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Sphingosine-1-Phosphate



At the beginning of this century, sphingosine-1-phosphate was an obscure lipid with no known function. Now it is recognized as one of the most crucial elements in both intra- and inter-cellular signalling, especially in animal cells, with innumerable biological effects; hundreds of publications appear every year on the topic. However, its functions cannot be described in isolation, and they must be considered together with those of the metabolically related sphingolipids ceramides, sphingoid bases and ceramide-1-phosphate, which have their own web pages here. Catabolism of sphingosine-1-phosphate is ultimately the means by which all sphingolipids are removed from cells.

Formula of sphingosine-1-phosphate


1.  Occurrence and Biosynthesis

Sphingosine-1-phosphate can be considered to be a zwitterionic lysophospholipid and an important cellular metabolite derived from ceramide that is synthesised de novo or as part of the sphingomyelin cycle in animal cells. It has also been found in insects, yeasts and plants. Although it is a minor lipid in quantitative terms, it has essential biological properties and it is an intermediate in the irreversible degradation of sphingolipids. For example, sphingosine-1-phosphate has vital roles in health and disease in that it affects cardiac function, vascular development, immune cell function, inflammation and cancer. Unlike the lysoglycerophospholipids, with which it has some functional kinship, it exists mainly as a single molecular species in most animals. However, in humans and mice, platelets contain dihydrosphingosine-1-phosphate in addition to sphingosine-1-phosphate, and there is some evidence that the two lipid species may have different and even opposing functions.

In most animal cells, sphingosine-1-phosphate occurs at concentrations in the low nanomolar range because of a rapid turnover. However, in plasma, it can reach a concentration of 200 nM in humans to 700 nM in mice. A high proportion (~60%) is found in intimate association with the lipoproteins, especially the high-density lipoproteins (HDL) and in particular the HDL3 subfamily, where it is bound to apolipoprotein M (apo M), a 25-kDa member of the lipocalin protein superfamily, which has a lipophilic binding pocket within the lipocalin structure; much of the remainder (~30%) is bound to albumin. Apo M has been termed a sphingosine-1-phosphate 'chaperone' that controls the levels of the lipid in blood. However, the highest concentrations are found in red blood cells.

The primary precursor is sphingomyelin, which is hydrolysed by sphingomyelinases to produce ceramides and these are in turn acted upon by the enzyme ceramidase, mainly in the lysosomes, to release sphingosine; this is phosphorylated by ATP-dependent sphingosine kinases, which catalyse the transfer of the γ-phosphate from a molecule of ATP onto the C1 hydroxyl group of sphingosine to form sphingosine-1-phosphate.

Biosynthesis of sphingosine-1-phosphate

There are in fact two sphingosine kinases, designated Types 1 and 2 (SPHK1 and SPHK2), which are part of a super-family of enzymes that includes ceramide kinase and diacylglycerol kinase. They are distributed ubiquitously in tissues, but are especially abundant in erythrocytes and epithelial cells. Although the enzymes differ substantially in size, they have a high degree of polypeptide sequence similarity, but with different developmental expression and tissue and subcellular distributions, suggesting that each has distinct and non-overlapping physiological functions. The type 1 sphingosine kinase is predominantly cytosolic and pro-survival, probably by inhibiting ceramide biosynthesis; it translocates to the plasma membrane upon activation and a significant fraction is released to the extracellular space. Thus, SPHK1 regulates the levels of sphingosine-1-phosphate in the cytosol and plasma membrane. The Type 2 enzyme is found mainly in the nucleus, with the potential to regulate gene expression, but it is also present in the cytosol, mitochondria, internal membranes and plasma membrane, depending on cell type, and it has a broader specificity with the ability to phosphorylate phytosphingosine and dihydrosphingosine as well as sphingosine, but it is not secreted and may be more important in the regulation of gene expression and of apoptosis. SPHK2 is the main form of the enzyme in platelets. The reverse reaction to synthesis can occur also by the action of sphingosine phosphatases (see below), and the enzymes act in concert to control the cellular concentrations of the metabolite, which are always low. In addition, the activity of SPHK1 is regulated by a number of factors including transcription, phosphorylation/dephosphorylation, protein-protein interactions, membrane accessibility and degradation.

Sphingosine-1-phosphate may also be synthesised on the inner leaflet of the plasma membrane, and it is able to cross this via the action of at least two transporter proteins (from the ATP-binding cassette (ABC) family) to interact with specific receptors on the surface of the same cell or on nearby cells, or it can be transported in plasma to more distant tissues. In addition, it can be produced in plasma by the hydrolysis of sphingosylphosphorylcholine by the enzyme autotaxin, which is best known for the production of lysophosphatidic acid.

It now seems certain that much of the sphingosine-1-phosphate in blood is synthesised in erythrocytes and platelets, where it is produced mainly by the action of sphingosine kinase on free sphingosine absorbed from plasma or derived from ceramide generated in the plasma membrane prior to secretion into plasma. Release from activated platelets requires calcium- and ATP-dependent transporters, while it is released constitutively in an ATP-dependent manner from erythrocytes possibly with the aid of an ATP-binding cassette (ABC) type transporter. In addition, a significant proportion of the sphingosine-1-phosphate in plasma, possibly as much as 40%, comes from the endothelial cells, which have an active transport mechanism from the interior of the cells with a passive export mechanism. Erythrocytes are also able to take up preformed sphingosine-1-phosphate from plasma, and they are well suited to function as a storage organ as they lack the relevant degradative enzymes; in effect they constitute a buffering system to maintain high levels in circulation.

Also, a high proportion of the sphingosine-1-phosphate in blood is solubilized by an interaction with albumin, although this does not have a specific binding site. As there is a high turnover of the lipid, it is assumed that the loose albumin complex is removed rapidly from plasma by the liver. Levels of sphingosine-1-phosphate in lymph fluid, where the source is the lymphatic endothelial cells, are four to five fold lower than plasma, while those in interstitial fluid are roughly 1000 fold lower, a gradient that is of great importance for directing immune cells to lymphoid organs and regulating their egress into blood and lymph.

Unlike most other sphingolipids, sphingosine-1-phosphate is not believed to participate in raft formation in membranes.


2.  Biological Functions

Like its precursors, sphingosine-1-phosphate is a potent messenger molecule that perhaps uniquely operates both intra- and inter-cellularly, i.e. it is both an autocrine and a paracrine agent, but with very different functions from ceramides, ceramide-1-phosphate and sphingosine. The balance between these various sphingolipid metabolites is important for health and has sometimes been termed the 'sphingolipid rheostat', although the readiness with which they can be interconverted does make it difficult to determine the true function or relative activity of each. In addition, there are differences between cell types under various conditions and the sphingolipid rheostat as a balancing mechanism may over-simplify the situation.

Sphingolipid rheostat

A general hypothesis is that this mechanism evolved early in the development of life to regulate cell survival under environmental stress. For example, within the cell (as a paracrine agent or first messenger) in contrast to ceramide and sphingosine, sphingosine-1-phosphate is pro-survival in that it promotes cellular division (mitosis) as opposed to cell death (apoptosis), which it inhibits in fact. However, it can also enhance apoptosis in some circumstances by promoting the formation of ceramide. In addition, it promotes autophagy, i.e. the controlled turnover of damaged organelles, proteins and invading microorganisms within cells while providing nutrients to maintain vital cellular functions. Intracellularly, sphingosine-1-phosphate functions to regulate calcium mobilization and cell growth in response to a variety of extracellular stimuli, and it is believed to be involved in the regulation of the immune and inflammatory responses of cytokines. It promotes the recruitment of lymphocytes to sites of inflammation.

As with the lysophospholipids, especially lysophosphatidic acid with which it has some structural similarities, sphingosine-1-phosphate exerts many of its extra-cellular effects (as an autocrine agent or second messenger) through acting as a ligand for specific receptors, in this instance five G protein-coupled receptors on cell surfaces (designated S1P1 to S1P5). Both sphingosine-1-phosphate and its dihydro analogue bind to them with a high affinity. In mammals, S1P1, S1P2, and S1P3 are found in all tissues, whereas S1P4 is restricted to lymphoid tissues and lung, and S1P5 to brain and skin. Of these, S1P1 is one of the most highly expressed of all G protein-coupled receptors, and it is now understood to exert its signalling functions as part of an enzyme complex that includes β-arrestins and other G proteins. Each receptor when activated, triggers distinctive signaling pathways and cellular responses, some of which can be antagonistic. The ligand-receptor interactions are important for the growth of new blood vessels, vascular maturation, cardiac development and immunity, and for directed cell movement. In addition, sphingosine-1-phosphate and its receptors regulate the biosynthesis of corticosteroid hormones and their function.

It appears that the cellular location of sphingosine-1-phosphate production may dictate its functions, even to the extent of producing opposing biological effects, although the two biosynthetic enzymes may compensate for each other, but only in part, if one is inhibited. Thus, there is evidence that cytosolic sphingosine-1-phosphate formed by the action of SPHK1 is pro-survival in that it stimulates cell proliferation and inhibits synthesis of ceramide de novo, while the SPHK2 isoform located in the endoplasmic reticulum promotes ceramide synthesis through the sphingosine salvage pathway. Loss of both enzymes is embryonically fatal. Scottish thistleMost functions of sphingosine-1-phosphate have been attributed to that generated by the action of SPHK1, with much less known of the role of SPHK2. In response to external stimuli, phosphorylated SPHK2 translocates into the nucleus and produces sphingosine-1-phosphate that binds to specific enzymes to regulate their activities. Sphingosine-1-phosphate produced in the nucleus increases the acetylation of lysine residues on histones, an essential process regulating gene transcription especially of pro-inflammatory genes. SPHK2 present in mitochondria is believed to be necessary for correct assembly of the cytochrome oxidase complex, and it may bind to phosphatidylinositol monophosphates, targeting them to intracellular membranes.

Sphingosine-1-phosphate is produced continuously and stored in relatively high concentrations in human platelets, erythrocytes, mast cells and monocytes, with erythrocytes as the primary source. Platelets do not express enzymes for degradation of this lipid, and it is only released upon external stimulation, for example by thrombin, a product of the coagulation process. This may be especially important when endothelial vessels are injured. Incidentally, the mechanism of secretion from human platelets activated in this way requires generation of thromboxane and is mediated via the thromboxane receptor, an interesting link between sphingolipid and eicosanoid metabolism. Export from many cell types is facilitated by a specific transporter termed 'spinster 2' (Spns2), and its inhibition influences inflammatory and autoimmune diseases. Spns2 exports sphingosine-1-phosphate from lymphatic endothelial cells and accounts for approximately 25–50% of that in plasma. A transport protein designated 'Mfsd2b' is expressed in erythrocytes and has a similar function. When the sphingosine-1-phosphate reaches the outer membrane, it is readily extracted by the chaperone apo M and incorporated into high-density lipoproteins; this provides at least 50% of the sphingosine-1-phosphate in plasma. In addition, the apo M may facilitate the interaction with the receptors.

The high levels of sphingosine-1-phosphate in blood and especially that bound to apolipoprotein M are important for the maintenance of vascular integrity. The vascular endothelium is the barrier that lines blood vessels and assists in cardiovascular homeostasis and blood flow, and in controlling the passage of leukocytes into and out of the bloodstream. Small dense HDL3 subfractions display potent vasorelaxing activity, presumably because of their content of sphingosine-1-phosphate. Also, plasma sphingosine-1-phosphate limits disruption of vascular endothelial monolayers, i.e. it protects the permeability barrier against such potentially disruptive molecules such as histamine or platelet-activating factor, while ceramide and lysophosphatidic acid increase vascular permeability. Damage to the endothelial barrier is involved in a number of disease states. It now known that sphingosine-1-phosphate is responsible for further beneficial clinical effects of HDL by stimulating the production of the potent anti-atherogenic and anti-inflammatory signalling molecule nitric oxide by this tissue.

Receptor-mediated sensing of elevated sphingosine-1-phosphate (and lysophosphatidic acid) levels in the blood and lymph serves as a general mechanism in the regulation of the proliferation, survival, differentiation and migration of many types of stem cells, but especially in the development of the vascular and nervous systems. It also regulates keratinocyte differentiation and epidermal homeostasis. Together with vitamin D, it controls the migratory behavior of circulating osteoclast precursors and thus mediates a critical point in bone homeostasis. Evidently, this lipid is an essential factor in embryogenesis.

In the brain and central nervous system, sphingosine-1-phosphate has important functions in neural development and survival, and it has a role in synaptic transmission by modulating the release of neurotransmitters. While it may have a protective role during autophagy in neurons, it is believed to have pro-inflammatory effects in glial cells. It has been implicated in hypersensitivity to spontaneous and thermal pain, although the results appear to be controversial. However, it has been established that sphingosine-1-phosphate and its receptor S1P3 are critical regulators of acute mechanical pain. The effects are mediated by fast-conducting mechanonociceptors, which close potassium channels and thence modulate the excitability of neurones.


3.  Sphingosine-1-Phosphate and Disease

The functions and malfunctions of sphingosine-1-phosphate signalling have been implicated in a number of disease states, including cardiovascular disease, cancer, multiple sclerosis, inflammatory bowel disease and influenza. For example, it may have a critical role in platelet aggregation and thrombosis. The relatively high concentration of the metabolite in HDL is now believed to have beneficial implications for atherogenesis, and it has been reported that decreased serum concentrations of sphingosine-1-phosphate are better markers of peripheral artery disease and carotid stenosis than is HDL cholesterol. In addition, sphingosine-1-phosphate activates the receptor S1P1 when bound to apo M, and their combined interactions may be of relevance to atherosclerosis. On the other hand, sphingosine 1-phosphate is an activator of vascular calcification (hardening of the arteries), an independent risk factor for cardiovascular disease. There is accumulating evidence that sphingosine-1-phosphate and its receptors regulate heart rate, blood flow in the coronary artery, and blood pressure. By secreting apolipoprotein M, the liver modulates the plasma levels of sphingosine-1-phosphate and dysregulation of its metabolism may contribute to the development of liver diseases.

Scottish thistleIn contrast, sphingosine-1-phosphate has a pro-inflammatory function in that in response to certain cytokines and bacterial lipopolysaccharides it induces up-regulation of the enzyme cyclooxygenase-2 (COX-2) and thence production of the prostaglandin PGE2. While SPHK1 upregulates the expression of pro-inflammatory factors in rheumatoid arthritis, SPKH2 is believed to be anti-inflammatory and controls platelet activation to limit or prevent arterial thrombosis after vascular injury, for example. Increased levels of sphingosine-1-phosphate are induced during chronic inflammation with the effect of decreasing the level of anticoagulants while inducing thrombin release to introduce blood clotting complications in some disease states including renal disease.

Sphingosine-1-phosphate has a key role in the immune system, for example by signalling newly made B and T lymphocytes to migrate from the bone marrow and thymus, respectively, to secondary lymphoid tissues including the spleen and lymph nodes where they may encounter foreign antigens. If they are not activated by an antigen, sphingosine-1-phosphate directs their circulation via other lymphoid tissues back into lymph and then into blood.

Like lysophosphatidic acid, it is a marker for certain types of cancer, and there is increasing evidence that its role in cell division or proliferation has an influence on the development of cancers. For example, in contrast to ceramide, it stimulates the growth, survival and migration of tumor cells and it is abundant in malignant tissue, especially breast, colon and brain cancers. In a glioblastoma, the sphingosine-1-phosphate concentration was found to be nine fold higher than in normal brain tissue, and there was a corresponding reduction in ceramide levels. Similarly, there is increased expression of SPHK1 in many different cancers, and this enzyme has been linked functionally with inflammation and the subsequent development of cancer, while SPHK2 has a role in B-cell acute lymphoblastic leukaemia and some other cancers. However, sphingosine-1-phosphate produced inside the cell by SPHK1, but not by SPHK2, is exported to the extracellular space with the aid of ABC transporters, and high levels are found in the interstitial fluid that bathes cancer cells in the tumour microenvironment. After secretion from cancer cells, it can be passed to non-cancer cells to promote the spread of the disease. Both sphingosine-1-phosphate and ceramide-1-phosphate are potent chemo-attractants for a variety of cell types with effects upon the trafficking of normal and malignant cells, thus promoting metastasis of cancer. This is currently a topic that is attracting great interest amongst medical researchers, and the potential for therapeutic intervention in sphingosine-1-phosphate metabolism, for example by selectively inhibiting biosynthesis by one or other of the sphingosine kinases, is under active investigation. On the other hand, this lipid is believed to have beneficial effects on wound healing by stimulating the proliferation of new cells that close the wound.

Structure of FingolimodLikewise, drugs that antagonize sphingosine-1-phosphate and its receptors are being tested clinically as immuno-suppressants to prevent rejection of kidney grafts and to reduce inflammatory and allergic responses. In particular, a molecule derived synthetically from considerations of the structures of sphingoid bases and termed 'fingolimod (or FTY720)' has been approved by the Food and Drug Administration (USA) as an oral treatment for relapsing and remitting multiple sclerosis, a condition caused by an autoimmune attack on the myelin sheaths of nerves. Within affected tissues, fingolimod is phosphorylated by SPHK2 and the resulting fingolimod-phosphate is released from cells as an agonist for sphingosine-1-phosphate receptors to cause immunosuppression. In addition, this drug has reached the phase II stage in clinical trials for amyotrophic lateral sclerosis, acute stroke and schizophrenia, and the phase I stage for Rett syndrome, colorectal and breast cancer and glioblastoma, while pre-clinical studies suggest that it have neuroprotective effects against a number of other diseases including Alzheimer's, Parkinson's and Huntington's diseases. Second generation analogues of fingolimod with greater specificity for particular receptors, especially the S1P1 receptor, are now at an early stage of testing both as primary therapies and as neuroprotective adjuvants to existing treatments. Sphingosine-1-phosphate receptors are viewed as promising therapeutic targets for other autoimmune diseases such as psoriasis, polymyositis and lupus.


4.  Sphingoid Base-1-Phosphates in Yeasts and Plants

In yeasts and plants, sphinganine, sphingosine, phytosphingosine and other long-chain bases are phosphorylated by kinases in a similar way to form the appropriate 1‑phosphate derivatives. As sphingosine-1-phosphate per se is rarely present in detectable amounts, it has been suggested that the term ‘long-chain (sphingoid) base-1-phosphates’ should be used in discussing the metabolism of these lipids in plants. On the other hand, sphingosine-1-phosphate can accumulate in leaves of the model plant Arabidopsis thaliana, when these are stressed by application of the fungal toxin fumonisin B1, although phytosphingosine-1-phosphate is a mediator of abscisic acid-mediated stomatal closure in this species; sphingosine-1-phosphate has the latter function in Commelina communis. Less is known of the function of these lipids in comparison to animal tissues and no receptors appear to have been found, but there is evidence that they are involved in such diverse processes as defence mechanisms, pathogenesis, calcium mobilization, membrane stability, and the response to drought or heat stress. In yeasts, phytosphingosine-1-phosphate also has a role in the regulation of genes required for mitochondrial respiration.


5.  Catabolism of Sphingosine-1-Phosphate and Sphingoid Bases

Long-chain bases can be regenerated from sphingosine-1-phosphate by the action of specific phosphatases (SPP1 and SPP2), located in the endoplasmic reticulum, and three lysophospholipid hydrolases. The balance between these catabolic activities, those of the sphingosine kinases and of sphingolipid resynthesis is tightly regulated. In mammalian cells, there is an unusual pathway for the salvage of sphingosine that requires its phosphorylation by SPHK2 (but not SPHK1) and then de-phosphorylation by a specific phosphatase for re-acylation to ceramide by ceramide synthase. Extracellular sphingosine-1-phosphate cannot be hydrolysed by the enzymes in the endoplasmic reticulum, but it is degraded by lysophospholipid phosphatase 3, an important enzyme in the catabolism of lysophosphatidic acid, to release sphingosine, which can then be taken up by cells for sphingolipid synthesis. However, in animals and plants, production of sphingosine-1-phosphate is also a key step in the catabolism of long-chain bases.

Within cells, the molecule is cleaved irreversibly in the endoplasmic reticulum by the enzyme sphingosine-1-phosphate lyase, which like the serine palmitoyltransferase involved in the synthesis of sphingoid bases requires pyridoxal 5’-phosphate as a cofactor. This enzyme catalyses the retro-aldol like cleavage of sphingosine-1-phosphate to to yield trans-2-hexadecenal and ethanolamine phosphate. In humans, it will only interact with the naturally occurring D-erythro-isomer of a long-chain base, and it will not react with sphinganine-1-phosphate. The comparable enzyme from rat liver extracts is much less regiospecific and can cleave a variety of different sphingoid bases including sphingosine-1-phosphate, sphinganine-1-phosphate, phytosphingosine-1-phosphate and sphingosine-1-phosphonate.

Degradation of sphingosine-1-phosphate

The reaction with sphingosine-1-phosphate lyase, which is found in many different organs and especially lymphoid tissues but not in erythrocytes, reduces the cellular levels of sphingosine and ceramide. As it is irreversible, it is ultimately the means by which all sphingolipids are removed from cells. Also, as a key enzyme in regulating the intracellular and circulating levels of sphingosine-1-phosphate, it is now seen as a potential target for pharmacological intervention.

trans-2-Hexadecenal produced in the reaction can enter into the β-oxidation pathway, or be reduced to the long-chain alcohol, or be converted via four reactions into palmitoyl-coA and incorporated into glycerolipids. In particular, the trans-2-enoyl-CoA reductase, responsible for the conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA, is a dual function enzyme involved in the production of very long-chain fatty acids. However, as an electrophilic α,β-unsaturated aldehyde, trans-2-hexadecenal also has the potential to interact with proteins via the Michael reaction; it is also known to have important signalling functions in that it induces cytoskeletal reorganization and apoptosis. The ethanolamine phosphate that is the other product of the reaction can be utilized for biosynthesis of phosphatidylethanolamine, so this reaction is a further important link between sphingolipid metabolism and that of the glycerophospholipids.

In plants and yeasts, phytosphingosine with an additional hydroxyl group in position 4 is catabolized in a similar way in the form of the 1-phosphate by a sphingosine-1-phosphate lyase to yield 2-hydroxy-hexadecanal, which is then subject to alpha-oxidation to form pentadecanoic acid (15:0) and thence further odd-chain isomers. This can be a significant pathway for the production of odd-chain fatty acids in yeasts.


6.  Analysis

Analysis of sphingosine-1-phosphate does present problems because of its high polarity and relatively low hydrophobicity. However, methods are available for quantitative extraction from tissues, and modern electrospray-ionization mass spectrometry techniques for detection and quantification afford high sensitivity and specificity.


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Lipid listings Credits/disclaimer Updated: October 17th, 2018 Author: William W. Christie LipidWeb icon