In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor-alpha (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase-2 (COX-2) induction as early as 2 h. This TNF-induced PGE2 production was inhibited by NS398, a COX-2 selective inhibitor. GC-MS analysis revealed that only COX-2-generated prostanoids were produced in response to TNF, thus providing further evidence of COX-2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX-2 induction and PGE2 production was evaluated. Sphingosine-1-phosphate (S1P) induced both COX-2 and PGE2 in a dose-responsive manner with an apparent ED50 of 100-300 nM. The related sphingolipid sphingosine also induced PGE2, though with much less efficacy. TNF induced a 3.5-fold increase in sphingosine-1-phosphate levels at 10 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK1 decreased (typically by 80%) SK1 protein and inhibited TNF-induced SK activity. Treatment of cells with RNAi to SK1 but not SK2 almost completely abolished the ability of TNF to induce COX-2 or generate PGE2. By contrast, cells treated with RNAi to S1P lyase or S1P phosphatase enhanced COX-2 induction leading to enhanced generation of PGE2. Treatment with SK1 RNAi also abolished the effects of exogenous sphingosine and ceramide on PGE2, revealing that the action of sphingosine and ceramide are due to intracellular metabolism into S1P. Collectively, these results provide novel evidence that SK1 and S1P are necessary for TNF to induce COX-2 and PGE2 production. Based on these findings, this study indicates that SK1 and S1P could be implicated in pathological inflammatory disorders and cancer.

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK1/S1P pathway also plays a critical role in regulation of cyclooxygenase-2 (COX-2), a well-established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK1 and COX-2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK1 staining was found in 21/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK1 staining was observed in normal mucosa. The increase in SK1 and COX-2 expression in AOM-induced rat colon adenocarcinoma was confirmed at the level of mRNA by real-time RT-PCR. In addition, it was found that 1) down-regulation of SK1 in HT-29 human colon cancer cells by small interfering RNA (siRNA) decreases COX-2 expression and PGE2 production; 2) overexpression of SK1 in RIE-1 rat intestinal epithelial cells induces COX-2 expression; and 3) S1P stimulates COX-2 expression and PGE2 production in HT-29 cells. These results suggest that the SK1/S1P pathway may play an important role in colon carcinogenesis, in part, by regulating COX-2 expression and PGE2 production.

Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr 54 and Asn 89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser 225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr 54 and Asn 89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions.Sphingosine 1-phosphate (S1P) 3 is a recently identified bioactive lipid that can act both extracellularly and intracellularly as a first and a second messenger, respectively (1). It has been shown that S1P is excreted into serum from platelets and binds members of the endothelial differentiation gene receptor family (S1P1-5) to activate cellular processes such as differentiation, migration, and mitogenesis (2). Intracellularly, S1P has been implicated in signaling cascades that lead to cytoskeletal changes, motility, release of intracellular Ca 2ϩ stores, and protection from apoptosis (3-6). S1P is formed from sphingosine (SPH) by sphingosine kinase (SK) and is degraded by S1P lyase and S1P phosphatases (1). In the resting state of cells, the balance between S1P formation and degradation maintains the low basal levels of S1P. However, cellular S1P levels have been shown to increase rapidly and transiently by agonists that activate SK, such as tumor necrosis factor-␣ (7, 8), platelet-derived growth factor (9), nerve growth factor (10, 11), muscarinic acetylcholine agonists (12), or phorbol esters (13,14).Two types of mammalian SKs (SK1 and SK2) have been characterized so far (15, 16), both of which are primarily cytosolic proteins. A recent study suggested that a phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces the protein kinase C (PKC)-mediated phosphorylation and the localization of SK1 to the plasma membrane in human embryonic kidney (HEK) 293 cells (17), which leads to enhanced release of S1P to the media. Subsequently, it was reported that PMA and tumor necrosis factor-␣ induced the phosphorylation of Ser 225 of SK1 through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, which resulted in pla...

Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was posttranscriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and ␥-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.Sphingolipids have received major attention over the past two decades as important mediators of signal transduction and cell regulation. Key lipids in the sphingolipid pathway are ceramide, sphingosine, and sphingosine 1-phosphate (S1P).

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C 6 -ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.

Sphingosine-1-phosphate (S1P) is a highly bioactive lipid that exerts numerous biological effects both intracellularly as a second messenger and extracellularly by binding to its G-protein-coupled receptors of the endothelial differentiation gene family (S1P receptors-(1-5)). Intracellularly, at least two enzymes, sphingosine kinase and S1P phosphatase, regulate the activity of S1P by governing the phosphorylation status of S1P. To study the regulation of S1P levels, we cloned the human isoform of S1P phosphatase 1 (hSPPase1). The hSPPase1 has 78% homology to the mouse SPPase at the amino acid level with 6 -8 possible transmembrane domains. Confocal microscopy revealed green fluorescent protein-tagged hSPPase1, expressed in either MCF7 or HEK293 cells, co-localized to endoplasmic reticulum with calreticulin. According to Northern blot analysis, hSPPase1 is expressed in most tissues, with the strongest levels found in the highly vascular tissues of placenta and kidney. Transient overexpression of hSPPase1 exhibited a 2-fold increase in phosphatase activity against S1P and dihydro-S1P, indicating that the expressed protein was functional. Small interfering RNA (siRNA) knockdown of endogenous hSPPase1 drastically reduced hSPPase1 mRNA levels, as confirmed by reverse transcription PCR, and resulted in an overall 25% reduction of in vitro phosphatase activity in the membrane fractions. Sphingolipid mass measurements in hSPPase1 siRNA knockdown cells revealed a 2-fold increase of S1P levels and concomitant decrease in sphingosine. In vivo labeling of hSPPase1 siRNA-treated cells showed accumulation of S1P within cells, as well as significantly increased secretion of S1P into the media, indicating that hSPPase1 regulates secreted S1P. In addition, siRNA-induced knockdown of hSPPase1 endowed resistance to tumor necrosis factor-␣ and the chemotherapeutic agent daunorubicin. Collectively, these data suggest that regulation of hSPPase1 with the resultant changes in cellular and secreted S1P could have important implications to cell proliferation, angiogenesis, and apoptosis.

(KYJ) S U M M A R Y Sphingosine kinase 1 (SK1) is a key enzyme critical to the sphingolipid metabolic pathway responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphate. SK1-mediated production of sphingosine-1-phosphate has been shown to stimulate such biological processes as cell growth, differentiation, migration, angiogenesis, and inhibition of apoptosis. In this study, cell type-specific immunolocalization of SK1 was examined in the bronchus/terminal bronchiole of the lung. Strong immunopositive staining was evident at the apical surface of pseudostratified epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, immature chondrocytes, type II alveolar cells, foamy macrophages, endothelial cells of blood vessels, and neural bundles.

Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with phagosomes containing live M.tb. To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1G82D, and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1S225A). The magnitude and kinetics of translocation of SK1G82D and SK1S225A to latex bead phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation.