Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.     

Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the “sphingolipid rheostat”, producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.     

Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.     

Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate

Sphingolipids are major lipid constituents of the eukaryotic plasma membrane. Without certain sphingolipids, cells and/or embryos cannot survive, indicating that sphingolipids possess important physiological functions that are not substituted for by other lipids. One such role may be signaling. Recent studies have revealed that some sphingolipid metabolites, such as long-chain bases (LCBs; sphingosine (Sph) in mammals), long-chain base 1-phosphates (LCBPs; sphingosine 1-phosphate (S1P) in mammals), ceramide (Cer), and ceramide 1-phosphate (C1P), act as signaling molecules. The addition of phosphate groups to LCB/Sph and Cer generates LCBP/S1P and C1P, respectively. These phospholipids exhibit completely different functions than those of their precursors. In this review, we describe recent advances in understanding the functions of LCBP/S1P and C1P in mammals and in the yeast Saccharomyces cerevisiae. Since LCB/Sph, LCBP/S1P, Cer, and C1P are mutually convertible, regulation of not only the total amount of the each lipid but also of the overall balance in cellular levels is important. Therefore, we describe in detail their metabolic pathways, as well as the genes involved in each reaction.     

Killing cancer cells by poly-drug elevation of ceramide levels: a hypothesis whose time has come?

Many papers have shown that sphingolipids control the balance in cells between growth and proliferation, and cell death by apoptosis. Sphingosine-1-phosphate (Sph1P) and glucosylceramide (GlcCer) induce proliferation processes, and ceramide (Cer), a metabolic intermediate between the two, induces apoptosis. In cancers, the balance seems to have come undone and it should be possible to kill the cells by enhancing the processes that lead to ceramide accumulation. The two control systems are intertwined, modulated by a variety of agents affecting the activities of the enzymes in Cer-GlcCer-Sph1P interdependence. It is proposed that successful cancer chemotherapy requires the use of many agents to elevate ceramide levels adequately. This review updates current knowledge of sphingolipid metabolism and some of the evidence showing that ceramide plays a causal role in apoptosis induction, as well as a chemotherapeutic agent.     

SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism

The potent sphingolipid metabolite sphingosine 1-phosphate is produced by phosphorylation of sphingosine catalyzed by sphingosine kinase (SphK) types 1 and 2. In contrast to pro-survival SphK1, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca(2+) transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK1 to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [(3)H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide, whereas SphK1 decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of sphingosine to ceramide in the recycling pathway and conversely, down-regulation of SphK1 increased it. Our results demonstrate that SphK1 and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of sphingosine 1-phosphate production dictates its functions.     

Sphingosine kinase signalling in immune cells: potential as novel therapeutic targets

During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca(2+) mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the potential for targeting SphKs to suppress inflammation and other pathological conditions.     

Sphingosine kinase type 1 inhibition reveals rapid turnover of circulating sphingosine 1-phosphate

S1P (sphingosine 1-phosphate) is a signalling molecule involved in a host of cellular and physiological functions, most notably cell survival and migration. S1P, which signals via a set of five G-protein-coupled receptors (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases) from Sph (sphingosine). Interfering RNA strategies and SphK1 (sphingosine kinase type 1)-null (Sphk1-/-) mouse studies implicate SphK1 in multiple signalling cascades, yet there is a paucity of potent and selective SphK1 inhibitors necessary to evaluate the effects of rapid onset inhibition of this enzyme. We have identified a set of submicromolar amidine-based SphK1 inhibitors and report using a pair of these compounds to probe the cellular and physiological functions of SphK1. In so doing, we demonstrate that our inhibitors effectively lower S1P levels in cell-based assays, but we have been unable to correlate SphK1 inhibition with changes in cell survival. However, SphK1 inhibition did diminish EGF (epidermal growth factor)-driven increases in S1P levels and Akt (also known as protein kinase B)/ERK (extracellular-signal-regulated kinase) phosphorylation. Finally, administration of the SphK1 inhibitor to wild-type, but not Sphk1-/-, mice resulted in a rapid decrease in blood S1P levels indicating that circulating S1P is rapidly turned over.     

Regulation by sphingolipids of the fate of FRTL-5 cells

Sphingolipids, including ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1-P) have recently emerged as signal-transducing molecules. Functionally, a distinguishing characteristic of these lipids is their apparent participation in pro- or anti-proliferative cell regulation pathways. In this study, we examined the involvement of sphingolipids in the fate of FRTL-5 thyroid follicular cells. We first examined the effects of sphingolipids on FRTL-5 cell viability. Sph and Cer induced apoptosis, as revealed by fluorescence microscopy of TUNEL-positive fragmented nuclei and 180-300 bp DNA fragmentation on agarose gel electrophoresis while Sph-1-P was confirmed to prevent FRTL-5 cell apoptosis induced by deprivation of serum and TSH, possibly via cell surface receptors. We then analysed the metabolism of radiolabelled Sph and C(6)-Cer (a synthetic cell-permeable Cer) in FRTL-5 cells by thin layer chromatography, followed by autoradiography. Sph was mainly metabolized to Cer, and then to sphingomyelin, while Sph conversion into Sph-1-P was hardly detected. These changes were not affected by stimulation of the cells with TSH. Our results indicate the involvement of sphingolipid mediators in the fate of FRTL-5 thyroid cells.     

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.     

The control of the balance between ceramide and sphingosine-1-phosphate by sphingosine kinase: oxidative stress and the seesaw of cell survival and death

Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological processes. Ceramides and sphingosine-1-phosphate (S1P) are signaling molecules that regulate cell fate decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S1P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called the sphingolipid rheostat or sphingolipid biostat. Sphingosine kinase, the enzyme that synthesizes S1P, is a crucial enzyme in regulation of the balance of these sphingolipids. Sphingosine kinase has been shown to play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence. This review summarizes the roles of sphingosine kinase signaling in these processes and the mechanisms mediating these responses. In addition, we discuss the evidence tying sphingosine kinase-mediated stress responses to the process of aging.     

Sphingosine Kinases/Sphingosine-1-Phosphate and Death Signalling in APP-Transfected Cells

It has been postulated that disturbances in the sphingolipid metabolism play a key role in the pathogenesis of Alzheimer’s disease (AD). An alteration in sphingosine kinases 1, 2 (SphK1/2) and sphingosine-1-phosphate (S1P) was recently reported in AD. However, the effect of AD-related amyloid beta (Aβ) peptides on SphK1/2 and the role of S1P in Aβ toxicity have not been fully elucidated. In this study the relationship between the Aβ concentration and SphK1/2 expression/activity was analysed in PC12 cells transfected with the Aβ precursor protein, wild-type (APP_wt) or bearing a double Swedish mutation (APP_sw). The role of SphK(s)/S1P in cell survival and death was also investigated. Our results indicated that endogenously liberated Aβ significantly decreases expression and activity of SphK1/2. The SphK(s) inhibitor (SKI II, 10 μM) decreased the viability of APP_wt, APP_sw as well as empty vector-transfected PC12 control cells. Our data demonstrated that expression of S1P receptor-1 (S1P1) was significantly reduced in APP-transfected cells. The effect of S1P applied exogenously was cell type-dependent. In control and APP_wt cells S1P reduced the effect of the SphK1 inhibitor on death signalling. Conversely, it decreased the survival of APP_sw cells and had no protective effect on cells treated with SKI II. Using the S1P1 agonist (SEW2871, 5 μM) and antagonist (W123, 20 μM), we demonstrated that the cytoprotective effect of S1P was receptor-independent. Summarising, we showed that Aβ peptides evoke down-regulation of gene expression and activity for SphK(s) and S1P1. Inhibition of SphK(s) significantly decreased cell survival. The effect of exogenous S1P depended on the concentration of Aβ peptides.     

New insights into the roles and regulation of SphK2 as a therapeutic target in cancer chemoresistance

Chemoresistance is a complicated process developed by most cancers and accounts for the majority of relapse and metastasis in cancer. The main mechanisms of chemoresistance phenotype include increased expression and/or activated drug efflux pumps, altered DNA repair, altered metabolism of therapeutics as well as impaired apoptotic signaling pathways. Aberrant sphingolipid signaling has also recently received considerable attention in chemoresistance. Sphingolipid metabolites regulate main biological processes such as apoptosis, cell survival, proliferation, and differentiation. Two sphingosine kinases, SphK1 and SphK2, convert sphingosine to sphingosine-1-phosphate, an antiapoptotic bioactive lipid mediator. Numerous evidence has revealed the involvement of activated SphK1 in tumorigenesis and resistance, however, contradictory results have been found for the role of SphK2 in these functions. In some studies, overexpression of SphK2 suppressed cell growth and induced apoptosis. In contrast, some others have shown cell proliferation and tumor promotion effect for SphK2. Our understanding of the role of SphK2 in cancer does not have a sufficient integrity. The main focus of this review will be on the re-evaluation of the role of SphK2 in cell death and chemoresistance in light of our new understanding of molecular targeted therapy. We will also highlight the connections between SphK2 and the DNA damage response. Finally, we will provide our insight into the regulatory mechanisms of SphKs by two main categories, micro and long, noncoding RNAs as the novel players of cancer chemoresistance.     

Novel analogs of D-e-MAPP and B13. Part 2: signature effects on bioactive sphingolipids

Novel isosteric analogs of the ceramidase inhibitors (1S,2R)-N-myristoylamino-phenylpropanol-1 (d-e-MAPP) and (1R,2R)-N-myristoylamino-4'-nitro-phenylpropandiol-1,3 (B13) with modified targeting and physicochemical properties were developed and evaluated for their effects on endogenous bioactive sphingolipids: ceramide, sphingosine, and sphingosine 1-phosphate (Cer, Sph, and S1P) in MCF7 cells as determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Time- and dose-response studies on the effects of these compounds on Cer species and Sph levels, combined with structure-activity relationship (SAR) data, revealed 4 distinct classes of analogs which were predominantly defined by modifications of the N-acyl-hydrophobic interfaces: N-acyl-analogs (class A), urea-analogs (class B), N-alkyl-analogs (class C), and omega-cationic-N-acyl analogs (class D). Signature patterns recognized for two of the classes correspond to the cellular compartment of action of the new analogs, with class D acting as mitochondriotropic agents and class C compounds acting as lysosomotropic agents. The neutral agents, classes A and B, do not have this compartmental preference. Moreover, we observed a close correlation between the selective increase of C(16)-, C(14)-, and C(18)-Cers and inhibitory effects on MCF7 cell growth. The results are discussed in the context of compartmentally targeted regulators of Sph, Cer species, and S1P in cancer cell death, emphasizing the role of C(16)-Cer. These novel analogs should be useful in cell-based studies as specific regulators of Cer-Sph-S1P inter-metabolism, in vitro enzymatic studies, and for therapeutic development.     

Arabidopsis sphingosine kinase and the effects of phytosphingosine-1-phosphate on stomatal aperture

Sphingolipids are a major component of membrane lipids and their metabolite sphingosine-1-phosphate (S1P) is a potent lipid mediator in animal cells. Recently, we have shown that the enzyme responsible for S1P production, sphingosine kinase (SphK), is stimulated by the phytohormone abscisic acid in guard cells of Arabidopsis (Arabidopsis thaliana) and that S1P is effective in regulating guard cell turgor. We have now characterized SphK from Arabidopsis leaves. SphK activity was mainly associated with the membrane fraction and phosphorylated predominantly the Delta4-unsaturated long-chain sphingoid bases sphingosine (Sph) and 4,8-sphingadienine, and to a lesser extent, the saturated long-chain sphingoid bases dihydrosphingosine and phytosphingosine (Phyto-Sph). 4-Hydroxy-8-sphingenine, which is a major sphingoid base in complex glycosphingolipids from Arabidopsis leaves, was a relatively poor substrate compared with the corresponding saturated Phyto-Sph. In contrast, mammalian SphK1 efficiently phosphorylated Sph, dihydrosphingosine, and 4,8-sphingadienine, but not the 4-hydroxylated long-chain bases Phyto-Sph and 4-hydroxy-8-sphingenine. Surface dilution kinetic analysis of Arabidopsis SphK with Sph presented in mixed Triton X-100 micelles indicated that SphK associates with the micellar surface and then with the substrate presented on the surface. In addition, measurements of SphK activity under different assay conditions combined with phylogenetic analysis suggest that multiple isoforms of SphK may be expressed in Arabidopsis. Importantly, we found that phytosphingosine-1-phosphate, similar to S1P, regulates stomatal apertures and that its action is impaired in guard cells of Arabidopsis plants harboring T-DNA null mutations in the sole prototypical G-protein alpha-subunit gene, GPA1.     

An oncogenic role of sphingosine kinase

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.     

鞘磷脂代谢与脑缺血

鞘磷脂特别是鞘脂是髓鞘的主要成分,高度集中在中枢神经系统。在生理和病理生理条件下,具有生物活性的鞘磷脂及其代谢产物以及信号传导过程的重要性正在逐步被人们所认识。鞘脂代谢产物鞘氨醇及其前体物质神经酰胺与细胞生长停滞和凋亡有关,而1-磷酸鞘氨醇与增强细胞增殖、分化和细胞生存以及调节细胞的生理和病理过程有关,具有细胞外第一信使和细胞内第二信使的双重功能。这三者之间的相互转换、鞘脂代谢物的相对水平以及细胞的命运,受到鞘氨醇激酶的活性的强烈影响。鞘氨醇激酶可催化磷酸鞘氨醇产生1-磷酸鞘氨醇。1-磷酸鞘氨醇在中枢神经系统中与G蛋白偶联受体家族结合对中枢神经系统发挥作用。本文对鞘磷脂代谢过程中的鞘氨醇激酶、1-磷酸鞘氨醇及其受体与脑缺血之间的关系进行概述。