Intracellular Sphingosine-1-phosphate 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.

Inhibition of Sphingosine Kinase 1 Reduces Sphingosine-1-Phosphate and Exacerbates Amyloid-Beta-Induced Neuronal Cell Death in Mixed-Glial-Cell Culture.

In Alzheimer's disease (AD) pathology, the accumulation of amyloid-beta (Aβ), a main component of senile plaques, activates glial cells and causes neuroinflammation. Excessive neuroinflammation results in neuronal dropouts and finally produces the symptoms of AD. Recent studies suggest that disorder in sphingosine-1-phosphate (S1P) metabolism, especially the decreased expression of sphingosine kinase (SK)1, followed by the reduction in the amount of S1P, can be a promotive factor in AD onset. Thus, we explored the possibility that dysregulated S1P metabolism affects AD through the altered function in glial cells. We evaluated the effect of PF-543, a pharmacological inhibitor of SK1, on the inflammatory responses by lipopolysaccharide (LPS)-activated glial cells, microglia, and astrocytes. The treatment with PF-543 decreased the intracellular S1P content in glial cells. The PF-543 treatment enhanced the nitric oxide (NO) production in the LPS-treated neuron/glia mixed culture. Furthermore, we found that the augmented production of NO and reactive oxygen species (ROS) in the PF-543-treated astrocytes affected the microglial inflammatory responses through humoral factors in the experiment using an astrocyte-conditioned medium. The PF-543 treatment also decreased the microglial Aβ uptake and increased the number of injured neurons in the Aβ-treated neuron/glia mixed culture. These results suggest that a decrease in the glial S1P content can exacerbate neuroinflammation and neurodegeneration through altered glial cell functions.

The fate of intracellular S1P regulates lipid droplet turnover and lipotoxicity in pancreatic beta-cells

Lipotoxicity has been considered the main cause of pancreatic beta-cell failure during type 2 diabetes development. Lipid droplets (LD) are believed to regulate the beta-cell sensitivity to free fatty acids (FFA), but the underlying molecular mechanisms are largely unclear. Accumulating evidence points, however, to an important role of intracellular sphingosine-1-phosphate (S1P) metabolism in lipotoxicity-mediated disturbances of beta-cell function. In the present study, we compared the effects of an increased irreversible S1P degradation (S1P-lyase, SPL overexpression) with those associated with an enhanced S1P recycling (overexpression of S1P phosphatase 1, SGPP1) on LD formation and lipotoxicity in rat INS1E beta-cells. Interestingly, although both approaches led to a reduced S1P concentration, they had opposite effects on the susceptibility to FFA. Overexpression of SGPP1 prevented FFA-mediated caspase-3 activation by a mechanism involving an enhanced lipid storage capacity and prevention of oxidative stress. In contrast, SPL overexpression limited LD biogenesis, content, and size, while accelerating lipophagy. This was associated with FFA-induced hydrogen peroxide formation, mitochondrial fragmentation, and dysfunction, as well as ER stress. These changes coincided with the upregulation of proapoptotic ceramides but were independent of lipid peroxidation rate. Also in human EndoC-βH1 beta-cells, suppression of SPL with simultaneous overexpression of SGPP1 led to a similar and even more pronounced LD phenotype as that in INS1E-SGPP1 cells. Thus, intracellular S1P turnover significantly regulates LD content and size and influences beta-cell sensitivity to FFA.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-β signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-β signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1β as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Beneficial Role of Erythrocyte Sphingosine 1-Phosphate in Preventing Renal Macrophage Polarization and Fibrosis through Combating HIF-1α-Dependent Creatine Phosphate Shuttle

Introduction: Erythrocytes, the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and function. Current evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O 2) delivery to counteract tissue hypoxia. Aim: To understand whether O 2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization and fibrosis. Methods and Results: Genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte, eSphK1 -/-) resulted in less O 2 delivery from erythrocytes, thus leading to severe renal hypoxia, damage and fibrosis in angiotensin II induced hypertensive kidney disease mouse model. Surprisingly, although plasma S1P level was reduced to 50% in Ang II-infused eSphK1 -/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules. Surprisingly, there was no significant difference of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1 -/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization. Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. We further validate this in vitro by 13C 6, 15N 4-labeled arginine tracer. Hypoxia (1~2% O 2) promoted increased 13C 2-labeled creatine (arginine downstream metabolite) in murine bone marrow derived macrophages (BMDMs) time dependently. At cellular and molecular level, we further demonstrated that hypoxia signaling via HIF-1a directly enhanced IL-4 induced M2 polarization of BMDMs as well as expression of Ckb (a gene encoding creatine shuttle), which can be reversed by Chrysin (an HIF-1α inhibitor). Preclinical studies revealed that chrysin rescued the severe renal damage in eSphK1 -/-mice manipulated with unilateral ureteral obstruction (UUO) and prevented development of chronic kidney disease (CKD) in the control mice. Translational research validated mouse findings in kidney tissue of CKD patients. Conclusion: Our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, renal macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and renal fibrosis.

Sphingosine 1-phosphate turnover in GtoPdb v.2023.1

S1P (sphingosine 1-phosphate) is a bioactive lipid which, after release from cells via certain transporters, acts as a ligand for a family of five S1P-specific G protein-coupled receptors (S1P1-5). However, it also has a number of intracellular targets. S1P is formed by the ATP-dependent phosphorylation of sphingosine, catalysed by two isoforms of sphingosine kinase (EC 2.7.1.91). It can be dephosphorylated back to sphingosine by sphingosine 1-phosphate phosphatase (EC 3.1.3) or cleaved into phosphoethanolamine and hexadecenal by sphingosine 1-phosphate lyase (EC 4.1.2.27). Recessive mutations in the S1P lyase (SPL) gene underlie a recently identified sphingolipidosis: SPL Insufficiency Syndrome (SPLIS). In general, S1P promotes cell survival, proliferation, migration, adhesion and inhibition of apoptosis. Intracellular S1P affects epigenetic regulation, endosomal processing, mitochondrial function and cell proliferation/senescence. S1P has myriad physiological functions, including vascular development, lymphocyte trafficking and neurogenesis. However, S1P is also involved in a number of diseases such as cancer, inflammation and fibrosis. Therefore, its GPCRs and enzymes of synthesis and degradation are a major focus for drug discovery.

Sphingosine-1-phosphate regulation of luteinising hormone-induced steroidogenesis and proliferation of bovine theca cells in vitro.

Sphingosine-1-phosphate (S1P) is synthesised by follicle granulosa cells under the influence of follicle-stimulating hormone and seems to be necessary for the biological effects of this gonadotrophin. To determine if luteinising hormone (LH) increases S1P production and if this sphingolipid, either induced by LH or added to culture media, regulates steroidogenesis and cell viability in bovine theca cells. We used bovine theca cell cultures treated with: S1P (0, 0.1, 1 and 10μM; Experiment 1), LH (0, 0.02, 0.2 and 2ngmL-1; Experiment 2) and LH (0.02ngmL-1) plus a sphingosine kinase inhibitor (SKI-178; 0, 5 and 10μM; Experiment 3). Treatment with S1P did not affect (P>0.05) theca cell viability or their ability to produce progesterone and testosterone. LH (0.02ngmL-1) increased (P<0.05) S1P production, and stimulated the expression of phosphorylated sphingosine kinase-1 (pSPHK1). However, the inhibition of SPHK1, by a specific SPHK1 inhibitor (SKI-178), reduced (P<0.05) cell viability and progesterone secretion. Additionally, the use of SKI-178 increased theca cell testosterone production (P<0.05). S1P added to culture media did not affect cell viability or steroid synthesis. However, LH stimulated the production of S1P, by increasing phosphorylation of SPHK1 in theca cells. This intracellular S1P was inhibitory on testosterone production but augmented progesterone and viable cell number. These results suggest a novel signalling pathway for LH in theca cells and underline the importance of S1P in the regulation of steroid synthesis.

Association of gut microbiota in the pathogenesis of acute respiratory distress syndrome and possible pharmacotherapy by sphingosine-1-phosphate

The Acute Respiratory Distress Syndrome (ARDS) is one of the major heterogeneous clinical manifestations of severe respiratory failure developing in response to pathogenetic mechanisms and several inflammatory insults. The translocation of gut microbiota has a crucial impact on the pathogenesis of ARDS. Hence, a deeper understanding of the interplay of the gut microbiota would allow shedding valuable insights into both the pathogenesis of ARDS and the development of effective therapeutic interventions. Moreover, the modulation of gut-lung axis could also play a pivotal role in minimizing the lung dysbiosis with gut microbiota. There is little question that greater study of the gut-lung axis in critically ill patients is required to establish causal links between the shifted microbiota, infections, inflammation, and acute lung damage. It is worth mentioning that the lack of effective preventative measures is one of the main reasons for the increased mortality rate of 30-40% in ARDS patients. Some antibiotics and nanosized drugs showed positive results in ARDS management to some extent in pre-clinical or even first stages of clinical trials but large-scale results had been controversial. However, Sphingosine-1-Phosphate (S1P) showed hopeful results in ARDS patients by facilitating both systemic and endothelial integrity. Numerous investigations have shown the immunological connection between the gastrointestinal tract and the respiratory system. As the SphKs /S1P /S1PL metabolic pathway is associated with a wide variety of human illnesses (including respiratory diseases), it should come as no surprise that influencing intracellular S1P levels would have therapeutic promise in reducing the severity of lung diseases like ARDS.

Erythrocyte Sphingosine 1 Phosphate Is Essential to Combat HIF1α-Dependent Creatine Shuttle, Macrophage Polarization and Fibrosis

Erythrocytes have long been considered as the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), which is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and polarization of tissue macrophages. Currently, emerging evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O2) delivery to counteract tissue hypoxia. However, whether O2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization, inflammation and fibrosis remains unrecognized. Here we report that genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte eSphK1-/-) resulted in less O2 delivery from erythrocytes, thus leading to severe hypoxic tissue microenvironment, kidney damage and fibrosis in a well-accepted pathological hypoxic model infused with angiotensin II (a potent vasoconstrictor known to be elevated in hypertensive patients). Surprisingly, although plasma S1P levels were reduced to 50% in Ang II-infused eSphK1-/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules (one of the most hypoxic sites with Ang II infusion). Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. Further studies demonstrated that no significant differences of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1-/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization in Ang II-infused eSphK1-/- mice. Instead, we revealed that reduced O2 offload from erythrocytes was previously unrecognized but essential to promote tissue M2 polarization and fibrosis by upregulating creatine shuttle to meet rapid intracellular energy needs when FAs, AAs and O2 were insufficient due to hypoxia in Ang II-infused eSphK1-/- mice. At cellular and molecular level, we demonstrated that hypoxia signaling via HIF-1a directly induced M2 polarization as well as expression of Slc6a8 (a gene encoding creatine transport), M2 specific marker gene and fibrotic marker gene in cultured murine BMDMs. Overall, our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, tissue macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and tissue fibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

New mechanism for mesenchymal stem cell microvesicle to restore lung permeability: intracellular S1P signaling pathway independent of S1P receptor-1

BackgroundMicrovesicles (MVs) derived from human bone marrow mesenchymal stem cell (MSC) were demonstrated to restore lung protein permeability and attenuate acute lung injury. In our previous study, we found that MSC MV increased sphingosine-1-phosphate (S1P) kinase1 mRNA levels in injured human lung microvascular endothelial cells (HLMVEC) significantly. However, the role of S1P signaling in MSC MV to restore lung protein permeability is unknown.MethodsIn this study, we hypothesized that MSC MV might restore lung permeability in part through increasing intracellular S1P signaling pathway in injured HLMVEC independent of S1P receptors. We used the transwell co-culture system to study the effect of MSC MV on protein permeability of Lipopolysaccharide (LPS) damaged HLMVEC.ResultsOur results showed that LPS significantly increased the permeability of HLMVEC to FITC-dextran (70 kDa) within 24 h. MSC MV restores this permeability and, to a large extent, prevents the cytoskeleton protein F-actin from recombining into “actin stress fibers,” and restores the positions of tight junctions and adhesion junctions in the damaged HLMVEC. This therapeutic effect of MSC MV was related to the increase in the S1P level in injured HLMVEC and was not eliminated when adding the antagonist of S1P receptor, suggesting that MSC MV to restore lung permeability was independent of S1P receptors on HLMVEC. Laser confocal further observed that Ca2+ mobilization and Rac1 activation in LPS injured HLMVEC were increased in parallel with the increase in intracellular S1P level after MSC MV treatment.ConclusionsIn short, MSC MV partially restored protein permeability across HLMVEC through the intracellular S1P signaling pathway independent of S1P receptor-1.

ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response

The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1’s kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL’s enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR–like ER kinase upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of ER and mitochondria to ER stress in mammalian cells.

Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood. To identify the genes within this S1P uptake and recycling pathway, we performed a genome-wide CRISPR/Cas9 KO screen using a positive-selection scheme with Shiga toxin, which binds a cell-surface glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes lethality upon internalization. The screen was performed in HeLa cells with their sphingolipid de novo pathway disabled so that Gb3 cell-surface expression was dependent on salvage of the sphingoid base of S1P taken up from the medium. The screen identified a suite of genes necessary for S1P uptake and the recycling of its sphingoid base to synthesize Gb3, including two lipid phosphatases, PLPP3 (phospholipid phosphatase 3) and SGPP1 (S1P phosphatase 1). The results delineate a pathway in which plasma membrane–bound PLPP3 dephosphorylates extracellular S1P to sphingosine, which then enters cells and is rephosphorylated to S1P by the sphingosine kinases. This rephosphorylation step is important to regenerate intracellular S1P as a branch-point substrate that can be routed either for dephosphorylation to salvage sphingosine for recycling into complex sphingolipid synthesis or for degradation to remove it from the sphingolipid synthesis pathway.

To Be or Not to Be: The Divergent Action and Metabolism of Sphingosine-1 Phosphate in Pancreatic Beta-Cells in Response to Cytokines and Fatty Acids.

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid with multiple functions conveyed by the activation of cell surface receptors and/or intracellular mediators. A growing body of evidence indicates its important role in pancreatic insulin-secreting beta-cells that are necessary for maintenance of glucose homeostasis. The dysfunction and/or death of beta-cells lead to diabetes development. Diabetes is a serious public health burden with incidence growing rapidly in recent decades. The two major types of diabetes are the autoimmune-mediated type 1 diabetes (T1DM) and the metabolic stress-related type 2 diabetes (T2DM). Despite many differences in the development, both types of diabetes are characterized by chronic hyperglycemia and inflammation. The inflammatory component of diabetes remains under-characterized. Recent years have brought new insights into the possible mechanism involved in the increased inflammatory response, suggesting that environmental factors such as a westernized diet may participate in this process. Dietary lipids, particularly palmitate, are substrates for the biosynthesis of bioactive sphingolipids. Disturbed serum sphingolipid profiles were observed in both T1DM and T2DM patients. Many polymorphisms were identified in genes encoding enzymes of the sphingolipid pathway, including sphingosine kinase 2 (SK2), the S1P generating enzyme which is highly expressed in beta-cells. Proinflammatory cytokines and free fatty acids have been shown to modulate the expression and activity of S1P-generating and S1P-catabolizing enzymes. In this review, the similarities and differences in the action of extracellular and intracellular S1P in beta-cells exposed to cytokines or free fatty acids will be identified and the outlook for future research will be discussed.

Therapeutic CFTR Correction Normalizes Systemic and Lung-Specific S1P Level Alterations Associated with Heart Failure.

Heart failure (HF) is among the main causes of death worldwide. Alterations of sphingosine-1-phosphate (S1P) signaling have been linked to HF as well as to target organ damage that is often associated with HF. S1P’s availability is controlled by the cystic fibrosis transmembrane regulator (CFTR), which acts as a critical bottleneck for intracellular S1P degradation. HF induces CFTR downregulation in cells, tissues and organs, including the lung. Whether CFTR alterations during HF also affect systemic and tissue-specific S1P concentrations has not been investigated. Here, we set out to study the relationship between S1P and CFTR expression in the HF lung. Mice with HF, induced by myocardial infarction, were treated with the CFTR corrector compound C18 starting ten weeks post-myocardial infarction for two consecutive weeks. CFTR expression, S1P concentrations, and immune cell frequencies were determined in vehicle- and C18-treated HF mice and sham controls using Western blotting, flow cytometry, mass spectrometry, and qPCR. HF led to decreased pulmonary CFTR expression, which was accompanied by elevated S1P concentrations and a pro-inflammatory state in the lungs. Systemically, HF associated with higher S1P plasma levels compared to sham-operated controls and presented with higher S1P receptor 1-positive immune cells in the spleen. CFTR correction with C18 attenuated the HF-associated alterations in pulmonary CFTR expression and, hence, led to lower pulmonary S1P levels, which was accompanied by reduced lung inflammation. Collectively, these data suggest an important role for the CFTR-S1P axis in HF-mediated systemic and pulmonary inflammation.

Sphingosine-1-phosphate and Sphingosine-1-phosphate receptors in the cardiovascular system: pharmacology and clinical implications.

Sphingosine-1-phosphate (S1P) is a lipid that binds and activates five distinct receptor subtypes, S1P1, S1P2, S1P3, S1P4, S1P5, widely expressed in different cells, tissues and organs. In the cardiovascular system these receptors have been extensively studied, but no drug acting on them has been approved so far for treating cardiovascular diseases. In contrast, a number of S1P receptor agonists are approved as immunomodulators, mainly for multiple sclerosis, because of their action on lymphocyte trafficking. This chapter summarizes the available information on S1P receptors in the cardiovascular system and discusses their potential for treating cardiovascular conditions and/or their role on the clinical pharmacology of drugs so far approved for non-cardiovascular conditions. Basic research has recently produced data useful to understand the molecular pharmacology of S1P and S1P receptors, regarding biased agonism, S1P storage, release and vehiculation and chaperoning by lipoproteins, paracrine actions, intracellular non-receptorial S1P actions. On the other hand, the approval of fingolimod and newer generation S1P receptor ligands as immunomodulators, provides information on a number of clinical observations on the impact of these drugs on cardiovascular system which need to be integrated with preclinical data. S1P receptors are potential targets for prevention and treatment of major cardiovascular conditions, including hypertension, myocardial infarction, heart failure and stroke.

Erythrocytes increase endogenous sphingosine 1-phosphate levels as an adaptive response to SARS-CoV-2 infection.

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Uncovering the 'sphinx' of sphingosine 1-phosphate signalling: from cellular events to organ morphogenesis.

ABSTRACTSphingosine 1‐phosphate (S1P) is a bioactive sphingolipid metabolite, functioning as a signalling molecule in diverse cellular processes. Over the past few decades, studies of S1P signalling have revealed that the physiological activity of S1P largely depends on S1P metabolizing enzymes, transporters and receptors on the plasma membrane, as well as on the intracellular proteins that S1P binds directly to. In addition to its roles in cancer signalling, immunity and inflammation, a large body of evidence has identified a close link of S1P signalling with organ morphogenesis. Here we discuss the vital role of S1P signalling in orchestrating various cellular events during organ morphogenesis through analysing each component along the extracellular and intracellular S1P signalling axes. For each component, we review advances in our understanding of S1P signalling and function from the upstream regulators to the downstream effectors and from cellular behaviours to tissue organization, primarily in the context of morphogenetic mechanisms. S1P‐mediated vesicular trafficking is also discussed as a function independent of its signalling function. A picture emerges that reveals a multifaceted role of S1P‐dependent pathways in the development and maintenance of organ structure and function.

The Role of Sphingomyelin Metabolism in the Protection of Rat Brain Microvascular Endothelial Cells by Mild Hypothermia.

Sphingomyelin, composed of ceramide (CER), sphingosine (Sph), and sphingosine-1-phosphate (S1P), is an essential structural component of cellular membranes and plays an important role in the signal transduction regulating cell proliferation, differentiation, and apoptosis. CER is mainly metabolized to Sph, and under the action of sphingosine kinases (SphKs), Sph produces S1P, which can be converted back to Sph by S1P phosphatase. It is suggested that the fate of cells is controlled partly by the interconversion of CER and intracellular S1P. SphK2 is considered the main kinase of S1P synthesis in the central nervous system. The objective of this study was to explore the hypothesis that SphK2 and sphingomyelin metabolism participated in the process of cell apoptosis and the protection of mild hypothermia. Rat brain microvascular endothelial cells were divided into groups for intervention of SphK2 inhibitor, SphK2 small interfering RNA (SiRNA) transfection, ischemia-reperfusion, and mild hypothermia. After interventions, cell apoptosis was detected by 4,6-diamino-2-phenyl indole (DAPI) and flow cytometry, the expression of apoptosis-related protein was detected by Western Blot, and SphK2 enzyme activity and the content of sphingomyelin were determined. ABC294640 and transfection of SphK2 SiRNA could increase apoptosis, accompanied by the increase of the expression of proapoptotic genes Caspase3 and Bax and the decrease of the expression of BCL-2. This effect could be partially reversed with mild hypothermia. Ischemia-reperfusion injury, transfection of SphK2 SiRNA, and the addition of ABC294640 could significantly inhibit the activity of SphK2, accompanied by the increase of CERs and the decrease of S1P. Mild hypothermia could reverse the changes of sphingolipids but have no significant effect on the activity of sphk2. Mild hypothermia can inhibit the occurrence of apoptosis and reverse the changes of apoptosis-related genes and sphingomyelin content induced by ischemia-reperfusion injury, but the effect on sphk2 enzyme activity was not significant.

Sgpl1 deletion elevates S1P levels, contributing to NPR2 inactivity and p21 expression that block germ cell development

Sphingosine phosphate lyase 1 (SGPL1) is a highly conserved enzyme that irreversibly degrades sphingosine-1-phosphate (S1P). Sgpl1-knockout mice fail to develop germ cells, resulting in infertility. However, the molecular mechanism remains unclear. The results of the present study showed that SGPL1 was expressed mainly in granulosa cells, Leydig cells, spermatocytes, and round spermatids. Sgpl1 deletion led to S1P accumulation in the gonads. In the ovary, S1P decreased natriuretic peptide receptor 2 (NPR2) activity in granulosa cells and inhibited early follicle growth. In the testis, S1P increased the levels of cyclin-dependent kinase inhibitor 1A (p21) and apoptosis in Leydig cells, thus resulting in spermatogenesis arrest. These results indicate that Sgpl1 deletion increases intracellular S1P levels, resulting in the arrest of female and male germ cell development via different signaling pathways.

Sphingosine-1-phosphate as a key player of insulin secretion induced by high-density lipoprotein treatment.

Beta cell failure is one of the most important features of type 2 diabetes mellitus (T2DM). High‐density lipoprotein (HDL) has been proposed to improve β‐cell function. However, the mechanisms involved in this process are still poorly understood. The aim of this study was to investigate the contribution of sphingosine‐1‐phosphate (S1P) in the impact of HDL treatment on insulin secretion by pancreatic β‐cells and to determine its mechanisms. Primary cultures of β‐cells isolated from rat were treated with or without HDL in the presence or absence of S1P pathway inhibitors and insulin secretion response was analyzed. The S1P content of HDL (HDL‐S1P) isolated from T2DM patients was analyzed and correlated to the HDL‐induced insulin secretion. The expression of genes involved in the biosynthesis of the insulin was also evaluated. HDL as well as S1P treatment enhanced glucose‐stimulated insulin secretion (GSIS). In HDL isolated from T2DM patients, while HDL‐S1P was strongly correlated to its pro‐secretory capacity (r = 0.633, p = 0.005), HDL‐cholesterol and apolipoprotein AI levels were not. HDL‐induced GSIS was blocked by the S1P1/3 antagonist but not by the S1P2 antagonist, and was also accompanied by increased intracellular S1P in β‐cells. We also observed that HDL improved GSIS without significant changes in expression levels of insulin biosynthesis genes. Our present study highlights the importance HDL‐S1P in GSIS in T2DM patients and demonstrates that HDL induces insulin secretion by a process involving both intra‐ and extra‐cellular sources of S1P independently of an effect on insulin biosynthesis genes.