Lisa C. Edsall

Molecular Cloning and Functional Characterization of Murine Sphingosine Kinase

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison withSaccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylatedd-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide,d,l-threo-dihydrosphingosine orN,N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma

Asthma is characterized by airway inflammation, remodeling, and hyperresponsiveness to contractile stimuli that promote airway constriction and wheezing. Here we present evidence that sphingosine 1‐phosphate (SPP) is a potentially important inflammatory mediator implicated in the pathogenesis of airway inflammation and asthma. SPP levels were elevated in the airways of asthmatic (but not control) subjects following segmental antigen challenge, and this increase was correlated with a concomitant increase in airway inflammation. Because human airway smooth muscle (ASM) cells expressed EDG receptors for SPP (EDG‐1, ‐3, ‐5, and ‐6), we examined whether SPP may play a role in airway inflammation and remodeling, by affecting ASM cell growth, contraction, and cytokine secretion. SPP is mitogenic and augments EGF‐ and thrombin‐induced DNA proliferation by increasing G1/S progression. SPP increased phosphoinositide turnover and intracellular calcium mobilization, the acute signaling events that affect ASM contraction. By modulating adenylate cyclase activity and cAMP accumulation, SPP had potent effects on cytokine secretion. Although SPP inhibited TNF‐α–induced RANTES release, it induced substantial IL‐6 secretion alone and augmented production of IL‐6 induced by TNF‐α. These studies are the first to associate SPP with airway inflammation and to identify SPP as an effective regulator of ASM growth, contraction and synthetic functions.

Inhibition of Neuronal Apoptosis by Docosahexaenoic Acid (22:6n-3) ROLE OF PHOSPHATIDYLSERINE IN ANTIAPOPTOTIC EFFECT

Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitrobiomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes.

Sphingosine 1-Phosphate-induced Cell Rounding and Neurite Retraction Are Mediated by the G Protein-coupled Receptor H218

Sphingosine 1-phosphate (SPP) is a lipid second messenger that also acts as a first messenger through the G protein-coupled receptor Edg-1. Here we show that SPP also binds to the related receptors H218 and Edg-3 with high affinity and specificity. SPP and sphinganine 1-phosphate bind to these receptors, whereas neither sphingosylphosphorylcholine nor lysophosphatidic acid compete with SPP for binding to either receptor. Transfection of HEK293 cells with H218 or edg-3, but not edg-1, induces rounded cell morphology in the presence of serum, which contains high levels of SPP. SPP treatment of cells overexpressing H218 cultured in delipidated serum causes cell rounding. A similar but less dramatic effect was observed in cells overexpressing Edg-3 but not with Edg-1. Cell rounding was correlated with apoptotic cell death, probably as a result of loss of attachment. Nerve growth factor-induced neuritogenesis in PC12 cells was inhibited by overexpression of H218 and to a lesser extent Edg-3. SPP treatment rapidly enhanced neurite retraction in PC12 cells overexpressing Edg-1, Edg-3, or H218. Thus, H218, and possibly Edg-3, may be the cell surface receptors responsible for cell rounding and neurite retraction induced by SPP. Moreover, the identification of these two additional SPP receptors indicates that a family of highly specific receptors exists that mediate different responses to SPP.

Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells

Sphingosine‐1‐phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G‐protein‐coupled SPP receptors, known as the endothelial differentiation gene‐1 (EDG‐1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C2‐ceramide. NGF, which protects PC12 cells from serum withdrawal‐induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF‐stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal‐induced activation of the stress activated protein kinase, c‐Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N‐dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface Gi‐coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.

Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells*

Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-xL had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C2-ceramide, induced cytochromec release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-xL overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells.

Activation of sphingosine kinase in pheochromocytoma PC12 neuronal cells in response to trophic factors

Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), dibutyryl cAMP and forskolin, known differentiating agents for pheochromocytoma PC12 cells, induced sustained activation of sphingosine kinase, the enzyme responsible for the formation of the sphingolipid second messenger, sphingosine‐1‐phosphate, which mediates the mitogenic effects of certain growth factors. In contrast, epidermal growth factor and insulin‐like growth factor‐1, which stimulate proliferation of PC12 cells, induced only small and transient increases in sphingosine kinase activity. Of the growth factors examined, NGF was the most potent activator of sphingosine kinase, inducing a 4‐fold increase in V max. Sphingosine kinase activity induced by NGF, but not FGF, was blocked by the protein kinase inhibitor K252a when added simultaneously, with minimal effect when added after 60 min. Thus, activation of sphingosine kinase may have an important role in neural differentiation.

Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCγ

Sphingosine‐1‐phosphate, a sphingo‐lipid metabolite, is involved in the mitogenic response of platelet‐derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild‐type or various mutant βPDGF receptors. Sphingosine ki‐nase was stimulated by PDGF in cells expressing wild‐type receptors but not in cells expressing kinase‐inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCγ, phosphatidylinositol 3‐kinase, Ras GTPase‐activating protein, or protein tyrosine phosphatase SHP‐2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine‐1021 of the PDGF receptor to phenylala‐nine, which impairs its association with PLCγ, abrogated PDGF‐induced activation of sphingosine ki‐nase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3‐ki‐nase and SHP‐2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor ‘add‐back’ mutants in which association with the Ras GTPase‐activating protein, phosphati‐dylinositol 3‐kinase, or SHP‐2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCγ (tyrosine‐1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the ty‐rosine residue responsible for binding of PLCγ is required for PDGF‐induced activation of sphin‐gosine kinase. Moreover, calcium mobilization downstream of PLCγ, but not protein kinase C activation, appears to be required for stimulation of sphin‐gosine kinase by PDGF.—Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet‐derived growth factor‐induced activation of sphin‐gosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCγ. FASEB J. 13, 1593–1600 (1999)

Involvement of Sphingosine Kinase in TNF-α-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 α (TNF-α) without affecting GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH4), a cofactor required for iNOS activity. TNF-α 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 BH4 biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-α on GTPCH expression and activity and BH4 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-α. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-α, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH4 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 BH4levels.

The Release of Polyunsaturated Fatty Acids and Their Lipoxygenation in the Brain

Stimulation of neuronal tissues with neurotransmitters results in the release of the polyunsaturated fatty acids 20:4n6 and 22:6n3. Astroglial cells hydrolyze 20:4n6 and 22:6n3 equally well under both stimulated and basal conditions. Despite the high abundance of 22:6n3 in neuronal membranes, 20:4n6 is preferentially hydrolyzed from neuronal cells. These results suggest that 22:6n3 may be of more physiological importance in neuronal membranes as a membrane component rather than as a released free fatty acid while in astroglia, release of 22:6n3 may also be a significant step involved in receptor-stimulated signaling processes. Oxygenation of these polyunsaturated fatty acids occurs in the brain. However, in contrast to the prevailing belief, lipid peroxidation rather than lipoxygenation is primarily responsible for their formation. In rodent brains, any significant lipoxygenation appears to occur only in the pineal. The production of hydroxylated polyunsaturated fatty acids in pineal may play a role in the pineal function especially in relation to melatonin synthesis.