Sphingosine-1-phosphate Receptor Expression Research Articles

Sphingosine-1-phosphate receptor 3 activation promotes sociability and regulates transcripts important for anxiolytic-like behavior

We previously demonstrated that sphingosine-1-phosphate receptor 3 (S1PR3) in the medial prefrontal cortex (mPFC) prevents reductions in sociability normally caused by stress. S1PR3 is a ubiquitously expressed G-protein coupled receptor that regulates immune system function, although its regulation of other biological processes is not well understood. Pharmacological activators of S1PR3 might provide important insights for understanding the neural substrates underlying sociability. Here we show that in mice, systemic injections of an S1PR3-specific agonist, CYM5541, promotes sociability in males and females whereas an S1PR3-specific antagonist, CAY10444, increases amygdala activation and increases social avoidance, particularly in females. S1PR3 expression is increased in the mPFC and dentate gyrus of females compared to males. RNA sequencing in the mPFC reveals that S1PR3 activation alters the expression of transcripts related to immune function, neurotransmission, transmembrane ion transport, and intracellular signaling. This work provides evidence that S1PR3 agonists, which have classically been used as immune modulators, might also be used to promote social behavior and, potentially, relieve symptoms of social anxiety. S1PR3 might be an important hub gene for mitigating maladaptive effects of stress as it reduces inflammatory processes, increases transcripts linked to anxiolytic neurotransmission, and promotes social behavior.

Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

BackgroundAbnormal sensory perception, particularly pain insensitivity (PAI), is a typical symptom of autism spectrum disorder (ASD). Despite the role of myelin metabolism in the regulation of pain perception, the mechanisms underlying ASD-related PAI remain unclear.MethodsThe pain-associated gene sphingosine-1-phosphate receptor 1 (S1PR1) was identified in ASD samples through bioinformatics analysis. Its expression in the dorsal root ganglion (DRG) tissues of BTBR ASD model mice was validated using RNA-seq, western blot, RT-qPCR, and immunofluorescence. Pain thresholds were assessed using the von Frey and Hargreaves tests. Patch-clamp techniques measured KCNQ/M channel activity and neuronal action potentials. The expression of S1PR1, KCNQ/M, mitogen-activated protein kinase (MAPK), and cyclic AMP/protein kinase A (cAMP/PKA) signaling proteins was analyzed before and after inhibiting the S1P-S1PR1-KCNQ/M pathway via western blot and RT-qPCR.ResultsThrough integrated transcriptomic analysis of ASD samples, we identified the upregulated gene S1PR1, which is associated with sphingolipid metabolism and linked to pain perception, and confirmed its role in the BTBR mouse model of ASD. This mechanism involves the regulation of KCNQ/M channels in DRG neurons. The enhanced activity of KCNQ/M channels and the decreased action potentials in small and medium DRG neurons were correlated with PAI in a BTBR mouse model of ASD. Inhibition of the S1P/S1PR1 pathway rescued baseline insensitivity to pain by suppressing KCNQ/M channels in DRG neurons, mediated through the MAPK and cAMP/PKA pathways. Investigating the modulation and underlying mechanisms of the non-opioid pathway involving S1PR1 will provide new insights into clinical targeted interventions for PAI in ASD.ConclusionsS1PR1 may contribute to PAI in the PNS in ASD. The mechanism involves KCNQ/M channels and the MAPK and cAMP/PKA signaling pathways. Targeting S1PR1 in the PNS could offer novel therapeutic strategies for the intervention of pain dysesthesias in individuals with ASD.

Fingolimod, a sphingosine-1-phosphate receptor modulator, prevents neonatal bronchopulmonary dysplasia and subsequent airway remodeling in a murine model.

Neonatal bronchopulmonary dysplasia (BPD) is associated with alveolar simplification and airway remodeling. Airway remodeling leads to deformation of airways characterized by peribronchial collagen deposition and hypertrophy of airway smooth muscle, which contribute to the narrowing of airways. Poorly developed lungs contribute to reduced lung function that deteriorates with the passage of time. We have earlier shown that sphingosine kinase 1 (SPHK 1)/sphingosine-1-phosphate (S1P)/S1P receptor1 (S1PR1) signaling plays a role in the pathogenesis of BPD. In this study, we investigated the role of fingolimod or FTY720, a known S1PR1 modulator approved for the treatment of multiple sclerosis in the treatment of BPD. Fingolimod promotes the degradation of S1PR1 by preventing its recycling, thus serving as the equivalent of an inhibitor. Exposure of neonatal mice to hyperoxia enhanced the expression of S1PR1 in both airways and alveoli as compared with normoxia. This increased expression of S1PR1 in the airways persisted into adulthood, accompanied by airway remodeling and airway hyperreactivity (AHR) after neonatal hyperoxia. Intranasal fingolimod at a much lower dose compared with the intraperitoneal route of administration during neonatal hyperoxia improved alveolarization in neonates and reduced airway remodeling and AHR in adult mice associated with improved lung function. The intranasal route was not associated with the lymphopenia seen with the intraperitoneal route of administration of the drug. An increase in S1PR1 expression in the airways was associated with an increase in the expression of enzyme lysyl oxidase (LOX) in the airways following hyperoxia, which was suppressed by fingolimod. This association warrants further investigation.NEW & NOTEWORTHY The role of the S1P receptor1 modulator, fingolimod, as an FDA-approved drug in preventing the recurrence of multiple sclerosis is established. Fingolimod prevented bronchopulmonary dysplasia (BPD) and its sequela of airway remodeling in a neonatal murine model. This protection was associated with the downregulation of lysyl oxidase signaling pathway. Fingolimod could be repurposed for the therapy of BPD.

Sphingosine-1 phosphate receptor 1 (S1PR1) expression maintains stemness of acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) arises from leukemia stem cells (LSCs) and is maintained by cells which have acquired features of stemness. We compared transcription profiles of AML cells with/without stem cell features defined as in vitro clonogenicity and serial engraftment in immune-deficient mice xenograft model. We used multi-parameter flow cytometry (MPFC) to separate CD34+ bone marrow-derived leukemia cells into sphingosine-1 phosphate receptor 1 (S1PR1)+ and S1PR1- fractions. Cells in the S1PR1+ fraction demonstrated significantly higher clonogenicity and higher engraftment potential compared with those in the S1PR1- fraction. In contrast, CD34+ bone marrow cells from normal samples showed reduced clonogenicity in the S1PR1+ fraction compared with the S1PR1- fraction. Inhibition of S1PR1 expression in an AML cell line reduced the colony-forming potential of KG1 cells. Transcriptomic analyses and rescue experiments indicated PI3K/AKT pathway and MYBL2 are downstream mediators of S1PR1-associated stemness. These findings implicate S1PR1 as a functional biomarker of LSCs and suggest its potential as a therapeutic target in AML treatment.

Endothelial cell sphingosine 1-phosphate receptor 1 restrains VE-cadherin cleavage and attenuates experimental inflammatory arthritis.

In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin "sheddase." Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.

S1PR3 inhibition protects against LPS-induced ARDS by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation

BackgroundInflammation and endothelial barrier dysfunction are the major pathophysiological changes in acute respiratory distress syndrome (ARDS). Sphingosine-1-phosphate receptor 3 (S1PR3), a G protein-coupled receptor, has been found to mediate inflammation and endothelial cell (EC) integrity. However, the function of S1PR3 in ARDS has not been fully elucidated.MethodsWe used a murine lipopolysaccharide (LPS)-induced ARDS model and an LPS- stimulated ECs model to investigate the role of S1PR3 in anti-inflammatory effects and endothelial barrier protection during ARDS.ResultsWe found that S1PR3 expression was increased in the lung tissues of mice with LPS-induced ARDS. TY-52156, a selective S1PR3 inhibitor, effectively attenuated LPS-induced inflammation by suppressing the expression of proinflammatory cytokines and restored the endothelial barrier by repairing adherens junctions and reducing vascular leakage. S1PR3 inhibition was achieved by an adeno-associated virus in vivo and a small interfering RNA in vitro. Both the in vivo and in vitro studies demonstrated that pharmacological or genetic inhibition of S1PR3 protected against ARDS by inhibiting the NF-κB pathway and improving mitochondrial oxidative phosphorylation.ConclusionsS1PR3 inhibition protects against LPS-induced ARDS via suppression of pulmonary inflammation and promotion of the endothelial barrier by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation, indicating that S1PR3 is a potential therapeutic target for ARDS.

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.

Low shear stress-induced blockage of autophagic flux impairs endothelial barrier and facilitates atherosclerosis in mice

Atherosclerosis preferentially occurs in areas with low shear stress (LSS) and oscillatory flow. LSS has been demonstrated to correlate with the development of atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1PR1), involving intravascular blood flow sensing, regulates vascular development and vascular barrier function. However, whether LSS affects atherosclerosis via regulating S1PR1 remains incompletely clear. In this study, immunostaining results of F-actin, β-catenin, and VE-cadherin indicated that LSS impaired endothelial barrier function in human umbilical vein endothelial cells (HUVECs). Western blot analysis showed that LSS resulted in blockage of autophagic flux in HUVECs. In addition, autophagy agonist Rapamycin (Rapa) antagonized LSS-induced endothelial barrier dysfunction, whereas autophagic flux inhibitor Bafilomycin A1 (BafA1) exacerbated it, indicating that LSS promoted endothelial barrier dysfunction by triggering autophagic flux blockage. Notably, gene expression analysis revealed that LSS downregulated S1PR1 expression, which was antagonized by Rapa. Selective S1PR1 antagonist W146 impaired endothelial barrier function of HUVECs under high shear stress (HSS) conditions. Moreover, our data showed that expression of GAPARAPL2, a member of autophagy-related gene 8 (Atg8) proteins, was decreased in HUVECs under LSS conditions. Autophagic flux blockage induced by GAPARAPL2 knockdown inhibited S1PR1, aggravated endothelial barrier dysfunction of HUVECs in vitro, and promoted aortic atherosclerosis in ApoE−/− mice in vivo. Our study demonstrates that autophagic flux blockage induced by LSS downregulates S1PR1 expression and impairs endothelial barrier function. GABARAPL2 inhibition is involved in LSS-induced autophagic flux blockage, which impairs endothelial barrier function via downregulation of S1PR1.

S1PR3 suppresses the inflammatory response and extracellular matrix degradation in human nucleus pulposus cells.

Sphingosine 1-phosphate receptor 3 (S1PR3) participates in the inflammatory response in multiple types of diseases. However, the biological role of S1PR3 in intervertebral disc degeneration and the underlying mechanism are unclear. The aim of the present study was to investigate the functional role and the mechanism of S1PR3 in lipopolysaccharide (LPS)-induced human nucleus pulposus cells. The expression of S1PR3 and Toll-like receptor (TLR) 2 in LPS-induced nucleus pulposus (NP) cells was investigated using western blotting. The Cell Counting Kit-8 assay was used to detect cell proliferation, and the levels of inflammatory factors were detected using ELISA. Flow cytometry and western blotting were used for the assessment of apoptosis. The deposition of extracellular matrix (ECM) proteins was investigated using reverse transcription-quantitative PCR and western blotting. In addition, western blotting was used to investigate the protein expression levels of phosphorylated (p)-STAT3, STAT3, p-JNK, JNK, p-ERK, ERK, p-p38 and p38associated with STAT3 and MAPK signaling. S1PR3 expression was reduced, while TLR2 expression was elevated in LPS-induced human nucleus pulposus cells (HNPC). S1PR3 overexpression increased HNPC viability, inhibited the inflammatory response and suppressed apoptosis. Meanwhile, S1PR3 overexpression regulated the expression of ECM-related proteins. Additionally, overexpression of S1PR3 inhibited the expression of the TLR2-regulated STAT3 and MAPK pathways in LPS-induced HNPCs. Furthermore, TLR2 overexpression partially offset the impacts of S1PR3 overexpression on HNPC viability, apoptosis level, inflammation and as ECM degradation. In conclusion, STAT3 overexpression suppressed viability injury, the inflammatory response and the level of apoptosis and alleviated ECM protein deposition in HNPCs through the TLR2/STAT3 and TLR2/MAPK pathways, which may offer a promising candidate for the amelioration of intervertebral disc degeneration.

Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.

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.

Transplantation of miR-193b-3p-Transfected BMSCs Improves Neurological Impairment after Traumatic Brain Injury through S1PR3-Mediated Regulation of the PI3K/AKT/mTOR Signaling Pathway.

The aim of the present study was to explore the molecular mechanisms by which miR-193b-3p-trans-fected bone marrow mesenchymal stem cells (BMSCs) transplantation improves neurological impairment after traumatic brain injury (TBI) through sphingosine-1-phosphate receptor 3 (S1PR3)-mediated regulation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway at the cellular and animal levels. BMSCs were transfected with miR-193b-3p. A TBI cell model was established by oxygen-glucose deprivation (OGD)-induced HT22 cells, and a TBI animal model was established by controlled cortical impact (CCI). Cell apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), and cell activity was detected by a cell counting kit 8 (CCK-8) assay. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of related proteins and genes. In this study, transfection of miR-193b-3p into BMSCs significantly enhanced BMSCs proliferation and differentiation. Transfection of miR-193b-3p reduced the levels of the interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-alpha (TNF-α) inflammatory factors in cells and mouse models, and it inhibited neuronal apoptosis, which alleviated OGD-induced HT22 cell damage and neural function damage in TBI mice. Downstream experiments showed that miR-193b-3p targeting negatively regulated the expression of S1PR3, promoted the activation of the PI3K/AKT/mTOR signaling pathway, and inhibited the levels of apoptosis and inflammatory factors, which subsequently improved OGD-induced neuronal cell damage and nerve function damage in TBI mice. However, S1PR3 overexpression or inhibition of the PI3K/AKT/mTOR signaling pathway using the IN-2 inhibitor weakened the protective effect of miR-193b-3p-transfected BMSCs on HT22 cells. Transplantation of miR-193b-3p-transfected BMSCs inhibits neurological injury and improves the progression of TBI in mice through S1PR3-mediated regulation of the PI3K/AKT/mTOR pathway.

Sphingosine-1-phosphate receptor 3 is a non-hormonal target to counteract endometriosis-associated fibrosis

ObjectiveTo study the molecular mechanisms responsible for fibrosis in endometriosis by investigating whether the protein expression levels of sphingosine-1-phosphate receptor 3 (S1PR3), one of the five specific receptors of the bioactive sphingolipid sphingosine 1-phosphate (S1P), correlated with fibrosis extent in endometriotic lesions and which are the cellular mechanisms involved in this process. DesignCase-control laboratory study and cultured endometriotic cells. SubjectsUniversity research institute and university hospital. Patient(s)A total of 33 women, with and without endometriosis, were included in the study. Interventions(s)Endometriotic lesions were obtained from women with endometriosis (endometrioma [OMA], n=8; deep infiltrating endometriosis [DIE], n=15, (urological n=5, gastrointestinal n=6, posterior n=4)) and control endometrium from healthy women, n=10, by means of laparoscopic/hysteroscopic surgery. The expression of S1PR3 was evaluated by immunohistochemistry and the extent of fibrosis by Masson’s trichrome staining. Human cultured epithelial endometriotic 12Z cells were used to evaluate the mechanisms involved in the profibrotic effect of S1PR3 activation. Main Outcome Measure(s)The expression of S1PR3 in endometriotic lesions positively correlated to endometriosis-associated fibrosis. In addition, S1P induced epithelial-mesenchymal transition (EMT)/fibrosis in epithelial endometriotic cells. By RNA interference and pharmacological approaches, the profibrotic effect of S1P was shown to rely on S1PR3, thus unveiling the molecular mechanism implicated in the profibrotic action of the bioactive sphingolipid. Result(s)The protein expression levels of S1PR3 resulted significantly augmented in the glandular sections of OMA and DIE of different localizations in respect to control endometrium and positively correlated with the extent of fibrosis. S1P was shown to have a crucial role in the onset of fibrosis in epithelial endometriotic cells, stimulating the expression of EMT and fibrotic markers. Genetic approaches highlighted that S1PR3 mediates the fibrotic effect of S1P. Downstream of S1PR3, ezrin and ERK1/2 signaling were found critically implicated in the EMT/fibrosis elicited by S1P. Conclusion(s)The S1PR3 receptor may represent a possible innovative pharmacological target for endometriosis.

Cardiovascular Safety of Ozanimod in Patients With Ulcerative Colitis: True North and Open-Label Extension Analyses

Evaluating cardiovascular safety of sphingosine 1-phosphate (S1P) receptor modulators is warranted due to S1P receptor expression on cardiomyocytes and vascular endothelial cells. This analysis reports the cardiovascular safety of ozanimod, an S1P receptor modulator, in patients with moderately to severely active ulcerative colitis from the phase 3 True North (TN) and open-label extension (OLE). All patients who received ozanimod in TN (n= 796) and all eligible TN patients who entered the OLE (n= 823) were included. Cardiovascular-related adverse events were evaluated in patients with up to 146 weeks of ozanimod exposure (2219 patient-years), which included 52 weeks during TN. On TN day 1, first-dose ozanimod resulted in a 0.2 beats per minute mean decrease in heart rate from pretreatment to hour 6; 2 patients experienced bradycardia, which resolved without treatment modification. Mean systolic and diastolic blood pressure increases of 5.1 and 2.2 mm Hg, respectively, were observed at TN week 52. No second-degree Mobitz type II atrioventricular block events were reported; 1 third-degree atrioventricular block unrelated to ozanimod occurred in the OLE. Cardiac and vascular treatment-emergent adverse events were infrequent (3.8% [31 of 823] and 8.5% [70 of 823]); no ozanimod-related cardiovascular deaths occurred. The incidences of deep-vein thrombosis (0.2%; 2 of 823), pulmonary embolism (0.2%; 2 of 823), and ischemic stroke (0.4%; 3 of 823) in the OLE were low. No new cardiovascular safety signals were identified, consistent with findings from previous ozanimod studies. There were few major adverse cardiovascular events or thromboembolic events, which were unrelated or unlikely related to ozanimod. Ozanimod has a well-tolerated cardiovascular safety profile when prescribed in accordance with the label. Clinical trial registry website and trial numbers: ClinicalTrials.gov numbers: NCT02435992 and NCT02531126.

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.

S1PR1 regulates ovarian cancer cell senescence through the PDK1-LATS1/2-YAP pathway

Cell senescence deters the activation of various oncogenes. Induction of senescence is, therefore, a potentially effective strategy to interfere with vital processes in tumor cells. Sphingosine-1-phosphate receptor 1 (S1PR1) has been implicated in various cancer types, including ovarian cancer. The mechanism by which S1PR1 regulates ovarian cancer cell senescence is currently elusive. In this study, we demonstrate that S1PR1 was highly expressed in human ovarian cancer tissues and cell lines. S1PR1 deletion inhibited the proliferation and migration of ovarian cancer cells. S1PR1 deletion promoted ovarian cancer cell senescence and sensitized ovarian cancer cells to cisplatin chemotherapy. Exposure of ovarian cancer cells to sphingosine-1-phosphate (S1P) increased the expression of 3-phosphatidylinositol-dependent protein kinase 1 (PDK1), decreased the expression of large tumor suppressor 1/2 (LATS1/2), and induced phosphorylation of Yes-associated protein (p-YAP). Opposite results were obtained in S1PR1 knockout cells following pharmacological inhibition. After silencing LATS1/2 in S1PR1-deficient ovarian cancer cells, senescence was suppressed and S1PR1 expression was increased concomitantly with YAP expression. Transcriptional regulation of S1PR1 by YAP was confirmed by chromatin immunoprecipitation. Accordingly, the S1PR1-PDK1-LATS1/2-YAP pathway regulates ovarian cancer cell senescence and does so through a YAP-mediated feedback loop. S1PR1 constitutes a druggable target for the induction of senescence in ovarian cancer cells. Pharmacological intervention in the S1PR1-PDK1-LATS1/2-YAP signaling axis may augment the efficacy of standard chemotherapy.

Clinical Significance of Sphingosine 1-phosphate Receptor 2 and Takeda G Protein-coupled Receptor 5 in Extrahepatic Cholangiocarcinoma Patients.

In biliary epithelial cells, two bile acid receptors, sphingosine 1-phosphate receptor 2 (S1PR2) and Takeda G protein-coupled receptor 5 (TGR5) have been reported to trigger cell proliferation, as well as neoplastic cell invasiveness. In this study, we aimed to investigate the clinical significance of S1PR2/ TGR5 expression in extrahepatic cholangiocarcinoma (CCA) patients. Patients who underwent surgical resection of extrahepatic CCA at Korea University Guro Hospital between 2002 and 2018 were included. Data on immunohistochemical staining and H-score of S1PR2 and TGR5 were evaluated using digital image analysis. A total of 115 cases of invasive CCA were analyzed. The H-score of S1PR2 showed a decrease in invasive CCA (p=0.052) but that of TGR5 showed a significant increase (p=0.02). Overall survival and disease-free survival were significantly lower in the low S1PR2 expression group (p<0.05) than in the control group; however, TGR5 expression was not significant (p=0.096). In multivariate analysis, low S1PR2 was only significant for poor prognosis. Low S1PR2 level was the only independent poor prognostic factor in patients with resected extrahepatic CCA.

HIV-1 Infection Results in Sphingosine-1-Phosphate Receptor 1 Dysregulation in the Human Thymus.

Regeneration of functional naïve T lymphocytes following the onset of human immunodeficiency virus (HIV) infection remains a crucial issue for people living with HIV (PLWH), even when adhering to antiretroviral therapy (ART). Thus far, reports on the impact of HIV-1 infection on the entry of thymic precursors and the egress of functional naïve T lymphocytes to and from the thymus are limited. We examined the impact of HIV-1 on Sphingosine-1-phosphate (S1P) signaling, which governs the egress of functional naïve thymocytes from the thymus to the periphery. Using in vitro experiments with primary human thymocytes and in vivo and ex vivo studies with humanized mice, we show that HIV-1 infection results in upregulation of the expression of S1P receptor 1 (S1PR1) in the human thymus. Intriguingly, this upregulation occurs during intrathymic infection (direct infection of the human thymic implant) as well as systemic infection in humanized mice. Moreover, considering the dysregulation of pro- and anti-inflammatory cytokines in infected thymi, the increased expression of S1PR1 in response to in vitro exposure to Interferon-Beta (IFN-β) and Tumor Necrosis Factor-Alpha (TNF-α) indicates that cytokine dysregulation following HIV infection may contribute to upregulation of S1PR1. Finally, an increased presence of CD3hiCD69- (fully mature) as well as CD3hiCD69+ (less mature) T cells in the spleen during HIV infection in humanized mice, combined with earlier expression of S1PR1 during thymocyte development, suggests that upregulation of S1PR1 may translate to increased or accelerated egress from the thymus. The egress of thymocytes that are not functionally mature from the thymus to peripheral blood and lymphoid organs may have implications for the immune function of PLWH.

Xiaoyin Jiedu Granules may alleviate psoriasis-like skin diseases in mice by regulating sphingosine 1-phosphate receptor expression and reducing Th17 cells

Sphingosine-1-phosphate (S1P) is associated with the onset and severity of psoriasis, a chronic inflammatory skin disease linked to innate and adaptive immune responses. This study explores the therapeutic effect of Xiaoyin Jiedu Granules, a combination of traditional Chinese medicines, on psoriasis-like skin lesions in mice and the underlying mechanism. We used imiquimod (IMQ) to induce psoriasis-like dermatitis in mice; the effects of Xiaoyin Jiedu Granules on S1P receptors (S1PRs) were investigated using histology and immunohistochemistry. The effects of Xiaoyin Jiedu Granules on the proliferation, differentiation, and activation of the NF-κB pathway in keratinocytes were verified using quantitative polymerase chain reaction (qPCR) and western blotting analyses. CD4+Th17 cells were screened using flow cytometry; the effects of Xiaoyin Jiedu Granules on the differentiation of Th17 cells and the content of related inflammatory factors were also verified. S1PR1-5 was highly expressed in psoriatic lesions. Xiaoyin Jiedu Granules significantly inhibited the secretion of proliferation-related proteins (K6, K16, K17, and IL-36γ) and proinflammatory cytokines (IL-17 and IL-22), transformation of Th17 cells, and activation of the NF-κB pathway and effectively alleviated IMQ-induced psoriasis-like dermatitis. Overall, our findings indicate that Xiaoyin Jiedu Granules have anti-inflammatory activity against S1PR expression, keratinocytes, and immune cells and can therefore mitigate psoriasis. Inhibiting the expression of S1PRs may be an effective treatment strategy against psoriasis.

S1PR1 attenuates pulmonary fibrosis by inhibiting EndMT and improving endothelial barrier function

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal disease of unknown etiology. Its pathological manifestations include excessive proliferation and activation of fibroblasts and deposition of extracellular matrix. Endothelial cell-mesenchymal transformation (EndMT), a novel mechanism that generates fibroblast during IPF, is responsible for fibroblast-like phenotypic changes and activation of fibroblasts into hypersecretory cells. However, the exact mechanism behind EndMT-derived fibroblasts and activation is uncertain. Here, we investigated the role of sphingosine 1-phosphate receptor 1 (S1PR1) in EndMT-driven pulmonary fibrosis. MethodsWe treated C57BL/6 mice with bleomycin (BLM) in vivo and pulmonary microvascular endothelial cells with TGF-β1 in vitro. Western blot, flow cytometry, and immunofluorescence were used to detect the expression of S1PR1 in endothelial cells. To evaluate the effect of S1PR1 on EndMT and endothelial barrier and its role in lung fibrosis and related signaling pathways, S1PR1 agonist and antagonist were used in vitro and in vivo. ResultsEndothelial S1PR1 protein expression was downregulated in both in vitro and in vivo models of pulmonary fibrosis induced by TGF-β1 and BLM, respectively. Downregulation of S1PR1 resulted in EndMT, indicated by decreased expression of endothelial markers CD31 and VE-cadherin, increased expression of mesenchymal markers α-SMA and nuclear transcription factor Snail, and disruption of the endothelial barrier. Further mechanistic studies found that stimulation of S1PR1 inhibited TGF-β1-mediated activation of the Smad2/3 and RhoA/ROCK1 pathways. Moreover, stimulation of S1PR1 attenuated Smad2/3 and RhoA/ROCK1 pathway-mediated damage to endothelial barrier function. ConclusionsEndothelial S1PR1 provides protection against pulmonary fibrosis by inhibiting EndMT and attenuating endothelial barrier damage. Accordingly, S1PR1 may be a potential therapeutic target in progressive IPF.