Sphingosine-1-phosphate Signaling Research Articles

Abstract 110: The Role Of Sphingosine-1-Phosphate In The Reverse Transendothelial Migration Of Aortic Intimal Myeloid Cells

CD11c + myeloid cells (MCs) reside in the normal aortic arch intima at regions predisposed to atherosclerosis. We showed that reverse transendothelial migration (RTM) of these intimal MCs into the arterial circulation is a protective immune response. RTM is triggered by systemic stimulation of cytokine and toll-like receptors (TLRs), e.g., TLR4 by lipopolysaccharide (LPS), and is inhibited by hypercholesterolemia and associated intimal MC lipid accumulation. We hypothesize that signaling by sphingosine-1-phosphate (S1P) and its receptors (S1PRs) is required for RTM of intimal MCs following systemic stimulation of TLRs, analogous to its role in immune cell trafficking in lymphoid tissues. In the lesser curvature of the aortic arch of wild type C57BL/6 mice, LPS induced the expression of S1P receptors 1 & 3, and sphingosine kinase 1 (SphK1). FTY720, a broad-spectrum S1P receptor modulator, blocked LPS-induced RTM, as determined by the enumeration of CD11c+ MCs by en face immunoconfocal microscopy. S1PR1 or S1PR3 inhibitors blocked RTM in a partial and additive manner, suggesting a role for both S1P receptors in intimal MC migration. Initial experiments show that Cre-mediated deletion of S1PR1 in myeloid cells partially blocks LPS-induced RTM, suggesting that myeloid-specific S1PR1 signaling is implicated in the migratory process. Furthermore, global loss or hematopoietic deficiency of SPHK1 reduced the abundance of CD11c+ MCs in the aortic intima, suggesting an additional role for myeloid S1P production in the homeostatic maintenance of resident aortic immune cells. Cre-mediated deletion of S1PR1 in endothelial cells also reduced the abundance of intimal MCs, providing further evidence that S1P signaling in the intima is important in maintaining the intimal MC population. Thus, functional and expression studies suggest that S1P signaling plays a role in both the maintenance of resident intimal MCs and LPS-induced RTM.

Pharmacological sphingosine-1 phosphate receptor 1 targeting in cigarette smoke-induced emphysema in mice.

Primarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01-1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.

Pharmacological Targeting of Sphingosine Kinases Impedes HIV-1 Infection of CD4 T Cells through SAMHD1 Modulation.

ABSTRACTSphingosine-1-phosphate (S1P) is a sphingolipid modulator of a myriad of cellular processes, and therapeutic targeting of S1P signaling is utilized clinically to treat multiple sclerosis. We have previously shown that functional antagonism of S1P receptors reduces cell-free, cell-to-cell, and latent HIV-1 infection in primary CD4 T cells. In this work, we examined whether targeting sphingosine kinase 1 or 2 (SPHK1/2) to inhibit S1P production would prevent infection using multiple HIV-1 primary isolates and infectious molecular clones. SPHK inhibition reduced HIV transmission between primary CD4 T cells in both cell-to-cell transmission and pretreatment coculture models. Mechanistically, pharmacological inhibition of SPHK reduced susceptibility to infection primarily by downregulating phosphorylated SAMHD1 (pSAMHD1), enhancing the activity of this innate HIV-1 restriction factor. Furthermore, genetic disruption of either SPHK1 or SPHK2 by CRISPR/Cas9 reduced phosphorylation of SAMHD1, demonstrating the role of these kinases in modulation of SAMHD1 activity. The effect of SPHK inhibition on limiting HIV-1 infection in CD4 T cells was observed irrespective of the biological sex or age of the donor, with neither variable significantly influencing the effectiveness of SPHK inhibition. Our results demonstrate that targeting SPHK inhibits transmission of HIV-1 via modulation of SAMHD1 phosphorylation to decrease permissiveness to infection in CD4 T cells and suggests that therapeutic targeting of this pathway early in infection enables development of strategies to prevent establishment of infection and hinder cell-to-cell transmission of HIV-1.IMPORTANCE HIV-1 infection, once established, requires lifelong treatment due to the ability of the virus to maintain latent infection in its host and become reactivated during an interruption in antiretroviral treatment (ART). Although preventing transmission and acquisition of HIV is an important goal, no ART thus far have exploited harnessing a component of the host immune system to combat transmission of the virus. We have previously shown that inhibition of sphingosine-1-phosphate (S1P) receptors, a component of S1P signaling, reduces HIV-1 infection in human CD4 T cells. We therefore investigated inhibition of sphingosine kinases, another element of this signaling system, in this work. We found that inhibition of sphingosine kinases 1 and 2 (SPHK1/2) could reduce HIV-1 transmission, both among CD4 T cells and between macrophages and CD4 T cells. Our research therefore suggests that therapeutic targeting of SPHK or S1P receptors may aid in the development of strategies to prevent establishment and transmission of HIV-1 infection among immune cells.

Sphingosine 1-phosphate pathway is dysregulated in adenomyosis

Research questionIs sphingosine 1-phosphate (S1P) pathway involved in the process of fibrosis in adenomyosis? DesignRNA was extracted from paraffin-embedded slices collected from the ectopic endometrium of patients with nodular adenomyosis (n = 27) and eutopic endometrium of healthy controls women (n = 29). Expression of genes involved in the metabolism and signalling of S1P, and actin-alpha-2 smooth muscle, encoded by ACTA2 gene, a gene involved in fibrogenesis, was evaluated by real-time polymerase chain reaction analysis. ResultsIn adenomyotic samples, the expression of sphingosine kinase 1 (SPHK1), the enzyme responsible for the synthesis of S1P, and of S1P phosphatase 2 (SGPP2), the enzyme responsible for the conversion of S1P back to sphingosine, was lower (P = 0.0006; P = 0.0015), whereas that of calcium and integrin-binding protein 1, responsible for membrane translocation of SPHK1, was higher (P = 0.0001) compared with healthy controls. In S1P signalling, a higher expression of S1P receptor S1P3 (P = 0.001), and a lower expression of S1P2 (P = 0.0019) mRNA levels, were found compared with healthy endometrium. In adenomyotic nodules, a higher expression of ACTA2 mRNA levels were observed (P = 0.0001), which correlated with S1P3 levels (P = 0.0138). ConclusionPresent data show a profound dysregulation of the S1P signalling axis in adenomyosis. This study also highlights that the bioactive sphingolipid might be involved in the fibrotic tract of the disease, correlated with the expression of ACTA2, suggesting its role as novel potential biomarker of adenomyosis.

The atypical sphingosine 1-phosphate variant, d16:1 S1P, mediates CTGF induction via S1P2 activation in renal cell carcinoma.

Sphingosine 1-phosphate (S1P) is a lipid mediator with numerous biological functions. The term 'S1P' mainly refers to the sphingolipid molecule with a long-chain sphingoid base of 18 carbon atoms, d18:1 S1P. The enzyme serine palmitoyltransferase catalyses the first step of the sphingolipid de novo synthesis using palmitoyl-CoA as the main substrate. After further reaction steps, d18:1 S1P is generated. However, also stearyl-CoA or myristoyl-CoA can be utilised by the serine palmitoyltransferase, which at the end of the S1P synthesis pathway, results in the production of d20:1 S1P and d16:1 S1P respectively. We measured these S1P homologues in mice and renal tissue of patients suffering from renal cell carcinoma (RCC). Our experiments highlight the relevance of d16:1 S1P for the induction of connective tissue growth factor (CTGF) in the human renal clear cell carcinoma cell line A498 and human RCC tissue. We show that d16:1 S1P versus d18:1 and d20:1 S1P leads to the highest CTGF induction in A498 cells via S1P2 signalling and that both d16:1 S1P and CTGF levels are elevated in RCC compared to adjacent healthy tissue. Our data indicate that d16:1 S1P modulates conventional S1P signalling by acting as a more potent agonist at the S1P2 receptor than d18:1 S1P. We suggest that elevated plasma levels of d16:1 S1P might play a pro-carcinogenic role in the development of RCC via CTGF induction.

Signaling sphingolipids are biomarkers for atopic dermatitis prone to disseminated viral infections

Life-threatening viral diseases such as eczema herpeticum (EH) and eczema vaccinatum (EV) occur in <5% of individuals with atopic dermatitis (AD). The diagnosis of AD, however, excludes all individuals with AD from smallpox vaccination. We sought to identify circulatory and skin lipid biomarkers associated with EH and EV. Stratum corneum and plasma samples from 15 subjects with AD and a history of EH, 13 age- and gender-matched subjects with AD and without EH history, and 13 healthy nonatopic (NA) controls were analyzed by liquid chromatography tandem mass spectrometry for sphingolipid content. Sphingosine-1-phosphate (S1P) and ceramide levels were validated in plasma samples from the Atopic Dermatitis Vaccinia Network/Atopic Dermatitis Research Network repository (12 NA, 12 AD, 23 EH) and plasma from 7 subjects with EV and 7 matched subjects with AD. S1P lyase was downregulated in human primary keratinocytes to evaluate its effect on herpes simplex virus 1 (HSV-1) replication invitro. The stratum corneum of patients with EH demonstrated significantly higher levels of free sphingoid bases than those in patients who were NA, indicating enhanced sphingolipid turnover in keratinocytes (P< .05). Plasma from 2 independent cohorts of patients with EH had a significantly increased S1P/ceramide ratio in subjects with EH versus those with AD and or who were NA (P< .01). The S1P level in plasma from subjects with EV was twice the level in plasma from subjects with AD (mean= 1,533 vs 732 pmol/mL; P< .001). Downregulation of S1P lyase expression with silencing RNA led to an increased S1P level and doubled HSV-1 titer in keratinocytes. Our data point to long-term abnormalities in the S1P signaling system as a biomarker for previous disseminated viral diseases and a potential treatment target in recurring infections.

S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes.

Background: Adiponectin (Adn), released by adipocytes and other cell types such as skeletal muscle, has insulin-sensitizing and anti-inflammatory properties. Sphingosine 1-phosphate (S1P) is reported to act as effector of diverse biological actions of Adn in different tissues. S1P is a bioactive sphingolipid synthesized by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK) 1 and 2. Consolidated findings support the key role of S1P in the biology of skeletal muscle. Methods and Results: Here we provide experimental evidence that S1P signalling is modulated by globular Adn treatment being able to increase the phosphorylation of SK1/2 as well as the mRNA expression levels of S1P4 in C2C12 myotubes. These findings were confirmed by LC-MS/MS that showed an increase of S1P levels after Adn treatment. Notably, the involvement of S1P axis in Adn action was highlighted since, when SK1 and 2 were inhibited by PF543 and ABC294640 inhibitors, respectively, not only the electrophysiological changes but also the increase of oxygen consumption and of aminoacid levels induced by the hormone, were significantly inhibited. Conclusion: Altogether, these findings show that S1P biosynthesis is necessary for the electrophysiological properties and oxidative metabolism of Adn in skeletal muscle cells.

Sphingosine 1-phosphate receptor 1 governs endothelial barrier function and angiogenesis by upregulating endoglin signaling

BackgroundThe sphingosine 1-phosphate (S1P)/S1P receptor (S1pr) 1 signaling plays an essential role in regulating vascular integrity and angiogenesis. We have previously shown that cell-surface expression of endoglin (Eng) is sustained by S1P/S1pr1 signaling in endothelial cells (ECs). However, whether S1pr1 mediates Eng signaling, or vice versa, remains unknown.MethodsS1pr1 inhibitors were used to study whether pharmacological inhibition induces basal vascular leakage in vivo. An acute respiratory distress syndrome (ARDS) model was used to study whether S1pr1 inhibition evoked greater inflammation in lungs. A S1pr1 inhibitor, a bone morphogenetic protein 9 (BMP9) blocking antibody, or lentivirus-mediated expression of soluble extracellular domain of Eng (sEng) were used to test whether blocking both S1P/S1pr1 and BMP9/Eng signaling axes would impose any interaction in retinal angiogenesis. To clarify whether S1P and BMP9 function in a linear pathway, a study of trans-endothelial electrical resistance (TEER) measurement was carried out using a mouse islet EC line MS1; time course studies were executed to exam downstream effectors of S1P and BMP9 signaling pathways in ECs; two stable MS1 cell lines were generated, one with overexpression of human S1PR1 and the other with knockdown of Eng, to validate S1pr1 and Eng were the key players for the crosstalk. Inhibitor of extracellular regulated protein kinases (ERK) was used to check whether this signaling was involved in S1P-induced cell-surface localization of Eng.ResultsThe present study elucidated that S1pr1 and Eng are both pivotal for angiogenesis in the postnatal mouse retina, and that the activation of S1pr1 or Eng increases vascular barrier function. Activation of S1pr1 enhanced the phosphorylation of Smad family members 1, 5, and 8 (pSmad1/5/8), while the inhibition of S1pr1 reduced the levels of pSma1/5/8 induced by BMP9 treatment. Activation or loss of Eng did not affect S1pr1 signaling. Moreover, activation of ERK was involved in promoting EC-surface expression of Eng by S1pr1.ConclusionsOur data demonstrates for the first time that there exists a linear pathway of S1pr1-Eng signaling axis in ECs, which governs vascular homeostasis. Functional BMP9/Eng signaling requires S1P/S1pr1 activation, and S1pr1 signaling acts as a vascular protection mechanism upstream of Eng.

Targeting Chronic Inflammation of the Digestive System in Cancer Prevention: Modulators of the Bioactive Sphingolipid Sphingosine-1-Phosphate Pathway

Simple SummaryObesity, an ongoing global pandemic, is a major contributor to inflammation and cancers of the digestive system. High saturated fat diets and being overweight are associated with chronic inflammation and increased cancer risk. Signalling molecules made from saturated fats known as bioactive sphingolipids play essential roles in healthy gastrointestinal immunity. In excess, these sphingolipid molecules can compromise our immune system leading to chronic, low-grade inflammation within the digestive system preceding many metabolic diseases including cancer. Sphingosine-1-phosphate is a bioactive sphingolipid and, in excess, contributes to chronic inflammation. Drugs that block sphingosine-1-phosphate activity have the potential to prevent chronic inflammation and reduce gastrointestinal cancer risk. We review how disruption of the sphingosine-1-phosphate pathway contributes to gastrointestinal inflammation and cancer. We also discuss the use of modulators of the sphingospine-1-phosphate pathway in clinical trials and in the clinic as therapeutics for inflammatory gastrointestinal diseases with the benefit of reducing cancer risk.Incidence of gastrointestinal (GI) cancers is increasing, and late-stage diagnosis makes these cancers difficult to treat. Chronic and low-grade inflammation are recognized risks for most GI cancers. The GI mucosal immune system maintains healthy homeostasis and signalling molecules made from saturated fats, bioactive sphingolipids, play essential roles in healthy GI immunity. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid, is a key mediator in a balanced GI immune response. Disruption in the S1P pathway underlies systemic chronic metabolic inflammatory disorders, including diabetes and GI cancers, providing a strong rationale for using modulators of the S1P pathway to treat pathological inflammation. Here, we discuss the effects of bioactive sphingolipids in immune homeostasis with a focus on S1P in chronic low-grade inflammation associated with increased risk of GI carcinogenesis. Contemporary information on S1P signalling involvement in cancers of the digestive system, from top to bottom, is reviewed. Further, we discuss the use of novel S1P receptor modulators currently in clinical trials and their potential as first-line drugs in the clinic for chronic inflammatory diseases. Recently, ozanimod (ZeposiaTM) and etrasimod have been approved for clinical use to treat ulcerative colitis and eosinophilic oesophagitis, respectively, which may have longer term benefits in reducing risk of GI cancers.

Sphingosine Kinase-1 Is Overexpressed and Correlates with Hypoxia in Osteosarcoma: Relationship with Clinicopathological Parameters.

Simple SummaryHypoxia has been recognized as a hallmark of solid tumors and a negative prognostic factor for response to therapeutics and survival of patients. Studies have demonstrated that the Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway regulates the expression of the HIF-1 transcription factor in a number of solid tumor models, but no data are available in osteosarcoma characterized by hypoxia. The objectives of the present study were (i) to assess the contribution of SphK1/S1P signaling in regulating HIF-1α expression under hypoxia in various osteosarcoma cell models, (ii) quantify SphK1 enzymatic activity in biopsies of osteosarcoma, and (iii) examine the relationship between SphK1, S1P receptor 1 (S1P1) and hypoxia (GLUT-1) in 130 cases of osteosarcoma by immunohistochemistry. Our data suggest that the SphK1/S1P signaling might represent a potential target to investigate in osteosarcoma patients, considering that fingolimod, which inhibits SphK1 and the S1P1 receptor, is now reconsidered for repurposing in cancer.The Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway is overexpressed in various cancers, and is instrumental for the adaptation to hypoxia in a number of solid tumor models, but no data are available in osteosarcoma. Here we report that SphK1 and the S1P1 receptor are involved in HIF-1α accumulation in hypoxic osteosarcoma cells. FTY720 (Fingolimod), which targets SphK1 and S1P1, prevented HIF-1α accumulation, and also inhibited cell proliferation in both normoxia and hypoxia unlike conventional chemotherapy. In human biopsies, a significant increase of SphK1 activity was observed in cancer compared with normal bones. In all sets of TMA samples (130 cases of osteosarcoma), immunohistochemical analysis showed the hypoxic marker GLUT-1, SphK1 and S1P1 were expressed in tumors. SphK1 correlated with the GLUT-1 suggesting that SphK1 is overexpressed and correlates with intratumoral hypoxia. No correlation was found between GLUT-1 or SphK1 and response to chemotherapy, but a statistical difference was found with increased S1P1 expression in patients with poor response in long bone osteosarcomas. Importantly, multivariate analyses showed that GLUT-1 was associated with an increased risk of death in flat bone, whereas SphK1 and S1P1 were associated with an increased risk of death in long bones.

Sphingolipid Metabolism and Signaling in Endothelial Cell Functions.

The endothelium, inner layer of blood vessels, constitutes a metabolically active paracrine, endocrine, and autocrine organ, able to sense the neighboring environment and exert a variety of biological functions important to preserve the health of vasculature, tissues, and organs. Sphingolipids are both fundamental structural components of the eukaryotic membranes and signaling molecules regulating a variety of biological functions. Ceramide and sphingosine-1-phosphate (S1P), bioactive sphingolipids, have emerged as important regulators of cardiovascular functions in health and disease. In this review we discuss recent insights into the role of ceramide and S1P biosynthesis and signaling in regulating endothelial cell functions, in health and diseases. We also highlight advances into the mechanisms regulating serine palmitoyltransferase, the first and rate-limiting enzyme of de novo sphingolipid biosynthesis, with an emphasis on its inhibitors, ORMDL and NOGO-B. Understanding the molecular mechanisms regulating the sphingolipid de novo biosynthesis may provide the foundation for therapeutic modulation of this pathway in a variety of conditions, including cardiovascular diseases, associated with derangement of this pathway.

Plasma Sphingosine-1-Phosphate Levels Are Associated with Progression of Estrogen Receptor-Positive Breast Cancer.

Although numerous experiments revealed an essential role of a lipid mediator, sphingosine-1-phosphate (S1P), in breast cancer (BC) progression, the clinical significance of S1P remains unclear due to the difficulty of measuring lipids in patients. The aim of this study was to determine the plasma concentration of S1P in estrogen receptor (ER)-positive BC patients, as well as to investigate its clinical significance. We further explored the possibility of a treatment strategy targeting S1P in ER-positive BC patients by examining the effect of FTY720, a functional antagonist of S1P receptors, on hormone therapy-resistant cells. Plasma S1P levels were significantly higher in patients negative for progesterone receptor (PgR) expression than in those positive for expression (p = 0.003). Plasma S1P levels were also significantly higher in patients with larger tumor size (p = 0.012), lymph node metastasis (p = 0.014), and advanced cancer stage (p = 0.003), suggesting that higher levels of plasma S1P are associated with cancer progression. FTY720 suppressed the viability of not only wildtype MCF-7 cells, but also hormone therapy-resistant MCF-7 cells. Targeting S1P signaling in ER-positive BC appears to be a possible new treatment strategy, even for hormone therapy-resistant patients.

Sphingosine 1‐phosphate (S1P) analogue FTY720 decreases ceramide levels and ameliorates memory performance in AD mice model

AbstractBackgroundNo effective therapeutic drugs were found for Alzheimer’s disease (AD), which focuses on amyloid plaques and neurofibrillary tangles in the past decades. Sphingolipid changes in AD patients and animals are getting more attention, especially the shift between ceramide and S1P in the brain, proposing an essential role in the amyloid‐β formation, neuroinflammation, and neuronal apoptosis in AD pathophysiology. Fingolimod (FTY720) is a drug that resembles sphingosine‐1‐phosphate (S1P) chemical structure and bioactivity. Therefore, we hypothesized that by increasing S1P signaling by means of FTY720, sphingolipid disbalance could be restored in the brain, and memory impairment observed in AD transgenic mouse models would be prevented.MethodTwo doses of FTY720 (0.1 mg/kg and 0.5 mg/kg daily) were given by oral gavage for 3 months to transgenic mouse models of AD on human apolipoprotein (APOE) APOE4 or APOE3 genetic background. During the drug administration, blood was withdrawn at different time points, and animals were challenged with behavioral tests for memory locomotion and anxiety. Brain was then collected for sphingolipids analysis by mass spectrometry and Aβ quantification.ResultSphingolipid analysis in the cortex showed that ceramide subsets elevated in AD mice. In the plasma, ceramides and sphingomyelins overall diminish in concentration over time. The behavioral tests analysis suggested that especially the low dose of FTY720 mildly prevented memory impairment in E4FAD and reduced anxiety behavior in APOE4 mice.ConclusionOn cerebral sphingolipids, Fingolimod shifted the pathway towards reducing ceramide, especially in the cortex. Our data are supportive of targeting the sphingolipid metabolism as a valid pathway for developing new therapeutic drugs for AD.

Sphingosine-1-Phosphate Signaling in Ischemic Stroke: From Bench to Bedside and Beyond.

Sphingosine-1-phosphate (S1P) signaling is being increasingly recognized as a strong modulator of immune cell migration and endothelial function. Fingolimod and other S1P modulators in ischemic stroke treatment have shown promise in emerging experimental models and small-scale clinical trials. In this article, we will review the current knowledge of the role of S1P signaling in brain ischemia from the aspects of inflammation and immune interventions, sustaining endothelial functions, regulation of blood-brain barrier integrity, and functional recovery. We will then discuss the current and future therapeutic perspectives of targeting S1P for the treatment of ischemic stroke. Mechanism studies would help to bridge the gap between preclinical studies and clinical practice. Future success of bench-to-bedside translation shall be based on in depth understanding of S1P signaling during stroke and on the ability to have a fine temporal and spatial regulation of the signal pathway.

Sphingosine-1-phosphate receptor 1 agonist SEW2871 alters membrane properties of late-firing somatostatin expressing neurons in the central lateral amygdala

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide spectrum of biological processes including apoptosis, immune response and inflammation. Here, we sought to understand how S1P signaling affects neuronal excitability in the central amygdala (CeA), which is a brain region associated with fear learning, aversive memory, and the affective dimension of pain. Because the G-protein coupled S1P receptor 1 (S1PR1) has been shown to be the primary mediator of S1P signaling, we utilized S1PR1 agonist SEW2871 and S1PR1 antagonist NIBR to determine a potential role of S1PR1 in altering the cellular physiology of neurons in the lateral division of the CeA (CeL) that share the neuronal lineage marker somatostatin (Sst). CeL-Sst neurons play a critical role in expression of conditioned fear and pain modulation. Here we used transgenic breeding strategies to identify fluorescently labeled CeL-Sst neurons for electrophysiological recordings. Using principal component analysis, we identified two primary subtypes of Sst neurons within the CeL in both male and female mice. We denoted the two types regular-firing (type A) and late-firing (type B) CeL-Sst neurons. In response to SEW2871 application, Type A neurons exhibited increased input resistance, while type B neurons displayed a depolarized resting membrane potential and voltage threshold, increased current threshold, and decreased voltage height. NIBR application had no effect on CeL Sst neurons, indicating the absence of tonic S1P-induced S1PR1. Our findings reveal subtypes of Sst neurons within the CeL that are uniquely affected by S1PR1 activation, which may have implications for how S1P alters supraspinal circuits.

Modulation of Neurolipid Signaling and Specific Lipid Species in the Triple Transgenic Mouse Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia in aging populations. Recently, the regulation of neurolipid-mediated signaling and cerebral lipid species was shown in AD patients. The triple transgenic mouse model (3xTg-AD), harboring βAPPSwe, PS1M146V, and tauP301L transgenes, mimics many critical aspects of AD neuropathology and progressively develops neuropathological markers. Thus, in the present study, 3xTg-AD mice have been used to test the involvement of the neurolipid-based signaling by endocannabinoids (eCB), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (S1P) in relation to the lipid deregulation. [35S]GTPγS autoradiography was used in the presence of specific agonists WIN55,212-2, LPA and CYM5442, to measure the activity mediated by CB1, LPA1, and S1P1 Gi/0 coupled receptors, respectively. Consecutive slides were used to analyze the relative intensities of multiple lipid species by MALDI Mass spectrometry imaging (MSI) with microscopic anatomical resolution. The quantitative analysis of the astrocyte population was performed by immunohistochemistry. CB1 receptor activity was decreased in the amygdala and motor cortex of 3xTg-AD mice, but LPA1 activity was increased in the corpus callosum, motor cortex, hippocampal CA1 area, and striatum. Conversely, S1P1 activity was reduced in hippocampal areas. Moreover, the observed modifications on PC, PA, SM, and PI intensities in different brain areas depend on their fatty acid composition, including decrease of polyunsaturated fatty acid (PUFA) phospholipids and increase of species containing saturated fatty acids (SFA). The regulation of some lipid species in specific brain regions together with the modulation of the eCB, LPA, and S1P signaling in 3xTg-AD mice indicate a neuroprotective adaptation to improve neurotransmission, relieve the myelination dysfunction, and to attenuate astrocyte-mediated neuroinflammation. These results could contribute to identify new therapeutic strategies based on the regulation of the lipid signaling in familial AD patients.

S1P/S1PR1 Signalis Required for Optimal T-Cell Pathogenicity to Induce Gvhd By RegulatingDrp1/mTOR Axis

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative option for the treatment of hematological malignancies, which is primarily mediated by donor immune cells. Acute graft-versus-host disease (aGVHD) mainly induced by transplanted donor T cells is a major and life-threating complication leading to severe “cytokines storm” and multiple organ damage, which contributes to high morbidity and mortality and thus limits the success of allo-HCT. Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is synthesized from sphingosine by sphingosine kinase 1 (Sphk1) or Sphk2 and degraded by S1P lyase. S1P signal plays an important role in regulating biological functions and homeostasis of T lymphocytes, and thus has been considered as a therapeutic candidate against autoimmune disease. In current study, we demonstrated that Sphk1 but not Sphk2 is required for antigen-presenting cells (APC) to activate allogeneic T cells. Using murine allo-HCT models, we found that secretory S1P produced by Sphk1 in the recipients was required for the development of full-blown GVHD (Fig. 1 A-B). Consistently, S1PR1, a primary receptor for S1P, plays a critical role in the pathogenicity of donor T cells to induce GVHD (Fig. 1C). Using pharmacologic inhibitors, we demonstrated that specific inhibition of Sphk1 (PF543) or S1PR1 (W146) substantially attenuated GVHD while preserving graft-vs.-leukemia (GVL) effect (Fig. 1D). Mechanistically, S1P/S1PR1 signal facilitated T-cell activation and differentiation towards Th1/Th17 but away from Tregs (Fig. 1E) and also promoted T-cell migratory potential into GVHD target organs (Fig. 1F). S1P/S1PR1 signaling increased mitochondrial fission of pathogenic CD4 + T cells through PRKAA1 dependent Drp1 and phosphorylated S6 (pS6) activation (Fig. 1 G-H). Whereas CD8 + T cells were much less sensitive to S1P-S1PR1-PRKAA1-pS6/Drp1 axis, which likely contributed to the GVL maintenance when S1P/S1PR1 signaling is absent or inhibited (Fig. 1 D, G). Furthermore, clinical data demonstrated that patients with acute GVHD exhibited a comparable level of sphingosine but a significantly higher level of S1P, as compared to the patients without GVHD, suggesting a positive role of S1P in GVHD development in clinic (Fig. 1I). Finally, we validated the efficacy of inhibiting Sphk1/S1PR1 in GVHD prevention induced by human T cells in a xenograft model (Fig. 1J). Taken together, our results provide a rationale and novel mechanism of targeting Sphk1/S1PR1 in the prevention of GVHD and leukemia relapse after allo-HCT. This novel strategy may be translated in the clinic to benefit patients with hematologic malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Lysophosphatidate Promotes Sphingosine 1-Phosphate Metabolism and Signaling: Implications for Breast Cancer and Doxorubicin Resistance.

Lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) promote vasculogenesis, angiogenesis, and wound healing by activating a plethora of overlapping signaling pathways that stimulate mitogenesis, cell survival, and migration. As such, maladaptive signaling by LPA and S1P have major effects in increasing tumor progression and producing poor patient outcomes after chemotherapy and radiotherapy. Many signaling actions of S1P and LPA are not redundant; each are vital in normal physiology and their metabolisms differ. In the present work, we studied how LPA signaling impacts S1P metabolism and signaling in MDA-MB-231 and MCF-7 breast cancer cells. LPA increased sphingosine kinase-1 (SphK1) synthesis and rapidly activated cytosolic SphK1 through association with membranes. Blocking phospholipase D activity attenuated the LPA-induced activation of SphK1 and the synthesis of ABCC1 and ABCG2 transporters that secrete S1P from cells. This effect was magnified in doxorubicin-resistant MCF-7 cells. LPA also facilitated S1P signaling by increasing mRNA expression for S1P1 receptors. Doxorubicin-resistant MCF-7 cells had increased S1P2 and S1P3 receptor expression and show increased LPA-induced SphK1 activation, increased expression of ABCC1, ABCG2 and greater S1P secretion. Thus, LPA itself and LPA-induced S1P signaling counteract doxorubicin-induced death of MCF-7 cells. We conclude from the present and previous studies that LPA promotes S1P metabolism and signaling to coordinately increase tumor growth and metastasis and decrease the effectiveness of chemotherapy and radiotherapy for breast cancer treatment.

Macrophage Motility in Wound Healing Is Regulated by HIF-1α via S1P Signaling.

Accumulating evidence indicates that the molecular pathways mediating wound healing induce cell migration and localization of cytokines to sites of injury. Macrophages are immune cells that sense and actively respond to disturbances in tissue homeostasis by initiating, and subsequently resolving, inflammation. Hypoxic conditions generated at a wound site also strongly recruit macrophages and affect their function. Hypoxia inducible factor (HIF)-1α is a transcription factor that contributes to both glycolysis and the induction of inflammatory genes, while also being critical for macrophage activation. For the latter, HIF-1α regulates sphingosine 1-phosphate (S1P) to affect the migration, activation, differentiation, and polarization of macrophages. Recently, S1P and HIF-1α have received much attention, and various studies have been performed to investigate their roles in initiating and resolving inflammation via macrophages. It is hypothesized that the HIF-1α/S1P/S1P receptor axis is an important determinant of macrophage function under inflammatory conditions and during disease pathogenesis. Therefore, in this review, biological regulation of monocytes/macrophages in response to circulating HIF-1α is summarized, including signaling by S1P/S1P receptors, which have essential roles in wound healing.