Sphingosine-1-phosphate Transporter Research Articles

Deficiency of the sphingosine-1-phosphate (S1P) transporter Mfsd2b protects the heart against hypertension-induced cardiac remodeling by suppressing the L-type-Ca2+ channel.

The erythrocyte S1P transporter Mfsd2b is also expressed in the heart. We hypothesized that S1P transport by Mfsd2b is involved in cardiac function. Hypertension-induced cardiac remodeling was induced by 4-weeks Angiotensin II (AngII) administration and assessed by echocardiography. Ca2+ transients and sarcomere shortening were examined in adult cardiomyocytes (ACM) from Mfsd2b+/+ and Mfsd2b-/- mice. Tension and force development were measured in skinned cardiac fibers. Myocardial gene expression was determined by real-time PCR, Protein Phosphatase 2A (PP2A) by enzymatic assay, and S1P by LC/MS, respectively. Msfd2b was expressed in the murine and human heart, and its deficiency led to higher cardiac S1P. Mfsd2b-/- mice had regular basal cardiac function but were protected against AngII-induced deterioration of left-ventricular function as evidenced by ~ 30% better stroke volume and cardiac index, and preserved ejection fraction despite similar increases in blood pressure. Mfsd2b-/- ACM exhibited attenuated Ca2+ mobilization in response to isoprenaline whereas contractility was unchanged. Mfsd2b-/- ACM showed no changes in proteins responsible for Ca2+ homeostasis, and skinned cardiac fibers exhibited reduced passive tension generation with preserved contractility. Verapamil abolished the differences in Ca2+ mobilization between Mfsd2b+/+ and Mfsd2b-/- ACM suggesting that S1P inhibits L-type-Ca2+ channels (LTCC). In agreement, intracellular S1P activated the inhibitory LTCC phosphatase PP2A in ACM and PP2A activity was increased in Mfsd2b-/- hearts. We suggest that myocardial S1P protects from hypertension-induced left-ventricular remodeling by inhibiting LTCC through PP2A activation. Pharmacologic inhibition of Mfsd2b may thus offer a novel approach to heart failure.

Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells

Red blood cells (RBC) are the major carriers of sphingosine-1-phosphate (S1P) in blood. Here we show that variations in RBC S1P content achieved by altering S1P synthesis and transport by genetic and pharmacological means regulate glucose uptake and metabolic flux. This is due to S1P-mediated activation of the catalytic protein phosphatase 2 (PP2A) subunit leading to reduction of cell-surface glucose transporters (GLUTs). The mechanism dynamically responds to metabolic cues from the environment by increasing S1P synthesis, enhancing PP2A activity, reducing GLUT phosphorylation and localization, and diminishing glucose uptake in RBC from diabetic mice and humans. Functionally, it protects RBC against lipid peroxidation in hyperglycemia and diabetes by activating the pentose phosphate pathway. Proof of concept is provided by the resistance of mice lacking the S1P exporter MFSD2B to diabetes-induced HbA1c elevation and thiobarbituric acid reactive substances (TBARS) generation in diabetic RBC. This mechanism responds to pharmacological S1P analogues such as fingolimod and may be functional in other insulin-independent tissues making it a promising therapeutic target.

Imidazole-based sphingosine-1-phosphate transporter Spns2 inhibitors

Sphingosine-1-phosphate (S1P) is a chemotactic lipid that influences immune cell positioning. S1P concentration gradients are necessary for proper egress of lymphocytes from the thymus and secondary lymphoid tissues. This trafficking is interdicted by S1P receptor modulators, and it is expected that S1P transporter (Spns2) inhibitors, by reshaping S1P concentration gradients, will do the same. We previously reported SLF1081851 as a prototype Spns2 inhibitor, which provided a scaffold to investigate the importance of the oxadiazole core and the terminal amine. In this report, we disclose a structure–activity relationship study by incorporating imidazole as both a linker and surrogate for a positive charge in SLF1081851. In vitro inhibition of Spns2-dependent S1P transport in HeLa cells identified 7b as an inhibitor with an IC50 of 1.4 ± 0.3 µM. The SAR studies reported herein indicate that imidazolium can be a substitute for the terminal amine in SLF1081851 and that Spns2 inhibition is highly dependent on the lipid alkyl tail length.

Assay of Sphingosine 1-phosphate Transporter Spinster Homolog 2 (Spns2) Inhibitors.

The sphingosine-1-phosphate (S1P) pathway remains an active area of research for drug discovery because S1P modulators are effective medicine for autoimmune diseases such as multiple sclerosis and ulcerative colitis. As such, other nodes in the pathway can be probed for alternative therapeutic candidates. As S1P elicits its function in an 'outside-in' fashion, targeting the transporter, Spns2, which is upstream of the receptors, is of great interest. To support our medicinal chemistry campaign to inhibit S1P transport, we developed a mammalian cell-based assay. In this protocol, Spns2 inhibition is assessed by treating HeLa, U-937, and THP-1 cells with inhibitors and S1P exported in the extracellular milieu is quantified by LC-MS/MS. Our studies demonstrated that the amount of S1P in the media in inversely proportional to inhibitor concentration. The details of our investigations are described herein.

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.

The role of sphingosine-1-phosphate in the gut mucosal microenvironment and inflammatory bowel diseases.

Sphingosine-1-phosphate (S1P), a type of bioactive sphingolipid, can regulate various cellular functions of distinct cell types in the human body. S1P is generated intracellularly by the catalysis of sphingosine kinase 1/2 (SphK1/2). S1P is transferred to the extracellular environment via the S1P transporter, binds to cellular S1P receptors (S1PRs) and subsequently activates S1P-S1PR downstream signaling. Dysbiosis of the intestinal microbiota, immune dysregulation and damage to epithelial barriers are associated with inflammatory bowel disease (IBD). Generally, S1P mainly exerts a proinflammatory effect by binding to S1PR1 on lymphocytes to facilitate lymphocyte migration to inflamed tissues, and increased S1P was found in the intestinal mucosa of IBD patients. Notably, there is an interaction between the distribution of gut bacteria and SphK-S1P signaling in the intestinal epithelium. S1P-S1PR signaling can also regulate the functions of intestinal epithelial cells (IECs) in mucosa, including cell proliferation and apoptosis. Additionally, increased S1P in immune cells of the lamina propria aggravates the inflammatory response by increasing the production of proinflammatory cytokines. Several novel drugs targeted at S1PRs have recently been used for IBD treatment. This review provides an overview of the S1P-S1PR signaling pathway and, in particular, summarizes the various roles of S1P in the gut mucosal microenvironment to deeply explore the function of S1P-S1PR signaling during intestinal inflammation and, more importantly, to identify potential therapeutic targets for IBD in the SphK-S1P-S1PR axis.

Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

An LC-MS-based workflow measures the export activity of S1P transporters

Sphingosine-1-phosphate (S1P) is an active signaling metabolite synthesized by blood cells, exported into blood stream, and can trigger many downstream signaling pathways with disease implications. Understanding how S1P is transported is of great values for dissecting the function of S1P, but most existing methods for measuring S1P transporter activity use radioactive substrates or involve multiple workup steps, hindering their broader uses. In this study, we develop a workflow combining sensitive LC-MS measurement and a cell-based transporter protein system to measure the export activity of S1P transporter proteins. Our workflow demonstrated good applications in studying different S1P transporters SPNS2 and MFSD2B, WT and mutated protein, and different protein substrates. In summary, we provide a simple yet versatile workflow for measuring the export activity of S1P transporters, facilitating future studies of S1P transport mechanism and drug development.

S1P promotes corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model.

Corneal disease was the most critical cause of vision loss. This study aimed to research a new method and provide a theoretical basis for treating corneal injury. A mice corneal epithelial injury model was constructed by the method of mechanical curettage. Models were treated with sphingosine 1-phosphate (S1P) and si-Spns2. An immunofluorescence assay was used to detect βIII-tubulin. The expressions of neurotrophic factor, S1P transporter, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway-related proteins were detected by western blot. Hematoxylin–eosin staining was processed to detect the effect of SIP on corneal repair in mice. si-Spns2 inhibited the effect of S1P. S1P significantly repaired the corneal injury, while si-Spns2 treatment made it more severe. Moreover, S1P could significantly increase the levels of NGF, BDNF, GDNF, Spns2, and p-ERK1/2. si-Spns2 inhibits the effect of S1P in the expression of these proteins. S1P significantly increased axonal differentiation of trigeminal ganglion neurons, which was inhibited after si-Spns2 treatment. S1P promoted corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model. Treatment of corneal injury by S1P may be an effective approach.

Proton-driven alternating access in a spinster lipid transporter

Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange.

Sphingosine 1-phosphate signaling in perivascular cells enhances inflammation and fibrosis in the kidney.

Chronic kidney disease (CKD), characterized by sustained inflammation and progressive fibrosis, is highly prevalent and can eventually progress to end-stage kidney disease. However, current treatments to slow CKD progression are limited. Sphingosine 1-phosphate (S1P), a product of sphingolipid catabolism, is a pleiotropic mediator involved in many cellular functions, and drugs targeting S1P signaling have previously been studied particularly for autoimmune diseases. The primary mechanism of most of these drugs is functional antagonism of S1P receptor-1 (S1P1) expressed on lymphocytes and the resultant immunosuppressive effect. Here, we documented the role of local S1P signaling in perivascular cells in the progression of kidney fibrosis using primary kidney perivascular cells and several conditional mouse models. S1P was predominantly produced by sphingosine kinase 2 in kidney perivascular cells and exported via spinster homolog 2 (Spns2). It bound to S1P1 expressed in perivascular cells to enhance production of proinflammatory cytokines/chemokines upon injury, leading to immune cell infiltration and subsequent fibrosis. A small-molecule Spns2 inhibitor blocked S1P transport, resulting in suppression of inflammatory signaling in human and mouse kidney perivascular cells in vitro and amelioration of kidney fibrosis in mice. Our study provides insight into the regulation of inflammation and fibrosis by S1P and demonstrates the potential of Spns2 inhibition as a treatment for CKD and potentially other inflammatory and fibrotic diseases that avoids the adverse events associated with systemic modulation of S1P receptors.

Mfsd2b and Spns2 are essential for maintenance of blood vessels during development and in anaphylactic shock.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.

Programming to S1PR1+ Endothelial Cells Promotes Restoration of Vascular Integrity.

[Figure: see text].

Sphingosine 1-phosphate signaling in uterine fibroids: implication in activin A pro-fibrotic effect

To explore the link between sphingosine 1-phosphate (S1P) signaling and leiomyoma and the possible S1P cross-talk with the fibrotic effect of activin A. Case-control laboratory study. University institute and university hospital. Patients with uterine fibroids (n = 26). Tissue specimens of leiomyoma and normal myometrium were obtained from patients undergoing myomectomy or total hysterectomy. Expression of mRNA levels of the enzyme involved in S1P metabolism, S1P receptors, and S1P transporter Spns2 was evaluated in matched leiomyoma/myometrium specimens and cell populations. The effects of inhibition of S1P metabolism and signaling was evaluated on activin A-induced fibrotic action in leiomyoma cell lines. The expression of the enzymes responsible for S1P formation, sphingosine kinase (SK) 1 and 2, and S1P2, S1P3, and S1P5 receptors was significantly augmented in leiomyomas compared with adjacent myometrium. In leiomyoma cells, but not in myometrial cells, activin A increased mRNA expression levels of SK1, SK2, and S1P2. The profibrotic action of activin A was abolished when SK1/2 were inhibited or S1P2/3 were blocked. Finally, S1P augmented by itself mRNA levels of fibrotic markers (fibronectin, collagen 1A1) and activin A in leiomyomas but not in myometrial cells. This study shows that S1P signaling is dysregulated in uterine fibroids and involved in activin A-induced fibrosis, opening new perspectives for uterine fibroid treatment.

A Glimpse of the Structural Biology of the Metabolism of Sphingosine-1-Phosphate.

As a key sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays crucial roles in vascular and immune systems. It regulates angiogenesis, vascular integrity and homeostasis, allergic responses, and lymphocyte trafficking. S1P is interconverted with sphingosine, which is also derived from the deacylation of ceramide. S1P levels and the ratio to ceramide in cells are tightly regulated by its metabolic pathways. Abnormal S1P production causes the occurrence and progression of numerous severe diseases, such as metabolic syndrome, cancers, autoimmune disorders such as multiple sclerosis, and kidney and cardiovascular diseases. In recent years, huge advances on the structure of S1P metabolic pathways have been accomplished. In this review, we have got a glimpse of S1P metabolism through structural and biochemical studies of: sphingosine kinases, S1P transporters and S1P receptors, and the development of therapeutics targeting S1P signaling. The progress we summarize here could provide fresh perspectives to further the exploration of S1P functions and facilitate the development of therapeutic molecules targeting S1P signaling with improved specificity and therapeutic effects.

Erythrocytes efficiently utilize exogenous sphingosines for S1P synthesis and export via Mfsd2b

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein–coupled receptors, designated as S1P1–5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions.

Decreased serum level of sphingosine-1-phosphate: a novel predictor of clinical severity in COVID-19.

The severity of coronavirus disease 2019 (COVID‐19) is a crucial problem in patient treatment and outcome. The aim of this study is to evaluate circulating level of sphingosine‐1‐phosphate (S1P) along with severity markers, in COVID‐19 patients. One hundred eleven COVID‐19 patients and forty‐seven healthy subjects were included. The severity of COVID‐19 was found significantly associated with anemia, lymphocytopenia, and significant increase of neutrophil‐to‐lymphocyte ratio, ferritin, fibrinogen, aminotransferases, lactate dehydrogenase (LDH), C‐reactive protein (CRP), and D‐dimer. Serum S1P level was inversely associated with COVID‐19 severity, being significantly correlated with CRP, LDH, ferritin, and D‐dimer. The decrease in S1P was strongly associated with the number of erythrocytes, the major source of plasma S1P, and both apolipoprotein M and albumin, the major transporters of blood S1P. Not last, S1P was found to be a relevant predictor of admission to an intensive care unit, and patient’s outcome. Circulating S1P emerged as negative biomarker of severity/mortality of COVID‐19 patients. Restoring abnormal S1P levels to a normal range may have the potential to be a therapeutic target in patients with COVID‐19.

Abstract 13667: Programming to S1PR1 + Endothelial Cell Population Promotes the Restoration of Vascular Integrity in vivo

Introduction: Increased endothelial permeability and failure to repair is the hallmark of several vascular diseases including acute lung injury (ALI). However, little is known about the intrinsic pathways that activate the endothelial cell (EC) regenerative programs facilitating thereby tissue repair. Studies have invoked a crucial role of sphingosine-1-phosphate (S1P) in resolving endothelial hyperpermeability through activation of the G-protein coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1). Hypothesis: We postulate that S1PR1 + EC serve as an endogenous means to prevent endothelial injury. Methods: Studies were made using EC-S1PR1 null mice and S1PR1-GFP reporter mice to trace the generation and characteristics of S1PR1 + EC by exploiting immuno-histochemical analysis and FACS. RNA-seq analysis was performed to identify the genetic signature of S1PR1 + EC. Combination of genetic and pharmacological strategies were included for mechanistic study. Transplantation of S1PR1 + EC and edema measurement was performed in EC-S1PR1 null mice. Results: We observed in a mouse model of endotoxemia that LPS via generation of S1P induced the programming of S1PR1 lo EC to S1PR1 + EC, comprising 80% of lung EC. Their generation preceded the vascular repair phase and these cells were required for reestablishing the endothelial barrier function. Thus, conditional deletion of S1PR1 in EC spontaneously increased lung vascular permeability. RNA-seq analysis of S1PR1 + EC showed enrichment of genes regulating S1P synthesis and transport, sphingosine kinase 1 (SPHK1) and SPNS2, respectively, as well as transcription factors EGR1 and STAT3. EGR1 and STAT3 were essential for transcribing SPHK1 and SPNS2, respectively to increase S1P concentration that served to amplify S1PR1 + EC transition. Transplantation of S1PR1 + EC into injured lung vasculature of EC-S1PR1 -/- mice restored endothelial integrity. Conclusions: Findings illustrate that generation of a specialized S1PR1 + EC population has the potential to activate key endothelial regenerative program mediating vascular endothelial repair raising the possibility of activating this pathway to restore vascular homeostasis in inflammatory lung injury.

Modulation of sphingosine 1-phosphate by hepatobiliary cholesterol handling.

Hepatobiliary cholesterol handling, mediated by Niemann-Pick C1-like 1 protein (NPC1L1) and ABCG5/8, is well-known to contribute to the homeostasis of cholesterol. We attempted to elucidate the impact of hepatobiliary cholesterol handling on the homeostasis of sphingolipids and lysophospholipids, especially sphingosine 1-phosphate (S1P). We induced the overexpression of NPC1L1 or ABCG5/8 in the mouse liver. Hepatic NPC1L1 overexpression increased the plasma and hepatic S1P levels, while it decreased the biliary S1P levels, and all of these changes were inhibited by ezetimibe. The ability of HDL to activate Akt in the endothelial cells was augmented by hepatic NPC1L1 overexpression. NPC1L1-mediated S1P transport was confirmed by both in vitro and in vivo studies conducted using C17 S1P, an exogenous S1P analog. Upregulation of apolipoprotein M (apoM) was involved in these modulations, although apoM was not necessary for these modulations. Moreover, the increase in the plasma S1P levels also observed in ABCG5/8-overexpressing mice was dependent on the elevation of the plasma apoM levels. In regard to other sphingolipids and lysophospholipids, ceramides were similarly modulated by NPC1L1 to S1P, while other lipids were differently influenced by NPC1L1 or ABCG5/8 from S1P. Hepatobiliary cholesterol handling might also regulate the functional lipids, such as S1P.

Mfsd2a and Spns2 are essential for sphingosine-1-phosphate transport in the formation and maintenance of the blood-brain barrier.

To maintain brain homeostasis, a unique interface known as the blood-brain barrier (BBB) is formed between the blood circulation and the central nervous system (CNS). Major facilitator superfamily domain-containing 2a (Mfsd2a) is a specific marker of the BBB. However, the mechanism by which Mfsd2a influences the BBB is poorly understood. In this study, we demonstrated that Mfsd2a is essential for sphingosine-1-phosphate (S1P) export from endothelial cells in the brain. We found that Mfsd2a and Spinster homolog 2 (Spns2) form a protein complex to ensure the efficient transport of S1P. Furthermore, the S1P-rich microenvironment in the extracellular matrix (ECM) in the vascular endothelium dominates the formation and maintenance of the BBB. We demonstrated that different concentrations of S1P have different effects on BBB integrity. These findings help to unravel the mechanism by which S1P regulates BBB and also provide previously unidentified insights into the delivery of neurological drugs in the CNS.