Sphingosine-1-phosphate Receptor Research Articles

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

Abstract 11818: The Beneficial Effects of Ceramide on Human Microvascular Endothelial Function is Dependent on S1P/S1PR1 Signaling Axis

Despite the known damaging effects of elevated intracellular ceramides on human microvascular function, our recent work showed that loss of ceramide-formation through neutral sphingomyelinase (NSmase) promotes endothelial dysfunction in arterioles from healthy adults. This presents as a switch in the endothelial-derived mediator responsible for eliciting dilation to flow (flow-induced dilation; FID), from the vasoprotective nitric oxide (NO) to pro-inflammatory hydrogen peroxide (H 2 O 2 ). The mechanism through which ceramide exerts beneficial effects on the vasculature remains poorly understood. One of ceramide’s metabolites, sphingosine-1-phosphate (S1P), has been shown to stimulate NO production through the activation of S1P receptor 1 (S1PR1). Thus, we hypothesize that ceramide promotes NO-mediated FID through its metabolism to S1P and activation of S1PR1. Arterioles (100-250μm) from healthy adults were dissected from discarded surgical adipose tissue. Vessels were cannulated in organ chambers, pre-constricted with endothelin-1, and changes in internal diameter in response to increases in flow or C2-ceramide was measured via videomicroscopy. Difference between mean % dilation ±SE with two-way ANOVA is reported. Agonism of S1PR1 (CYM5442, 1μM, 30min) during NSmase inhibition (GW4869, 4μM, 30min) restored dependency on NO for FID, as L-NAME (100μM; NO synthase inhibitor) diminished FID (GW+CYM, n=4 vs GW+CYM+L-NAME, n=4: -70.3±20.0, p=0.01). A similar switch to NO-mediated FID was seen with C2-ceramide (10μM)+GW4869 treatment (GW+C2, n=5 vs GW+C2+L-NAME, n=4: -52.5±5.9, p<0.001). Inhibition of S1PR1 (W146, 10μM, 30min) induced a switch from NO to H 2 O 2 mediated FID, as catalase (500U/ml; W146, n=5 vs W146+Catalase, n=4: -34.0±7.9; p=0.01) but not L-NAME (W146, n=5 vs W146+L-NAME, n=4; -1.4±9.9, p=0.89) impaired FID. Acute ceramide-induced dilation (1min) was diminished in the presence of L-NAME (C2, n=5 vs C2+L-NAME, n=4: -33.40±10.06, p=0.02) and W146 (C2, n=5 vs C2+W146, n=4; -39.7±5.9, p=0.01). Together, these data suggest that ceramide induces NO-signaling through the activation of S1PR1 and provide mechanistic insight into how ceramide may exert beneficial effects within the human microvascular endothelium.

MicroRNA-10a enrichment in factor VIIa–released endothelial extracellular vesicles: potential mechanisms

BackgroundFactor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. ObjectivesTo investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. MethodsActivation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. ResultsFVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage. ConclusionOur data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

Modulating intestinal barrier function by sphingosine-1-phosphate receptor 1 specific agonist SEW2871 attenuated ANIT-induced cholestatic hepatitis via the gut-liver axis

Bile acid (BA) homeostasis throughout the enterohepatic circulation system is a guarantee of liver physiological functions. BA circulation disorders is one of the characteristic clinical manifestations of cholestasis, and have a closely relationship with intestinal barrier function, especially ileum. Here, our in vivo and in vitro studies showed that intestinal tight junctions (TJs) were disrupted by α-naphthylisothiocyanate (ANIT), which also down-regulated the protein expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the top of villus of mice ileum. Activating S1PR1 by specific agonist SEW2871 could improve TJs via inhibiting ERK1/2/LKB1/AMPK signaling pathway in the ileum of ANIT-treated mice and ANIT-cultured Caco-2 cells. SEW2871 not only regained ileum TJs by activating S1PR1 in the epithelial cells of ileum mucosa, but also recovered ileum barrier function, which was further verified by the recovered BA homeostasis in mice ileum (content and tissue) by using of high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). Subsequently, the improved intestinal injury and inflammation further strengthened that SEW2871 modulated intestinal barrier function in ANIT-treated mice. Finally, our data revealed that along with the down-regulated levels of serum lipopolysaccharides (LPS), SEW2871 improved liver function and relieved hepatitis via blocking TLR4/MyD88/NF-kB signaling pathway in ANIT-treated mice. In conclusion, these results demonstrated that activating intestinal S1PR1 by SEW2871 could modulate intestinal barrier function, leading to the improvement of cholestatic hepatitis in ANIT-treated mice via the “gut-liver” axis.

CD8 T cell tolerance results from eviction of immature autoreactive cells from the thymus.

CD8 T cell tolerance is thought to result from clonal deletion of autoreactive thymocytes before they differentiate into mature CD8 T cells in the thymus. However, we report that, in mice, CD8 T cell tolerance instead results from premature thymic eviction of immature autoreactive CD8 thymocytes into the periphery, where they differentiate into self-tolerant mature CD8 T cells. Premature thymic eviction is triggered by T cell receptor (TCR)-driven down-regulation of the transcriptional repressor Gfi1, which induces expression of sphingosine-1-phosphate receptor-1 (S1P1) on negatively selected immature CD8 thymocytes. Thus, premature thymic eviction is the basis for CD8 T cell tolerance and is the mechanism responsible for the appearance in the periphery of mature CD8 T cells bearing autoreactive TCRs that are absent from the thymus.

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.

Post-vaccination SARS-Cov-2 T-cell receptor repertoires in patients with multiple sclerosis and related disorders.

Attenuation in post-vaccination SARS-CoV-2 humoral responses has been demonstrated in people treated with either anti-CD20 therapies or sphingosine-1-phosphate (S1P) receptor modulators. In the setting of disease modifying therapy (DMT) use, humoral response may not correlate with effective immunity, and analysis of vaccine-mediated SARS-CoV-2-specific memory T-cell responses is crucial. While vaccination in patients treated with anti-CD20 agents leads to deficient antibody production, emerging data from live cell assays suggests intact T-cell responses to vaccination. We evaluated post-vaccination SARS-CoV-2 T-cell receptor (TCR) repertoires in DMT-treated patients using the ImmunoSeqR assay, an assay that does not require live cells. Adults 18-80 years old without prior COVID-19, with neuroimmune conditions, who had been vaccinated with two doses of Pfizer-BioNTech or Moderna mRNA vaccines at least 3 weeks and up to 6 months prior, were recruited. Whole blood was obtained for immunosequencing, and matched serum was obtained for humoral analysis. Immunosequencing of the CDR3 regions of human TCRβ chains was completed using the immunoSEQR Assay (Adaptive Biotechnologies). TCR sequences were mapped across a set of TCR sequences reactive to SARS-CoV-2. Clonal diversity (breadth) and frequency (depth) of TCRs specific to SARS-CoV-2 spike protein were calculated and relationships with clinical variables were assessed. Forty patients were recruited into the study, aged 25-77, and 27 female. 37 had MS, 2 had neuromyelitis optica spectrum disorder (NMOSD), and 1 had hypophysitis. Subjects treated with anti-CD20 agents and S1P receptor modulators had severely attenuated humoral responses, but SARS-CoV-2-spike-specific TCR clonal depth and breadth were robust across all treatment classes except S1P modulators. No spike-specific or non-spike-specific SARS-CoV-2-associated TCRs were found in those treated with S1P modulators (p=0.002 for both breadth and depth). Subjects treated with fumarates exhibited somewhat lower spike TCR breadth than subjects treated with other or no DMTs (median 2.27×10^-5 for fumarates and 4.96×10^-5 for all others, p=0.008), but no statistically significant difference was demonstrated with spike TCR depth. No other significant associations with DMT type were found. We found no significant correlations between depth or breadth and age, duration of treatment, type of vaccination, or time interval since vaccination. This is the first study to characterize post-vaccination SARS-CoV-2 TCR repertoires in DMT-treated individuals. We demonstrated a dichotomous response to SARS-CoV-2 vaccination in anti-CD20-treated patients, with severely attenuated humoral response but intact TCR depth and breadth. It is unclear to what degree each arm of the adaptive immune system impacts post-vaccine immunity, both from the standpoint of incidence of post-vaccine infections and that of infection severity, and further clinical studies are necessary. S1P modulator-treated subjects exhibited both severely attenuated humoral responses and absent spike-specific TCR depth and breadth, information which is crucial for counseling of patients on these agents. Our methodology can be used in larger studies to determine the benefit of repeated vaccination doses, including those that are modified to better target modern or seasonal variants, without the use of live cell assays.

Tertiary lymphoid structures as local perpetuators of organ-specific immune injury: implication for lupus nephritis.

In response to inflammatory stimuli in conditions such as autoimmune disorders, infections and cancers, immune cells organize in nonlymphoid tissues, which resemble secondary lymphoid organs. Such immune cell clusters are called tertiary lymphoid structures (TLS). Here, we describe the potential role of TLS in the pathogenesis of autoimmune disease, focusing on lupus nephritis, a condition that incurs major morbidity and mortality. In the kidneys of patients and animals with lupus nephritis, the presence of immune cell aggregates with similar cell composition, structure, and gene signature as lymph nodes and of lymphoid tissue-inducer and -organizer cells, along with evidence of communication between stromal and immune cells are indicative of the formation of TLS. TLS formation in kidneys affected by lupus may be instigated by local increases in lymphorganogenic chemokines such as CXCL13, and in molecules associated with leukocyte migration and vascularization. Importantly, the presence of TLS in kidneys is associated with severe tubulointerstitial inflammation, higher disease activity and chronicity indices, and poor response to treatment in patients with lupus nephritis. TLS may contribute to the pathogenesis of lupus nephritis by increasing local IFN-I production, facilitating the recruitment and supporting survival of autoreactive B cells, maintaining local production of systemic autoantibodies such as anti-dsDNA and anti-Sm/RNP autoantibodies, and initiating epitope spreading to local autoantigens. Resolution of TLS, along with improvement in lupus, by treating animals with soluble BAFF receptor, docosahexaenoic acid, complement inhibitor C4BP(β-), S1P1 receptor modulator Cenerimod, dexamethasone, and anti-CXCL13 further emphasizes a role of TLS in the pathogenesis of lupus. However, the mechanisms underlying TLS formation and their roles in the pathogenesis of lupus nephritis are not fully comprehended. Furthermore, the lack of non-invasive methods to visualize/quantify TLS in kidneys is also a major hurdle; however, recent success in visualizing TLS in lupus-prone mice by photon emission computed tomography provides hope for early detection and manipulation of TLS.

Does cardiovascular risk matter in IBD patients?

Cardiovascular and thromboembolic risks are increasing in the population as a whole and therefore also in inflammatory bowel disease (IBD) patients. Obesity is a worldwide challenge also affecting the IBD population, and a causal association with Crohn's disease may exist. IBD itself, particularly when active, is also associated with a significant risk of thromboembolic and cardiovascular events such as myocardial infarction and stroke. Cardiovascular risk is also a significant consideration when using Janus kinase (JAK) inhibitors and sphingosine 1 phosphate (S1P) receptor modulators to treat IBD. JAK inhibitors - such as tofacitinib - are associated with several cardiovascular and venous thromboembolic risks, including hypertension and alterations in lipid profiles - specifically, increased LDL cholesterol and triglycerides - which may contribute to atherosclerosis and cardiovascular disease. S1P receptor modulators pose a slightly different set of cardiovascular risks. Initially, these drugs can cause transient bradycardia and atrioventricular (AV) block, leading to bradycardia. Moreover, they may induce QT interval prolongation, which increases the risk of life-threatening arrhythmias such as torsades de pointes. Some patients may also experience hypertension as a side effect. In this context, IBD healthcare providers need to be alert to the assessment of cardiovascular risk - particularly as cardiovascular events appear to be confined to specific patient groups with pre-existing risk factors. In addition, the potential for S1P modulator drug interactions requires a higher level of vigilance in patients with polypharmacy compared to biologics. Cardiovascular risk is not static, and updated assessment will need to become part of the routine in many IBD units.

Targeting the SPHK1/S1P/S1PR2 axis ameliorates GH-secreted pituitary adenoma progression.

Growth hormone-secreted pituitary adenoma (GHPA) is a prominent subtype of pituitary adenoma (PA) associated with progressive somatic disfigurement, various complications, and elevated mortality rates. Existing treatment options have limited efficacy, highlighting the urgent need for novel pharmacological interventions. Previous studies have revealed that sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptors (S1PRs) signalling have critical roles in the tumour microenvironment, but their role in GHPA remains unclear. We performed integrative analyses including bioinformatics analyses, functional studies, and clinical validation to investigate the pathological roles of SPHK1/S1P and evaluated the effectiveness of the S1P receptor 2 (S1PR2) inhibitor JTE-013 in GHPA treatment. SPHK1/S1P signalling is abnormally expressed in patients with GHPA. Knockdown of SPHK1 suppresses S1P-mediated cell proliferation in GH3 Cells. Mechanistically, S1P inhibits apoptosis and autophagy while promoting the secretion of Growth Hormone (GH) by binding to the S1P receptor subtype 2 (S1PR2) in GH3 cells. Moreover, the function of S1PR2 in GH3 cells is mediated by the downstream Akt-Creb pathway. We then identify the S1PR2 as a novel target for therapeutic intervention in GHPA. Systemic administration of the potent and selective S1PR2 antagonist, JTE-013, significantly reduces both tumour size and GH secretion. Importantly, we identify preoperative serum S1P levels as a biomarker predicting poor prognosis in GHPA patients at follow-up. Our study shows that blocking SPHK1/S1P/S1PR2 axis can ameliorate the progression of GHPA, providing evidence of a promising therapeutic target for GHPA.

Quantitative Analysis of S1PR1 Expression in the Postmortem Multiple Sclerosis Central Nervous System.

Multiple sclerosis (MS) is an immune-mediated disease that is characterized by demyelination and inflammation in the central nervous system (CNS). Previous studies demonstrated that sphingosine-1-phosphate receptor (S1PR) modulators effectively inhibit S1PR1 in immune cell trafficking and reduce entry of pathogenic cells into the CNS. Studies have also implicated a nonimmune, inflammatory role of S1PR1 within the CNS in MS. In this study, we explored the expression of S1PR1 in the development and progression of demyelinating pathology of MS by quantitative assessment of S1PR1 expression using our S1PR1-specific radioligand, [3H]CS1P1, in the postmortem human CNS tissues including cortex, cerebellum, and spinal cord of MS cases and age- and sex-matched healthy cases. Immunohistochemistry with whole slide scanning for S1PR1 and various myelin proteins was also performed. Autoradiographic analysis using [3H]CS1P1 showed that the expression of S1PR1 was statistically significantly elevated in lesions compared to nonlesion regions in the MS cases, as well as normal healthy controls. The uptake of [3H]CS1P1 in the gray matter and nonlesion white matter did not significantly differ between healthy and MS CNS tissues. Saturation autoradiography analysis showed an increased binding affinity (Kd) of [3H]CS1P1 to S1PR1 in both gray matter and white matter of MS brains compared to healthy brains. Our blocking study using NIBR-0213, a S1PR1 antagonist, indicated [3H]CS1P1 is highly specific to S1PR1. Our findings demonstrated the activation of S1PR1 and an increased uptake of [3H]CS1P1 in the lesions of MS CNS. In summary, our quantitative autoradiography analysis using [3H]CS1P1 on human postmortem tissues shows the feasibility of novel imaging strategies for MS by targeting S1PR1.

The GPCR adaptor protein Norbin regulates S1PR1 trafficking and the morphology, cell cycle and survival of PC12 cells

Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking.

Benefits of sphingosine-1-phosphate receptor modulators in relapsing MS estimated with a treatment sequence model

BackgroundThree sphingosine-1-phosphate receptor (S1PR) modulators are currently available as disease-modifying therapies (DMTs) for relapsing MS in the Netherlands (i.e. fingolimod, ozanimod and ponesimod). We aimed to identify which S1PR modulator yields the highest benefit from a health-economic and societal perspective during a patient's lifespan. MethodsIncorporating Dutch DMT list prices, we used the ErasmusMC/iMTA MS model to compare DMT sequences, including S1PR modulators and eight other DMT classes, for treatment-naïve patients with relapsing MS in terms of health outcomes (number of lifetime relapses, time to Expanded Disability Status Scale (EDSS) 6, lifetime quality-adjusted life years (QALYs)) and cost-effectiveness (net health benefit (NHB)). We estimated the influence of list price and EDSS progression on cost-effectiveness outcomes. ResultsIn deterministic and probabilistic analysis, DMT sequences with ponesimod have lower lifetime costs and higher QALYs resulting in a higher average NHB compared to sequences with other S1PR modulators. Ponesimod remains the most cost-effective S1PR modulator when EDSS progression is class-averaged. Given the variable effects on disability progression, list price reductions could make fingolimod but not ozanimod more cost-effective than ponesimod. ConclusionOur model favours ponesimod among the S1PR modulators for the treatment of relapsing MS. This implies that prioritizing ponesimod over other S1PR modulators translates into a more efficacious spending of national healthcare budget without reducing benefit for people with MS. Prioritizing cost-effective choices when counselling patients contributes to affordable and accessible MS care.

Long non-coding RNA HOX transcript antisense intergenic RNA depletion protects against alcoholic hepatitis through the microRNA-148a-3p/sphingosine 1-phosphate receptor 1 axis.

The aggravating role of long noncoding RNA (lncRNA) HOTAIR has been indicated in liver injury caused by hepatic ischemia/reperfusion. However, under the condition of alcoholic hepatitis (AH), its effects remain unclear. The present study aimed to examine the effect of lncRNA HOTAIR on hepatic stellate cell viability and apoptosis during liver injury caused by AH. In the liver tissues of AH rats, HOTAIR and S1PR1 were overexpressed, and microRNA (miR)-148a-3p was poorly expressed. Loss-of-function assays revealed that silencing of HOTAIR alleviated liver injury in AH by inhibiting the activated phenotype of hepatic stellate cells, inflammation, and fibrosis. Using the bioinformatics databases, dual-luciferase, RIP, and FISH assays, we observed that HOTAIR was mainly localized in the cytoplasm of hepatic stellate cells, and HOTAIR could bind specifically to miR-148a-3p. In addition, miR-148a-3p could target S1PR1 expression. Rescue experiments showed that silencing of miR-148a-3p or overexpression of S1PR1 reversed the alleviating effects of HOTAIR silencing on liver injury. Taken together, our findings revealed that HOTAIR regulates hepatic stellate cell proliferation via the miR-148a-3p/S1PR1 axis in liver injury, which may serve as the basis for developing novel therapeutic strategies to treat AH.

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.

JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission.

Pulmonary fibrosis may be due to the proliferation of fibroblasts and the aggregation of extracellular matrix, resulting in the stimulation of inflammation damage, destroying lung tissue structure, seriously affecting the patient's respiratory function, and even leading to death. We investigated the role and mechanism of JTE-013 in attenuating bleomycin (BLM)-induced pulmonary fibrosis. BLM-induced pulmonary fibrosis was established in mice. Type 2 alveolar epithelial cells (MLE-12) were stimulated with sphingosine monophosphate (S1P) in vitro. JTE-013, an S1PR2 (sphingosine 1-phosphate receptor 2) antagonist, and Verteporfin were administered in vivo and in vitro. IL-4, IL-5, TNF-α, and IFN-γ were measured by ELISA. IL-4 and IFN-γ positive cells were detected by flow cytometry. Inhibition of S1PR2 with JTE-013 significantly ameliorated BLM-induced pathological changes and inflammatory cytokine levels. JTE-013 also significantly reduced the expression of RHOA/YAP pathway proteins and mitochondrial fission protein Drp1, apoptosis, and the colocalization of α-SMA with YAP, Drp1, and Tom20, as detected by immunohistochemistry, immunofluorescence staining, TUNEL, and Western blot. In vitro, S1PR2 and YAP knockdown downregulated RHOA/YAP pathway protein expression, Drp1 phosphorylation, and Drp1 translocation, promoted YAP phosphorylation and phenotypic transformation of MFN2, and inhibited the up-regulation of mitochondrial membrane potential, reactive oxygen species production, and cell apoptosis (7.13% vs. 18.14%), protecting the integrity of the mitochondrial dynamics. JTE-013 also inhibited the expression of fibrosis markers α-SMA, MMP-9, and COL1A1, and alleviated the symptoms of pulmonary fibrosis. Conclusively, JTE-013 has great anti-pulmonary fibrosis potential by regulating RHOA/YAP and mitochondrial fusion/fission.

Imidazole-based sphingosine-1-phosphate transporter Spns2 inhibitors

Sphingosine-1-phosphate (S1P) is a chemotactic lipid that influences immune cell positioning. S1P concentration gradients are necessary for proper egress of lymphocytes from the thymus and secondary lymphoid tissues. This trafficking is interdicted by S1P receptor modulators, and it is expected that S1P transporter (Spns2) inhibitors, by reshaping S1P concentration gradients, will do the same. We previously reported SLF1081851 as a prototype Spns2 inhibitor, which provided a scaffold to investigate the importance of the oxadiazole core and the terminal amine. In this report, we disclose a structure–activity relationship study by incorporating imidazole as both a linker and surrogate for a positive charge in SLF1081851. In vitro inhibition of Spns2-dependent S1P transport in HeLa cells identified 7b as an inhibitor with an IC50 of 1.4 ± 0.3 µM. The SAR studies reported herein indicate that imidazolium can be a substitute for the terminal amine in SLF1081851 and that Spns2 inhibition is highly dependent on the lipid alkyl tail length.

Structural determinants of sphingosine-1-phosphate receptor selectivity.

Fingolimod, the prodrug of fingolimod-1-phosphate (F1P), was the first sphingosine-1-phosphate receptor (S1PR) modulator approved for multiple sclerosis. F1P unselectively targets all five S1PR subtypes. While agonism (functional antagonism via receptor internalization) at S1PR1 leads to the desired immune modulatory effects, agonism at S1PR3 is associated with cardiac adverse effects. This motivated the development of S1PR3 -sparing compounds and led to a second generation of S1PR1,5 -selective ligands like siponimod and ozanimod. Our method combines molecular dynamics simulations and three-dimensional pharmacophores (dynophores) and enables the elucidation of S1PR subtype-specific binding site characteristics, visualizing also subtle differences in receptor-ligand interactions. F1P and the endogenous ligand sphingosine-1-phosphate bind to the orthosteric pocket of all S1PRs, but show different binding mode dynamics, uncovering potential starting points for the development of subtype-specific ligands. Our study contributes to the mechanistic understanding of the selectivity profile of approved drugs like ozanimod and siponimod and pharmaceutical tool compounds like CYM5541.

SAT039 Chronic Glucocorticoid Exposure Induced A S1PR2-Rorc Axis To Enhance Hepatic Gluconeogenesis

Abstract Disclosure: R.A. Lee: None. M. Chang: None. A. Tsay: None. Y. Lee: None. D. Li: None. N. Yiv: None. S. Tian: None. J. Wang: None. Glucocorticoids are steroid hormones whose circulating levels are elevated in response to stress to maintain metabolic homeostasis. Glucocorticoids are also used to treat inflammatory and autoimmune diseases due to their potent anti-inflammatory and immunomodulatory activities. However, chronic glucocorticoid treatment causes various metabolic disorders, such as hyperglycemia and insulin resistance. Glucocorticoids convey their functions through an intracellular glucocorticoid receptor (GR) which is a transcriptional regulator. While GR stimulates hepatic gluconeogenic gene transcription, additional mechanisms activated specifically by chronic glucocorticoid exposure can further enhance hyperglycemia. We found that chronic glucocorticoid exposure elevated hepatic production of sphingosine-1-phosphate (S1P) and hepatic knockdown of sphingosine-1-phosphate receptor 2 (S1PR2) attenuated chronic glucocorticoid-induced glucose intolerance and hepatic gluconeogenesis while having no effect on insulin signaling. Hepatic S1PR2 knockdown reduced GR occupancy on glucocorticoid response elements (GREs) of gluconeogenic genes, such as phosphoenolpyruvate carboxykinase (Pck1) and the catalytic subunit of glucose 6 phosphatase (G6pc), thus reducing their expressions. Interestingly, RAR-related orphan receptor C (Rorc) expression was reduced in the liver of glucocorticoid treated hepatic S1PR2 knockdown mice. We found that Rorc was also recruited to the promoter of Pck1 upon glucocorticoid treatment and reducing Rorc activity decreased the GR occupancy at the GRE of Pck1. In contrast, overexpressing Rorc in the liver of hepatic S1PR2 knockdown mice restored glucocorticoid-induced glucose intolerance, GR recruitment to GREs of gluconeogenic genes, and gluconeogenic gene expression. Overall, we propose that chronic glucocorticoid exposure induces a novel S1PR2-Rorc axis to promote the transcription of gluconeogenic genes to enhance hyperglycemia. Presentation: Saturday, June 17, 2023

S854 Sphingosine 1-Phosphate Receptor Modulators as an Induction and Maintenance Therapy for Ulcerative Colitis: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

S854 Sphingosine 1-Phosphate Receptor Modulators as an Induction and Maintenance Therapy for Ulcerative Colitis: A Systematic Review and Meta-Analysis of Randomized Controlled Trials