Sphingosine 1-phosphate Analog Research Articles

PGE2 and NO Mediate the Inhibitory Effect Exerted by FTY720P at 15 Minutes on Colonic Na+/K+ ATPase

Inflammatory Bowel Disease was shown to be accompaniedwith higher levels of sphingosine‐1‐ phosphate (S1P) and lower Na+/K+ ATPase activity. This work was undertaken to see if a cause‐effect relationship exists between S1P and the ATPase, and in case it does to determine the main mediators involved, using Caco‐2 cells as a model and fingolimod phosphate (FTY720P), a S1P analogue. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the Na+/K+ATPase. Western blot analysis showed that sphingosine‐1‐phosphate receptor 2 (S1PR2) is the mostly expressed receptor in Caco‐2 cells. FTY720P, applied for 15 min, reduced significantly the activity of the Na+/K+ ATPase, with a maximal inhibition observed at a concentration of 7.5nM. The effect of FTY720P disappeared completely in presence of JTE‐013 (a specific blocker of S1PR2) and indomethacin (an inhibitor of cyclooxygenase enzymes), and was mimicked by CYM5520 (a S1PR2 agonist), prostaglandin E2 (PGE2), and sulprostone (an agonist of E prostanoid receptor 3 (EP3)). Carboxy‐PTIO a nitric oxide scavenger, abrogated the effect of FTY720P as well as that of PGE2. Treating the cells with Glyco‐SNAP (nitric oxide donor) reduced significantly the ATPase activity. It was concluded that FTY720‐P inhibits the ATPase by binding to S1PR2 leading to PGE2 production. PGE2 binds to EP3 receptors and increases nitric oxide levels resulting in an inhibitory effect on the Na+/K+ ATPase.Support or Funding InformationUniversity Research BoardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FTY720‐P Activates Hepatic Na+/K+ ATPase via S1PR3, PKC and PGE2

BackgroundAlterations in hepatocytes' volume contribute to the pathophysiology of several hepatic disorders including liver insufficiency, diabetic ketoacidosis, hyper‐catabolism and infection. A key regulator of ionic homeostasis and hepatocellular volume is the Na+/K+ ATPase which establishes a sodium electro‐chemical gradient involved in a wide array of cellular activities. Emerging evidence ascribes a role for sphingosine 1‐phosphate (S1P) in the development and progression of liver diseases. The sphingolipid signal via 5 different types of G protein coupled sphingosine 1‐phosphate receptors (S1PR1‐S1PR5), and induces many cellular responses. Previous studies in our lab showed that S1P promotes at 2hrs, an increase in the Na+/K+ ATPase activity in HepG2 (Hepatic carcinoma cell line) cells, but its signaling pathways was not determined. FTY720‐P, a S1P analogue, has been approved recently by US‐FDA as an oral treatment for relapsing form of multiple sclerosis. The drug is a S1P receptor agonist and has been shown to exert some side effects on different organs like the heart. Its side effects on the liver have not been however sufficiently studied.AimThe aim of this work is investigate the effect of FTY720P on hepatic Na+/K+ ATPase and identify the signaling pathway involved, using HepG2 cells as a model. An effect of the drug on liver Na+/K+ ATPase may alter ionic homeostasis and exert adverse effects leading to liver disease. Unraveling the mode of action of the drug would help in reducing the undesirable effects, by inhibiting any of the specific mediators involved.Methods and resultsHepG2 cells were incubated with FTY720P for 2 hrs and the activity of the pump was assayed by measuring the amount of inorganic‐phosphate in the presence and absence of ouabain, a specific inhibitor of the Na+/K+ ATPase. FTY720P induced a 2.5 fold increase in the activity of the Na+/K+ ATPase which was maintained in the presence of JTE‐013, a specific blocker of S1PR2, but disappeared completely in presence of CAY 10444, a specific S1PR3 antagonist. Inhibiting protein kinase C (PKC) and the cyclooxygenase (COX) enzymes with respectively calphostin and indomethacin abolished also the effect of FTY720P. Western blot analysis showed an increase in the expression of COX‐2 enzyme. The PKC activator Phorbol 12‐ myristate 13‐acetate (PMA), induced as FTY720P, a significant increase in the activity of the ATPase and a significant increase in the expression of COX‐2 enzyme. Exogenous prostaglandin E2 (PGE2), exerted a stimulatory effect on the ATPase which was maintained in presence of calphostin.ConclusionsIt was concluded that FTY720P, when applied for 2 hours, binds to S1PR3 and activates PKC, which increases directly or indirectly the expression of COX‐2 enzyme leading to PGE2 release. The latter exerts a stimulatory effect on the Na+/K+ ATPase through a pathway that still needs to be determined.Support or Funding InformationUniversity Research BoardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sphingolipids Signaling in Lamellipodia Formation and Enhancement of Endothelial Barrier Function.

Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury.

Combination treatment of FTY720 and cisplatin exhibits enhanced antitumour effects on cisplatin-resistant non-small lung cancer cells.

A major common medical treatment for lung carcinoma is cisplatin (DDP)-based therapy. However, the development or existence of chemoresistance frequently blocks its effectiveness. Currently, autophagy is recognised as a potential anticancer strategy, although there is controversy over its role in the development of cancer. In lung carcinoma, no studies of autophagy induced by FTY720, a sphingosine 1-phosphate analog and a novel immunosuppressant drug, have been published, while apoptosis has been shown to be induced by FTY720 in several cancer cell lines. We evaluated the effects of FTY720 on autophagy in A549 cells and studied the related mechanisms of cell autophagy and apoptosis in non-small cell lung carcinoma, including both DDP-resistant and -sensitive cells. The results revealed that FTY720 inhibited the growth and induced apoptosis in the A549/DDP cells in a time- and dose-dependent manner and the combination of FTY720 and DDP further enhanced apoptosis in these cells as determined by CCK-8 assay, western blotting and flow cytometry. Compared with the sensitive cell line A549, DDP-resistant A549/DDP cells showed a substantial increase in baseline autophagy as determined by increased LC puncta, and expression of LC3-I, LC3-II and Atg7 expression. DDP-induced apoptotic cell death was enhanced by the blockade of either siRNA-mediated knockdown of Atg7 genetic expression or a pharmacological inhibitor (3-MA). Moreover, the combination of FTY720 and DDP showed enhanced antitumour activity in vivo in lung cancer-bearing mice. Immunohistochemistry showed that the mice with lung carcinoma treated with FTY720 and DDP showed decreased expression of Atg7 and Ki67. Compared with monotherapy invivo and invitro, FTY720 in combination with DDP inhibited A549 cell growth more effectively. and these findings also show the influence of FTY720 in the induction of autophagy. Overall, the results indicated that FTY720 in combination with a DDP-based regime could enhance the effectiveness of lung carcinoma treatment.

FTY720 ameliorates renal fibrosis by simultaneously affecting leucocyte recruitment and TGF-β signalling in fibroblasts.

Renal fibrosis is the common final manifestation of chronic kidney diseases and usually results in end-stage renal failure. In this study, we evaluated the effect of fingolimod (FTY720), an analogue of sphingosine 1-phosphate (S1P), as a treatment for the unilateral ureteral obstruction (UUO)-induced renal fibrosis animal model. We treated mice with FTY720 at a dosage of 1 mg/kg/day by intragastric administration from day 1 until day 7. The control group received the same amount of saline. FTY720 reduced significantly the urine albumin/creatinine ratio (UACR) in treated UUO mice. FTY720 treatment also caused a significant decrease in interstitial expansion and collagen deposition in the kidney, accompanied by reduced mononuclear cell recruitment and inflammatory cytokine expression. In addition, the expression levels of the endothelial cell adhesion molecules P-selectin and vascular cell adhesion protein 1 (VCAM-1) were suppressed in the ligated kidney by FTY720 administration, suggesting reduced renal endothelial cell activation. Furthermore, in renal interstitial fibroblast normal rat kidney (NRK)-49F cells, FTY720 significantly affected transforming growth factor (TGF)-β-induced α-smooth muscle actin (SMA) expression and collagen synthesis by inhibiting both the Mothers against decapentaplegic homologue (Smad)2/3 and phosphatidylinositol 3-kinase/protein kinase B/glycogen synthase kinase 3 beta (PI3K/AKT/GSK3β) signalling pathways. S1P1 knock-down by siRNA reversed this effect significantly in our fibroblast cell culture model. Therefore, FTY720 attenuates renal fibrosis via two different mechanisms: first, FTY720 suppresses the synthesis of extracellular matrix in interstitial fibroblasts by interfering with TGF-β signalling; and secondly, FTY720 affects endothelial cell activation and chemokine expression, thereby reducing immune cell recruitment into the kidney.

Ligand chain length drives activation of lipid G protein-coupled receptors

Sphingosine-1-phosphate (S1P) is a lipid mediator that can activate five cell membrane G protein-coupled receptors (GPCRs) which carry a variety of essential functions and are promising drug targets. S1P is composed of a polar zwitterionic head-group and a hydrophobic alkyl chain. This implies an activation mechanism of its cognate receptor that must be significantly different from what is known for prototypical GPCRs (ie receptor to small hydrophilic ligands). Here we aim to identify the structural features responsible for S1P agonism by combining molecular dynamics simulations and functional assays using S1P analogs of different alkyl chain lengths. We propose that high affinity binding involves polar interactions between the lipid head-group and receptor side chains while activation is due to hydrophobic interactions between the lipid tail and residues in a distinct binding site. We observe that ligand efficacy is directly related to alkyl chain length but also varies with receptor subtypes in correlation with the size of this binding pocket. Integrating experimental and computational data, we propose an activation mechanism for the S1P receptors involving agonist-induced conformational events that are conserved throughout class A GPCRs.

FTY720 Attenuates Angiotensin II-Induced Podocyte Damage via Inhibiting Inflammatory Cytokines.

FTY720, a new chemical substance derived from the ascomycete Isaria sinclairii, is used for treating multiple sclerosis, renal cancer, and asthma. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite and exists in red blood cells. FTY720 is a synthetic S1P analog which can block S1P evoking physiological effects. Recently studies show that S1P was participating in activated inflammation cells induced renal injury. The objective of this study was to assess the protective effect of FTY720 on kidney damage and the potential mechanism of FTY720 which alleviate podocyte injury in chronic kidney disease. In this study, we selected 40 patients with IgA nephropathy and examined their clinical characteristics. Ang II-infusion rat renal injury model was established to evaluate the glomeruli and tubulointerstitial lesion. The result showed that the concentration of S1P in serum and urine was positively correlated with IgA nephropathy patients' renal injury. FTY720 could reduce renal histological lesions induced by Ang II-infusion in rats. Moreover, FTY720 decreased S1P synthesis in Ang II-infusion rats via downregulation of inflammatory cytokines including TNF-α and IL-6. In addition, FTY720 alleviated exogenous S1P-induced podocyte damage. In conclusion, FTY720 is able to attenuate S1P-induced podocyte damage via reducing inflammatory cytokines.

Effects of FTY720 on Lung Injury Induced by Hindlimb Ischemia Reperfusion in Rats

Background Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid mediator involved in modulating inflammatory process. We investigated the effects of FTY720, a structural analogue of S1P after phosphorylation, on lung injury induced by hindlimb ischemia reperfusion (IR) in rats. Methods Fifty Sprague-Dawley rats were divided into groups SM, IR, F3, F5, and F10. Group SM received sham operation, and bilateral hindlimb IR was established in group IR. The rats in groups F3, F5, and F10 were pretreated with 3, 5, and 10 mg/kg/d FTY720 for 7 days before IR. S1P lyase (S1PL), sphingosine kinase (SphK) 1, and SphK2 mRNA expressions, wet/dry weight (W/D), and polymorphonuclear/alveolus (P/A) in lung tissues were detected, and the lung injury score was evaluated. Results W/D, P/A, and mRNA expressions of S1PL, SphK1, and SphK2 were higher in group IR than in group SM, while these were decreased in both groups F5 and F10 as compared to IR (p < 0.05). The lung tissue presented severe lesions in group IR, which were attenuated in groups F5 and F10 with lower lung injury scores than in group IR (p < 0.05). Conclusions FTY720 pretreatment could attenuate lung injury induced by hindlimb IR by modulating S1P metabolism and decreasing pulmonary neutrophil infiltration.

A Rapid Fluorescence Assay for Measuring Sphingosine-1-Phosphate Transporter Activity in Erythrocytes.

Sphingosine-1-phosphate (S1P) is an intercellular signaling molecule that is present in the plasma and plays an important role in recruiting lymphocytes from the thymus and secondary lymphoid organs. Erythrocytes are the most abundant cells in the blood and substantially contribute to the S1P supply in the plasma by releasing intracellularly synthesized S1P via an S1P transporter. Thus, the S1P transporter in erythrocytes is a potential target for immuno-suppressing drugs.In this chapter, we describe a rapid method for measuring the activity of the erythrocyte S1P transporter by using the fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. This method does not require chromatography performed with high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry, or thin-layer chromatography methods. Furthermore, S1P transporter activity can be detected by measuring the increase in fluorescence intensity in the extracellular buffer without performing lipid extraction.

Ceramide and S1P Signaling in Embryonic Stem Cell Differentiation.

Bioactive sphingolipids are important regulators for stem cell survival and differentiation. Most recently, we have coined the term "morphogenetic lipids" for sphingolipids that regulate stem cells during embryonic and postnatal development. The sphingolipid ceramide and its derivative, sphingosine-1-phosphate (S1P), can act synergistically as well as antagonistically on embryonic stem (ES) cell differentiation. We show here simple as well as state-of-the-art methods to analyze sphingolipids in differentiating ES cells and discuss new protocols to use ceramide and S1P analogs for the guided differentiation of mouse ES cells toward neuronal and glial lineage.

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers.

Combination of ciclopirox olamine and sphingosine-1-phosphate as granulation enhancer in diabetic wounds.

Granulation tissue formation requires a robust angiogenic response. As granulation tissue develops, collagen fibers are deposited and compacted. Forces generated in the wake of this process drive wound contraction to reduce the wound area. In diabetics, both angiogenesis and wound contraction are diminished leading to impaired wound healing. To emulate this pathology and to address it pharmacologically, we developed a wound healing model in the diabetic Zucker fatty rat and tested a topical proangiogenic strategy combining antifungal agent ciclopirox olamine (CPX) and lysophospholipid sphingosine-1-phosphate (S1P) to promote diabetic wound closure. In vitro, we demonstrated that CPX + S1P up-regulates a crucial driver of angiogenesis, hypoxia-inducible factor-1, in endothelial cells. Injection of CPX + S1P into subcutaneously implanted sponges in experimental rats showed, in an additive manner, a fivefold increased endothelial infiltration and lectin-perfused vessel length. We developed a splinted diabetic rodent model to achieve low wound contraction rates that are characteristic for the healing mode of diabetic ulcers in humans. We discovered specific dorsal sites that allowed for incremental full-thickness excisional wound depths from 1 mm (superficial) to 3 mm (deep). This enabled us to bring down wound contraction from 51% in superficial wounds to 8% in deep wounds. While the effects of topical gel treatment of CPX + S1P were masked by the rodent-characteristic dominant contraction in superficial wounds, they became clearly evident in deep diabetic wounds. Here, a fivefold increase of functional large vessels resulted in accelerated granulation tissue formulation, accompanied by a 40% increase of compacted thick collagen fibers. This was associated with substantially reduced matrix metalloproteinase-3 and -13 expression. These findings translated into a fivefold increase in granulation-driven contraction, promoting diabetic wound closure. With CPX and S1P analogues already in clinical use, their combination presents itself as an attractive proangiogenic treatment to be repurposed for diabetic wound healing.

Differential elastic responses to barrier-altering agonists in two types of human lung endothelium

Vascular integrity is primarily determined by endothelial cell (EC) cytoskeletal structure that is differentially regulated by various stimuli. In this study, atomic force microscopy (AFM) was used to characterize structural and mechanical properties in the cytoskeleton of cultured human pulmonary artery EC (HPAEC) and human lung microvascular EC (HLMVEC) by determining elastic properties (Young's modulus) in response to endogenous barrier protective agents sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF), or the barrier disruptive molecule thrombin. Initial studies in unstimulated cells indicate higher baseline peripheral elastic modulus values in HPAEC (mean 2.9 KPa) than in HLMVEC (1.8 KPa). After 30 min of stimulation, S1P induced the highest Young's modulus increase (6.1 KPa) compared to the other barrier enhancing stimuli, HGF (5.8 KPa) and the pharmaceutical agent and S1P analog FTY720 (4.1 KPa). In contrast, the barrier disruptive agent thrombin decreased values from 2.5 KPa to 0.7 KPa depending on the cell type and treatment time. AFM topographical imaging supports these quantitative biophysical data regarding differential peripheral elastic properties in EC. Overall, these AFM studies provide novel insights into the biomechanical properties of human lung EC that regulate vascular barrier function and have potential applicability to pathophysiologic vascular leak syndromes such as acute lung injury.

Progress in orthopedic biomaterials and drug delivery

Musculoskeletal conditions, including arthritis, back pain, and skeletal injuries/trauma, affect 50 % of the adult population in the U.S.A., and account for the second greatest cause of disability worldwide (www.bjdonline.org). The aggregate total annual cost of musculoskeletal diseases was $796 billion in 2011. While most musculoskeletal injuries and diseases have lowmortality, they have a tremendous impact of quality of life and morbidity. Biomaterials have a long history in orthopedics—metal and plastic implants have been used over the last 60 years to treat end-stage joint degeneration and skeletal tissue loss, and alleviate the resulting pain and disability. First-generation orthopedic implants were designed to be biologically inert and selected primarily for their mechanical properties. However, they do not replicate the function of normal tissue and have a limited life span in the body, which is especially problematic in younger patients. Recent decades have focused on the development of biomaterials that are biodegradable and incorporate tissue-specific physicochemical cues to guide cell processes and tissue morphogenesis, with the goal of restoring natural tissue structure and function. These advances in biomaterials provide critical enabling technologies for the delivery of stem cells, gene therapies, and new biologics in the orthopedic field. This special issue highlights the recent advances and future directions in orthopedic biomaterials and drug delivery systems, and showcases emerging pharmaceutical and regenerative approaches to treat injuries, diseases, and disorders of the musculoskeletal system. Non-union bone fractures are a significant clinical challenge, and bone tissue engineering could have a key impact in improving current clinical options. Telvin et al. review the application of stem cells from various sources towards bone tissue engineering. The ability of transplanted stem cells to survive, continue to differentiate, and form bone tissue is key to making stem cell-mediated bone tissue regeneration approaches successful. The research article by Wen et al. precisely address this challenge. Their study compares the in vivo bone tissue forming ability of human bone marrow derived stem cells and human embryonic stem cells using osteoinductive mineralized materials as a scaffold. The article by Nyberg et al. reviews progress and challenges in achieving precise temporal and spatial control of growth factor presentation for functional bone tissue engineering ex vivo and in vivo. Ultimately, successful bone regeneration requires integration between the repair and native bone tissue. Botchway and colleagues address this critical issue, by modifying the surface of a bone allograft with FTY720, a synthetic analog of sphingosine 1-phosphate, to promote endogenous bone formation and osseous integration at the interface. Another key hurdle for the success of bone tissue engineering is vascularization in vivo. The review by Garcia and Garcia summarizes the current strategies and new developments targeted at vascularization of engineered bone, including growth factor and cell delivery, gene therapy, biomaterials, and combination approaches. The article by Tonello et al. demonstrates the potential of human placental matrix for promoting angiogenesis and its controlled release from biodegradable microparticles. In addition to bone, this special issue contains articles that highlight advances in cartilage tissue regeneration and osteoarthritis (OA) therapies. Osteoarthritis is the leading cause of * Blanka Sharma blanka.sharma@bme.ufl.edu

Effects of FTY720 (Fingolimod) on Proliferation, Differentiation, and Migration of Brain-Derived Neural Stem Cells.

Insufficient proliferation, differentiation, and migration are the main pitfalls of neural stem cells (NSCs) in reparative therapeutics for the central nervous system (CNS) diseases. The potent lipid mediator sphingosine-1-phosphate (S1P) regulates cells' biological behavior broadly in the CNS. However, the effects of activating S1P on NSCs are not quite clear. In the current study, FTY720 (Fingolimod), an analog of S1P, was employed to induce the proliferation, differentiation, and migration of cultured brain-derived NSCs. The results indicated that proliferation and migration ability of NSCs were promoted by FTY720. Though we observed no obvious neuron prefers differentiation of NSCs, there were more protoplasmic astrocytes developed in the presence of certain concentration of FTY720. This work gives more comprehensive understanding of how FTY720 affects NSCs.

Fingolimod induces neuroprotective factors in human astrocytes

BackgroundFingolimod (FTY720) is the first sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of multiple sclerosis. The phosphorylated active metabolite FTY720-phosphate (FTY-P) interferes with lymphocyte trafficking. In addition, it accumulates in the CNS and reduces brain atrophy in multiple sclerosis (MS), and neuroprotective effects are hypothesized.MethodsHuman primary astrocytes as well as human astrocytoma cells were stimulated with FTY-P or S1P. We analyzed gene expression by a genome-wide microarray and validated induced candidate genes by quantitative PCR (qPCR) and ELISA. To identify the S1P-receptor subtypes involved, we applied a membrane-impermeable S1P analog (dihydro-S1P), receptor subtype specific agonists and antagonists, as well as RNAi silencing.ResultsFTY-P induced leukemia inhibitory factor (LIF), interleukin 11 (IL11), and heparin-binding EGF-like growth factor (HBEGF) mRNA, as well as secretion of LIF and IL11 protein. In order to mimic an inflammatory milieu as observed in active MS lesions, we combined FTY-P application with tumor necrosis factor (TNF). In the presence of this key inflammatory cytokine, FTY-P synergistically induced LIF, HBEGF, and IL11 mRNA, as well as secretion of LIF and IL11 protein. TNF itself induced inflammatory, B-cell promoting, and antiviral factors (CXCL10, BAFF, MX1, and OAS2). Their induction was blocked by FTY-P. After continuous exposure of cells to FTY-P or S1P for up to 7 days, the extent of induction of neurotrophic factors and the suppression of TNF-induced inflammatory genes declined but was still detectable. The induction of neurotrophic factors was mediated via surface S1P receptors 1 (S1PR1) and 3 (S1PR3).ConclusionsWe identified effects of FTY-P on astrocytes, namely induction of neurotrophic mediators (LIF, HBEGF, and IL11) and inhibition of TNF-induced inflammatory genes (CXCL10, BAFF, MX1, and OAS2). This supports the view that a part of the effects of fingolimod may be mediated via astrocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0393-6) contains supplementary material, which is available to authorized users.

GluN2B-containing NMDA receptors are upregulated in plasma membranes by the sphingosine-1-phosphate analog FTY720P

Sphingosine-1-phosphate (S1P) is a ceramide derivative serving not only as a regulator of immune properties but also as a modulator of brain functions. To better understand the mechanism underlying the effects of S1P on brain functions, we investigated the potential impact of S1P receptor (S1PR) activation on NMDA receptor subunits. We used acute rat hippocampal slices as a model system, and determined the effects of the active phosphorylated S1P analog, fingolimod (FTY720P) on various NMDA receptors. Treatment with FTY720P significantly increased phosphorylation of GluN2B-containing NMDA receptors at Tyr1472. This effect appears rather specific, as treatment with FTY720P did not modify GluN2B-Tyr1336, GluN2B-Ser1480, GluN2A-Tyr1325 or GluN1-Ser897 phosphorylation. Pre-treatment of hippocampal slices with the compounds W146 and PP1 indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 epitopes was dependent on activation of S1PR subunit 1 (S1PR1) and Src/Fyn kinase, respectively. Cell surface biotinylation experiments indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 was also associated with increased levels of GluN1 and GluN2B subunits on membrane surface, whereas no change was observed for GluN2A subunits. We finally demonstrate that FTY720P is inclined to favor Tau and Fyn accumulation on plasma membranes. These results suggest that activation of S1PR1 by FTY720P enhances GluN2B receptor phosphorylation in rat hippocampal slices, resulting in increased levels of GluN1 and GluN2B receptor subunits in neuronal membranes through a mechanism probably involving Fyn and Tau.

Sphingosine-1-phosphate (S1P) inhibits TNFA-induced MMP-9 expression (TIME) through S1P2-mediated activation of PTEN

S1P regulates numerous functions in vascular smooth muscle cells (VSMC). We have shown that high S1P levels promoted neointima formation after carotid injury, a process that depends on matrix metalloproteinases (MMP). Here, we tested whether S1P affects basal and cytokine-induced MMP-9 activity in rat and mouse VSMC. S1P potently inhibited TIME as seen on gelatin zymography of the secreted enzyme and gene expression by real time PCR, respectively, without any effect on MMP-2. Using S1P receptor antagonists and VSMC isolated from S1P receptor knockout mice we have identified S1P2 as the S1P receptor responsible for TIME inhibition. S1P had no effect on IkBa phosphorylation and NF-kB activation by TNFa but potently inhibited Akt phosphorylation after TNFa that we found to be required for TIME. S1P inhibited PI3K/Akt by activating the Phosphatase and Tensin homolog (PTEN) pathway by engaging the S1P2 receptor as we could show using PI3K inhibitors, siRNA to PTEN and pharmacological PTEN blockade (bpVHO(pic)). The functional consequence of TIME inhibition by S1P was the abrogation of MMP-dependent TNFa-stimulated VSMC migration in Boyden chamber experiments. In vivo, downregulation of all S1P receptors by maintaining high S1P levels through pharmacological S1P lyase inhibition promoted neointima formation after carotid ligation, while the S1P analogue FTY720 had no effect. FTY720 acts on all S1P receptors except S1P2, and S1P2-deficient mice exhibit the same phenotype as S1P lyase-inhibited. Thus lack of MMP-9 inhibition due to downregulated or missing S1P2 may be responsible for increased neointima formation, a process shown to causally depend on MMP-9.

Synthesis and Evaluation of Fluorinated Fingolimod (FTY720) Analogues for Sphingosine-1-Phosphate Receptor Molecular Imaging by Positron Emission Tomography

Sphingosine-1-phosphate (S1P) is a lysophospholipid that evokes a variety of biological responses via stimulation of a set of cognate G-protein coupled receptors (GPCRs): S1P1-S1P5. S1P and its receptors (S1PRs) play important roles in the immune, cardiovascular, and central nervous systems and have also been implicated in carcinogenesis. Recently, the S1P analogue Fingolimod (FTY720) has been approved for the treatment of patients with relapsing multiple sclerosis. This work presents the synthesis of various fluorinated structural analogues of FTY720, their in vitro and in vivo biological testing, and their development and application as [(18)F]radiotracers for the study of S1PR biodistribution and imaging in mice using small-animal positron emission tomography (PET).

Sphingosin-1-phosphate Receptor 1: a Potential Target to Inhibit Neuroinflammation and Restore the Sphingosin-1-phosphate Metabolism.

Recent evidence suggests that an extreme shift may occur in sphingosine metabolism in neuroinflammatory contexts. Sphingosine 1-phosphate (S1P)-metabolizing enzymes (SMEs) regulate the level of S1P. We recently found that FTY720, a S1P analogue, and SEW2871, a selective S1P receptor 1 (S1P1) agonist, provide protection against neural damage and memory deficit in amyloid beta (Aβ)-injected animals. This study aimed to evaluate the effects of these two analogues on the expression of SMEs as well as their anti-inflammatory roles. Rats were treated with intracerebral lipopolysaccharide (LPS) or Aβ. Memory impairment was assessed by Morris water maze and the effects of drugs on SMEs as well as inflammatory markers, TNF- α and COX-II, were determined by immunoblotting. Aβ and LPS differentially altered the expression profile of SMEs. In Aβ-injected animals, FTY720 and SEW2871 treatments exerted anti-inflammatory effects and restored the expression profile of SMEs, in parallel to our previous findings. In LPS animals however, in spite of anti-inflammatory effects of the two analogues, only FTY720 restored the levels of SMEs and prevented memory deficit. The observed ameliorating effects of FTY720 and SEW7821 can be partly attributed to the interruption of the vicious cycle of abnormal S1P metabolism and neuro-inflammation. The close imitation of the FTY720 effects by SW2871 in Aβ-induced neuro-inflammation may highlight the attractive role of S1P1 as a potential target to restore S1P metabolism and inhibit inflammatory processes.