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The potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P), is produced by 2 sphingosine kinase isoenzymes, SphK1 and SphK2. Expression of SphK1 is up-regulated in cancers, including leukemia, and associated with cancer progression. A screen of sphingosine analogs identified (2R,3S,4E)-N-methyl-5-(4'-pentylphenyl)-2-aminopent-4-ene-1,3-diol, designated SK1-I (BML-258), as a potent, water-soluble, isoenzyme-specific inhibitor of SphK1. In contrast to pan-SphK inhibitors, SK1-I did not inhibit SphK2, PKC, or numerous other protein kinases. SK1-I decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of Bcl-2. Lethality of SK1-I was reversed by caspase inhibitors and by expression of Bcl-2. SK1-I not only decreased S1P levels but concomitantly increased levels of its proapoptotic precursor ceramide. Conversely, S1P protected against SK1-I-induced apoptosis. SK1-I also induced multiple perturbations in activation of signaling and survival-related proteins, including diminished phosphorylation of ERK1/2 and Akt. Expression of constitutively active Akt protected against SK1-I-induced apoptosis. Notably, SK1-I potently induced apoptosis in leukemic blasts isolated from patients with acute myelogenous leukemia but was relatively sparing of normal peripheral blood mononuclear leukocytes. Moreover, SK1-I markedly reduced growth of AML xenograft tumors. Our results suggest that specific inhibitors of SphK1 warrant attention as potential additions to the therapeutic armamentarium in leukemia.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-sphingosine kinase (SphK) inhibitor N,N-dimethylsphingosine to block S1P formation surprisingly caused a large increase in expression of SphK1 concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydrosphingosine, also induced apoptosis and produced dramatic increases in SphK1 expression. However, up-regulation of SphK1 was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK1 expression and activity and promoted S1P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK1 and secretion of S1P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S1P potently stimulated chemotaxis of monocytic THP-1 and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK1 to produce and secrete S1P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes including proliferation, survival, and migration, as well as angiogenesis and allergic responses. S1P levels inside cells are tightly regulated by the balance between its synthesis by sphingosine kinases and degradation. S1P is interconvertible with ceramide, which is a critical mediator of apoptosis. It has been postulated that the ratio between S1P and ceramide determines cell fate. Activation of sphingosine kinase by a variety of agonists increases intracellular S1P, which in turn can function intracellularly as a second messenger or be secreted out of the cell and act extracellularly by binding to and signaling through S1P receptors in autocrine and/or paracrine manners. Recent studies suggest that this "inside-out" signaling by S1P may play a role in many human diseases, including cancer, atherosclerosis, inflammation, and autoimmune disorders such as multiple sclerosis. In this review we summarize metabolism of S1P, mechanisms of sphingosine kinase activation, and S1P receptors and their downstream signaling pathways and examine relationships to multiple disease processes. In particular, we describe recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod), S1P receptor agonists, sphingosine kinase inhibitors, and anti-S1P monoclonal antibody.

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.

Sphingosine-1-phosphate (S1P) and cannabinoid receptors are G-protein-coupled receptors that mediate the effects of S1P and endocannabinoids, respectively. Cannabinoid receptors also mediate the effects of Delta9-tetrahydrocannabinol, the primary psychoactive ingredient in marijuana, whereas S1P receptors contribute to the immunosuppressant effects of 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720). FTY720 is a sphingosine analog that can prevent renal graft rejections and suppress a variety of autoimmune disorders in animal models and clinical trials. We now report that both FTY720 and sphingosine interact with CB1 but not CB2 cannabinoid receptors. FTY720 and sphingosine inhibited the binding of the CB1-selective antagonist [3H]N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide ([3H]SR141716A) and the cannabinoid agonist [3H](-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP55,940) in a concentration-dependent manner in both CB1-expressing cell lines and mouse cerebellum. However, these compounds did not significantly alter [3H]CP55,940 binding to CB2 receptors. In G-protein activation assays, FTY720 and sphingosine inhibited the maximal stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding by the cannabinoid agonist R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN55,212-2) in a concentration-dependent manner, and this antagonist effect was not mimicked by S1P. FTY720 and sphingosine also inhibited activation of extracellular signal-regulated kinases 1 and 2 and Akt by WIN55,212-2 in intact Chinese hamster ovary (CHO) cells expressing CB1 receptors and attenuated WIN55,212-2-stimulated internalization of a fluorescence-tagged CB1 receptor in CHO cells. Moreover, both FTY720 and sphingosine produced rightward shifts in the concentration-effect curves of cannabinoid agonists for G-protein activation, indicating that they act as competitive CB1 antagonists. These results suggest that the CB1 receptor could be a novel target of FTY720 and that sphingosine could be an endogenous CB11 antagonist.

Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-1 (PAI-1) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-1 expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-1 via sequential activation of c-Src, protein kinase C delta (PKCδ), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine-1-phosphate. EGF induced rapid phosphorylation of c-Src and PKCδ and concomitant translocation of PKCδ as well as SphK1 to the plasma membrane. Down-regulation of PKCδ abolished EGF-induced SphK1 translocation and up-regulation of PAI-1 by EGF; whereas, down-regulation of PKCα had no effect on the EGF-induced PAI-1 activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK1 and PKCδ to the plasma membrane and up-regulation of PAI-1 expression. Furthermore, SphK1 was indispensable for both EGF-induced c-Jun phosphorylation and PAI-1 expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCδ and SphK1 that have all been implicated in regulating motility and invasion of glioma cells. [ABSTRACT FROM AUTHOR]

The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled sphingosine-1-phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720. [Copyright &y& Elsevier]