Unraveling the complexities of sphingosine-1-phosphate function: the mast cell model

Sphingosine-1-phosphate (S1P) is a lipid mediator involved in diverse biological processes, from vascular and neural development to the regulation of lymphocyte trafficking. Many of its functions are regulated by five widely expressed S1P G-protein-coupled receptors (S1P(1-5)). S1P is produced mostly intracellularly, thus, much of its potential as an autocrine and paracrine mediator depends on how, when, and where it is generated or secreted out of the cells. However, S1P can also have intracellular activity independent of its receptors, adding to the complexity of S1P function. The mast cell, a major effector cell during an allergic response, has proven instrumental towards understanding the complex regulation and function of S1P. Antigen (Ag) engagement of the IgE receptor in mast cells stimulates sphingosine kinases, which generate S1P and are involved in the activation of calcium fluxes critical for mast cell responses. In addition, mast cells secrete considerable amounts of S1P upon activation, thus affecting the surrounding tissues and recruiting inflammatory cells. Export of S1P is also involved in the autocrine transactivation of S1P receptors present in mast cells. The in vivo response of mast cells, however, is not strictly dependent on their ability to generate S1P, but they are also affected by changes in S1P in the environment previous to Ag challenge. This review will discuss the recent advances towards understanding the intricacies of S1P generation, secretion and regulation in mast cells. In addition, how S1P receptors are activated and their involvement in mast cell functions will also be covered, including new insights on the role of S1P in the mast cell-mediated allergic response of systemic anaphylaxis.     

Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood. To identify the genes within this S1P uptake and recycling pathway, we performed a genome-wide CRISPR/Cas9 KO screen using a positive-selection scheme with Shiga toxin, which binds a cell-surface glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes lethality upon internalization. The screen was performed in HeLa cells with their sphingolipid de novo pathway disabled so that Gb3 cell-surface expression was dependent on salvage of the sphingoid base of S1P taken up from the medium. The screen identified a suite of genes necessary for S1P uptake and the recycling of its sphingoid base to synthesize Gb3, including two lipid phosphatases, PLPP3 (phospholipid phosphatase 3) and SGPP1 (S1P phosphatase 1). The results delineate a pathway in which plasma membrane–bound PLPP3 dephosphorylates extracellular S1P to sphingosine, which then enters cells and is rephosphorylated to S1P by the sphingosine kinases. This rephosphorylation step is important to regenerate intracellular S1P as a branch-point substrate that can be routed either for dephosphorylation to salvage sphingosine for recycling into complex sphingolipid synthesis or for degradation to remove it from the sphingolipid synthesis pathway.     

Export and functions of sphingosine-1-phosphate

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), has emerged as a critical player in a number of fundamental biological processes and is important in cancer, angiogenesis, wound healing, cardiovascular function, atherosclerosis, immunity and asthma, among others. Activation of sphingosine kinases, enzymes that catalyze the phosphorylation of sphingosine to S1P, by a variety of agonists, including growth factors, cytokines, hormones, and antigen, increases intracellular S1P. Many of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface in an autocrine and/or paracrine manner. Therefore, understanding the mechanism by which intracellularly generated S1P is released out of cells is both interesting and important. In this review, we will discuss how S1P is formed and released. We will focus particularly on the current knowledge of how the S1P gradient between tissues and blood is maintained, and the role of ABC transporters in S1P release.     

Epithelial cell extrusion requires the sphingosine-1-phosphate receptor 2 pathway

To maintain an intact barrier, epithelia eliminate dying cells by extrusion. During extrusion, a cell destined for apoptosis signals its neighboring cells to form and contract a ring of actin and myosin, which squeezes the dying cell out of the epithelium. Here, we demonstrate that the signal produced by dying cells to initiate this process is sphingosine-1-phosphate (S1P). Decreasing S1P synthesis by inhibiting sphingosine kinase activity or by blocking extracellular S1P access to its receptor prevented apoptotic cell extrusion. Extracellular S1P activates extrusion by binding the S1P(2) receptor in the cells neighboring a dying cell, as S1P(2) knockdown in these cells or its loss in a zebrafish mutant disrupted cell extrusion. Because live cells can also be extruded, we predict that this S1P pathway may also be important for driving delamination of stem cells during differentiation or invasion of cancer cells.     

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

Sphingosine kinase and sphingosine-1-phosphate in liver pathobiology

Over 20?years ago, sphingosine-1-phosphate (S1P) was discovered to be a bioactive signaling molecule. Subsequent studies later identified two related kinases, sphingosine kinase 1 and 2, which are responsible for the phosphorylation of sphingosine to S1P. Many stimuli increase sphingosine kinase activity and S1P production and secretion. Outside the cell, S1P can bind to and activate five S1P-specific G protein-coupled receptors (S1PR1–5) to regulate many important cellular and physiological processes in an autocrine or paracrine manner. S1P is found in high concentrations in the blood where it functions to control vascular integrity and trafficking of lymphocytes. Obesity increases blood S1P levels in humans and mice. With the world wide increase in obesity linked to consumption of high-fat, high-sugar diets, S1P is emerging as an accomplice in liver pathobiology, including acute liver failure, metabolic syndrome, control of blood lipid and glucose homeostasis, nonalcoholic fatty liver disease, and liver fibrosis. Here, we review recent research on the importance of sphingosine kinases, S1P, and S1PRs in liver pathobiology, with a focus on exciting insights for new therapeutic modalities that target S1P signaling axes for a variety of liver diseases.     

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.     

Generation and metabolism of bioactive sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingophospholipid that has been implicated in the regulation of vital biological processes. Abundant evidence indicates that S1P acts as both an intracellular messenger and an extracellular ligand for a family of five specific G protein-coupled S1P receptors (S1PRs). Cellular levels of S1P are tightly regulated in a spatio-temporal manner through its synthesis catalyzed by sphingosine kinases (SphKs) and degradation by S1P lyase (SPL) and specific S1P phosphohydrolases. Over the past decade, the identification and cloning of genes encoding S1P metabolizing enzymes has increased rapidly. Overexpression and deletion of these enzymes has provided important insights into the intracellular and the "inside-out" functions of S1P. The purpose of this review is to summarize the current knowledge of S1P metabolizing enzymes, their enzymatic properties, and their roles in the control of cellular functions by S1P.     

Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca²⁺ from thapsigargin-sensitive stores. Here, we have studied Ca²⁺ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca²⁺]_i was elevated and agonist-induced [Ca²⁺]_i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca²⁺. Importantly, [Ca²⁺]_i increases and Ca²⁺ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca²⁺ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca²⁺ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca²⁺]_i increase and the other with a slower and prolonged [Ca²⁺]_i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca²⁺]_i increases, reflecting overall Ca²⁺ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca²⁺ storage and elevated basal [Ca²⁺]_i. S1P metabolism thus plays a role not only in acute Ca²⁺ mobilization but also in long-term regulation of Ca²⁺ homeostasis.     

Intracellular generation of sphingosine 1-phosphate in human lung endothelial cells: role of lipid phosphate phosphatase-1 and sphingosine kinase 1

Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of (32)P-labeled HPAECs to S1P or sphingosine (Sph) increased the intracellular accumulation of [(32)P]S1P in a dose- and time-dependent manner. The S1P formed in the cells was not released into the medium. The exogenously added S1P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S1P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-14, an inhibitor of LPPs. HPAECs expressed LPP1-3, and overexpression of LPP-1 enhanced the hydrolysis of exogenous [(3)H]S1P to [(3)H]Sph and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.     

Effects of sphingosine-1-phosphate and lysophosphatidic acid on human osteoblastic cells

The effects of the lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) were studied in human primary osteoblastic cells and the human osteosarcomal cell lines, G292 and MG-63. The studies focused on the role of the Gi protein in the regulation of S1P and LPA-induced proliferation, the effects of the phospholipids on alkaline phosphatase, an early marker of osteoblastic cell proliferation, and the presence of edg receptors. Proliferation was assessed by 3H-thymidine incorporation. Short-term incubation with S1P or LPA induced increases in proliferation that were attenuated in the presence of the Gi inhibitor, pertussis toxin. Alkaline phosphatase activity was measured with a spectrophotometric assay. Biphasic effects of S1P and LPA were observed with the nature of the response dependent upon the cell type, concentration of test agent and the time period of incubation. RTPCR studies revealed that edg-1,2,4,5 receptors are present in the primary normal osteoblastic cells, the MG63 and G292 cells. Only the G292 cells expressed the edg-3 receptor to any significant extent.     

Sphingosine 1-phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling

Sphingosine 1-phosphate (S1P), produced by sphingosine kinase (SPHK), acts both by intracellular and extracellular modes. We evaluated the role of SPHK1 and S1P in osteoclastogenesis using bone marrow-derived macrophage (BMM) single and BMM/osteoblast coculture systems. In BMM single cultures, the osteoclastogenic factor receptor activator of NF-kappaB ligand (RANKL) upregulated SPHK1 and increased S1P production and secretion. SPHK1 siRNA enhanced and SPHK1 overexpression attenuated osteoclastogenesis via modulation of p38 and ERK activities, and NFATc1 and c-Fos levels. Extracellular S1P had no effect in these cultures. These data suggest that intracellular S1P produced in response to RANKL forms a negative feedback loop in BMM single cultures. In contrast, S1P addition to BMM/osteoblast cocultures greatly increased osteoclastogenesis by increasing RANKL in osteoblasts via cyclooxygenase-2 and PGE(2) regulation. S1P also stimulated osteoblast migration and survival. The RANKL elevation and chemotactic effects were also observed with T cells. These results indicate that secreted S1P attracts and acts on osteoblasts and T cells to augment osteoclastogenesis. Taken together, S1P plays an important role in osteoclastogenesis regulation and in communication between osteoclasts and osteoblasts or T cells.     

Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor-beta and mediates TIMP-1 up-regulation

Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (approximately 2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK1 may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-beta. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-beta. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF-beta cooperated in TIMP-1 up-regulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-1 gene.     

Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.

Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.     

Sphingosine-1-phosphate lyase is involved in the differentiation of F9 embryonal carcinoma cells to primitive endoderm

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.     

Synthesis of fluorinated agonist of sphingosine-1-phosphate receptor 1

The bioactive metabolite sphingosine-1-phosphate (S1P), a product of sphingosine kinases (SphKs), mediates diverse biological processes such as cell differentiation, proliferation, survival and angiogenesis. A fluorinated analogue of S1P receptor agonist has been synthesized by utilizing a ring opening reaction of oxacycles by a lithiated difluoromethylphosphonate anion as the key reaction. In vitro activity of this S1P analogue is also reported.     

Regulation of metabolism and transport of sphingosine-1-phosphate in mammalian cells

Sphingosine-1-phosphate (S1P), which is generated from the sphingosine kinase-catalyzed phosphorylation of sphingosine, is now recognized as a critical regulator of many kinds of physiological and pathological processes, including cancer, cardiovascular function, and diabetes. It can also trigger a wide variety of biological effect, such as cell movement, differentiation, survival, inflammation, immunity, calcium homeostasis, and angiogenesis. As we know, a number of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface or intracellular targets. However, the synthesis and the secretion of S1P are regulated by various endogenetic or ectogenous stimuli and involve many kinds of enzymes and transporters. In this review, we discuss the regulation of S1P synthesis by many kinds of enzymes and mainly introduce the process of ceramide to S1P. Moreover, S1P deterioration is important balance in physiologic adjustment. We also describe the role of verified or potential transporters in S1P release in detail.     

Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.     

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase,hSPP2

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.     

Enzymatic measurement of sphingosine 1-phosphate.

Sphingosine 1-phosphate (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including sphingosine. Extracted SPP is efficiently converted to sphingosine by alkaline phosphatase treatment. Sphingosine thus formed is then quantitatively phosphorylated to [(32)P]SPP using recombinant sphingosine kinase and [gamma-(32)P]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0. 5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/10(6) cells. Concurrent measurements of sphingosine levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC12 cells after treatment with exogenous sphingosine or with nerve growth factor which stimulates sphingosine kinase activity.