Sphingosine-1-Phosphate

While sphingosine-1-phosphate was first identified as an intermediate in sphingoid base catabolism in the laboratory of W. Stoffel in 1970, it was rediscovered but as "a novel lipid, involved in cellular proliferation" by Prof. Sarah Spiegel and colleagues in 1991. Now, it is recognized as a crucial element in both intra- and inter-cellular signalling, especially in animal cells, with innumerable biological effects, and hundreds of publications appear every year on the topic. Its functions cannot be described in isolation but 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, as part of the sphingolipid rheostat. Catabolism of sphingosine-1-phosphate is ultimately the mechanism for removal of all sphingolipids from cells.

Formula of sphingosine-1-phosphate

1. Occurrence and Biosynthesis in Animals

Sphingosine-1-phosphate is a zwitterionic lysophospholipid and an important cellular metabolite derived from ceramide that is synthesised de novo or as part of the sphingomyelin cycle or sphingolipid rheostat (see below) in animal cells. Analogues have been found in insects, yeasts and plants. Although it is a minor lipid in quantitative terms, it has essential biological properties with 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, but in humans and mice, platelets contain dihydrosphingosine-1-phosphate as well as sphingosine-1-phosphate, and there is some evidence that the two lipid species may have some 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, but in plasma, it can reach concentrations 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. When not bound to proteins, sphingosine-1-phosphate behaves in aqueous media as a soluble amphiphile with a critical micellar concentration of ~12 μM. Apo M has been termed a sphingosine-1-phosphate 'chaperone' that controls the levels of the lipid in plasma, although the highest concentrations are found in red blood cells. In contrast to most other sphingolipids, sphingosine-1-phosphate per se is not believed to participate in formation of cholesterol-sphingolipid enriched regions (rafts) in membranes, although many of the proteins involved in its biosynthesis and signalling functions are located in these membrane microdomains.

The primary precursor is sphingomyelin, which is hydrolysed by sphingomyelinases to produce ceramides and these are in turn acted upon by ceramidases (5 family members) to release sphingosine. While this occurs in a constitutive manner in the endosomal/lysosomal compartment, those ceramidases that take part in the production of sphingosine-1-phosphate are present in other membranes in the vicinity of relevant effectors. Finally, sphingosine is phosphorylated by ATP-dependent sphingosine kinases, which catalyse the transfer of 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, or SK1/2), 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. SPHK1 is predominantly cytosolic, although to a lesser extent in the endoplasmic reticulum and Golgi, and it is considered to be pro-survival, probably by inhibiting ceramide biosynthesis. Upon activation by stimuli that include growth factors, hormones and cytokines, it translocates to raft microdomains in the plasma membrane where it interacts with phosphatidylserine and phosphatidic acid and with proteins of the calmodulin family. A significant fraction is released to the extracellular space. Thus, SPHK1 controls the levels of sphingosine-1-phosphate in the cytosol and plasma membrane, while its activity is regulated by several factors including transcription, phosphorylation/dephosphorylation, protein-protein interactions, membrane accessibility and degradation.

SPHK2 is found mainly in the nucleus where it has the potential to regulate gene expression, but upon activation, it can translocate to 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 (and Fingolimod - see below) as well as sphingosine. As it is not secreted, it may be more important in the regulation of gene expression and of apoptosis. SPHK2 is the main form of the enzyme in platelets and the only form in the central nervous system.

Both enzymes have important roles in controlling the sphingosine-1-phosphate gradient between cells and in circulation and so impinge upon innumerable functions of the lipid, but they can have opposing functions, depending on their cellular locations. Thus, sphingosine-1-phosphate that results from the activation of SPHK1 contributes to cell growth and proliferation, whereas activation of SPHK2 can lead to the inhibition of cell growth and induce apoptosis. Sphingosine-1-phosphate synthesised on the inner leaflet of the plasma membrane can cross this via the action of at least two transporter proteins, spinster homologue 2 (SPNS2) and Mfsd2b, from the ATP-binding cassette (ABC) family, or it can be transported in plasma to more distant tissues to interact with specific receptors on the surface of the same cell or on nearby cells. SPNS2 is important in endothelial cells, while Mfsd2b is abundant in the plasma membranes of red blood cells and platelets, and in spleen and bone marrow.

In animals, the reverse reaction to synthesis occurs by the action of sphingosine phosphatases as part of the 'sphingolipid rheostat' (see below), and the enzymes act in concert to control the cellular concentrations of the metabolite, which are always low.

Sphingosine-1-phosphate in the circulation: It now seems certain that much of the sphingosine-1-phosphate in blood is synthesised in erythrocytes, platelets, mast cells and monocytes. In platelets, sphingosine-1-phosphate is produced mainly by the action of SPHK2 on free sphingosine absorbed from plasma or derived from ceramide generated in the plasma membrane from sphingomyelin. Resting platelets store sphingosine-1-phosphate in α-granules in the inner leaflet of the plasma membrane and secrete it upon activation by thrombin, a product of the coagulation process, during injury and inflammation by degranulation and binding to its receptor. 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. Platelets can thus provide sphingosine-1-phosphate rapidly as required as in the case of an injury with consequent platelet activation, when the transporter MRP4 in the plasma membrane actively exports it from the cell before much is bound to albumin. On the other hand, platelets may also influence immune cell functions over a longer term in inflammation-driven vascular diseases.

Erythrocytes produce sphingosine-1-phosphate using SPHK1 and export it constitutively into blood in an ATP-dependent manner by means of a protein major facilitator superfamily transporter 2b or 'MFSD2B' and utilizing a proton gradient to facilitate its release. This occurs partly in free form but mainly by extraction by the protective chaperone apo M with incorporation into high-density lipoproteins (HDL) to provide more than 60% of the sphingosine-1-phosphate in plasma, with the remainder bound to albumin and VLDL. Erythrocytes can take up preformed sphingosine-1-phosphate from plasma, and they are believed to function mainly as a storage organ as they lack the relevant degradative enzymes. In effect, they constitute a buffering system to maintain appropriate levels in the circulation.

A proportion of the sphingosine-1-phosphate in plasma comes from lymphatic endothelial cells, which have an active transport mechanism from the interior of the cells with the aid of the transporter SPNS2; inhibition of the synthesis of this protein influences inflammatory and autoimmune diseases. Both proteins are present in other cell types where they can function in the importation of this lipid as well as export, although a high proportion is hydrolysed at the plasma membrane to sphingosine, which can be reutilized within the cell for synthesis of sphingosine-1-phosphate and other sphingolipids. Levels of sphingosine-1-phosphate in lymph fluid are four to five-fold lower than plasma, while those in interstitial fluid are roughly 1000-fold lower. Some sphingosine-1-phosphate may be produced within plasma by the hydrolysis of sphingosylphosphorylcholine by the enzyme autotaxin, better known for the production of lysophosphatidic acid from lysophosphatidylcholine.

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 well as with the HDL. There is a high turnover of the lipid in the circulation, and it has been determined that the liver is the primary site of clearance as well as being a major site for its synthesis. At the surface of hepatocytes, it is hydrolysed by lipid phosphate phosphatase 3 (LPP3) to sphingosine, which is taken up into the cells and converted back to sphingosine-1-phosphate by SPHK2; ultimately, it is catabolized as described below. It has been suggested that this may be a general mechanism whereby sphingosine-1-phosphate gradients are shaped.

2. Membrane Receptors and 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. In an extracellular mode, sphingosine-1-phosphate transported out of cells binds selectively to G protein-coupled receptors (GPCRs), which trigger a cascade of downstream signalling pathways. Alternatively, it can act directly on intracellular targets to mediate or coordinate cellular activities. It has very different functions from other sphingolipid metabolites such as ceramides, ceramide-1-phosphate and sphingosine, and the balance between them has sometimes been termed the 'sphingolipid rheostat', although the readiness with which they can be interconverted can make it difficult to determine the true function or relative activity of each. There are differences between cell types under various conditions and the description of the sphingolipid rheostat may be an over-simplification as a balancing mechanism, though one that is important for health.

Sphingolipid rheostat

A general hypothesis is that this mechanism evolved early in the development of life to regulate cell survival under environmental stress. 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. When produced in the nucleus by SPHK2, it delays senescence of cells, probably by binding to the telomerase reverse transcriptase (hTERT) to promote telomere maintenance. In contrast, it can enhance apoptosis in some circumstances by promoting the formation of ceramide, and 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. The concentration of sphingosine-1-phosphate in cells or the gradient in its concentration within or between tissues is regulated closely by a combination of synthesis de novo and catabolism (see below).

As with the lysophospholipids, especially lysophosphatidic acid with which it has some structural similarities and is often compared, 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 that are designated S1PR₁ to S1PR₅. Both sphingosine-1-phosphate and its dihydro analogue bind to them with a high affinity. These receptors are all differentially expressed and are coupled to one or more further G proteins (G_i, G_12/13 and G_q), resulting in multiple different downstream messaging targets. In mammals, S1PR₁ and S1PR₂ are found in all tissues, often located within raft microdomains or caveolae, with S1PR₁ (coupled to G_i) usually the most highly expressed. S1PR₃ is expressed highly in the heart, lung, kidney and spleen and is located in the plasma membrane, while S1PR₄ is restricted to blood cells and lymphoid tissues. S1PR₅ is relatively abundant in brain, skin and natural killer (NK) cells, and it is one of the main regulators of the egress of natural killer cells from both bone marrow and spleen into the blood.

Each receptor when activated, triggers distinctive signalling 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, organ morphogenesis, directed cell movement and for the regulation of corticosteroid hormone biosynthesis and function. Sphingosine-1-phosphate acts directly on intracellular targets such as histone deacetylase and prohibitin 2 to mediate multiple signalling cascades.

Many of the effects are mediated ultimately by the regulation of intracellular calcium fluxes by various mechanisms. As one example, sphingosine-1-phosphate binds to and activates its receptors that couple to calcium signalling through the activation of phospholipase C and thence the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol triphosphate and diacylglycerols, followed by activation of the inositol triphosphate receptor and release of calcium from intracellular calcium stores. Sphingosine-1-phosphate may bind to phosphatidylinositol monophosphates, targeting them to intracellular membranes.

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 if one is inhibited, but only in part. 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 thistle Most 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, which inhibits DNA synthesis with effects upon subsequent cellular events. 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, and it is believed to be involved in the regulation of the immune and inflammatory responses of cytokines. SPHK2 present in mitochondria is necessary for correct assembly of the cytochrome oxidase complex, and reductions of SPHK2 levels result in defective mitochondrial respiration and can lead eventually to metabolic diseases.

The high levels of sphingosine-1-phosphate in blood and especially that bound to apolipoprotein M are important for the maintenance of vascular integrity. Apo M protects the lipid from phosphatases and facilitates the interaction with the receptors, and it is of key importance in down-stream signalling while contributing to shaping the anti-inflammatory properties of HDL. While sphingosine-1-phosphate bound to albumin interacts with receptors S1PR₁ to S1PR₃, there appear to be some functional differences in its effects. 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. A relatively high concentration of sphingosine-1-phosphate is maintained in blood and lymph in contrast to low levels in intracellular or interstitial fluids, and this gradient in concentration is utilized to facilitate the exit of lymphocytes from lymphoid organs and direct them to sites of inflammation.

Plasma sphingosine-1-phosphate limits disruption of vascular endothelial monolayers, i.e., it protects the permeability barrier against potentially disruptive molecules such as histamine or platelet-activating factor. Damage to endothelial barriers, such as the blood-brain barrier and placenta, can result from a deficiency in sphingosine-1-phosphate, and this is a factor in many inflammatory disease states including sepsis. In contrast, ceramide and lysophosphatidic acid increase vascular permeability.

Small dense HDL₃ subfractions display potent vasorelaxing activity, presumably because of their content of sphingosine-1-phosphate. 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. Evidently, this lipid is an essential factor in embryogenesis, for example in the formation, positioning and function of the primitive heart tube. It also regulates keratinocyte differentiation and epidermal homeostasis. Together with vitamin D, it controls the migratory behaviour of circulating osteoclast precursors and thus mediates a critical point in bone homeostasis; systemically elevated blood levels may be an independent risk factor for osteoporosis-related fractures.

3. Sphingosine-1-Phosphate and Disease

The functions and malfunctions of sphingosine-1-phosphate signalling have been implicated in many pathological conditions in humans, including cardiovascular disease, cancer, neurological diseases, multiple sclerosis, metabolic disease, inflammatory bowel disease and viral infections such as influenza. High levels of circulating sphingosine-1-phosphate and ceramides have been correlated with pregnancy disorders. The effects of sphingosine-1-phosphate are often opposed to those of ceramides.

Inflammation and cardiovascular disease: As a blood-born lipid modulator, sphingosine-1-phosphate may have a critical role in platelet aggregation and thrombosis. The relatively high concentration of this lipid in plasma high-density lipoprotein (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 (though less easily measured). The plasma concentration of sphingosine-1-phosphate, especially that bound to HDL, is decreased in patients with diabetes with negative effects upon cardiomyopathy, an important risk factor for heart failure. As this lipid activates the receptor S1PR₁ when bound to apo M, 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 thistle In 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 mediators in rheumatoid arthritis, SPKH2 is reported to be anti-inflammatory and controls platelet activation to limit or prevent arterial thrombosis after vascular injury. 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. On the other hand, studies with specific enzyme inhibitors suggest that SPKH2 can have pro-inflammatory effects, and it is involved in the production of inflammatory cytokines in the skin when triggered by mechanical stress or bacterial invasion.

Cancer: Like lysophosphatidic acid, sphingosine-1-phosphate 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. It promotes the expression and release of interleukin 8 (IL-8), which is a potent angiogenic factor, and thus it has a critical role in the growth and spread of cancers by enhancing the availability of nutrients and oxygen. In contrast to ceramide, it stimulates the growth, survival (delay of senescence) and migration of tumour 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. A key therapeutic aim is to ensure that these two sphingolipids are balanced to optimize cell survival and proliferation, so that the pro-apoptotic signalling of ceramides outweighs the effects of sphingosine-1-phosphate signalling in glioma cells to enhance tumour cell death.

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 many other cancers, including those of the prostate, breast, ovaries, pancreas, lung and kidney. 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; the transporter SPNS2 is important to this process. This is currently a topic that is attracting great interest amongst medical researchers, and the potential for therapeutic intervention in sphingosine-1-phosphate metabolism by selectively inhibiting biosynthesis by one or other of the sphingosine kinases or by targeting the receptors is under active investigation (e.g., fingolimod, see below). 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.

Immune system: Sphingosine-1-phosphate has a key role in the immune system 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. Diminished circulating levels of the lipid are seen in patients with sepsis, and it is a factor in autoimmune diseases such as rheumatoid arthritis and related diseases involving joints and bone, while the receptors for sphingosine-1-phosphate and relevant enzymes are viewed as promising therapeutic targets for other autoimmune diseases such as psoriasis, polymyositis and lupus. Sphingosine-1-phosphate induces differentiation of keratinocytes in skin, and inhibition of the catabolic enzyme S1P-lyase (see below) is a therapeutic target for the treatment of psoriasis vulgaris.

Sphingosine-1-phosphate and enzymes involved in its metabolism have important protective functions in some virus infections by regulating virus replication and/or controlling the innate defence of the host. Sphingosine-1-phosphate lyase inhibits replication of the influenza virus by promoting antiviral interferon activity, and it has been demonstrated that both SPHK1 and SPHK2 can regulate the replication or pathogenicity of many other viruses, including respiratory viruses, enteroviruses, hepatitis viruses and herpes viruses. In contrast, some viruses appear to divert sphingosine-1-phosphate metabolism to their own benefit.

Neurological diseases: There is much more sphingosine-1-phosphate in the brain than in any other organ, and this lipid is crucial to the regulation of diverse processes in the brain, including neural development, differentiation, migration and survival, while it functions in synaptic transmission by modulating the release of neurotransmitters. On the other hand, its role in neurodegenerative processes is debated with some reports of a neuroprotective function and others of neurotoxic effects, and while it may be protective 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, although it does seem to have been established that sphingosine-1-phosphate and its receptor S1PR₃ 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 neurons.

Receptor-mediated signalling by both sphingosine-1-phosphate and lysophosphatidic acid is certainly critical for the pathogenicity of diverse neurological diseases, including such as neuropathic pain, systemic sclerosis, spinal cord injury, multiple sclerosis, traumatic brain injury and neuropsychiatric disorders, although it is not certain that changes in concentration of these lipids in some of these disorders are a cause or an effect of neuronal malfunction. In contrast, sphingosine-1-phosphate is reported to act as an inhibitor of neuronal apoptosis and to be protective against cellular damage induced by oxidative stress in the brain. It may inhibit the aggregation of aberrant amyloid-β peptide and the formation of neurofibrillary tangles with the resultant neuro-inflammation, which results in neuronal death in Alzheimer’s disease.

Structure of Fingolimod Fingolimod: Drugs that antagonize sphingosine-1-phosphate and its receptors are being tested clinically as immuno-suppressants to prevent rejection of kidney grafts, to reduce inflammatory and allergic responses, especially in neurological diseases, and as anti-cancer agents. 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 by inducing a marked decrease in circulating lymphocytes where the S1PR₁ receptor is expressed on the cell surface.

Fingolimod has multiple anti-cancer activities by preventing the formation of sphingosine-1-phosphate and inhibiting its activity. In mouse breast cancer models, FTY720 treatment reduces tumour growth and metastasis while improving the response to conventional therapies and reducing neuropathic pain, and it is under consideration as a simultaneous adjuvant treatment of triple negative breast cancer. In consequence, 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.

Pre-clinical studies suggest that fingolimod has neuroprotective effects against Alzheimer's, Parkinson's and Huntington's diseases. While sphingosine-1-phosphate prevents photoreceptor and ganglion cell degeneration, it can promote inflammation in age-related macular degeneration, glaucoma and pro-fibrotic disorders, and fingolimod preserves neuronal viability and retinal function. Second generation analogues of fingolimod (siponimod, ozanimod and ponesimod) with greater specificity for particular receptors, especially the S1PR₁ receptor, are undergoing advanced clinical testing both as primary therapies and as neuroprotective adjuvants to existing treatments. Siponimod has been approved for the treatment of secondary progressive multiple sclerosis.

Dihydrosphingosine(sphinganine)-1-phosphate may have functions in various disease states that differ from the sphingosine analogue, and they include cardiovascular disease, cancer, and lung, liver and kidney diseases.

4. Sphingoid Base-1-Phosphates in Plants, Fungi and Bacteria

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 with three such kinases identified in Arabidopsis. 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. Phytosphingosine-1-phosphate is a mediator of abscisic acid-mediated stomatal closure in Arabidopsis, but sphingosine-1-phosphate and sphingadienine-1-phosphate have this function in Commelina communis and rice, respectively. 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. As in animals, they inhibit the process of apoptosis.

Sphingosine-1-phosphate and dihydrosphingosine-1-phosphate have been detected in pathogenic fungi (Cryptococcus species). In yeasts, phytosphingosine-1-phosphate has a role in the regulation of genes required for mitochondrial respiration.

The oral anaerobic bacterium Porphyromonas gingivalis, which synthesises a variety of sphingolipids, utilises DhSphK1, a protein that shows high similarity to a eukaryotic sphingosine kinase, to synthesise dihydrosphingosine-1-phosphate, which may have signalling functions in its human host.

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

The balance between catabolic activities and those of the sphingosine kinases and of sphingolipid resynthesis is tightly regulated. 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 of three lysophospholipid hydrolases. 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 acylation by ceramide synthase to regenerate ceramide. Extracellular sphingosine-1-phosphate is degraded at the plasma membrane 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 synthesis of complex sphingolipids or resynthesis of intracellular sphingosine-1-phosphate.

Degradation of sphingosine-1-phosphate

In animals and plants, production of sphingosine-1-phosphate is a key step in the catabolism of long-chain bases. Within cells, sphingosine-1-phosphate 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 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, but the analogous 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. As the reaction is irreversible, it is ultimately the mechanism for removal of all sphingolipids from cells.

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. Because it is a key enzyme in regulating the intracellular and circulating levels of sphingosine-1-phosphate, it is seen as a potential target for pharmacological intervention.

trans-2-Hexadecenal produced in the reaction can enter the β-oxidation pathway, or it can be reduced to the long-chain alcohol or converted via four reactions into palmitoyl-coA for incorporation into glycerolipids. In the last of these, 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. As an electrophilic α,β-unsaturated aldehyde, trans-2-hexadecenal has the potential to interact with proteins via the Michael reaction, and it is known to have important signalling functions in that it induces cytoskeletal reorganization and apoptosis. Failure to oxidize this to palmitic acid is believed to be the cause of the rare genetic condition Sjögren-Larsson syndrome. The ethanolamine phosphate that is the other product of the reaction can be utilized for biosynthesis of phosphatidylethanolamine, and this is essential in the protozoan parasite Trypanosoma brucei, where the enzyme with sphingosine-1-phosphate lyase activity is in mitochondria not the endoplasmic reticulum. 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 phytosphingosine-1-phosphate lyase (DPL1) 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 production of odd-chain fatty acids in yeasts. There is also a plant phosphatase, phyto-S1P phosphatase (SPPASE).

6. Analysis

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

	© Author: William W. Christie
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