Sphingosine-1-phosphate (S1P) is a naturally occurring bioactive sphingolipid metabolite. Sphingolipids are essential structural constituents of the plasma membrane of all eukaryotic organisms and are also produced by some bacteria. In response to a wide range of stressful stimuli, membrane sphingomyelin and to lesser extent other complex sphingolipids are rapidly metabolized to the bioactive sphingolipid intermediate, ceramide and subsequently to sphingosine (Hannun and Obeid 2008). Phosphorylation of sphingosine by two sphingosine kinases (SphKs) results in the formation of S1P. It has emerged as a potent signaling molecule that regulates diverse cellular processes including cell proliferation, survival, differentiation, and migration (Spiegel and Milstien 2003). S1P plays a critical role in several physiological processes including lymphocyte trafficking, vascular development, and inflammation. S1P exerts its biological actions both in cell-extrinsic and cell-intrinsic manner. S1P acts as ligand for five G protein-coupled receptors (GPCRs) known as S1P receptors (S1PRs) 1–5 (S1P1–5). S1P also acts as an intracellular second messenger. In 2002, it was discovered that the phosphorylated form of FTY720, an immunosuppressive compound, is an analog of S1P and binds to four out of five S1PRs (Mandala et al. 2002). Subsequent to this discovery, intense research by many groups has provided a comprehensive understanding of the role of S1P signaling in lymphocyte development, immune cell migration, and inflammation (Spiegel and Milstien 2011). In addition to its role in immune cell trafficking and signaling, S1P functions as an oncogenic lipid that promotes tumor growth and progression. S1P plays a critical role in the regulation of many essential biological and developmental processes (Proia and Hla 2015). Not surprisingly, both the deficiency of S1P and its excessive accumulation are involved in pathological conditions including cancer, vascular abnormalities, inflammation, and autoimmune diseases and are likely to be linked to human congenital defects. Conversely, targeting the S1P signaling pathways and S1P metabolism appears promising as a therapeutic strategy for a number of human diseases.
Structure, Biosynthesis, and Metabolism
S1P is synthesized by phosphorylation of its precursor sphingolipid, sphingosine. All sphingolipids contain a sphingoid or long-chain base (LCB) backbone. The term sphingoid base refers to a group of related compounds that contain two hydroxyl groups at the C1 and C3 positions and an amino group at the C2 position (Hannun and Obeid 2008). The most prevalent of sphingoid bases are sphinganine [(2S,3R)-2-aminooctadece-1,3-diol] and sphingenine [(2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol]. In mammalian cells, the most common sphingoid base is (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol, which is also called D-erythro-sphingosine or just sphingosine. Other sphingoid bases vary in alkyl chain length and branching, the number and position of double bonds, the presence of additional hydroxyl groups, and other features (Hannun and Obeid 2008). In fungi and plants, D-ribo-phytosphingosine (phytosphingosine) is predominant, and it carries an additional hydroxyl group at C4. D-erythro-dihydrosphingosine (dihydrosphingosine or sphinganine) is also detectable in most organisms. Sphingoid bases vary in chain length among species, with mammals carrying primarily C18; yeast, C16, C18, and C20; fly, C14 and C16; and nematode, C17. Some species, such as nematodes, contain unusual branch chain sphingoid bases. Unless otherwise stated, S1P biosynthesis, metabolism, and functions have been referred to the mammalian S1P.
Biosynthesis and Metabolism
S1P levels in the tissues and plasma are tightly regulated. SphK1 is the primary enzyme responsible for S1P generation and is mainly localized in the cytosol. Activation of SphK1 is regulated by many factors such as its intracellular localization and epigenetic or posttranslational modification. SphK1 can be activated by wide variety of growth factors including platelet-derived growth factor, vascular endothelial growth factor, epidermal growth factor, hepatocyte growth factor, and tumor necrosis factor-α (TNF-α) (Gao et al. 2015). Upon stimulation by growth factors, cytokines, and lipopolysaccharide, SphK1 translocates to the plasma membrane. SphK1-generated S1P has been implicated in many pathophysiological conditions including vascular permeability, angiogenesis, inflammation, invasion, metastasis, and chemoresistance. Compared to SphK1, SphK2 initially received less attention, however, recent studies have revealed the latter’s critical role in cardiac development, endothelial cell barrier integrity, platelet aggregation, and liver regeneration (Adams et al. 2016).
Occurrence, Source, and Transport
Although S1P is generated by most cell types in mammals, its level in most tissues is low. This is in contrast to plasma where S1P is found in relatively high concentration. In humans, plasma S1P concentrations vary between 100 and 400 nM (Ksiazek et al. 2015), whereas plasma S1P concentrations are higher in mice (0.47–1.35 μM). Erythrocytes and platelets are major contributor of blood S1P, as both of these cell types are exposed to circulating sphingosine, contain SphKs activity, and lack most of the S1P-degrading enzymes. In lymph, S1P is produced by radiation-resistant cells of non-hematopoietic origin, presumably lymphatic endothelial cells. In blood, most of plasma S1P is transported bound to high-density lipoproteins (HDL) (50–60%). The remaining part binds to albumin (30–40%), low-density lipoproteins (∼8%), and very-low-density lipoproteins (2–3%). Apolipoprotein M (ApoM), a lipocalin, is the carrier of S1P in HDL. ApoM acts as an S1P chaperone which may also protect S1P from degradation and facilitate presentation to receptors. Albumin-bound S1P and free S1P are susceptible to degradation, whereas HDL-bound S1P is more stable (Ksiazek et al. 2015). Many of beneficial effects of HDL have been ascribed to ApoM-S1P component. Circulating S1P is cleared rapidly; the half-life of albumin-bound S1P in blood is ∼17 min.
Mechanism of Action
To date, five S1P receptors EDG1/S1P1, EDG5/S1P2, EDG3/S1P3, EDG6/S1P4, and EDG8/S1P5 that bind to S1P and dihydro-S1P have been identified in vertebrates (Hanson and Peach 2014). In mammalian cells, S1PRs are ubiquitous, but their expression patterns vary among the different tissues. Each S1PR couples to different heterotrimeric G proteins. For example, S1P1 and S1P4 couple mainly to Gi; S1P2 and S1P3 activate Gi, Gq, and G12/13; and S1P5 binds to Gi and G12/13. After coupling to G proteins, these receptors either activate or inhibit downstream signaling pathways including extracellular signal-regulated kinase, c-Jun N-terminal kinase, phosphatidylinositol 3-kinase, phospholipase C, Rac, Rho, cyclic AMP, and phospholipase D (Spiegel and Milstien 2003). The functional response of each cell to S1P varies depending on S1PR expression and intracellular signaling configuration. By activating these receptors, S1P regulates diverse biological processes including vascular development, angiogenesis, and immunity. The crystal structure of S1P1 suggests a potential mechanism for ligand access to the GPCR that involves insertion of S1P into the outer leaflet of the plasma membrane followed by lateral diffusion to enter the binding pocket (Hanson and Peach 2014). Until recently, S1P receptors were not studied in invertebrates. However, recently two S1P receptors, S1P1 and S1P4, have been identified in Botryllus schlosseri, a basal chordate (Kassmer et al. 2015).
S1P regulates many biological processes including cell survival, DNA damage repair, calcium mobilization, and stress response by acting as an intracellular second messenger. Many studies conducted in lower organisms such as yeast, flies, slime molds, nematodes, and plants that lack identifiable S1PRs revealed that S1P regulates important developmental processes through direct intracellular signaling. In fact, S1P has a universal signaling role in calcium mobilization in yeast, plants, and mammals (Spiegel and Milstien 2011). However, the question of how S1P mediates its intracellular signaling effects remained unanswered, and specific intracellular targets of S1P remained elusive. Few direct intracellular S1P targets have been identified. For example, S1P directly binds to histone deacetylase (HDAC) 1 and 2, TNF receptor-associated factor 2 (TRAF2), p21-activated protein kinase 1, and prohibitin 2 (Spiegel and Milstien 2011). Whether these or other intracellular interactions are responsible for the function of S1P in simple metazoans remains to be determined.
S1P Signaling in Plants and Microbes
LCBs are actively phosphorylated by nuclear protein kinases of tobacco BY-2 cells, suggesting the presence of SphK-like activity in plants. The Δ4-unsaturated LCB D-erythro-sphingosine and the saturated LCB D-ribo-phytosphingosine increase free calcium levels in the nucleus of BY-2 cells. A homologue of SphK1, which phosphorylates sphingosine, phytosphingosine, and LCBs, has been identified in Arabidopsis. Deficiency of SphK1 in Arabidopsis decreases the sensitivity of stomata to abscisic acid but enhanced the rate of seed germination (Lynch et al. 2009). Further, S1P-degrading enzyme, LCB-1-phosphate lyase, has been shown to play a role in the dehydration stress response in Arabidopsis. Phytosphingosine and S1P induce stomatal closure in epidermis of pea (Pisum sativum) by raising the levels of nitric oxide and pH in guard cells.
Legionella pneumophila is an intracellular pathogen that can cause a severe pneumonia in humans. In L. pneumophila, S1P lyase (LpSpl) has been identified as an effector protein which is translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. LpSpl targets the host sphingosine biosynthesis to inhibit macrophage autophagy. Similarly, Bukholderia pseudomallei produces an S1P lyase that enables its pathogenicity by removing host S1P and thereby preventing the inflammatory response of macrophages. Structure of a prokaryotic homologue of S1P lyase (StSpl) from Symbiobacterium thermophilum has also been elucidated, which paves the way for determining the structure of SPL.
S1P in Development
Expression of SphK is essential for Leishmania parasites to remove toxic metabolites, to survive stressful conditions, and to cause disease in mice. Trypanosoma brucei, SphK (TbSPHK), is constitutively but differentially expressed during the life cycle of T. brucei. TbSPHK is involved in G1-to-S cell cycle progression, organelle positioning, and maintenance of cell morphology of T. brucei. The colonial ascidian Botryllus schlosseri continuously regenerates entire bodies in an asexual budding process. Germ cell precursors from Botryllus schlosseri express S1P1, and S1P signaling is essential for homing of germ cells to newly developing bodies (Kassmer et al. 2015). The maternal-zygotic sphk2 mutant (MZsphk2) of zebrafish exhibits a defect in the cardiac progenitor migration and a concomitant decrease in S1P level, leading to a two-heart phenotype (cardia bifida). This phenotype is identical to that of zygotic mutants of the S1P transporter spns2 and S1PR s1pr2, indicating that the Sphk2-Spns2-S1pr2 axis regulates the cardiac progenitor migration in zebrafish (Proia and Hla 2015).
S1P signaling in fruit fly is required for development of reproductive organs and flight muscles. Drosophila Sply mutants that lack functional S1P lyase and thereby accumulate LCBs and LCB phosphates exhibit multiple developmental abnormalities, including supernumerary spermathecae, degenerative ovaries, and severely reduced testes. Moreover, sply mutants are flightless, have reduced viability and have defects in nephrocytes. SphKs and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of rhodopsin and transient receptor potential channel. Further, genetic elevation of S1P suppresses dystrophic muscle phenotypes in Drosophila (Donati et al. 2013). In Caenorhabditis elegans, a nematode, inhibition of S1P lyase by RNA interference leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage.
In mammalian system, S1P1 expression in endothelial cells and cardiomyocytes is essential for the development of cardiovascular system (Borodzicz et al. 2015). Similarly, both S1P synthesis and S1P1 expression are required for embryonic neurogenesis. S1P produced by SphK2 in the megakaryocytes promotes thrombopoiesis. Furthermore, S1P1 promotes the growth of pro-platelet strings in the bloodstream and the shedding of platelets into the circulation (Thuy et al. 2014). Deaf mouse mutants have revealed an important role of S1P signaling in normal auditory function. Mutation of the S1P transporter Spns2 leads to rapidly progressive loss of auditory sensitivity, and three independent null mutations in the S1pr2 have been identified that cause severe elevations in auditory thresholds before 4 weeks old of age in mice. Few genetic mutations, for example c.323G>C (p.Arg108Pro) and c.419A>G (p.Tyr140Cys) in S1pr2 gene in humans have been linked with progressive hearing loss.
S1P in Health and Disease
S1P in Immunity
In a landmark study by Mandala and colleagues, it was shown that FTY720, an immunosuppressive agent, is an analog of sphingosine and after phosphorylation in vivo it (phosphorylated compound of FTY720) binds to four of five S1PRs and induces lymphopenia (Mandala et al. 2002). A plethora of immunological investigations have now revealed the major impact of S1P signaling on the mammalian immune system. S1P signaling through its receptors was found to regulate the trafficking and migration of many bone-marrow-derived immune cells including T and B lymphocytes, natural killer T cells, dendritic cells, macrophages, neutrophils, mast cells, and hematopoietic progenitors cells (Spiegel and Milstien 2011; Proia and Hla 2015). Pharmacological and genetic approaches combined with sophisticated in vivo imaging of immune cells have provided novel insights regarding the role of S1P1 signaling in mature lymphocyte egress from the thymus and secondary lymph nodes into the circulation. Due to high levels of plasma and lymph S1P and low S1P levels in lymphoid tissues, an S1P gradient exists between plasma and lymphoid organs. Different cell types may be responsible for generating the gradient, such as in thymus where dendritic cells produce the gradient at the site of T cell egress. Mature thymocytes expressing high levels of S1P1 sense the gradient and migrate toward the blood stream. Inhibition or genetic deficiency of S1P-catabolizing enzymes, such as S1P lyase or LPP3, raises thymic S1P levels, disrupting the S1P gradient and resulting in blockade of exit of mature thymocytes, thereby producing lymphopenia (Schwab et al. 2005; Baeyens et al. 2015).
S1P3 promotes leukocyte rolling by mobilizing endothelial P-selectin, whereas S1P4 is required for plasmacytoid dendritic cell differentiation. In addition to its role in leukocyte migration, S1P signaling contributes to T lymphocyte differentiation, allergy, inflammation, endothelial barrier integrity in anaphylactic shock, and other immune functions. S1P mediates chemotaxis of macrophages in vitro and in vivo via S1P3 and causes atherosclerosis by promoting inflammatory macrophage recruitment and altering smooth muscle cell behavior. S1P is also involved in mast cell and eosinophil and dendritic cell recruitment in asthma (Spiegel and Milstien 2011; Proia and Hla 2015). A human recessive genetice disorder charcterized by nephrotic syndrome, adrenal insufficiency, immunodeficiency, neurological defects and acanthosis has recently been described which is caused by mutation of the human S1P lyase gene.
S1P in Cell Survival and Cell Death
The metabolic balance between ceramide, sphingosine, and S1P plays an important role in determining cell fate. In general, ceramide and sphingosine accumulation results in growth arrest and/or induce cell death, whereas S1P has a mitogenic function and inhibits apoptosis. S1P inhibits ceramide-induced apoptosis in many cell types (Pyne and Pyne 2010; Carroll et al. 2015). Moreover, overexpression of SphK1 inhibits apoptosis and induces chemoresistance, whereas overexpression of S1P lyase which depletes intracellular S1P promotes apoptosis in response to DNA-damaging agents and ionizing radiation (Santos and Lynch 2015; Adams et al. 2016). S1P increases the expression of antiapoptotic proteins Bcl-2 and Mcl1 and downregulates proapoptotic Bcl-2 family proteins Bad and Bax. In vivo, S1P protects the small intestine from radiation-induced endothelial apoptosis, and it also prevents radiation-induced cell death in germ cells (Pyne and Pyne 2010). However, the rheostat model has recently been challenged due to some interesting findings. For example, under certain conditions, S1P can induce autophagy, similar to ceramide. However, the cellular outcome of S1P-induced autophagy is different than the ceramide-induced autophagy, because S1P-induced autophagy promotes cell survival, whereas ceramide-induced autophagy leads to cell death (Pyne and Pyne 2010). There is also evidence that S1P pools generated by SphK1 versus SphK2 have opposite effects on cellular growth and apoptosis. SphK2 contains a 9-amino acid motif similar to that present in BH3-only proapoptotic proteins. Notably, S1P generated by SphK2 in the mitochondria directly binds with the proapoptotic protein BAK and induces the latter’s oligomerization. In contrast, endogenous SphK2 has been implicated in chemoresistance, cell proliferation, and cell migration (Pyne and Pyne 2010; Adams et al. 2016).
S1P in Cancer
S1P signaling has a significant impact on the processes of carcinogenesis, cancer progression, and drug and radiation resistance patterns. Many components of S1P signaling pathways including SphKs, S1P lyase, and S1PRs have been mechanistically linked with cancer cell aggressiveness, invasion, metastasis, and chemoresistance. SphK1 is overexpressed in various types of human cancers including prostate, gastric, breast, lung, and colon cancer, glioma, and non-Hodgkin’s lymphoma (Pyne and Pyne 2010; Degagne et al. 2014). Further, overexpression of SphK1 in mouse fibroblast induces transformation through a Ras-dependent pathway, demonstrating that SphK 1 is an oncogene. Elevation of SphK1 expression in cancers is often associated with clinical grade of tumors and chemoresistance. Inhibition of SphK1 enzyme activity or genetic knockdown blocks tumor progression in many types of cancers and induces apoptosis and autophagy (Pyne and Pyne 2010; Adams et al. 2016). On the contrary, S1P lyase expression is downregulated in melanomas and colon and ovarian cancer and may act as a tumor suppressor gene (Degagne et al. 2014). S1P promotes tumor invasion, neovascularization, and metastasis by activating its cognate receptors. However, no consistent pattern of S1P receptor gene expression has been associated with clinical tumor grade. Notably, mice lacking S1pr2 develop diffuse large B-cell lymphoma, and several mutations have been reported in S1pr2 in diffuse large B-cell lymphoma patients.
Therapeutic Targeting of S1P Signaling Pathways
Due to the central involvement of S1P signaling in immunity and tumorigenesis, substantial activity in the pharmaceutical industry has developed around the goals of targeting S1P (the monoclonal antibodies developed by LPath), S1PRs (S1PR-specific agonist and antagonist compounds generated by many research groups), SphKs (inhibitors), and S1P lyase (inhibitors) for therapeutic purposes (Santos and Lynch 2015; Proia and Hla 2015; Gao et al. 2015). Notably, FTY720 (fingolimod, trade name Gilenya, Novartis), a synthetic analog of sphingosine, has been approved by US Federal Drug Administration in relapsing-multiple sclerosis patients. It has reduced the rate of relapses in relapsing-remitting multiple sclerosis by approximately one-half over a two-year period. S1P-specific monoclonal antibody, sphingomab (ASONEP; LPath, Inc.), reduces tumor progression in various mouse models of cancer. Sonepcizumab, a humanized version of sphingomab, was tested in phase II clinical trial for late-stage (unresectable and refractory) disease of renal carcinoma; however, the clinical trial has been terminated. Several S1P receptor-specific modulators (CS077, BAF312, ponesimod, and ceralifimod) have completed phase I clinical trial, and these drugs may be useful for autoimmune diseases. Ozanimod, a selective S1P receptor modulator, has been entered in phase III clinical trial for the management of multiple sclerosis. LX2931 (Lexicon Inc.), an oral S1P lyase inhibitor, has completed phase II clinical trial in rheumatoid arthritis patients. Several SphK inhibitors are being tested in mouse models for cancer, inflammation, and sepsis. ABC294640 (trade name, Yeliva; RedHill Biopharma), a selective Sphk2 inhibitor, has successfully completed safe study, and phase II study has been planned in multiple myeloma and hepatocellular carcinoma patients. As more potent and selective agents become available, their evaluation in preclinical models and clinical trials will reveal the true potential of targeting S1P signaling for therapeutic purposes.
S1P signaling regulates myriad biological functions, and each year new targets are being identified. In fact, over the last two decades, S1P has become one of the most highly investigated signaling lipids. Although the S1P signaling field has advanced tremendously, some important questions remained unanswered. For example, the exact role of S1P in regulating diverse physiological functions in lower eukaryotes in the absence of identifiable S1P receptors is still an enigma. Direct intracellular binding of S1P to TRAF2 and HDAC has been shown recently. However, it is highly likely that additional intracellular targets remain to be discovered. FTY720 has been approved for clinical use in relapsing-multiple sclerosis patients, providing the first major impact of the S1P pathway on medical care. Whether FTY720 will be efficacious and safe in other conditions such as chronic inflammatory demyelinating neuropathy, which is the peripheral counterpart to multiple sclerosis, in other autoimmune diseases, or in other conditions such as regenerative diseases, radioprotection, and cancer are still outstanding questions. While it seems that FTY720’s main function is by receptor downregulation, there is evidence that agonism of S1PRs contributes to some of its beneficial effects. It is hoped that other S1PR agonists/antagonists will be useful in various clinical contexts. As biological and physiological research continues to reveal the functions of S1P signaling, the clinical contexts in which S1P signaling can be targeted for patient benefit will also likely expand.
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