Sphingosine-1-phosphate Signaling Pathway Research Articles

The contribution of the sphingosine 1-phosphate signaling pathway to chronic kidney diseases: recent findings and new perspectives.

Chronic kidney disease (CKD) is a multifactorial condition with diverse etiologies, such as diabetes mellitus, hypertension, and genetic disorders, often culminating in end-stage renal disease (ESRD). A hallmark of CKD progression is kidney fibrosis, characterized by the excessive accumulation of extracellular matrix components, for which there is currently no effective anti-fibrotic therapy. Recent literature highlights the critical role of sphingosine 1-phosphate (S1P) signaling in CKD pathogenesis and renal fibrosis. This review provides an in-depth analysis of the latest findings on S1P metabolism and signaling in renal fibrosis and in specific CKDs, including diabetic nephropathy (DN), lupus nephritis (LN), focal segmental glomerulosclerosis (FSGS), Fabry disease (FD), and IgA nephropathy (IgAN). Emerging studies underscore the therapeutic potential of modulating S1P signaling with receptor modulators and inhibitors, such as fingolimod (FTY720) and more selective agents like ozanimod and cenerimod. Additionally, the current knowledge about the effects of established kidney protective therapies such as glucocorticoids and SGLT2 and ACE inhibitors on S1P signaling will be summarized. Furthermore, the review highlights the potential role of S1P as a biomarker for disease progression in CKD models, particularly in Fabry disease and diabetic nephropathy. Advanced technologies, including spatial transcriptomics, are further refining our understanding of S1P's role within specific kidney compartments. Collectively, these insights emphasize the need for continued research into S1P signaling pathways as promising targets for CKD treatment strategies.

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.

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.

P172 Spatial Transcriptomics Characterization of S1P Pathway Genes in Ulcerative Colitis and Crohn’s Disease Colon Tissues

Abstract Background Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory diseases of the gastrointestinal tract, related to the confluence of environmental and immunological factors in genetically predisposed individuals. Activated T and B cells migrate from the lymphoid tissue into the circulation through the interaction of Sphingosine-1-phosphate (S1P) and the S1P1 receptor (S1P1), which can perpetuate inflammation and lead to tissue damage. Targeting of cellular migratory pathways has emerged as a novel therapeutic paradigm in IBD. The aim of this study is to characterize the S1P pathway-related genes and signaling pathways driving the pathogenesis of UC and CD. Methods Spatial transcriptomics analysis was performed in health volunteer (HV, n=2), UC (n=4) and CD (n=2) FFPE colon tissues using the GeoMX platform and NanoString whole transcriptome atlas (WTA) assay. The WTA panel was run on 8 samples stained with antibodies to S1P1, pan Cytokeratin, and CD45. Six regions of interests (ROIs) were selected in each sample based on the staining and each ROI were split into two areas (CD45+ or CD45-). Differential gene expression and enriched pathways analysis including S1P pathway genes were performed in CD45+ and CD45- segments in HV, UC and CD colon tissues. Results A total of 6085 and 6054 differentially expressed genes were identified from the CD45+ segments in UC and CD respectively, compared to HV colon (P=0.05), including the genes that are involved in T cell activation (e.g., HLA-DPA1, CCDC13), immune cell migration (e.g., ADAM15, CCL5) and tissue damage (e.g., FABP6, PDK4). Interestingly, significant differences were identified in UC and CD in S1P pathway gene expression. Specifically, SMPD1, a gene involved in generating ceramide and regulating inflammatory responses, was the most significantly up-regulated gene from both CD45+ and CD45- compartments in UC compared to HV colon. While CERS2 and CERS6, genes related to S1P metabolism, were significantly down-regulated genes in CD45+ and CD45- compartment in UC respectively, as compared to HV colon. Additionally, RTN4 (participates in the intestinal epithelial barrier function) and PLPP3 (dephosphorylates extracellular S1P to sphingosine) were most significantly down-regulated genes from CD45+ segments in both UC and CD compared to HV colon. Conclusion In summary, our study successfully validated the GeoMx transcriptome analysis approach in FFPE colon tissue and assessed the S1P pathway gene signature difference in HV, UC and CD colon tissues supporting the pathway’s role in IBD. Results could also facilitate the identification of potential S1P pathway UC and CD disease biomarkers.

Response to Fingolimod in Multiple Sclerosis Patients Is Associated with a Differential Transcriptomic Regulation.

Fingolimod is an immunomodulatory sphingosine-1-phosphate (S1P) analogue approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). The identification of biomarkers of clinical responses to fingolimod is a major necessity in MS to identify optimal responders and avoid the risk of disease progression in non-responders. With this aim, we used RNA sequencing to study the transcriptomic changes induced by fingolimod in peripheral blood mononuclear cells of MS-treated patients and their association with clinical response. Samples were obtained from 10 RRMS patients (five responders and five non-responders) at baseline and at 12 months of fingolimod therapy. Fingolimod exerted a vast impact at the transcriptional level, identifying 7155 differentially expressed genes (DEGs) compared to baseline that affected the regulation of numerous signaling pathways. These DEGs were predominantly immune related, including genes associated with S1P metabolism, cytokines, lymphocyte trafficking, master transcription factors of lymphocyte functions and the NF-kB pathway. Responder and non-responder patients exhibited a differential transcriptomic regulation during treatment, with responders presenting a higher number of DEGs (6405) compared to non-responders (2653). The S1P, NF-kB and TCR signaling pathways were differentially modulated in responder and non-responder patients. These transcriptomic differences offer the potential of being exploited as biomarkers of a clinical response to fingolimod.

The role of sphingosine-1-phosphate in the development and progression of Parkinson's disease.

Parkinson's disease (PD) could be viewed as a proteinopathy caused by changes in lipids, whereby modifications in lipid metabolism may lead to protein alterations, such as the accumulation of alpha-synuclein (α-syn), ultimately resulting in neurodegeneration. Although the loss of dopaminergic neurons in the substantia nigra is the major clinical manifestation of PD, the etiology of it is largely unknown. Increasing evidence has highlighted the important role of lipids in the pathophysiology of PD. Sphingosine-1-phosphate (S1P), a signaling lipid, has been suggested to have a potential association with the advancement and worsening of PD. Therefore, better understanding the mechanisms and regulatory proteins is of high interest. Most interestingly, S1P appears to be an important target to offers a new strategy for the diagnosis and treatment of PD. In this review, we first introduce the basic situation of S1P structure, function and regulation, with a special focus on the several pathways. We then briefly describe the regulation of S1P signaling pathway on cells and make a special focused on the cell growth, proliferation and apoptosis, etc. Finally, we discuss the function of S1P as potential therapeutic target to improve the clinical symptoms of PD, and even prevent the progression of the PD. In the context of PD, the functions of S1P modulators have been extensively elucidated. In conclusion, S1P modulators represent a novel and promising therapeutic principle and therapeutic method for PD. However, more research is required before these drugs can be considered as a standard treatment option for PD.

Role of the S1P Signaling Pathway in the Pathogenesis of Angioimmunoblastic T-Cell Lymphoma

Peripheral T cell lymphomas (PTCLs) are a group of aggressive lymphoid malignancies originating from mature T cells. Angioimmunoblastic T-cell lymphomas (AITL) represent 20% to 30% of all PTCL diagnoses and are associated with autoimmune features, poor response to chemotherapy, and dismal prognosis. Molecular profiling of AITL has identified T-follicular helper (TFH) cells as the cell of origin of AITL, while mutational analysis has identified frequent alterations in epigenetic regulators ( TET2, DNMT3A, and IDH2) and elements of the TCR pathway as drivers of AITL transformation. Our group identified the highly recurrent RHOA G17V mutation as a defining hallmark of AITL and other T-cell lymphomas of TFH-cell origin (Palomero et al., 2014). Using a novel conditional knockin mouse model, we demonstrated that expression of Rhoa G17V in CD4+ T-cells induces TFH cell specification and promotes AITL lymphomagenesis in the context of loss of Tet2 (Cortes et al., 2018). Recently, we have identified that Rhoa G17V regulates the expression of the sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) one of the five members of the receptor family of S1P, which plays an essential role in immune response and lymphocyte trafficking. During thymic T-cell development, expression of Rhoa G17V in CD4+ cells inhibited S1PR1 downregulation after the double positive stage leading to altered positive selection, reduced CD4+ single positive cell numbers and premature thymic egress of autorreactive T-cells which results in the development of systemic inflammation and autoimmunity. In our Tet2 −/− RHOA G17V AITL tumor model, constitutive activation of S1P receptors supported activation of the STAT3 and NFkB pathway and enhanced migration of malignant cells to peripheral organs. Mechanistically, expression of Rhoa G17V in mature CD4+ T-cells impaired re-phosphorylation of the Ezrin-Radoxin-Moesin (ERM) complex independently of ROCK signaling and led to the activation and increased migration of CD4+ T-cells. Importantly, blockade of S1PR1 signaling by fingolimod induced anti-tumor activity in Tet2 −/− RHOA G17V lymphoma-bearing mice in vivo with significant reductions in tumor burden associated with increased apoptosis and decreased STAT3 phosphorylation. Furthermore, analysis of a panel of primary tumors from AITL patients using a multiplexed staining panel and PhenoImager platform revealed significantly higher S1PR1 expression in AITL tumor cells than normal TFH cells from benign reactive lymph nodes. Our findings highlight the role of S1P signaling pathway in the pathogenesis of RHOA G17V-driven AITLs and the potential therapeutic benefit of targeting this pathway.

Bone marrow mesenchymal stem cells therapy regulates sphingolipid and glycerophospholipid metabolism to promote neurological recovery in stroke rats: A metabolomics analysis

Bone marrow mesenchymal stem cells (BMSCs) have therapeutic potential in the subacute/chronic phase of acute ischemic stroke (AIS), but the underlying mechanisms are not yet fully elucidated. There is a knowledge gap in understanding the metabolic mechanisms of BMSCs in stroke therapy. In this study, we administered BMSCs intravenously 24 h after reperfusion in rats with transient cerebral artery occlusion (MCAO). The treatment with BMSCs for 21 days significantly reduced the modified neurological severity score of MCAO rats (P < 0.01) and increased the number of surviving neurons in both the striatum and hippocampal dentate gyrus region (P < 0.01, respectively). Moreover, BMSCs treatment resulted in significant enhancements in various structural parameters of dendrites in layer V pyramidal neurons in the injured hemispheric motor cortex, including total length (P < 0.05), number of branches (P < 0.05), number of intersections (P < 0.01), and spine density (P < 0.05). Then, we performed plasma untargeted metabolomics analysis to study the metabolic changes of BMSCs on AIS. There were 65 differential metabolites identified in the BMSCs treatment group. Metabolic profiling analysis revealed that BMSCs modulate abnormal sphingolipid metabolism and glycerophospholipid metabolism, particularly affecting core members such as sphingomyelin (SM), ceramide (Cer) and sphingosine-1-phosphate (S1P). The metabolic network analysis and pathway-based compound-reaction-enzyme-gene network analysis showed that BMSCs inhibited the Cer-induced apoptotic pathway and promoted the S1P signaling pathway. These findings suggest that the enhanced effects of BMSCs on neuronal survival and synaptic plasticity after stroke may be mediated through these pathways. In conclusion, our study provides novel insight into the potential mechanisms of BMSCs treatment in stroke and sheds light on the possible clinical translation of BMSCs.

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction

Abstract Background/Introduction Platelet activation on ruptured or eroded atherosclerotic lesions leads to thrombus formation and ischemic events. Hence, platelet inhibition is the cornerstone of primary and secondary prevention in patients with cardiovascular disease. Recently, non-canonical effects of platelets in multiple disease processes were revealed. However, others have demonstrated the opposite: a protective and regenerative role of platelets during myocardial healing. Sphingosine-1-phosphate (S1P) is an important bioactive lipid released from platelets upon activation and has potent cardioprotective properties in AMI when acutely administered or kept constitutively high due to pharmacological or genetic inhibition of its degradation. In humans, S1P levels in plasma are dynamically regulated during AMI. Purpose We hypothesized that platelets may be an important source of the S1P burst during AMI and that platelet-derived S1P is relevant for the extent of myocardial damage due to AMI. Methods We have addressed these questions in experimental AMI in mice lacking components of the S1P synthesis, release and signaling pathways. In additon, we conducted a hypothesis generating, prospective, monocentric, time-series, translational analysis in 127 patients with ST-elevation myocardial infarction (STEMI) to analyze the association between S1P plasma concentration and infarct size. Results Injection of cell-free supernatant of activated platelets (SNT+) prior AMI in mice led to decreased infarct size after 24 h of reperfusion as compared to supernatant of non-activated platelets (SNT-). This reduction of infarct size by SNT+ was associated with reduced neutrophil accumulation and reduced apoptosis after AMI. S1P-concentrations in SNT+ was 25% higher as compared to SNT-. S1P concentrations in SNT- of mice lacking Sphingosinkinase-1 (SphK1) were 70% lower and did not increase in SNT+. Accordingly, SNT+ of SphK1- deficient platelets failed to reduce infarct size. Deficiency of platelet S1P exporter Mfsd2b shows increased infarct size during in vivo AMI compared to littermate controls. SNT+ of WT mice led to improved outcome after ischemia in a model of Langendorff-perfused hearts. This was blunted by inhibition of S1P receptor 1 and genetic cardiomyocyte deficiency of S1PR1. Platelet S1P release during activation was reduced by P2Y12 inhibition but preserved during GPIIb/IIIa (tirofiban) inhibition. This translated to blunted cardioprotection by SNT+ of cangrelor treated platelets but sustained cardioprotection during tirofiban treatment. Plasma S1P concentrations was associated with cardiovascular death and infarct size in patients with ST-elevation myocardial infarction. Conclusion We have identified the mechanisms underlying platelet-associated cardioprotection and finally, revealed the importance of platelet-released S1P on admission in patients with ST-elevation myocardial infarction (STEMI) regarding to infarct size and long-term prognosis.

The Sphingosine 1-Phosphate Axis: an Emerging Therapeutic Opportunity for Endometriosis.

Endometriosis is a common condition in women of reproductive age, but its current interventions are unsatisfactory. Recent research discovered a dysregulation of the sphingosine 1-phosphate (S1P) signaling pathway in endometriosis and showed a positive outcome by targeting it. The S1P axis participates in a series of fundamental pathophysiological processes. This narrative review is trying to expound the reported and putative (due to limited reports in this area for now) interactions between the S1P axis and endometriosis in those pathophysiological processes, to provide some perspectives for future research. In short, S1P signaling pathway is highly activated in the endometriotic lesion. The S1P concentration has a surge in the endometriotic cyst fluid and the peritoneal fluid, with the downstream dysregulation of its receptors. The S1P axis plays an essential role in the migration and activation of the immune cells, fibrosis, angiogenesis, pain-related hyperalgesia, and innervation. S1P receptor (S1PR) modulators showed an impressive therapeutic effect by targeting the different S1P receptors in the endometriosis model, and many other conditions resemble endometriosis. And several of them already got approval for clinical application in many diseases, which means a drug repurposing direction and a rapid clinical translation for endometriosis treatments.

Chicoric acid ameliorates palmitate-induced sphingosine 1-phosphate signaling pathway in the PBMCs of patients with newly diagnosed type 2 diabetes.

Sphingosine 1-phosphate (S1P) signaling pathway is involved in the pathogenesis of type 2 diabetes (T2D). So, targeting S1P signaling pathway could be considered as potential therapeutic target for T2D. The aim of this study was to investigate the effects of palmitate and chicoric acid (CA) on S1P signaling pathway in peripheral blood mononuclear cells (PBMCs) from newly diagnosed patients with T2D. 20 newly diagnosed patients with T2D, aged 40-60 years, were enrolled in the study. PBMCs were isolated and then treated as follows: control groups (untreated, treated with BSA 1% for 12h), CA groups (treated with 50 μM CA for 6h), palmitate groups (treated with 500 μM palmitate for 12h), palmitate + CA groups (treated with 500 μM palmitate for 12h and then treated with 50 μM CA for 6h). Finally, sphingosine kinase 1 (SPHK1) and sphingosine 1-phosphate receptor 1 (S1PR1) genes expression were evaluated by real-time PCR and S1PR1 protein levels were quantified using ELISA. Palmitate significantly increased SPHK1 and S1PR1 genes expression and S1PR1 protein levels in PBMCs of patients with diabetes. However, CA ameliorates palmitate-increased SPHK1 and S1PR1 genes expression and S1PR1 levels in these cells. These data indicate that CA could be considered as a novel S1P signaling pathway inhibitor through down regulation of SPHK1 and S1PR1.

Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages.

Radiotherapy for head-and-neck cancers frequently causes long-term hypofunction of salivary glands that severely compromises quality of life and is difficult to treat. Here, we studied effects and mechanisms of Sphingosine-1-phosphate (S1P), a versatile signaling sphingolipid, in preventing irreversible dry mouth caused by radiotherapy. Mouse submandibular glands (SMGs) were irradiated with or without intra-SMG S1P pretreatment. The saliva flow rate was measured following pilocarpine stimulation. The expression of genes related to S1P signaling and radiation damage was examined by flow cytometry, immunohistochemistry, quantitative RT-PCR, Western blotting, and/or single-cell RNA-sequencing. S1P pretreatment ameliorated irradiation-induced salivary dysfunction in mice through a decrease in irradiation-induced oxidative stress and consequent apoptosis and cellular senescence, which is related to the enhancement of Nrf2-regulated anti-oxidative response. In mouse SMGs, endothelial cells and resident macrophages are the major cells capable of producing S1P and expressing the pro-regenerative S1P receptor S1pr1. Both mouse SMGs and human endothelial cells are protected from irradiation damage by S1P pretreatment, likely through the S1pr1/Akt/eNOS axis. Moreover, intra-SMG-injected S1P did not affect the growth and radiosensitivity of head-and-neck cancer in a mouse model. These data indicate that S1P signaling pathway is a promising target for alleviating irradiation-induced salivary gland hypofunction.

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.

Sphingosine-1-Phosphate (S1P) and S1P Signaling Pathway Modulators, from Current Insights to Future Perspectives.

Sphingosine-1-phosphate (S1P) and S1P receptors (S1PR) are bioactive lipid molecules that are ubiquitously expressed in the human body and play an important role in the immune system. S1P-S1PR signaling has been well characterized in immune trafficking and activation in both innate and adaptive immune systems. Despite this knowledge, the full scope in the pathogenesis of autoimmune disorders is not well characterized yet. From the discovery of fingolimod, the first S1P modulator, until siponimod, the new molecule recently approved for the treatment of secondary progressive multiple sclerosis (SPMS), there has been a great advance in understanding the S1P functions and their involvement in immune diseases, including multiple sclerosis (MS). Modulation on S1P is an interesting target for the treatment of various autoimmune disorders. Improved understanding of the mechanism of action of fingolimod has allowed the development of the more selective second-generation S1PR modulators. Subtype 1 of the S1PR (S1PR1) is expressed on the cell surface of lymphocytes, which are known to play a major role in MS pathogenesis. The understanding of S1PR1’s role facilitated the development of pharmacological strategies directed to this target, and theoretically reduced the safety concerns derived from the use of fingolimod. A great advance in the MS treatment was achieved in March 2019 when the Food and Drug Association (FDA) approved Siponimod, for both active secondary progressive MS and relapsing–remitting MS. Siponimod became the first oral disease modifying therapy (DMT) specifically approved for active forms of secondary progressive MS. Additionally, for the treatment of relapsing forms of MS, ozanimod was approved by FDA in March 2020. Currently, there are ongoing trials focused on other new-generation S1PR1 modulators. This review approaches the fundamental aspects of the sphingosine phosphate modulators and their main similarities and differences.

Slit-Robo signalling establishes a Sphingosine-1-phosphate gradient to polarise fin mesenchyme.

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identifies a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the apical ectodermal ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate the coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

Sphingolipids Imbalanced in Brain Microvessel of Predisposed Reduced Uterine Perfusion Mice

Preeclampsia (PE), a hypertension disorder, is one of the most serious complications that can occur during pregnancy. It affects at least 2‐8% of pregnancies, causing up to 76,000 maternal deaths and 500,000 infant fatalities. PE negatively impacts the vasculature of multiple body systems and has significant neurovascular implications including reversible cerebral vasoconstriction syndrome and ischemic stroke. Although PE resolves after delivery, long term cerebrovascular effects remain. Women who have experienced PE are estimated to have an 80% increased risk of future stroke. Although the consequences of PE are extensive and prevalent, its full etiology is not yet known. It is suspected that cerebrovascular dysfunction plays a significant role in these critical ramifications of PE. Interestingly, the ceramide and sphingosine‐1‐phosphate (S1P) pathway is known to regulate many of the systems that are altered in PE. Specifically, ceramide and S1P are involved in regulation of blood pressure, vascular tone, and vessel permeability in addition to other cell and tissue processes. Because of the significant involvement of ceramide, S1P, and their signaling constituents in endothelial cell and vascular integrity, variations within this pathway may explain the link between PE and cerebrovascular disease. We hypothesize that the ceramide and S1P signaling pathway is linked to dysfunctional brain vasculature in mice that had a PE‐induced pregnancy. This study aims to prove the ceramide and S1P signaling pathway is altered in the cerebral microvasculature of PE murine models. C57BL female mice underwent reduced uterine perfusion pressure (RUPP) surgery at 10 weeks old to simulate PE or sham surgery. Brain microvessels (BMVs) were isolated from these mice dams nine months following delivery. Two step reverse transcription qPCR was performed to assess the mRNA expression of the various ceramide and S1P signaling components. It was found that RUPP mice BMVs express significantly less sphingosine kinase 1 (SphK1) in comparison to sham BMVs. RUPP mice BMVs also demonstrated significantly increased sphingosine‐1‐phosphate lyase 1 (Sgpl1) and phospholipid phosphatase 1 (Plpp1). RUPP mice BMVs exhibited decreased expression of ceramide kinase and sphingosine kinase 2 (Sphk2) as well. SphK1 and Sphk2 are key enzymes in producing S1P, an active signaling lipid responsible for maintaining the blood brain barrier, cerebrovascular integrity, and angiogenesis. Sgpl1 is responsible for the degradation of S1P into its inactive form, therefore contributing to endothelial apoptosis and diminished vessel integrity. Plpp1 catalyzes the production of pro‐apoptotic ceramide and was discovered to promote the metabolism of S1P. Ceramide kinase is a crucial enzyme in producing anti‐apoptotic ceramide‐1‐phosphate. These discrepancies in ceramide and S1P signaling pathway expression may contribute to impaired cerebrovascular health, providing insight into the increased incidence of stroke with PE.

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.

Mechanisms of sphingosine-1-phosphate (S1P) signaling on excessive stress-induced root resorption during orthodontic molar intrusion.

The aim of this study was to investigate cementocyte mechanotransduction during excessive orthodontic intrusive force-induced root resorption and the role of S1P signaling in this process. Fifty-four 12-week-old male Wistar rats were randomly divided into 3 groups: control group (Control), intrusive stress application group (Stress), and intrusive stress together with S1PR2-specific antagonist injection group (Stress + JTE). A rat molar intrusion model was established on animals in the Stress and the Stress + JTE groups. The animals in the Stress + JTE group received daily intraperitoneal (i.p.) injection of S1PR2 antagonist JTE-013, while the Control and Stress groups received only the vehicle. Histomorphometric, immunohistochemical, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were performed after euthanizing of the rats. Root resorption was promoted in the Stress group with increased volumes of resorption pits and amounts of molar intrusion compared with the Control group. The expression levels of cementogenic- and cementoclastic-related factors were affected under excessive intrusive force. Immunohistochemical staining and qRT-PCR analysis showed promoted S1P signaling activities during molar intrusion. Western blot analysis indicated decreased nuclear translocation of β-catenin under excessive intrusive force. Through the administration of JTE-013, S1P signaling activity was suppressed and excessive intrusive force-induced root resorption was reversed. The regulation of S1P signaling could also influence the nuclear translocation of β-catenin and the expressions of cementogenic- and cementoclastic-related factors. Root resorption was promoted under excessive orthodontic intrusive force due to the disruption of cementum homeostasis. S1P signaling pathway might play an important role in cementocyte mechanotransduction in this process. The S1P signaling might be a promising therapeutic target for novel therapeutic approaches to prevent external root resorption caused by excessive orthodontic intrusive force.