Sphingosine-1-phosphate Signaling Research Articles

Role of Sphingosine-1-Phosphate (S1P) Signaling in Diabetes-Induced Cognitive Decline in Wistar Rats

Background and objective: The study of the S1P signaling pathway in the context of diabetes offers a novel perspective for understanding the mechanisms underlying cognitive decline and developing new therapeutic strategies. Material and methods: The study involved 24 Wistar rats, divided into three groups: group 1 was given distilled water and served as a control group; group 2 was made up of rats made diabetic and given distilled water; group 3 was made up of rats made diabetic and treated with D-erythro-dihydrosphingosine (SPK1 and SPK2 inhibitor). Behavioral tests included object recognition and maze tests, and Rt-PCR was used to assess sphingosine-1-phosphate expression. Results: The study revealed significant differences in weight between rats, with a notable decrease observed from day 7 onwards. It also revealed significant differences in the time spent exploring objects and the mental influence of diabetes on the acquisition of spatial knowledge. The study also revealed that diabetes affects working memory and inhibits S1P expression in the hippocampal region. Conclusion: The study reveals significant differences in exploratory behavior and memory capacity between diabetic and normal rats, highlighting the negative effects of diabetes on memory.

TMIC-57. EFFECT OF HYPOXIA ON SPHINGOSINE-1-PHOSPHATE (S1P) METABOLIZING ENZYMES IN DIFFUSE INTRINSIC PONTINE GLIOMA CELL LINES

Abstract Diffuse Midline Glioma H3K27 altered (DMG) is a devastating and incurable paediatric brain tumour. Previously a bioinformatic approach was taken to evaluate genes involved in the sphingosine-1-phosphate (S1P) biosynthesis pathway in diffuse intrinsic pontine gliomas (DIPG). S1P is known to interact with epigenetic partners (HDAC1) as well as play a role in proliferation, immune cell trafficking, angiogenesis and apoptosis. As a part of a broad bioinformatic approach evaluating the expression of genes associated with the sphingomyelin (SM) biosynthesis pathway, S1P metabolism, S1P receptors and downstream targets, 14 of 25 genes were found to be dysregulated in DIPG relative to normal brain (p<0.0002). Herein we focus on the imbalance of the S1P metabolic enzymes sphingosine kinase 2 (SPHK2) and sphingosine-1-phosphate lyase (SGPL1). Bioinformatic data revealed higher levels of SGPL1 and lower levels of SPHK2 compared with normal brain. DIPG cell lines (SU-DIPG-IV, VUMC-DIPG-08, VUMC-DIPG-A) were used to validate gene and protein expression and to evaluate changes following exposure to hypoxic conditions. Confocal images reveal an increase in nuclear SPHK2 staining in cells cultured for 24- and 48-hours under hypoxic conditions. HDAC1 immunostaining was localised in the nucleus. SGPL1 immunostaining, while mostly cytoplasmic, nuclear staining was observed. qRT-PCR assays were used to evaluate changes in mRNA expression in SPHK2, SGPL1 and HDAC1 in DIPG cell lines incubated under hypoxic conditions. The Pfaffl method was used to analyse gene expression relative to the housekeeping gene TBP. Under hypoxic conditions, the levels of SPHK2 and HDAC1 were higher in DIPG cells cultured in hypoxic conditions for 48 hours compared to DIPG cells cultured in normoxic conditions, whereas SGPL1 expression was lower in hypoxic treated cells. Current experiments targeting the modulation of these S1P-metabolizing enzymes are underway to determine the role of the S1P signalling pathway and determine the potential advantage of targeting this pathway in DIPG.

Role of Sphingosine-1-Phosphate Signaling Pathway in Pancreatic Diseases.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolic product produced via the phosphorylation of sphingosine by sphingosine kinases (SPHKs), serving as a powerful modulator of various cellular processes through its interaction with S1P receptors (S1PRs). Currently, this incompletely understood mechanism in pancreatic diseases including pancreatitis and pancreatic cancer, largely limits therapeutic options for these disorders. Recent evidence indicates that S1P significantly contributes to pancreatic diseases by modulating inflammation, promoting pyroptosis in pancreatic acinar cells, regulating the activation of pancreatic stellate cells, and affecting organelle functions in pancreatic cancer cells. Nevertheless, no review has encapsulated these advancements. Thus, this review compiles information about the involvement of S1P signaling in exocrine pancreatic disorders, including acute pancreatitis, chronic pancreatitis, and pancreatic cancer, as well as prospective treatment strategies to target S1P signaling for these conditions. The insights presented here possess the potential to offer valuable guidance for the implementation of therapies targeting S1P signaling in various pancreatic diseases.

Sphingosine-1-Phosphate Signalling Inhibition Suppresses Th1-Like Treg Generation by Reversing Mitochondrial Uncoupling.

Inflammatory environments induce the generation of dysfunctional IFNγ+T-bet+FOXP3+ Th1-like Tregs, which show defective function and are found in autoimmune conditions including multiple sclerosis (MS). The pathways that control the generation of Th1-like Tregs are not well understood. Sphingosine-1-phosphate (S1P) signalling molecules are upregulated in Th1-like Tregs, and invivo S1P inhibition with Fingolimod (FTY720) inhibits the expression of genes responsible for Treg plasticity in MS patients. However, the underlying mechanisms are unknown. Here we show that S1P signalling inhibition by FTY720 inhibits the generation of Th1-like Tregs and rescues their suppressive function. These effects are mediated by a decrease in mTORC1 signalling and reversal of the mitochondrial uncoupling that Tregs undergo during their reprogramming into Th1-like Tregs invitro. Finally, these results are validated in invivo-generated Th1-like Tregs, as Tregs from MS patients treated with FTY720 display decreased Th1-like Treg frequency, increased suppressive function and mitochondrial metabolism rebalance. These results highlight the involvement of mitochondrial uncoupling in Treg reprogramming and identify S1P signalling inhibition as a target to suppress the generation of dysfunctional Th1-like Tregs.

Targeting Sphingosine-1-Phosphate Signaling to Prevent the Progression of Aortic Valve Disease.

Aortic valve disease (AVD) is associated with high mortality and morbidity. To date, there is no pharmacological therapy available to prevent AVD progression. Because valve calcification is the hallmark of AVD and S1P (sphingosine-1-phosphate) plays an important role in osteogenic signaling, we examined the role of S1P signaling in aortic stenosis disease. AVD progression and its consequences for cardiac function were examined in a murine wire injury-induced AVD model with and without pharmacological and genetic modulation of S1P production, degradation, and receptor signaling. S1P was measured by LC-MS. Calcification of valvular interstitial cells and their response to biomechanical stress were analyzed in the context of S1P signaling. Human explanted aortic valves from patients undergoing aortic valve replacement and cardiovascular magnetic resonance imaging were analyzed for S1P by LC-MS. Raising S1P concentrations in mice with injury-induced AVD by pharmacological inhibition of its sole degrading enzyme S1P lyase vastly enhanced AVD progression and impaired cardiac function resembling human disease. In contrast, low S1P levels caused by SphK1 (sphingosine kinase 1) deficiency potently attenuated AVD progression. We found S1P/S1PR2 (S1P receptor 2) signaling to be responsible for the adverse S1P effect because S1PR2-deficient mice were protected against AVD progression and its deterioration by high S1P. It is important to note that pharmacological S1PR2 inhibition administered after wire injury successfully prevented AVD development. Mechanistically, biomechanical stretch stimulated S1P production by SphK1 in human valvular interstitial cells as measured by C17-S1P generation, whereas S1P/S1PR2 signaling induced their osteoblastic differentiation and calcification through osteogenic RUNX2/OPG signaling and the GSK3β-Wnt-β-catenin pathway. In patients with AVD, stenotic valves exposed to high wall shear stress had higher S1P content and increased SphK1 expression. Increased systemic or local S1P levels lead to increased valvular calcification. S1PR2 antagonists and SphK1 inhibitors may offer feasible pharmacological approaches to human AVD in prophylactic, disease-modifying or relapse-preventing manners.

Sphingosine-1-phosphate signaling regulates the ability of Müller glia to become neurogenic, proliferating progenitor-like cells.

The purpose of these studies is to investigate how Sphingosine-1-phosphate (S1P) signaling regulates glial phenotype, dedifferentiation of Müller glia (MG), reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs. We found that S1P-related genes are highly expressed by retinal neurons and glia, and levels of expression were dynamically regulated following retinal damage. S1PR1 is highly expressed by resting MG and is rapidly downregulated following acute retinal damage. Drug treatments that activate S1PR1 or increase levels of S1P suppressed the formation of MGPCs, whereas treatments that inhibit S1PR1 or decreased levels of S1P stimulated the formation of MGPCs. Inhibition of S1PR1 or SPHK1 significantly enhanced the neuronal differentiation of the progeny of MGPCs. Further, ablation of microglia from the retina, wherein the formation of MGPCs in damaged retinas is impaired, has a significant impact upon expression patterns of S1P-related genes in MG. Inhibition of S1PR1 and SPHK1 partially rescued the formation of MGPCs in damaged retinas missing microglia. Finally, we show that TGFβ/Smad3 signaling in the resting retina maintains S1PR1 expression in MG. We conclude that the S1P signaling is dynamically regulated in MG and MGPCs and activation of S1P signaling depends, in part, on signals produced by reactive microglia.

Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans-Golgi network

The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional glycoprotein not only play roles in IGF-II degradation and pro-TGFβ activation but binding to and transport M6P-bearing lysosomal enzymes from the trans-Golgi network (TGN) or the cell surface to lysosomes. At present, information regarding a retrograde transport of IGF-II/M6P receptor from endosomes to the TGN is still limited. We show here that a continuous ligand-dependent activation of sphingosine 1-phosphate receptor type 3 (S1P3R) on the endosomal membranes is required for subsequent recycling back of cargo-unloaded IGF-II/M6P receptors to the TGN. We have further clarified that Gq coupled with S1P3R plays a critical role in the activation of casein kinase 2, which phosphorylates and keeps PACS1 connector protein active for the association with IGF-II/M6P receptors, which enables transport carrier formation with the aid of other adaptor proteins toward the TGN. These findings shed light on the molecular mechanism underlying how continuous activation of the S1P receptor and subsequent downstream Gq signaling regulates the retrograde transport of the empty IGF-II/M6P receptors back to the TGN.

Deciphering the role of sphingosine 1-phosphate in central nervous system myelination and repair.

Sphingosine 1-phosphate (S1P) is a bioactive lipid of the sphingolipid family and plays a pivotal role in the mammalian nervous system. Indeed, S1P is a therapeutic target for treating demyelinating diseases such as multiple sclerosis. Being part of an interconnected sphingolipid metabolic network, the amount of S1P available for signalling is equilibrated between its synthetic (sphingosine kinases 1 and 2) and degradative (sphingosine 1-phosphate lyase) enzymes. Once produced, S1P exerts its biological roles via signalling to a family of five G protein-coupled S1P receptors 1-5 (S1PR1-5). Despite significant progress, the precise roles that S1P metabolism and downstream signalling play in regulating myelin formation and repair remain largely opaque and somewhat controversial. Genetic or pharmacological studies adopting various model systems identify that stimulating S1P-S1PR signalling protects myelin-forming oligodendrocytes after central nervous system (CNS) injury and attenuates demyelination invivo. However, evidence to support its role in remyelination of the mammalian CNS is limited, although blocking S1P synthesis sheds light on the role of endogenous S1P in promoting CNS remyelination. This review focuses on summarising the current understanding of S1P in CNS myelin formation and repair, discussing the complexity of S1P-S1PR interaction and the underlying mechanism by which S1P biosynthesis and signalling regulates oligodendrocyte myelination in the healthy and injured mammalian CNS, raising new questions for future investigation.

Zonation and ligand and dose dependence of sphingosine 1-phosphate receptor-1 signalling in blood and lymphatic vasculature.

Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for the endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet, as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC autonomous S1P production, it is unclear if relative reductions in circulating S1P can cause endothelial dysfunction. It is also unclear how EC S1PR1 insufficiency, whether induced by deficiency in circulating ligand or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. We here fine map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell-type-specific and relative deficiencies in S1P production to define ligand source and dose dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries, and a subset of high-endothelial venules (HEVs). Similar zonation was observed for albumin extravasation in EC S1PR1-deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic ECs, S1PR1 engagement was high in collecting vessels and lymph nodes and low in blind-ended capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signalling in lymphatics and HEV, haematopoietic cells provided ∼90% of plasma S1P and sustained signalling in resistance arteries and lung capillaries. S1PR1 signalling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.

S1P Signaling Genes as Prominent Drivers of BCR-ABL1-Independent Imatinib Resistance and Six Herbal Compounds as Potential Drugs for Chronic Myeloid Leukemia.

Imatinib (IM), a breakthrough in chronic myeloid leukemia (CML) treatment, is accompanied by discontinuation challenges owing to drug intolerance. Although BCR-ABL1 mutation is a key cause of CML resistance, understanding mechanisms independent of BCR-ABL1 is also important. This study investigated the sphingosine-1-phosphate (S1P) signaling-associated genes (SphK1 and S1PRs) and their role in BCR-ABL1-independent resistant CML, an area currently lacking investigation. Through comprehensive transcriptomic analysis of IM-sensitive and IM-resistant CML groups, we identified the differentially expressed genes and found a notable upregulation of SphK1, S1PR2, and S1PR5 in IM-resistant CML. Functional annotation revealed their roles in critical cellular processes such as proliferation and GPCR activity. Their network analysis uncovered significant clusters, emphasizing the interconnectedness of the S1P signaling genes. Further, we identified interactors such as BIRC3, TRAF6, and SRC genes, with potential implications for IM resistance. Additionally, receiver operator characteristic curve analysis suggested these genes' potential as biomarkers for predicting IM resistance. Network pharmacology analysis identified six herbal compounds-ampelopsin, ellagic acid, colchicine, epigallocatechin-3-gallate, cucurbitacin B, and evodin-as potential drug candidates targeting the S1P signaling genes. In summary, this study contributes to efforts to better understand the molecular mechanisms underlying BCR-ABL1-independent CML resistance. Moreover, the S1P signaling genes are promising therapeutic targets and plausible new innovation avenues to combat IM resistance in cancer clinical care in the future.

The placenta in fetal death: molecular evidence of dysregulation of inflammatory, proliferative, and fetal protective pathways

BackgroundIt is estimated that over 2 million cases of fetal death occur worldwide every year, but, despite the high incidence, several basic and clinical characteristics of this disorder are still unclear. Placenta is suggested to play a central role in fetal death. Placenta produces hormones, cytokines and growth factors that modulate functions of the placental-maternal unit. Fetal death has been correlated with impaired secretion of some of these regulatory factors. Objective(s)The aim of the present study was to evaluate, in placentas collected from fetal death, the gene expression of inflammatory, proliferative and protective factors. Study designCases of fetal death in singleton pregnancy were retrospectively selected, excluding pregnancies complicated by fetal anomalies, gestational diabetes, intrauterine growth restriction and moderate to severe maternal diseases. A group of placentas collected from healthy singleton term pregnancies were used as controls. Groups were compared regarding maternal and gestational age, fetal sex and birth weight. Placental mRNA expression of inflammatory (IL-6), proliferative (Activin A, TGF-β1) and regulatory (VEGF, VEGFR2, ATP-binding cassette (ABC) transporters ABCB1 and ABCG2, sphingosine 1-phosphate (S1P) signaling pathway) markers was conducted using real-time PCR. Statistical analysis and graphical representation of the data were performed using the GraphPad Prism 5 software. For the statistical analysis, Student’s t-test was used, and P values < 0.05 were considered significant. ResultsPlacental mRNA expression of IL-6 and VEGFR2 resulted significantly higher in the fetal death group compared to controls (P<0.01), while activin A, ABCB1 and ABCG2 expression resulted significantly lower (P<0.01). A significant alteration in the S1P signaling pathway was found in the fetal death group, with an increased expression of the specific receptor isoforms sphingosine 1-phosphate receptor 1, 3 and 4 (S1P1, S1P3, S1P4) and of sphingosine kinase 2 (SK2), one of the enzyme isoforms responsible for S1P synthesis (P<0.01). Conclusion(s): The present study confirmed a significantly increased expression of placental IL-6 and VEGFR2 mRNA, and for the first time showed an increased expression of S1P receptors and SK2 as well as a decreased expression of activin A and of selected ATP-binding cassette transporters, suggesting that multiple inflammatory and protective factors are deranged in placenta of fetal death.

Virtual Screening, Molecular Docking, and Molecular Dynamics Simulation Studies on Potential Phytochemicals as Sphingosine Kinase 1 Inhibitors for Cancer Therapy

Sphingosine kinases (SphKs) as lipid kinases catalyze the phosphorylation of sphingosine (Sph) to sphingosine-1-phosphate (S1P). Targeting the S1P signaling pathway is a significant strategy for many human diseases. Herein, we evaluated main prenylated bioactive components of a medicinal plant and performed a virtual screening study with flavonoid compounds and then, molecular docking and molecular dynamics (MD) simulation for the targeted cancer therapy. In silico ADMET and drug-likeness results were determined by BIOVIA Discovery Studio (DS). Molecular docking and molecular dynamics (MD) simulations were carried out by using Glide/SP and Desmond of Maestro with the filtered ligands. Glide/SP docking results showed higher binding affinity with xanthohumol (XN), 8-prenylnaringenin (8-PN), and neobavaisoflavone against SphK1. Three hits displayed strong hydrogen binding between the specific amino acid residues of targeting SphK1. There were no significant structural changes between SphK1-XN and SphK1-neobavaisoflavone complexes during 200 ns MD simulation analysis performed by GROMACS. Root-mean square deviation (RMSD) average values of XN- and neobavaisoflavone-protein complexes were compared to free SphK1 and were found as 0.2626 nm, 0.2589 nm, and 0.2508 nm, respectively. As a result, XN and 8-PN, and neobavaisoflavone have been determined as potential inhibitor candidates of SphK1 to examine for further in vitro and in vivo studies.

Sphingosine-1-Phosphate Promotes Resiliency to External Stress in the Human Microvasculature

Exposure to external stress (e.g. high pressure, high glucose) promotes human microvascular endothelial dysfunction and reduces the overall magnitude of flow-induced dilation (FID). We have previously shown that treatment with sphingosine-1-phosphate (S1P), a sphingolipid known to activate pro-survival pathways in endothelial cells, restores nitric oxide (NO) as the primary mediator of flow-induced dilation (FID) in arterioles from patients with coronary artery disease. Whether pre-treatment with S1P can prevent stress-induced microvascular endothelial dysfunction remains unknown. Our hypothesis is that pre-conditioning with S1P will promote resiliency to external stress (high intraluminal pressure) and prevent stress-induced reduction of FID. Human resistance arterioles (100-250μm) from otherwise healthy adults (one risk factor or less for coronary artery disease) were dissected from discarded surgical adipose tissue and treated with S1P (1μM) or vehicle control for 16-20 hr prior to the flow experiment. Vessels were cannulated for videomicroscopy and stressed with elevated intraluminal pressure (150mmHg, 30 min) then pre-constricted with endothelin-1 prior to initiating flow to elicit dilation. Arterioles treated with vehicle and exposed to high intraluminal pressure exhibited an overall reduction in vasodilatory capacity to flow (37.3%±8.6) compared to non-stressed, vehicle treated microvessels (66.1%±5.5). However, the ability to dilate to flow was preserved in microvessels pre-treated with S1P and exposed to high pressure (74.8%±6.7) compared to vehicle and high pressure (37.3%±8.6). These data suggest that S1P-activated pathways may foster resilience by reversing stress-induced microvascular endothelial function. Targeting S1P signaling may offer a novel therapeutic strategy to prevent microvascular dysfunction and the myriad diseases it causes, such as large vessel atherosclerosis, and heart failure with preserved ejection fraction. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Imaging and molecular features of adenomyosis after menopause

ObjectivesTo explore the imaging features and the molecular characterization of adenomyosis after menopause. Study designAn observational cross-sectional study was performed in a group of postmenopausal patients undergoing a transvaginal ultrasound (TVUS) (n = 468). Among those presenting the US criteria for adenomyosis, also confirmed by magnetic resonance imaging (MRI), previous menstrual symptoms, gynecological and obstetric history were reviewed. In a subgroup undergoing hysterectomy, uterine specimens were analyzed by histology and expression of genes implicated in the epithelial-mesenchymal transition, inflammation and fibrosis, including the sphingosine-1-phosphate (S1P) pathway, was evaluated and compared to matched non-menopausal adenomyosis specimens. Main outcome measuresDirect and indirect US features of adenomyosis according to Morphological Uterus Sonographic Assessment at TVUS. Molecular characterization of postmenopausal versus pre-menopausal adenomyosis samples. ResultsAccording to TVUS and MRI, adenomyosis was identified in 49 patients (10.4 %). On US, diffuse adenomyosis was the most common phenotype, whereas internal adenomyosis with diffuse pattern and asymmetric type was the most prevalent on MRI. Molecular analysis showed that adenomyosis lesions express markers of epithelial-mesenchymal transition, inflammation and fibrosis also in postmenopausal women. By comparing the results with those from pre-menopausal samples, the expression of α smooth muscle actin (αSMA), a marker of fibrosis, was significantly greater after menopause, and altered S1P catabolism and signaling were observed. ConclusionsAdenomyosis may be identified in postmenopausal women by imaging, either TVUS or MRI, and fibrosis is one of the key features on molecular analysis.

Abstract 2659: Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response

Abstract Introduction: Immunotherapy has revolutionized the landscape of cancer treatment; however, to date, only limited aspects of the tumor-immune interaction have been exploited successfully, e.g., relief of immune checkpoints, increase of tumor-associated antigen presentation. Although it is well-known that trafficking and entry of T cells from the circulation into tumors is often impaired, leading to an immune “cold”, checkpoint inhibitor-refractory state, there are no available therapies that address this important aspect of antitumor immunity. Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive lipid that has been shown to regulate lymphocyte egress from lymph nodes through its interaction with one of its several receptors, S1P1. Clinically, tumor expression levels of the sphingosine-1-phosphate-producing enzyme sphingosine kinase 1 (SPHK1) strongly and negatively correlates with survival of melanoma patients after immune checkpoint inhibitor (ICI) therapy, and pre-clinically correlates with reduced tumor infiltrating lymphocytes (TILs). We therefore hypothesize that aberrant S1P signaling in tumors and in their draining lymph nodes (TDLN) causes immune exclusion by impeding lymphocyte egress from the TDLN, ultimately restricting immune cell infiltration of tumors. Methods: We tested the therapeutic efficacy of an engineered S1P-degrading enzyme, S1P lyase (S1PL), on established (~100mm^3) syngeneic B16, CT26, and MC38 murine tumors. Using flow cytometry, we performed immune profiling of systemic, TDLN, and intratumoral T cell populations, assessing both activation status and expression of the S1P1 receptor. Results: Treatment of tumor-bearing animals with S1PL depleted systemic S1P, and dramatically increased both trafficking of lymphocytes out of TDLNs and T cell infiltration of tumors relative to control-treated animals. Mice treated with S1PL evinced a higher proportion of S1P1+ circulating and intratumoral T cells relative to control-treated mice, suggesting that the increase in liberation of T cells from the TDLN was effected through the S1P/S1P1 axis. This increased lymphocyte trafficking correlated with strong inhibition of tumor growth when S1PL was administered as a single agent. Conclusions: Therapeutic administration of S1PL may represent an attractive strategy for modulating lymphocyte trafficking to improve the efficacy of therapies that rely on the adaptive immune system to exert antitumor activity. This approach may be particularly potent in settings wherein tumor-mediated immune exclusion is a dominant method of immune escape. Citation Format: Alessandra Araujo, Max Rodnick-Smith, Ranya Al-Khaledy, George Georgiou, Mark Badeaux, Everett Stone. Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2659.

Targeting Sphingosine-1-Phosphate Signaling in Breast Cancer.

In recent years, newly emerging therapies, such as immune checkpoint inhibitors and antibody-drug conjugates, have further improved outcomes for breast cancer patients. However, recurrent and metastatic breast cancer often eventually develops resistance to these drugs, and cure is still rare. As such, the development of new therapies for refractory breast cancer that differ from conventional mechanisms of action is necessary. Sphingosine-1-phosphate (S1P) is a key molecule with a variety of bioactive activities, including involvement in cancer cell proliferation, invasion, and metastasis. S1P also contributes to the formation of the cancer microenvironment by inducing surrounding vascular- and lymph-angiogenesis and regulating the immune system. In this article, we outline the basic mechanism of action of S1P, summarize previous findings on the function of S1P in cancer cells and the cancer microenvironment, and discuss the clinical significance of S1P in breast cancer and the therapeutic potential of targeting S1P signaling.

The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis.

Canonical Wnt and sphingosine-1-phosphate (S1P) signaling pathways are highly conserved systems that contribute to normal vertebrate development, with key consequences for immune, nervous, and cardiovascular system function; despite these functional overlaps, little is known about Wnt/β-catenin-S1P cross-talk. In the vascular system, both Wnt/β-catenin and S1P signals affect vessel maturation, stability, and barrier function, but information regarding their potential coordination is scant. We report an instance of functional interaction between the two pathways, including evidence that S1P receptor 1 (S1PR1) is a transcriptional target of β-catenin. By studying vascular smooth muscle cells and arterial injury response, we find a specific requirement for the β-catenin carboxyl terminus, which acts to induce S1PR1, and show that this interaction is essential for vascular remodeling. We also report that pharmacological inhibition of the β-catenin carboxyl terminus reduces S1PR1 expression, neointima formation, and atherosclerosis. These findings provide mechanistic understanding of how Wnt/β-catenin and S1P systems collaborate during vascular remodeling and inform strategies for therapeutic manipulation.

Rewiring Endothelial Sphingolipid Metabolism to Favor S1P Over Ceramide Protects From Coronary Atherosclerosis.

Growing evidence correlated changes in bioactive sphingolipids, particularly S1P (sphingosine-1-phosphate) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining lesion-prone areas plays a pivotal role in atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood. We used an established model of coronary atherosclerosis in mice, combined with sphingolipidomics, RNA-sequencing, flow cytometry, and immunostaining to investigate the contribution of sphingolipid metabolism and signaling to endothelial cell (EC) activation and dysfunction. We demonstrated that hemodynamic stress induced an early metabolic rewiring towards endothelial sphingolipid de novo biosynthesis, favoring S1P signaling over ceramides as a protective response. This finding is a paradigm shift from the current belief that ceramide accrual contributes to endothelial dysfunction. The enzyme SPT (serine palmitoyltransferase) commences de novo biosynthesis of sphingolipids and is inhibited by NOGO-B (reticulon-4B), an ER membrane protein. Here, we showed that NOGO-B is upregulated by hemodynamic stress in myocardial EC of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and humans. We demonstrated that mice lacking NOGO-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of NOGO-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective endothelial transcriptional signature. These data demonstrated that hemodynamic stress induced a protective rewiring of sphingolipid metabolism, favoring S1P over ceramide. NOGO-B deletion sustained the rewiring of sphingolipid metabolism toward S1P protecting EC from activation under hemodynamic stress and refraining coronary atherosclerosis. These findings also set forth the foundation for sphingolipid-based therapeutics to limit atheroprogression.

A Crosstalk between the Receptor Tyrosine Kinase-Like Orphan Receptors ROR1/2 and S1P Signaling Pathways in Lung Cancer.

Availability of multimodal treatment strategies, including targeted therapies and immunotherapies, have improved the survival of non-small cell lung carcinoma (NSCLC). However, some patients still progress or respond poorly due to inherent resistance, acquired resistance, or lack of druggable driver mutations. Sphingosine-1-phosphate (S1P) and receptor tyrosine kinase-like orphan receptor (ROR1/2) signaling pathways are activated during lung carcinogenesis. In this study, we have evaluated the crosstalk of S1P and ROR1/2 signaling pathways in lung cancer cells. S1P treatment of lung cancer cells decreases ROR1 and ROR2 transcript levels. While treatment with PF-543, a pharmacological SphK1 inhibitor or genetic knockdown of SPHK1 by shRNA, raises ROR1 and ROR2. Furthermore, simultaneous inhibition of SphK1 along with ROR1 reduced the migration of lung cancer cells. These findings demonstrate the reciprocal regulation of both pathways, suggesting that both pathways have an inverse relation i.e, in the absence of one pathway, another pathway may take charge of the other pathway. Therefore, simultaneously targeting both pathways could serve as a potential therapeutic target for lung cancer treatment.

How do sphingosine-1-phosphate affect immune cells to resolve inflammation?

Inflammation is an important immune response of the body. It is a physiological process of self-repair and defense against pathogens taken up by biological tissues when stimulated by damage factors such as trauma and infection. Inflammation is the main cause of high morbidity and mortality in most diseases and is the physiological basis of the disease. Targeted therapeutic strategies can achieve efficient toxicity clearance at the inflammatory site, reduce complications, and reduce mortality. Sphingosine-1-phosphate (S1P), a lipid signaling molecule, is involved in immune cell transport by binding to S1P receptors (S1PRs). It plays a key role in innate and adaptive immune responses and is closely related to inflammation. In homeostasis, lymphocytes follow an S1P concentration gradient from the tissues into circulation. One widely accepted mechanism is that during the inflammatory immune response, the S1P gradient is altered, and lymphocytes are blocked from entering the circulation and are, therefore, unable to reach the inflammatory site. However, the full mechanism of its involvement in inflammation is not fully understood. This review focuses on bacterial and viral infections, autoimmune diseases, and immunological aspects of the Sphks/S1P/S1PRs signaling pathway, highlighting their role in promoting intradial-adaptive immune interactions. How S1P signaling is regulated in inflammation and how S1P shapes immune responses through immune cells are explained in detail. We teased apart the immune cell composition of S1P signaling and the critical role of S1P pathway modulators in the host inflammatory immune system. By understanding the role of S1P in the pathogenesis of inflammatory diseases, we linked the genomic studies of S1P-targeted drugs in inflammatory diseases to provide a basis for targeted drug development.