A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.     

Role of sphingosine-1-phosphate receptors in vascular injury of inflammatory bowel disease

Sphingosine-1-phosphate receptors (S1PRs) have an impact on the intestinal inflammation of inflammatory bowel disease (IBD) by regulating lymphocyte migration and differentiation. S1PR modulators as an emerging therapeutic approach are being investigated for the treatment of IBD. However, the role of S1PRs in intestinal vessels has not drawn much attention. Intestinal vascular damage is one of the major pathophysiological features of IBD, characterized by increased vascular density and impaired barrier function. S1PRs have pleiotropic effects on vascular endothelial cells, including proliferation, migration, angiogenesis and barrier homeostasis. Mounting evidence shows that S1PRs are abnormally expressed on intestinal vascular endothelial cells in IBD. Unexpectedly, S1PR modulators may damage intestinal vasculature, for example increase intestinal bleeding; therefore, S1PRs are thought to be involved in the regulation of intestinal vascular function in IBD. However, little is understood about how S1PRs regulate intestinal vascular function and participate in the initiation and progression of IBD. In this review, we summarize the pathogenic role of S1PRs in and the underlying mechanisms behind the intestinal vascular injury in IBD in order for improving IBD practice including S1PR-targeted therapies.     

S1P对心肌细胞的保护作用研究

目的：探究1-磷酸鞘氨醇（S1P）对缺氧／复氧乳鼠心肌细胞的保护作用及其分子机制。方法：在大鼠乳鼠心肌细胞原代培养基础上,应用液体石蜡覆盖法制备心肌细胞缺氧／复氧模型,采用流式细胞术PI染色法和流式细胞术罗丹明123染色法检测S1P对缺氧／复氧心肌细胞的细胞凋亡和线粒体膜电位的影响;Western Blot分析法检测S1P作用后的心肌细胞p-Akt1蛋白水平变化,并且观察PI3K（磷脂酰肌醇3-激酶）阻断剂渥曼青霉素（wommamin）对S1P上述作用的影响。结果：在S1P的影响下,缺氧／复氧心肌细胞的凋亡率显著下降（P〈0．01）,线粒体膜电位的去极化被明显抑制（P〈0．05）,p-Akt1水平明显升高（P〈0．01）,wormannin能够部分阻断S1P的上述效应。结论：S1P能够显著抑制缺氧／复氧引起的心肌细胞凋亡,其机制可能与S1P激活PDK-Akt信号通路进而稳定线粒体膜电位有关。     

Phosphorylated FTY720 promotes astrocyte migration through sphingosine-1-phosphate receptors

Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.     

Sphingosine kinase,sphingosine-1-phosphate,and apoptosis

The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 51 (1991) 1613] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. 10 (2000) 1527]. Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (1999) 301], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion [Science 291 (2001) 1800] to regulate blood vessel formation [J. Clin. Invest. 106 (2000) 951]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) 1109]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis.     

The role of sphingosine-1-phosphate in skeletal muscle: Physiology,mechanisms, and clinical perspectives

Sphingolipids were discovered more than a century ago and were simply considered as a class of cell membrane lipids for a long time. However, after the discovery of several intracellular functions and their role in the control of many physiological and pathophysiological conditions, these molecules have gained much attention. For instance, the sphingosine-1-phosphate (S1P) is a circulating bioactive sphingolipid capable of triggering strong intracellular reactions through the family of S1P receptors (S1PRs) spread in several cell types and tissues. Recently, the role of S1P in the control of skeletal muscle metabolism, atrophy, regeneration, and metabolic disorders has been widely investigated. In this review, we summarized the knowledge of S1P and its effects in skeletal muscle metabolism, highlighting the role of S1P/S1PRs axis in skeletal muscle regeneration, fatigue, ceramide accumulation, and insulin resistance. Finally, we discussed the physical exercise role in S1P/S1PRs signaling in skeletal muscle cells, and how this nonpharmacological strategy may be prospective for future investigations due to its ability to increase S1P levels.     

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren’s syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.     

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.     

Evidence for regulated monoacylglycerol acyltransferase expression and activity in human liver

Intrahepatic lipid accumulation is extremely common in obese subjects and is associated with the development of insulin resistance and diabetes. Hepatic diacylglycerol and triacylglycerol synthesis predominantly occurs through acylation of glycerol-3-phosphate. However, an alternative pathway for synthesizing diacylglycerol from monoacylglycerol acyltransferases (MGAT) could also contribute to hepatic glyceride pools. MGAT activity and the expression of the three genes encoding MGAT enzymes (MOGAT1, MOGAT2, and MOGAT3) were determined in liver biopsies from obese human subjects before and after gastric bypass surgery. MOGAT expression was also assessed in liver of subjects with nonalcoholic fatty liver disease (NAFLD) or control livers. All MOGAT genes were expressed in liver, and hepatic MGAT activity was readily detectable in liver lysates. The hepatic expression of MOGAT3 was highly correlated with MGAT activity, whereas MOGAT1 and MOGAT2 expression was not, and knockdown of MOGAT3 expression attenuated MGAT activity in a liver-derived cell line. Marked weight loss following gastric bypass surgery was associated with a significant reduction in MOGAT2 and MOGAT3 expression, which were also overexpressed in NAFLD subjects. These data suggest that the MGAT pathway is active and dynamically regulated in human liver and could be an important target for pharmacologic intervention for the treatment of obesity-related insulin resistance and NAFLD.     

Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.51: 2546–2559.     

鞘氨醇-1-磷酸与免疫细胞的调节

鞘氨醇-1-磷酸（sphingosine-1 phosphate,S1P）是来源于鞘脂代谢途径的多效性信号分子,其代谢受到多种因素调控。S1P由细胞内的鞘氨醇激酶（sphingosine kinases,SphKs）催化鞘氨醇的磷酸化而合成,可通过转运蛋白释放至细胞外。S1P可通过在胞外结合其特异性G蛋白偶联受体及胞内作用而调节多种重要生物学效应。作为细胞外介质和细胞内信使,S1P在免疫系统中也发挥重要的调节作用。S1P参与免疫细胞的迁移、增殖、分化及死亡细胞清除等过程。本文对S1P的代谢以及其对于免疫细胞的调节作用进行综述。     

Sphingosine-1-phosphate metabolism: A structural perspective

Abstract

Sphingolipids represent an important class of bioactive signaling lipids which have key roles in numerous cellular processes. Over the last few decades, the levels of bioactive sphingolipids and/or their metabolizing enzymes have been realized to be important factors involved in disease development and progression, most notably in cancer. Targeting sphingolipid-metabolizing enzymes in disease states has been the focus of many studies and has resulted in a number of pharmacological inhibitors, with some making it into the clinic as therapeutics. In order to better understand the regulation of sphingolipid-metabolizing enzymes as well as to develop much more potent and specific inhibitors, the field of sphingolipids has recently taken a turn toward structural biology. The last decade has seen the structural determination of a number of sphingolipid enzymes and effector proteins. In these terms, one of the most complete arms of the sphingolipid pathway is the sphingosine-1-phosphate (S1P) arm. The structures of proteins involved in the function and regulation of S1P are being used to investigate further the regulation of said proteins as well as in the design and development of inhibitors as potential therapeutics.     

An update on sphingosine-1-phosphate receptor 1 modulators

Sphingolipids represent an essential class of lipids found in all eukaryotes, and strongly influence cellular signal transduction. Autoimmune diseases like asthma and multiple sclerosis (MS) are mediated by the sphingosine-1-phosphate receptor 1 (S1P₁) to express a variety of symptoms and disease patterns. Inspired by its natural substrate, an array of artificial sphingolipid derivatives has been developed to target this specific G protein-coupled receptor (GPCR) in an attempt to suppress autoimmune disorders. FTY720, also known as fingolimod, is the first oral disease-modifying therapy for MS on the market. In pursuit of improved stability, bioavailability, and efficiency, structural analogues of this initial prodrug have emerged over time. This review covers a brief introduction to the sphingolipid metabolism, the mechanism of action on S1P₁, and an updated overview of synthetic sphingosine S1P₁ agonists.     

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.     

Effects of sphingosine-1-phosphate on pacemaker activity of interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P_1,3,4,5 agonist) and SEW 2871 (an S1P₁ agonist) had no effects on pacemaker activity. Suramin (an S1P₃ antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P₂ antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P₂ in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH₂-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca²⁺-free solution or thapsigargin (a Ca²⁺-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca²⁺ ([Ca²⁺]_i) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca²⁺]_i oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P₂ via regulation of external and internal Ca²⁺ and mitogenactivated protein kinase activation.     

SphK1 mediates hepatic inflammation in a mouse model of NASH induced by high saturated fat feeding and initiates proinflammatory signaling in hepatocytes

Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.