Sphingosine 1-phosphate Binding Research Articles

SPHK1/S1PR1/PPAR-α axis restores TJs between uroepithelium providing new ideas for IC/BPS treatment.

Interstitial cystitis/bladder pain syndrome (IC/BPS) represents a chronic, aseptic inflammatory bladder condition with an unclear etiology and few therapeutic options. A composite barrier structure composed of the uroepithelium and glycosaminoglycan layer forms on the bladder's inner surface to block urine and other harmful substances. Dysfunction of this barrier may initiate the pathogenesis of IC/BPS. Sphingosine-1-phosphate (S1P) plays a crucial role in forming tight junctions. Perfusion of S1P into the bladder restored uroepithelial tight junctions in mice with cyclophosphamide-induced acute cystitis and ameliorated symptoms of the lower urinary tract. Mice lacking sphingosine kinase 1 (SHPK1) exhibited more severe bladder injuries and dysfunction. Concurrent in vitro experiments elucidated S1P's protective effects and its role as a primary messenger through SPHK1 and S1P receptor 1 (S1PR1) knockdown. This study identifies a novel mechanism whereby S1P binding to S1PR1 activates the PPAR-α pathway, thereby enhancing cholesterol transport and restoring tight junctions between uroepithelial cells. These findings elucidate the regulatory role of S1P in the bladder epithelial barrier and highlight a promising therapeutic target for IC/BPS.

Activating transcription factor-3 orchestrates the modulation of vascular anti-contractile activity and relaxation by governing the secretion of HDL-bound sphingosine-1-phosphate in perivascular adipose tissue.

Perivascular adipose tissues (PVATs) play a critical role in modulating vascular homeostasis and protecting against cardiovascular dysfunction-mediated blood pressure dysregulation. We demonstrated that the activating transcription factor-3 (Atf3) gene in the PVAT is crucial for improving vascular wall tension abnormalities; however, its protective mechanism remains unclear. Herein, we aim to determine whether ATF3 regulates PVAT-derived relaxing factor (PVDRF) biosynthesis and if its secretion contributes to vasorelaxation. This study employed an in vivo animal model using global Atf3-deficient mice, in vitro blood vessel myography, and biochemical analyses to evaluate ATF3-mediated PVDRF release and reactivity in the vasculature. Wild-type (WT) mouse thoracic aortic PVAT extracts significantly induced resting tone dilation and attenuated vasoconstrictor-induced contractile responses compared to Atf3-/- mice. Heat-stable PVAT extracts from WT mice caused sustained and reproducible vasodilation without tachyphylaxis in control aortic rings. Biochemical evaluation of PVDRF release revealed that Atf3-/- mice had lower sphingosine-1-phosphate (S1P) and HDL cholesterol (HDL-C) levels than WT mice. Furthermore, PVAT extracts from WT mice induced long-lasting vasorelaxation, which was significantly inhibited by the S1P3 receptor antagonist TY52156 and scavenger receptor class B type 1 receptor antagonist glyburide. ATF3 within the PVAT can modulate vascular function by strengthening sphingosine kinase 1 (sphk1)-S1P-S1P3 receptor lipid signalling and stimulating S1P binding to HDL to form the vasodilator HDL-S1P. ATF3 is an essential modulator for maintaining the physiological function of PVAT, providing a novel target for treatment of obesity-related cardiovascular diseases.

The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.

S1P/S1PR2 promote pancreatic stellate cell activation and pancreatic fibrosis in chronic pancreatitis by regulating autophagy and the NLRP3 inflammasome

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule that governs various functions by embedding its receptor, S1PR, in different cells. Chronic pancreatitis (CP) is characterized by pancreatic fibrosis via activation of pancreatic stellate cells (PSCs). However, the effect of S1P on CP and PSC activation is still unknown. Here, we conducted a series of experiments to explore the effect of S1P on a CP rat model and primary cultured PSCs. In vivo, CP was induced by intravenous injection of dibutyltin dichloride. S1P was administered at a dosage of 200 μg/kg body weight per day by intraperitoneal injection. After 4 weeks, serum, plasma and pancreas samples were collected for molecular analysis and histological detection. In vitro, PSCs were isolated and cultured for treatment with different doses of S1P. 3MA and MCC950 were used to determine the effect of S1P on PSC activation by regulating autophagy and the NLRP3 inflammasome. JTE013 and Si–S1PR2 were applied to verify that the functions of S1P were realized by combining with S1PR2. Cells were collected for RT‒PCR, western blotting and immunofluorescence. The results showed that S1P was increased in the plasma and pancreatic tissue of CP rats. When S1P was administered to CP rats, the function and histomorphology of the pancreas were severely impaired. In addition, S1P promoted PSC activation, heightened autophagy and enhanced the NLRP3 inflammasome in vivo and in vitro. Moreover, S1PR2 mediated the effect of S1P on PSC activation by regulating autophagy and the NLRP3 inflammasome sequentially. In conclusion, S1P binding to S1PR2 promoted PSC activation and pancreatic fibrosis in CP by regulating autophagy and the NLRP3 inflammasome. These findings provide a theoretical basis for targeting S1P/S1PR2 to treat pancreatic fibrosis and further suggest that considering the role of autophagy and the NLRP3 inflammasome may help with the treatment pancreatic fibrosis.

The relationship between sphingosine-1-phosphate receptor 2 and epidermal growth factor in migration and invasion of oral squamous cell carcinoma

Sphingosine-1-phosphate (S1P) is a lipid mediator and its binding to the S1P receptor 2 (S1PR2) is reported to regulate cytoskeletal organization. Epidermal growth factor (EGF) has been shown to induce migration and invasion in tumour cells. Since binding of S1P to S1PR2 and EGF to the EGF receptors exhibit some overlapping functionality, this study aimed to determine whether S1PR2 was involved in EGF-induced migration and invasion of oral squamous cell carcinoma (OSCC) lines and to identify any potential crosstalk between the two pathways. Migration was investigated using the scratch wound assay while invasion was studied using the transwell invasion and multicellular tumour spheroid (MCTS) assays. Activity of Rac1, a RhoGTPase, was measured using G-LISA (small GTPase activation assays) while S1P production was indirectly measured via the expression of sphingosine kinase (Sphk). S1PR2 inhibition with 10 µM JTE013 reduced EGF-induced migration, invasion and Rac1 activity, however, stimulation of S1PR2 with 10 µM CYM5478 did not enhance the effect of EGF on migration, invasion or Rac1 activity. The data demonstrated a crosstalk between EGF/EGFR and S1P/S1PR2 pathways at the metabolic level. S1PR2 was not involved in EGF production, but EGF promoted S1P production through the upregulation of Sphk1. In conclusion, OSCC lines could not migrate and invade without S1PR2 regulation, even with EGF stimulation. EGF also activated S1PR2 by stimulating S1P production via Sphk1. The potential for S1PR2 to control cellular motility may lead to promising treatments for OSCC patients and potentially prevent or reduce metastasis.

Receptor-dependent effects of sphingosine-1-phosphate (S1P) in COVID-19: the black side of the moon.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection leads to hyper-inflammation and amplified immune response in severe cases that may progress to cytokine storm and multi-organ injuries like acute respiratory distress syndrome and acute lung injury. In addition to pro-inflammatory cytokines, different mediators are involved in SARS-CoV-2 pathogenesis and infection, such as sphingosine-1-phosphate (S1P). S1P is a bioactive lipid found at a high level in plasma, and it is synthesized from sphingomyelin by the action of sphingosine kinase. It is involved in inflammation, immunity, angiogenesis, vascular permeability, and lymphocyte trafficking through G-protein coupled S1P receptors. Reduction of the circulating S1P level correlates with COVID-19 severity. S1P binding to sphingosine-1-phosphate receptor 1 (S1PR1) elicits endothelial protection and anti-inflammatory effects during SARS-CoV-2 infection, by limiting excessive INF-α response and hindering mitogen-activated protein kinase and nuclear factor kappa B action. However, binding to S1PR2 opposes the effect of S1PR1 with vascular inflammation, endothelial permeability, and dysfunction as the concomitant outcome. This binding also promotes nod-like receptor pyrin 3 (NLRP3) inflammasome activation, causing liver inflammation and fibrogenesis. Thus, higher expression of macrophage S1PR2 contributes to the activation of the NLRP3 inflammasome and the release of pro-inflammatory cytokines. In conclusion, S1PR1 agonists and S1PR2 antagonists might effectively manage COVID-19 and its severe effects. Further studies are recommended to elucidate the potential conflict in the effects of S1P in COVID-19.

Sphingosine 1-Phosphate Modulation in Inflammatory Bowel Diseases: Keeping Lymphocytes Out of the Intestine.

Inflammatory bowel diseases (IBDs) are chronic and disabling conditions that, uncontrolled, lead to irreversible bowel damage and associated comorbidities. Despite the new era of biological therapies, IBDs remain not curative. The treatment purpose is to induce endoscopic remission, reduce the progression of the disease and improve the quality of life. Optimal and early treatment could enable the prevention of their complications. Small molecules, administrated as oral agents, have the capacity of overcoming the limitations of biologic agents (i.e., parenteral administration, rapidity of action and primary and secondary non-responsiveness). Of special interest are results from the use of oral sphingosine 1-phosphate (S1P) receptor modulators (ozanimod, etrasimod, fingolimod and laquinimod), based on S1P activities to target lymphocyte recirculation in the mucosa, acting as immunosuppressive agents. Most S1P modulators are reported to be safe and effective in the treatment of both UC and CD. High and satisfactory rates of clinical remission as well as endoscopic improvement and remission can be achieved with these molecules. Safety alarms remain rather low, although the S1P binding to two of its G protein-coupled receptors, 2 and 3 (S1PR2 and S1PR3), may be associated with cardiovascular risks. Cost-effectiveness studies and head-to-head trials are needed to better define their place in therapy. This review summarizes these emerging data published by PubMed and EMBASE databases and from ongoing clinical trials on the safety and efficacy of selectivity of S1P modulators in the treatment of IBD.

Structural insights into sphingosine-1-phosphate receptor activation

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step “shallow to deep” transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

ST-2191, an Anellated Bismorpholino Derivative of Oxy-Fingolimod, Shows Selective S1P1 Agonist and Functional Antagonist Potency In Vitro and In Vivo.

Sphingosine 1-phosphate (S1P) is an extensively studied signaling molecule that contributes to cell proliferation, survival, migration and other functions through binding to specific S1P receptors. The cycle of S1P1 internalization upon S1P binding and recycling to the cell surface when local S1P concentrations are low drives T cell trafficking. S1P1 modulators, such as fingolimod, disrupt this recycling by inducing persistent S1P1 internalization and receptor degradation, which results in blocked egress of T cells from the secondary lymphoid tissues. The approval of these compounds for the treatment of multiple sclerosis has placed the development of S1PR modulators in the focus of pharmacological research, mostly for autoimmune indications. Here, we report on a novel anellated bismorpholino derivative of oxy-fingolimod, named ST-2191, which exerts selective S1P1 agonist and functional antagonist potency. ST-2191 is also effective in reducing the lymphocyte number in mice, and this effect is not dependent on phosphorylation by sphingosine kinase 2 for activity. These data show that ST-2191 is a novel S1P1 modulator, but further experiments are needed to analyze the therapeutic impact of ST-2191 in animal models of autoimmune diseases.

In silico Docking Studies of Fingolimod and S1P1 Agonists.

The sphingosine-1-phosphate receptor 1 (S1P1), originally the endothelial differentiation gene 1 receptor (EDG-1), is one of five G protein–coupled receptors (GPCRs) S1P1–5 that bind to and are activated by sphingosine-1-phosphate (S1P). The lipid S1P is an intermediate in sphingolipid homeostasis, and S1P1 is a major medical target for immune system modulation; agonism of the receptor produces a myriad of biological responses, including endothelial cell barrier integrity, chemotaxis, lymphocyte trafficking/targeting, angiogenesis, as well as regulation of the cardiovascular system. Use of in silico docking simulations on the crystal structure of S1P1 allows for pinpointing the residues within the receptor’s active site that actively contribute to the binding of S1P, and point to how these specific interactions can be exploited to design more effective synthetic analogs to specifically target S1P1 in the presence of the closely related receptors S1P2, S1P3, S1P4, and S1P5. We examined the binding properties of the endogenous substrate as well as a selection of synthetic sphingosine-derived S1P1 modulators of S1P1 with in silico docking simulations using the software package Molecular Operating Environment® (MOE®). The modeling studies reveal the relevance of phosphorylation, i.e., the presence of a phosphate or phosphonate moiety within the substrate for successful binding to occur, and indicate which residues are responsible for S1P1 binding of the most prominent sphingosine-1-phosphate receptor (S1PR) modulators, including fingolimod and its structural relatives. Furthermore, trends in steric preferences as for the binding of enantiomers to S1P1 could be observed, facilitating future design of receptor-specific substrates to precisely target the active site of S1P1.

S1P/S1P Receptor Signaling in Neuromuscolar Disorders.

The bioactive sphingolipid metabolite, sphingosine 1-phosphate (S1P), and the signaling pathways triggered by its binding to specific G protein-coupled receptors play a critical regulatory role in many pathophysiological processes, including skeletal muscle and nervous system degeneration. The signaling transduced by S1P binding appears to be much more complex than previously thought, with important implications for clinical applications and for personalized medicine. In particular, the understanding of S1P/S1P receptor signaling functions in specific compartmentalized locations of the cell is worthy of being better investigated, because in various circumstances it might be crucial for the development or/and the progression of neuromuscular diseases, such as Charcot–Marie–Tooth disease, myasthenia gravis, and Duchenne muscular dystrophy.

Identification of ApoA4 as a sphingosine 1-phosphate chaperone in ApoM- and albumin-deficient mice

HDL-bound ApoM and albumin are protein chaperones for the circulating bioactive lipid, sphingosine 1-phosphate (S1P); in this role, they support essential extracellular S1P signaling functions in the vascular and immune systems. We previously showed that ApoM- and albumin-bound S1P exhibit differences in receptor activation and biological functions. Whether the physiological functions of S1P require chaperones is not clear. We examined ApoM-deficient, albumin-deficient, and double-KO (DKO) mice for circulatory S1P and its biological functions. In albumin-deficient mice, ApoM was upregulated, thus enabling S1P functions in embryonic development and postnatal adult life. The Apom:Alb DKO mice reproduced, were viable, and exhibited largely normal vascular and immune functions, which suggested sufficient extracellular S1P signaling. However, Apom:Alb DKO mice had reduced levels (∼25%) of plasma S1P, suggesting that novel S1P chaperones exist to mediate S1P functions. In this study, we report the identification of ApoA4 as a novel S1P binding protein. Recombinant ApoA4 bound to S1P, activated multiple S1P receptors, and promoted vascular endothelial barrier function, all reflective of its function as a S1P chaperone in the absence of ApoM and albumin. We suggest that multiple S1P chaperones evolved to support complex and essential extracellular signaling functions of this lysolipid mediator in a redundant manner.

Molecular Mechanism of S1P Binding and Activation of the S1P1 Receptor.

Sphingosine-1-phosphate (S1P) is a lipidic mediator in mammals that functions either as a second messenger or as a ligand. In the latter case, it is transported by its HDL-associated apoM carrier and circulated in blood where it binds to specific S1P receptors on cell membranes and induces downstream reactions. Although S1P signaling pathways are essential for many biological processes, they are poorly understood at the molecular level. Here, the solved crystal structures of the S1P1 receptor were used to evaluate molecular dynamics (MD) simulations to generate greater detailed molecular insights into the mechanism of S1P signaling. The MD simulations provided observations at the coarse-grained and atomic levels indicating that S1P may access the receptor binding pocket directly from solvents. Lifting of the bulky N-terminal cap region of the receptor precedes initial S1P binding. Glu1213.29 guides S1P penetration, and together with Arg2927.34 is responsible for the stabilization of S1P in the binding pocket, which is consistent with experimental predictions. The complete binding of S1P is followed by receptor activation, wherein Trp2696.48 moves toward the transmembrane helix (TM) 7, resulting in the formation of an enhanced hydrogen bond network in the lower region of TM7. The distance between TM3 and TM6 is subsequently increased, resulting in the opening of the intracellular binding pocket that enables G protein binding. Further analysis of the force distribution network in the receptor yielded a detailed molecular understanding of the signal transmission network that is activated upon agonist binding.

Modulation of Cu2+ Binding to Sphingosine-1-Phosphate by Lipid Charge.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that is thought to participate in the regulation of many physiological processes and may play a key role in several diseases. Herein, we found that Cu2+ binds tightly to supported lipid bilayers (SLBs) containing S1P. Specifically, we demonstrated via fluorescence assays that Cu2+-S1P binding was bivalent and sensitive to the concentration of S1P in the SLB. In fact, the apparent equilibrium dissociation constant, KDApp, tightened by a factor of 132 from 4.5 μM to 34 nM as the S1P density was increased from 5.0 to 20 mol %. A major driving force for this apparent tightening was the more negative surface potential with increasing S1P concentration. This potential remained unaltered upon Cu2+ binding at pH 7.4 because two protons were released for every Cu2+ that bound. At pH 5.4, however, Cu2+ could not outcompete protons for the amine and no binding occurred. Moreover, at pH 9.4, the amine was partially deprotonated before Cu2+ binding and the surface potential became more positive on binding. The results for Cu2+-S1P binding were reminiscent of those for Cu2+-phosphatidylserine binding, where a carboxylate group helped to deprotonate the amine. In the case of S1P, however, the phosphate needed to bear two negative charges to facilitate amine deprotonation in the presence of Cu2+.

Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

Telomerase activation protects cells from telomere damage by delaying senescence and inducing cell immortalization, whereas telomerase inhibition mediates rapid senescence or apoptosis. However, the cellular mechanisms that determine telomere damage-dependent senescence versus apoptosis induction are largely unknown. Here, we demonstrate that telomerase instability mediated by silencing of sphingosine kinase 2 (SPHK2) and sphingosine 1-phosphate (S1P), which binds and stabilizes telomerase, induces telomere damage-dependent caspase-3 activation and apoptosis, but not senescence, in p16-deficient lung cancer cells or tumors. These outcomes were prevented by knockdown of a tumor-suppressor protein, transcription factor 21 (TCF21), or by ectopic expression of WT human telomerase reverse transcriptase (hTERT) but not mutant hTERT with altered S1P binding. Interestingly, SphK2-deficient mice exhibited accelerated aging and telomerase instability that increased telomere damage and senescence via p16 activation especially in testes tissues, but not in apoptosis. Moreover, p16 silencing in SphK2-/- mouse embryonic fibroblasts activated caspase-3 and apoptosis without inducing senescence. Furthermore, ectopic WT p16 expression in p16-deficient A549 lung cancer cells prevented TCF21 and caspase-3 activation and resulted in senescence in response to SphK2/S1P inhibition and telomere damage. Mechanistically, a p16 mutant with impaired caspase-3 association did not prevent telomere damage-induced apoptosis, indicating that an association between p16 and caspase-3 proteins forces senescence induction by inhibiting caspase-3 activation and apoptosis. These results suggest that p16 plays a direct role in telomere damage-dependent senescence by limiting apoptosis via binding to caspase-3, revealing a direct link between telomere damage-dependent senescence and apoptosis with regards to aging and cancer.

Sphingosine 1 Phosphate Regulates Store‐Operated Calcium Entry through binding to STIM1

A growing body of evidence suggests that sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) pathway contributes to vascular dysfunction through dysregulation of Ca2+ mobilization. We demonstrated recently that intracellular S1P triggers store‐operated Ca2+ (SOC) entry and independent of G protein‐coupled S1P receptors suggesting SOC as intracellular target for S1P. However, the mechanism by which S1P modulates SOC entry is not well defined. SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. While STIM function as Ca2+ sensors within the ER, the Orai proteins constitute the pore‐forming unit of SOC in PM. We first investigated the role of SK1/S1P on STIM1/Orai1 assembly by confocal microscopy. Pretreating transiently transfected HEK293 cells with SK1 specific inhibitor inhibited angiotensin‐induced colocalization of GFP.STIM1 and mCherry Orai1. Similarly, using BRET assay, inhibition of SK1 decreased NET BRET values of STIM1‐FlAsH and Orai1‐Rluc in transiently transfected HEK293 cells stimulated with GPCR agonists. Our molecular modeling simulation predicted direct binding of S1P to EF/SAM1 domain of STIM1 molecule. This is initially assessed by in vitro binding assay revealed binding of biotinylated S1P to STIM1 in the membrane fractions of vascular smooth muscle cell lysates. We then used circular dichroism spectroscopy (CD) to measure the thermostability of the EF/SAM1 protein in the presence or absence of S1P. Addition of S1P to EF/SAM1 induced a consistent left shift indicative of lower melting temperature (Tm) compared to vehicle control. To further validate direct binding, we prepared 15N‐labeled EF/SAM1 for the NMR spectroscopy. A five point titration series with S1P caused perturbations of residue specific chemical shift due to binding interactions between EF/SAM1 and S1P using 2D TROSY (Transverse Relaxation Optimized Spectroscopy). Collectively, our data provide evidences of intracellular role for S1P in regulation of SOC through direct binding to STIM1.Support or Funding InformationAmerican Heart Association 17GRNT33700222 (HME)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Unexpected profile of sphingolipid contents in blood and bone marrow plasma collected from patients diagnosed with acute myeloid leukemia

BackgroundImpaired apoptotic pathways in leukemic cells enable them to grow in an uncontrolled way. Moreover, aberrations in the apoptotic pathways are the main factor of leukemic cells drug resistance.MethodsTo assess the presence of potential abnormalities that might promote dysfunction of leukemic cells growth, HPLC system was used to determine sphingosine (SFO), sphinganine (SFA), sphingosine-1-phosphate (S1P) and ceramide (CER) concentration in the blood collected from patients diagnose with acute myeloblastic leukemia (AML; n = 49) and compare to values of control (healthily) group (n = 51). Additionally, in AML group concentration of SFO, SFA, S1P and CER was determined in bone marrow plasma and compared to respective values in blood plasma. The concentration of S1P and CER binding protein – plasma gelsolin (GSN) was also assessed in collected samples using immunoblotting assay.ResultsWe observed that in AML patients the average SFO, SFA and CER concentration in blood plasma was significantly higher (p < 0.001) compare to control group, when blood plasma S1P concentration was significantly lower (p < 0.001). At the same time the CER/S1P ratio in AML patient (44.5 ± 19.4) was about 54% higher compare to control group (20.9 ± 13.1). Interestingly the average concentration of S1P in blood plasma (196 ± 13 pmol/ml) was higher compare to its concentration in plasma collected from bone marrow (154 ± 21 pmol/ml).ConclusionsWe hypothesize that changes in profile of sphingolipids concentration and some of their binding protein partners such as GSN in extracellular environment of blood and bone marrow cells in leukemic patients can be targeted to develop new AML treatment method(s).

Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma.

The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-β1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-β1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth.

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling.

Regulation of Store‐Operated Calcium Channels by Sphingosine 1 Phosphate

Several studies have implicated sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) axis in mobilization of intracellular Ca2+ and defective vascular tone. We and other independent investigators demonstrated recently that SK1/S1P mediates transmembrane Ca2+ flux through store‐operated Ca2+ channel (SOC). SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. STIM function as Ca2+ sensors within the ER, and Orai proteins at PM. Depletion of Ca2+ from ER leads to oligomerization and translocation of STIM to bind Orai at the PM to activate Ca2+ flux. We found that S1P regulates SOC through yet undefined intracellular mechanism that does not involve activation of G protein‐coupled S1P receptors. Here, we have studied the mechanism by which SK/S1P mediates activation of SOC. Molecular modeling predicted the binding of S1P with STIM1 of SOC in monomer and dimer forms. Unlike biotin ceramide and biotin controls, only biotinylated S1P bound to endogenous STIM1 in the membrane fraction of cell lysates that was inhibited with competitive cold S1P suggesting specificity of S1P binding to STIM1. Intriguingly, direct mutation of the predicted S1P binding sites to STIM1 markedly inhibited angiotensinII (AngII) and intracellular S1P‐dependent Ca2+ flux without affecting Ca2+ release from intracellular stores. To examine the effect of SK1/S1P on STIM1/Orai1 assembly by BRET assay, we generated STIM1‐FlAsH and Orai1‐Rluc plasmids. We found that NET BRET values of AngII‐dependent stimulated HEK293 cells transiently transfected with STIM1‐FlAsH and Orai1‐Rluc were significantly inhibited by SK1 inhibitor. Functionally, transfecting STIM1 mutants in HEK293 cells inhibited cell proliferation compared to vehicle control. These findings suggest the requirement of intracellular S1P for activation of SOC via modulation of STIM1 and provide novel mechanistic and functional insights into pathophysiology of vascular dysfunction.