Lysophosphatidic acid can support the formation of membranous structures and an increase in MBP mRNA levels in differentiating oligodendrocytes.

During development, differentiating oligodendrocytes progress in distinct maturation steps from premyelinating to myelinating cells. Such maturing oligodendrocytes express both the receptors mediating signaling via extracellular lysophosphatidic acid (LPA) and the major enzyme generating extracellular LPA, namely phosphodiesterase-Ialpha/autotaxin (PD-Ialpha/ATX). However, the biological role of extracellular LPA during the maturation of differentiating oligodendrocytes is currently unclear. Here, we demonstrate that application of exogenous LPA induced an increase in the area occupied by the oligodendrocytes' process network, but only when PD-Ialpha/ATX expression was down-regulated. This increase in network area was caused primarily by the formation of membranous structures. In addition, LPA increased the number of cells positive for myelin basic protein (MBP). This effect was associated by an increase in the mRNA levels coding for MBP but not myelin oligodendrocyte glycoprotein (MOG). Taken together, these data suggest that LPA may play a crucial role in regulating the later stages of oligodendrocyte maturation.

A rapid and sensitive method to measure secretion of sphingosine-1-phosphate.

The serum-borne, bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P), regulates numerous important physiological and pathological processes, mainly acting through specific cell surface G-protein-coupled receptors. Although many mammalian cells can produce S1P, there is little information as to how it is secreted to reach its receptors. Progress in elucidating this mechanism has been hampered by the difficulty of measuring very low levels of S1P. This chapter describes a simple, rapid method to measure S1P export from cells. It also discusses the current knowledge of how S1P is exported out of cells and its physiological significance.

The immunomodulator FTY720 has a direct cytoprotective effect in oligodendrocyte progenitors.

The immunomodulator 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (FTY720) has promising therapeutic effects in multiple sclerosis (MS), a degenerative disease in which demyelination of the central nervous system is accompanied by death of oligodendrocytes (OLGs), the myelin-producing cells. In vivo phosphorylation of FTY720 generates an agonist for G protein-coupled receptors for sphingosine-1-phosphate, a lipid mediator that plays a crucial role in the stimulation of OLG survival by neurotrophin-3 (NT-3). The mechanisms underlying the action of FTY720 in MS are not clearly understood, although the effects of this drug in autoimmune diseases are thought to stem from its ability to reduce lymphocyte infiltration and inflammation. Interestingly, we now found that FTY720 also has a direct effect on OLG progenitors. Treatment of these cells with FTY720 causes activation of extracellular signal-regulated kinase 1/2 and Akt, accompanied by protection from apoptosis. However, FTY720 also arrested OLG differentiation. Importantly, this effect was counteracted by NT-3, which not only enhanced the survival of OLG progenitors induced by FTY720 but also stimulated their maturation. Altogether, these observations suggest that in addition to its immunosuppressive functions, FTY720 could also have a beneficial effect in MS by direct action on OLG progenitors. However, the finding that FTY720 blocks the differentiation of these cells raises the question of whether MS therapies with FTY720 should include the use of differentiation-enhancing factors such as NT-3. This approach would ensure both protection of existing OLG progenitor pools against immune-mediated insults as well as stimulation of remyelination by enhancing the maturation of these cells.

Sphingosine-1-phosphate and the immunosuppressant, FTY720-phosphate, regulate detrusor muscle tone.

Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth muscle (detrusor) exhibits spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS. We have now uncovered a prominent role for the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and contraction. S1P-induced contraction of detrusor muscle was dependent on stretch and intracellular calcium. Although detrusor expresses the S1P receptors S1P1 and S1P2, only S1P2 appeared to be involved in S1P-induced contraction, since SEW2871 (S1P1 agonist) and dihydro-S1P (potent agonist for all S1P receptors except S1P2) were poor contractile agents. In agreement, the S1P2 antagonist JTE013 inhibited S1P-induced contraction. The fast, transient muscle contraction (phasic) mediated by S1P was dependent on phospholipase C (PLC) whereas the slower, sustained contraction (tonic) was not. Surprisingly, the immunosuppressant FTY720-phosphate, an agonist for all S1P receptors except S1P2, had distinct contractile properties and also induced slow, sustained contraction. Thus, FTY720-phosphate and/or S1P may regulate calcium channels in an S1P receptor-independent manner. Collectively, our results demonstrate that S1P may regulate detrusor smooth muscle tone and suggest that dysregulation of complex S1P signaling might contribute to OBS.

Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells.

Ceramide-1-phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro-apoptotic ceramide. To examine the role of endogenous C1P, expression of ceramide kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that ceramide kinase determines the balance between pro-apoptotic ceramide and anti-apoptotic C1P to regulate cell fate, reminiscent of its function in plants.

The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors.

FTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A2 (PLA2)-mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA2 or ERK1/2 activation, suggesting that it does not interfere with FcepsilonRI-mediated events leading to cPLA2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA2alpha activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA2alpha and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis.

Role of ABCC1 in export of sphingosine-1-phosphate from mast cells.

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that S1P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S1P release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses.

The histone deacetylase inhibitor LAQ824 induces human leukemia cell death through a process involving XIAP down-regulation, oxidative injury, and the acid sphingomyelinase-dependent generation of ceramide.

Determinants of differentiation and apoptosis induction by the novel histone deacetylase inhibitor (HDACI) LAQ824 were examined in human leukemia cells (U937 and Jurkat). Exposure of U937 cells to a low concentration of LAQ824 (30 nM) resulted in a delayed (2 h) increase in reactive oxygen species (ROS), induction of p21(WAF1/CIP1), pRb dephosphorylation, growth arrest of cells in G(0)/G(1) phase, and differentiation. On the other hand, exposure of cells to a higher concentration of LAQ824 (75 nM) resulted in the early (30 min) generation of ROS, arrest of cells in G(2)/M phase, down-regulation of XIAP (at the transcriptional level) and Mcl-1 (through a caspase-mediated process), the acid sphingomyelinase-dependent generation of ceramide, and profound mitochondrial injury, caspase activation, and apoptosis. LAQ824-induced lethality in U937 cells did not involve the extrinsic apoptotic pathway, nor was it associated with death receptor up-regulation; instead, it was markedly inhibited by ectopic expression of Bcl-2, Bcl-x(L), XIAP, and Mcl-1. The free radical scavenger N-acetyl cysteine blocked LAQ824-mediated ROS generation, mitochondrial injury, Mcl-1 down-regulation, ceramide generation, and apoptosis, suggesting a primary role for oxidative injury in LAQ824 lethality. Together, these findings indicate that LAQ824-induced lethality represents a multifactorial process in which LAQ824-mediated ROS generation is necessary but not sufficient to induce apoptosis, and that the degree of XIAP and Mcl-1 down-regulation and ceramide generation determines whether this agent engages a maturation rather than an apoptotic program.

A novel acylglycerol kinase that produces lysophosphatidic acid modulates cross talk with EGFR in prostate cancer cells.

The bioactive phospholipids, lysophosphatidic acid (LPA) and phosphatidic acid (PA), regulate pivotal processes related to the pathogenesis of cancer. Here, we report characterization of a novel lipid kinase, designated acylglycerol kinase (AGK), that phosphorylates monoacylglycerol and diacylglycerol to form LPA and PA, respectively. Confocal microscopy and subcellular fractionation suggest that AGK is localized to the mitochondria. AGK expression was up-regulated in prostate cancers compared with normal prostate tissues from the same patient. Expression of AGK in PC-3 prostate cancer cells markedly increased formation and secretion of LPA. This increase resulted in concomitant transactivation of the EGF receptor and sustained activation of extracellular signal related kinase (ERK) 1/2, culminating in enhanced cell proliferation. AGK expression also increased migratory responses. Conversely, down-regulating expression of endogenous AGK inhibited EGF- but not LPA-induced ERK1/2 activation and progression through the S phase of the cell cycle. Hence, AGK can amplify EGF signaling pathways and may play an important role in the pathophysiology of prostate cancer.

Coadministration of histone deacetylase inhibitors and perifosine synergistically induces apoptosis in human leukemia cells through Akt and ERK1/2 inactivation and the generation of ceramide and reactive oxygen species.

Interactions between histone deacetylase inhibitors (HDACIs) and the alkyl-lysophospholipid perifosine were examined in human leukemia cells. Coadministration of sodium butyrate, suberoylanilide hydroxamic acid (SAHA), or trichostatin with perifosine synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and a marked decrease in cell growth in U937 as well as HL-60 and Jurkat leukemia cells. These events were associated with inactivation of extracellular signal-regulated kinase (ERK) 1/2 and Akt, p46 c-jun-NH2-kinase (JNK) activation, and a pronounced increase in generation of ceramide and reactive oxygen species (ROS). They were also associated with up-regulation of Bak and a marked conformational change in Bax accompanied by membrane translocation. Ectopic expression of Bcl-2 delayed but was ultimately ineffective in preventing perifosine/HDACI-mediated apoptosis. Enforced expression of constitutively active mitogen-activated protein kinase kinase (MEK) 1 or myristoylated Akt blocked HDACI/perifosine-mediated ceramide production and cell death, suggesting that MEK/ERK and Akt inactivation play a primary role in these phenomena. However, inhibition of JNK activation (e.g., by the JNK inhibitor SP600125) did not attenuate sodium butyrate/perifosine-induced apoptosis. In addition, the free radical scavenger N-acetyl-L-cysteine attenuated ROS generation and apoptosis mediated by combined treatment. Finally, the acidic sphingomyelinase inhibitor desipramine attenuated HDACI/perifosine-mediated ceramide and ROS production as well as cell death. Together, these findings indicate that coadministration of HDACIs with perifosine in human leukemia cells leads to Akt and MEK/ERK disruption, a marked increase in ceramide and ROS production, and a striking increase in mitochondrial injury and apoptosis. They also raise the possibility that combining these agents may represent a novel antileukemic strategy.

Expression of SphK1 impairs degranulation and motility of RBL-2H3 mast cells by desensitizing S1P receptors.

Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting a variety of biologically active substances, including sphingosine-1 phosphate (S1P). Sphingosine kinase 1 (SphK1) and formation of S1P, which leads to transactivation of S1P receptors and their downstream signaling pathways, regulates mast-cell functions initiated by cross-linking of the high-affinity immunoglobulin E (IgE) receptor FcepsilonRI. Surprisingly, overexpression of SphK1 in rat basophilic leukemia (RBL)-2H3 mast cells impaired degranulation as well as migration toward antigen. These effects were reversed by serum withdrawal, yet the increased formation and secretion of S1P were the same as in the presence of serum. Nonetheless, serum increased localization of SphK1 at the plasma membrane. This restricted formation of S1P induced internalization and desensitization of S1P receptors on the surface of mast cells as determined by confocal immunofluorescence microscopy, aberrant S1P receptor signaling, and lack of S1P receptor coupling to G proteins. Serum starvation, which significantly reduced membrane-associated SphK1 activity, restored S1P receptor functions. Our results have important implications for mast-cell migration and degranulation as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream.

Effect of a membrane-targeted sphingosine kinase 1 on cell proliferation and survival.

Numerous extracellular stimuli activate SK1 (sphingosine kinase type 1) to catalyse the production of sphingosine 1-phosphate, a bioactive lipid that functions as both an extracellular ligand for a family of G-protein-linked receptors and as a putative intracellular messenger. Phorbol esters, calcium or immunoglobulin receptors stimulate SK1 by promoting its translocation to the plasma membrane, which brings it into proximity both to its substrate (i.e. sphingosine) and to activating acidic phospholipids (e.g. phosphatidylserine). To evaluate the consequence of SK translocation, we generated an SK1-derivative tagged with a myristoylation sequence (Myr-SK1) on its N-terminus and overexpressed the construct in 3T3-L1 fibroblasts using recombinant retrovirus. Myr-SK1 overexpression increased SK activity by more than 50-fold in crude membranes, while only stimulating cytoplasmic SK activity by 4-fold. In contrast, the overexpression of WT-SK1 (wild-type SK1), as well as that of a construct containing a false myristoylation sequence (A2-Myr-SK1), markedly increased SK activity in both membrane and cytoplasmic compartments. Immunofluorescence confirmed that Myr-SK1 preferentially localized at the plasma membrane, whereas WT-SK1 and A2-Myr-SK1 partitioned in cytoplasmic/perinuclear cellular regions. Surprisingly, Myr-SK1 overexpression significantly decreased the rates of cell proliferation by delaying exit from G0/G1 phase. Moreover, expression of Myr-SK1 but not WT-SK1 or A2-Myr-SK1 protected cells from apoptosis induced by serum withdrawal. Collectively, these findings reveal that altering the subcellular location of SK1 has marked effects on cell function, with plasma membrane-associated SK having a potent inhibitory effect on the G1-S phase transition.

Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension.

The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

Modulation of adaptive immune responses by sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) has long been recognized as a mediator of a variety of cell functions. A growing body of evidence has accumulated demonstrating its role in cell migration and as a mediator of growth factor-induced events. In recent years, it has become apparent that S1P also mediates many cytokine and chemokine functions. Cells of the immune system function and migrate in response to a complex network of cytokines and chemokines, and the outcome is determined by the interplay of the effects of these molecules on the target cell. S1P may be a bona fide component of these networks and influence the responses of cells to these immune modulators.

The histone deacetylase inhibitor MS-275 interacts synergistically with fludarabine to induce apoptosis in human leukemia cells.

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-beta-D-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27(KIP1), cyclins A, E, and D(1), and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH(2)-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger L-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

Deoxycholic acid activates the c-Jun N-terminal kinase pathway via FAS receptor activation in primary hepatocytes. Role of acidic sphingomyelinase-mediated ceramide generation in FAS receptor activation.

We have shown previously that bile acids can activate the JNK pathway and down-regulate cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis. In this study, the mechanism(s) by which deoxycholic acid (DCA) activates the JNK pathway were examined. FAS receptor (FAS-R) and acidic sphingomyelinase (ASM)-deficient hepatocytes were resistant to DCA-induced activation of the JNK pathway. Activation of the JNK pathway (2-3-fold) in response to tumor necrosis factor-alpha was similar in both wild-type and FAS-R(-/-) hepatocytes. In wild-type and FAS-R(-/-) hepatocytes, ceramide elevation was detected as early as 2 min and peaked at 10 min after DCA treatment. In contrast, ASM(-/-) hepatocytes were defective in DCA-induced ceramide generation. Treatment with DCA resulted in movement of FAS-R to the cell surface, which was blocked upon treatment with brefeldin A. However, brefeldin A failed to block DCA-mediated JNK activation in wild-type hepatocytes. DCA-induced JNK activation was independent of either the epidermal growth factor receptor activation or free radical generation. Addition of ASM to rat hepatocytes activated JNK and down-regulated CYP7A1 mRNA levels. In conclusion, these results show that DCA activates JNK and represses CYP7A1 mRNA levels in primary hepatocytes via an ASM/FAS-R-dependent mechanism that is independent of either the epidermal growth factor receptor or free radical generation.

The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2.

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.

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis.

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.

Sphingosine-1-phosphate: dual messenger functions.

The sphingolipid metabolite sphingosine-1-phosphate (S1P) is a serum-borne lipid that regulates many vital cellular processes. S1P is the ligand of a family of five specific G protein-coupled receptors that are differentially expressed in different tissues and regulate diverse cellular actions. Much less is known of the intracellular actions of S1P. It has been suggested that S1P may also function as an intracellular second messenger to regulate calcium mobilization, cell growth and suppression of apoptosis in response to a variety of extracellular stimuli. Dissecting the dual actions and identification of intracellular targets of S1P has been challenging, but there is ample evidence to suggest that the balance between S1P and ceramide and/or sphingosine levels in cells is an important determinant of cell fate.

Involvement of sphingosine kinase in TNF-alpha-stimulated tetrahydrobiopterin biosynthesis in C6 glioma cells.

In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor alpha (TNF-alpha) without affecting GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a cofactor required for iNOS activity. TNF-alpha also stimulates sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (SPP), a further metabolite of ceramide. Several clones of C6 cells, expressing widely varying levels of sphingosine kinase, were used to examine the role of SPP in regulation of GTPCH and BH(4) biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-alpha on GTPCH expression and activity and BH(4) biosynthesis. In contrast, enforced expression of sphingosine kinase had no effect on iNOS expression or NO formation. Furthermore, N,N-dimethylsphingosine, a potent sphingosine kinase inhibitor, completely eliminated the increased GTPCH activity and expression induced by TNF-alpha. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-alpha, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH(4) biosynthesis. However, both SPP and dihydro-SPP markedly stimulated ERK 1/2 in C6 cells, which express cell surface SPP receptors. Interestingly, although this ERK activation was blocked by PD98059, which also reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity. Collectively, these results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH(4) levels.