An update on sphingosine-1-phosphate receptor 1 modulators

Sphingolipids represent an essential class of lipids found in all eukaryotes, and strongly influence cellular signal transduction. Autoimmune diseases like asthma and multiple sclerosis (MS) are mediated by the sphingosine-1-phosphate receptor 1 (S1P₁) to express a variety of symptoms and disease patterns. Inspired by its natural substrate, an array of artificial sphingolipid derivatives has been developed to target this specific G protein-coupled receptor (GPCR) in an attempt to suppress autoimmune disorders. FTY720, also known as fingolimod, is the first oral disease-modifying therapy for MS on the market. In pursuit of improved stability, bioavailability, and efficiency, structural analogues of this initial prodrug have emerged over time. This review covers a brief introduction to the sphingolipid metabolism, the mechanism of action on S1P₁, and an updated overview of synthetic sphingosine S1P₁ agonists.     

Synthesis of fluorinated agonist of sphingosine-1-phosphate receptor 1

The bioactive metabolite sphingosine-1-phosphate (S1P), a product of sphingosine kinases (SphKs), mediates diverse biological processes such as cell differentiation, proliferation, survival and angiogenesis. A fluorinated analogue of S1P receptor agonist has been synthesized by utilizing a ring opening reaction of oxacycles by a lithiated difluoromethylphosphonate anion as the key reaction. In vitro activity of this S1P analogue is also reported.     

Roles of sphingosine-1-phosphate signaling in angiogenesis

Sphingosine-1-phosphate (S1P) is a blood-borne lipid mediator with pleiotropic biological activities. S1P acts via the specific cell surface G-protein-coupled receptors, S1P(1-5). S1P(1) and S1P(2) were originally identified from vascular endothelial cells (ECs) and smooth muscle cells, respectively. Emerging evidence shows that S1P plays crucial roles in the regulation of vascular functions, including vascular formation, barrier protection and vascular tone via S1P(1), S1P(2) and S1P(3). In particular, S1P regulates vascular formation through multiple mechanisms; S1P exerts both positive and negative effects on angiogenesis and vascular maturation. The positive and negative effects of S1P are mediated by S1P(1) and S1P(2), respectively. These effects of S1P(1) and S1P(2) are probably mediated by the S1P receptors expressed in multiple cell types including ECs and bone-marrow-derived cells. The receptor-subtype-specific, distinct effects of S1P favor the development of novel therapeutic tactics for antitumor angiogenesis in cancer and therapeutic angiogenesis in ischemic diseases.     

Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P₁) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P₁ in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P₁ proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P₁ are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P₁ signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.     

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.     

Immunohistochemical localization of endothelin in human vascular endothelial cells

The cellular localization of endothelin (ET), a novel vasoconstrictor peptide, was studied in human vascular tissues by immunohistochemistry. Distinct and diffuse staining for ET-like immunoreactivity was demonstrated in the cytoplasm of vascular endothelial cells, but not in smooth muscle cells or adventitial fibroblasts. The specificity was confirmed by the negative results following immunoabsorption. These findings suggest that human vascular endothelial cells function as an endocrine and/or paracrine cells for ET secretion.     

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren’s syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.     

Molecular mechanisms underlying the heterogeneous barrier responses of two primary endothelial cell types to sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.     

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.     

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y₁₀ receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y₁₀ receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca²⁺ increases in the CHO cells stably expressing the P2Y₁₀ fused with a G_16α protein. These Ca²⁺ responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y₁₀ receptor into the P2Y₁₀-CHO cells effectively blocked both S1P- and LPA-induced Ca²⁺ increases. RT-PCR analysis showed that the mouse P2Y₁₀ was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y₁₀ receptor is the first receptor identified as a dual lysophospholipid receptor.     

Role of sphingosine-1-phosphate receptors in vascular injury of inflammatory bowel disease

Sphingosine-1-phosphate receptors (S1PRs) have an impact on the intestinal inflammation of inflammatory bowel disease (IBD) by regulating lymphocyte migration and differentiation. S1PR modulators as an emerging therapeutic approach are being investigated for the treatment of IBD. However, the role of S1PRs in intestinal vessels has not drawn much attention. Intestinal vascular damage is one of the major pathophysiological features of IBD, characterized by increased vascular density and impaired barrier function. S1PRs have pleiotropic effects on vascular endothelial cells, including proliferation, migration, angiogenesis and barrier homeostasis. Mounting evidence shows that S1PRs are abnormally expressed on intestinal vascular endothelial cells in IBD. Unexpectedly, S1PR modulators may damage intestinal vasculature, for example increase intestinal bleeding; therefore, S1PRs are thought to be involved in the regulation of intestinal vascular function in IBD. However, little is understood about how S1PRs regulate intestinal vascular function and participate in the initiation and progression of IBD. In this review, we summarize the pathogenic role of S1PRs in and the underlying mechanisms behind the intestinal vascular injury in IBD in order for improving IBD practice including S1PR-targeted therapies.     

Implication of sphingosine-1-phosphate signaling in diseases: molecular mechanism and therapeutic strategies

Sphingosine-1-phosphate signaling is emerging as a critical regulator of cellular processes that is initiated by the intracellular production of bioactive lipid molecule, sphingosine-1-phosphate. Binding of sphingosine-1-phosphate to its extracellular receptors activates diverse downstream signaling that play a critical role in governing physiological processes. Increasing evidence suggests that this signaling pathway often gets impaired during pathophysiological and diseased conditions and hence manipulation of this signaling pathway may be beneficial in providing treatment. In this review, we summarized the recent findings of S1P signaling pathway and the versatile role of the participating candidates in context with several disease conditions. Finally, we discussed its possible role as a novel drug target in different diseases.     

Glomerular proliferation during early stages of diabetic nephropathy is associated with local increase of sphingosine-1-phosphate levels

In this study, the effects of short-term diabetes (4 days) on rat renal glomerular cells proliferation and the potential involvement of sphingolipids in this process were investigated. Immunohistochemical analysis showed that streptozotocin (STZ)-induced diabetes promoted increased intra-glomerular hyperplasia, particularly marked for mesangial cells. This was associated with a concomitant increase in neutral ceramidase and sphingosine-kinase activities and the accumulation of the pro-proliferative sphingolipid sphingosine-1-phosphate, in glomeruli isolated from kidney cortex of STZ-treated rats. These results suggest a possible involvement of sphingolipid metabolites in the glomerular proliferative response during the early stages of diabetic nephropathy.     

GVI phospholipase A2 role in the stimulatory effect of sphingosine-1-phosphate on TRPC5 cationic channels

The Transient Receptor Potential Canonical 5 (TRPC5) protein forms calcium-permeable cationic channels that are stimulated by G protein-coupled receptor agonists. The signaling pathways of such agonist effects are poorly understood. Here we investigated the potential for involvement of lysophosphatidylcholine (LPC) and arachidonic acid generated by group 6 (GVI) phospholipase A2 (PLA2) enzymes, focusing on stimulation of TRPC5 by sphingosine-1-phosphate (S1P) which acts via a pertussis toxin-sensitive (Gi/o protein) pathway without Ca²⁺-release. Experiments were on HEK 293 cells containing conditional expression of human TRPC5. Channel activity was recorded using an intracellular calcium indicator or whole-cell patch-clamp and PLA2 activity was detected using ³H-arachidonic acid. S1P stimulated PLA2 and TRPC5 activities. Both effects were suppressed by the GVI PLA2 inhibitor bromoenol lactone. Knock-down of GVI PLA2 by RNA interference suppressed channel activity evoked by S1P whereas activity evoked by the direct channel stimulator LPC was unaffected. Arachidonic acid did not stimulate the channels. Prior exposure of channels to LPC but not arachidonic acid suppressed channel activity evoked by S1P but not gadolinium, a putative direct stimulator of the channels. The data suggest roles of LPC and GVI PLA2 in S1P-evoked TRPC5 activity.     

Ras/Raf1-dependent signal in sphingosine-1-phosphate-induced tube formation in human coronary artery endothelial cells

Since we recently reported that high density lipoprotein, which contains the bioactive lipid sphingosine-1-phosphate (S1P) [Arterioscler. Thromb. Vasc. Biol. 23 (2003) 802], induced human coronary artery endothelial cell (HCEC) tube formation mediated by a Ras/Raf/ERK (extracellular signal-activated kinase) pathway, we thought that it would be very important to evaluate whether the signal in S1P-induced tube formation is Ras-dependent or -independent. In an in vitro model of HCEC tube formation on a matrix gel, S1P-induced tube formation. ERK1/2 inhibitor (PD98059) and pertussis toxin (PTX) suppressed S1P-induced tube formation. S1P activated phospho(p)-ERK1/2, while dominant-negative RasN17 blocked S1P-induced p-ERK1/2. Moreover, RasN17 inhibited S1P-induced tube formation. S1P activated Ras/Raf1 by Ras pull-down assay and this effect was inhibited by PTX. These results demonstrate that Ras/Raf1-dependent ERK activation mediated by PTX-sensitive G protein-coupled receptors may be a potent signal in S1P-induced HCEC tube formation.     

A house divided: Ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P(1-5). S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P(1), S1P(2) and S1P(3) all contribute positively to S1P-stimulated glioma cell proliferation, with S1P(1) being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P(5) blocks glioma cell proliferation, and inhibits ERK activation. S1P(1) and S1P(3) enhance glioma cell migration and invasion. S1P(2) inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P(2) also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P(2)-stimulated glioma invasion. Thus, while S1P(2) decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix.     

Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.     

Design,synthesis, and in vitro bioactivity evaluation of fluorine-containing analogues for sphingosine-1-phosphate 2 receptor

Twenty eight new aryloxybenzene analogues were synthesized and their in vitro binding potencies toward S1PR2 were determined using a [³²P]S1P competitive binding assay. Out of these new analogues, three compounds, 28c (IC₅₀ = 29.9 ± 3.9 nM), 28e (IC₅₀ = 14.6 ± 1.5 nM), and 28g (IC₅₀ = 38.5 ± 6.3 nM) exhibited high binding potency toward S1PR2 and high selectivity over the other four receptor subtypes (S1PR1, 3, 4, and 5; IC₅₀ > 1000 nM). Each of the three potent compounds 28c, 28e, and 28g contains a fluorine atom that will allow to develop F-18 labeled PET radiotracers for imaging S1PR2.