</em>P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl<em>1</em> deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S<em>1</em>P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S<em>1</em>P through S<em>1</em>P transporter spinster homolog 2 (SPNS2), S<em>1</em>P receptor activation, JAK2/STAT3-dependent miR-<em>1</em>8<em>1</em>b-<em>1</em> induction, and silencing of miR-<em>1</em>8<em>1</em>b-<em>1</em> target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S<em>1</em>P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S<em>1</em>P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S<em>1</em>P or promote S<em>1</em>P metabolism through the actions of SPL.

The mechanism by which locally delivered <em>sphingosine</em> analogs regulate host response to localized viral infection has never been addressed. In this report, we show that intratracheal delivery of the chiral <em>sphingosine</em> analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) or its <em>phosphate</em> ester inhibits the T-cell response to influenza virus infection. In contrast, neither intraperitoneal delivery of AAL-R nor intratracheal instillation of the non-phosphorylatable stereoisomer AAL-S suppressed virus-specific T-cell response, indicating that in vivo phosphorylation of AAL-R and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulation in lungs is essential for immunomodulation. Intratracheal delivery of water-soluble S<em>1</em>P(<em>1</em>) receptor agonist at doses sufficient to induce systemic lymphopenia did not inhibit virus-specific T-cell response, indicating that S<em>1</em>P(<em>1</em>) is not involved in the immunosuppressive activities of AAL-R and that immunosuppression acts independently of naive lymphocyte recirculation. Accumulation of dendritic cells (DCs) in draining lymph nodes was inhibited by intratracheal but not intraperitoneal delivery of AAL-R. Direct modulation of DCs is demonstrated by the impaired ability of virus-infected bone marrow-derived DCs treated in vitro with AAL-R to trigger in vivo T-cell response after adoptive transfer to the airways. Thus, our results suggest that locally delivered <em>sphingosine</em> analogs induce immunosuppression by modulating S<em>1</em>P receptors other than S<em>1</em>P(<em>1</em>) or S<em>1</em>P(2) on dendritic cells in the lungs after influenza virus infection.

Pericytes occur in tumour blood vessels and are critical for the development of a functional vascular network. Targeting tumour pericytes is a promising anti-angiogenic therapy but requires identifying the mechanisms of their recruitment in tumour and addressing whether these mechanisms can be selectively harnessed. Among the pathways involved in pericyte recruitment during embryonic development, the contribution of platelet-derived growth factor B and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> is confirmed in tumour angiogenesis. The effect of angiopoietin <em>1</em> depends on the tumour model. Transforming growth factor-beta<em>1</em> enhances tumour vascularization and microvessel maturation. Recent reports suggest a participation of matrix metalloproteinases (MMP) in tumour pericyte recruitment that is consistent with the effect of certain MMPs in the development of microvasculature in embryonic development and in in vitro models of vascular remodelling. Here, we discuss the possibility for MMPs to contribute to pericyte recruitment at six levels: (<em>1</em>) direct promotion of pericyte invasion by extracellular matrix degradation; (2) stimulation of pericyte proliferation and protection against apoptosis by modification of the ECM; (3) activation of pericytes through the release of growth factor bound to the ECM; (4) transactivation of angiogenic cell surface receptor; (5) propagation of angiogenic signalling as cofactor; and (6) recruitment of bone marrow-derived stem cells.

Acute lung injury (ALI) attributable to sepsis or mechanical ventilation and subacute lung injury because of ionizing radiation (RILI) share profound increases in vascular permeability as a key element and a common pathway driving increased morbidity and mortality. Unfortunately, despite advances in the understanding of lung pathophysiology, specific therapies do not yet exist for the treatment of ALI or RILI, or for the alleviation of unremitting pulmonary leakage, which serves as a defining feature of the illness. A critical need exists for new mechanistic insights that can lead to novel strategies, biomarkers, and therapies to reduce lung injury. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a naturally occurring bioactive sphingolipid that acts extracellularly via its G protein-coupled S<em>1</em>P<em>1</em>-5 as well as intracellularly on various targets. S<em>1</em>P-mediated cellular responses are regulated by the synthesis of S<em>1</em>P, catalyzed by <em>sphingosine</em> kinases <em>1</em> and 2, and by the degradation of S<em>1</em>P mediated by lipid <em>phosphate</em> phosphatases, S<em>1</em>P phosphatases, and S<em>1</em>P lyase. We and others have demonstrated that S<em>1</em>P is a potent angiogenic factor that enhances lung endothelial cell integrity and an inhibitor of vascular permeability and alveolar flooding in preclinical animal models of ALI. In addition to S<em>1</em>P, S<em>1</em>P analogues such as 2-amino-2-(2-[4-octylphenyl]ethyl)-<em>1</em>,3-propanediol (FTY720), FTY720 <em>phosphate</em>, and FTY720 phosphonates offer therapeutic potential in murine models of lung injury. This translational review summarizes the roles of S<em>1</em>P, S<em>1</em>P analogues, S<em>1</em>P-metabolizing enzymes, and S<em>1</em>P receptors in the pathophysiology of lung injury, with particular emphasis on the development of potential novel biomarkers and S<em>1</em>P-based therapies for ALI and RILI.

Evidence from clinical, animal and cell culture studies demonstrates that increased autotaxin (ATX) expression is responsible for enhancing tumor progression, cell migration, metastases, angiogenesis and chemo-resistance. These effects depend mainly on the rapid formation of lysophosphatidate (LPA) by ATX. Circulating LPA has a half-life of about 3 min in mice and it is degraded by the ecto-activities of lipid <em>phosphate</em> phosphatases (LPPs). These enzymes also hydrolyze extracellular <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a potent signal for cell division, survival and angiogenesis. Many aggressive tumor cells express high ATX levels and low LPP activities. This favors the formation of locally high LPA and S<em>1</em>P concentrations. Furthermore, LPPs attenuate signaling downstream of the activation of G-protein coupled receptors and receptor tyrosine kinases. Therefore, we propose that the low expression of LPPs in many tumor cells makes them hypersensitive to growth promoting and survival signals that are provided by LPA, S<em>1</em>P, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). One of the key signaling pathways in this respect appears to be activation of phospholipase D (PLD) and phosphatidate (PA) production. This is required for the transactivations of the EGFR and PDGFR and also for LPA-induced cell migration. PA also increases the activities of ERK, mTOR, myc and <em>sphingosine</em> kinase-<em>1</em> (SK-<em>1</em>), which provide individual signals for cells division, survival, chemo-resistance and angiogenesis. This review focuses on the balance of signaling by bioactive lipids including LPA, phosphatidylinositol 3,4,5-tris<em>phosphate</em>, PA and S<em>1</em>P versus the action of ceramides. We will discuss how these lipid mediators interact to produce an aggressive neoplastic phenotype.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which acts as both the extracellular and intracellular messenger, exerts pleiotropic biological activities including regulation of formation of the vasculature, vascular barrier integrity, and lymphocyte trafficking. Many of these S<em>1</em>P actions are mediated by five members of the G protein-coupled S<em>1</em>P receptors (S<em>1</em>P(<em>1</em>) -S<em>1</em>P(5) ) with overlapping but distinct coupling to heterotrimeric G proteins. The biological activities of S<em>1</em>P are based largely on the cellular actions of S<em>1</em>P on migration, adhesion, and proliferation. Notably, S<em>1</em>P often exhibits receptor subtype-specific, bimodal effects in these cellular actions. For example, S<em>1</em>P(<em>1</em>) mediates cell migration toward S<em>1</em>P, that is, chemotaxis, via G(i) /Rac pathway whereas S<em>1</em>P(2) mediates inhibition of migration toward a chemoattractant, that is, chemorepulsion, via G(<em>1</em>2/<em>1</em>3) /Rho pathway, which induces Rac inhibition. In addition, S<em>1</em>P(<em>1</em>) mediates stimulation of cell proliferation through the G(i) -mediated signaling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and ERK whereas S<em>1</em>P(2) mediates inhibition of cell proliferation through mechanisms involving G(<em>1</em>2/<em>1</em>3) /Rho/Rho kinase/PTEN-dependent Akt inhibition. These differential effects of S<em>1</em>P receptor subtypes on migration and proliferation lead to bimodal regulation of various biological responses. An observed biological response is likely determined by an integrated outcome of the counteracting signals input by S<em>1</em>P receptor subtypes. More recent studies identified the new intracellular targets of S<em>1</em>P including the inflammatory signaling molecule TRAF2 and histone deacetylases HDAC<em>1</em> and HDAC2. These interactions of S<em>1</em>P regulate NF-κB activity and gene expression, respectively. Development of S<em>1</em>P receptor agonists and antagonists with improved receptor subtype-selectivity, inhibitors, or modulators of sphingolipid-metabolizing enzymes, and their optimal drug delivery system provide novel therapeutic tactics.

<em>Sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) is a lipid kinase that catalyzes the conversion of <em>sphingosine</em> to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S<em>1</em>P levels in cells, SphK<em>1</em> emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK<em>1</em> in the apo form and in complexes with a substrate <em>sphingosine</em>-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK<em>1</em> structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK<em>1</em> recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), the product of <em>sphingosine</em> kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S<em>1</em>P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S<em>1</em>P can mobilize Ca(2+) in intact cells independently of S<em>1</em>P-GPCR. Within seconds, S<em>1</em>P generated by the photolysis of caged S<em>1</em>P raised the intracellular free Ca(2+) concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S<em>1</em>P was either blocked or absent. Ca(2+) transients induced by photolysis of caged S<em>1</em>P were caused by Ca(2+) mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S<em>1</em>P.

OBJECTIVE

Endothelial activation and monocyte adhesion to endothelium are key events in inflammation. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a sphingolipid that binds to G protein-coupled receptors on endothelial cells (ECs). We examined the role of S<em>1</em>P in modulating endothelial activation and monocyte-EC interactions in vivo.

RESULTS

We injected C57BL/6J mice intravenously with tumor necrosis factor (TNF)-alpha in the presence and absence of the S<em>1</em>P<em>1</em> receptor agonist SEW287<em>1</em> and examined monocyte adhesion. Aortas from TNF-alpha-injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-alpha plus SEW287<em>1</em>-treated mice had few monocytes bound (P<0.000<em>1</em>). Using siRNA, we found that inhibiting the S<em>1</em>P<em>1</em> receptor in vascular ECs blocked the ability of S<em>1</em>P to prevent monocyte-EC interactions in response to TNF-alpha. We examined signaling pathways downstream of S<em>1</em>P<em>1</em> and found that <em>1</em>00 nM S<em>1</em>P increased phosphorylation of Akt and decreased activation of c-jun.

CONCLUSIONS

Thus, we provide the first evidence that S<em>1</em>P signaling through the endothelial S<em>1</em>P<em>1</em> receptor protects the vasculature against TNF-alpha-mediated monocyte-EC interactions in vivo.

We demonstrate here a new concept termed "oncogene tolerance" whereby human EGF receptor 2 (HER2) increases <em>sphingosine</em> kinase <em>1</em> (SK<em>1</em>) expression in estrogen receptor-positive (ER(+)) MCF-7 HER2 cells and SK<em>1</em>, in turn, limits HER2 expression in a negative-feedback manner. The HER2-dependent increase in SK<em>1</em> expression also limits p2<em>1</em>-activated protein kinase <em>1</em> (p65 PAK<em>1</em>) and extracellular signal regulated kinase <em>1</em>/2 (ERK-<em>1</em>/2) signaling. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> signaling via S<em>1</em>P(3) is also altered in MCF-7 HER2 cells. In this regard, S<em>1</em>P binding to S<em>1</em>P(3) induces a migratory phenotype via an SK<em>1</em>-dependent mechanism in ER(+) MCF-7 Neo cells, which lack HER2. This involves the S<em>1</em>P stimulated accumulation of phosphorylated ERK-<em>1</em>/2 and actin into membrane ruffles/lamellipodia and migration. In contrast, S<em>1</em>P failed to promote redistribution of phosphorylated ERK-<em>1</em>/2 and actin into membrane ruffles/lamellipodia or migration of MCF-7 HER2 cells. However, a migratory phenotype in these cells could be induced in response to S<em>1</em>P when SK<em>1</em> expression had been knocked down with a specific siRNA or when recombinant PAK<em>1</em> was ectopically overexpressed. Thus, the HER2-dependent increase in SK<em>1</em> expression functions to desensitize the S<em>1</em>P-induced formation of a migratory phenotype. This is correlated with improved prognosis in patients who have a low HER<em>1</em>-3/SK<em>1</em> expression ratio in their ER(+) breast cancer tumors compared to patients that have a high HER<em>1</em>-3/SK<em>1</em> expression ratio.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are two well-studied lysophospholipids that are known to be important regulators of cellular events. Their actions are mediated by activating a family of G-protein coupled receptors present in many cell types and tissues. These receptors have diverse biological roles owing to the heterogeneity of their signal transduction pathways. Many of these receptors are expressed in subsets of cells in the developing and mature mammalian nervous system and are thought to have important functions in its formation and maintenance. They are also widely expressed within other organ systems such as the immune system. Growing interest in the field has stimulated the development of a number of molecules that act as agonists or antagonists to LPA and S<em>1</em>P receptors. These molecules may lead to the development of new therapeutic compounds. Indeed, one such compound (FTY720) is currently in clinical trials for use in preventing transplant rejection and treating multiple sclerosis. The purpose of this manuscript is to: <em>1</em>) review effects elicited by LPA and S<em>1</em>P on cells and tissues with a particular emphasis on the nervous system, 2) examine possible roles of these lipids in the development of disease, and 3) summarize the existing literature describing their agonists/antagonists.

Although it is routine to predict the blood or plasma pharmacokinetics of compounds for man based upon preclinical studies, the real value of such predictions only comes when linked to drug effects. In the first example, the immunomodulator, FTY720, the first <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor agonist, stimulates the sequestration of lymphocytes into lymph nodes thus removing cells from blood circulation. A prior physiology-based pharmacokinetic model fitted the concentration-time course of FTY720 in rats. This was connected to an indirect response model of the lymphocyte system to characterise the cell trafficking effects. The IC(50) of FTY720 was different in the rat compared with the monkey; man was assumed to be similar to the monkey. The systemic lymphocyte half-lives were also different between species. To make predictions of the pharmacodynamic behaviour for man, two elements are required, i) systemic exposure, in this case from an upscaled physiology based model, and ii) an estimate of lymphocyte turnover in man, gained from the literature from other drug treatments. Predictions compared well with clinical results. The second example is the monoclonal antibody Xolair, designed to bind immunoglobulin E for atopic diseases. A mechanism based two-site binding model described the kinetics of both Xolair and endogenous IgE. This model has been reused for other monoclonal antibodies designed to bind fluid-phase ligands. Sensitivity analysis shows that if differences across species in the kinetics of the endogenous system are not accounted for, then pharmacokinetic/pharmacodynamic models may give misleading predictions of the time course and extent of the response.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), a polar sphingolipid metabolite, has received much attention recently as an extracellular mediator and an intracellular second messenger. It regulates a wide range of biological responses such as cell growth, death, differentiation, and migration. Recent identification of plasma membrane receptors and the cloning of SPP metabolizing enzymes have increased our understanding of the biology of SPP synthesis and action. However, controversy exists regarding the mode of action of this molecule. EDG-<em>1</em> and related G-protein-coupled receptors were identified recently as plasma membrane receptors for SPP. In light of this recent discovery, many of the functions of SPP previously thought to be due to intracellular second messenger action should be reevaluated. In addition, signaling properties and functions of the three known receptors for SPP need to be fully delineated. The structures and the evolutionary conservation of SPP metabolizing enzymes from yeast to mammals support the hypothesis that SPP also plays a role as an intracellular second messenger. However, definitive assignment of the intracellular role of SPP awaits purification/molecular cloning of elusive intracellular receptors. Better knowledge of the molecular basis of SPP action is needed to assess the physiological and pathophysiological significance of this bioactive lipid mediator.