Sphingosine 1-phosphate Receptor Type Research Articles

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

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

Dissociation between the anti-allodynic effects of fingolimod (FTY720) and desensitization of S1P1 receptor-mediated G-protein activation in a mouse model of sciatic nerve injury

Sphingosine-1-phosphate (S1P) receptor (S1PR) agonists, such as fingolimod (FTY720), alleviate nociception in preclinical pain models by either activation (agonism) or inhibition (functional antagonism) of S1PR type-1 (S1PR1). However, the dose-dependence and temporal relationship between reversal of nociception and modulation of S1PR1 signaling has not been systematically investigated. This study examined the relationship between FTY720-induced antinociception and S1PR1 adaptation using a sciatic nerve chronic constriction injury (CCI) model of neuropathic pain in male and female C57Bl/6J mice. Daily injections of FTY720 for 14 days dose-dependently reversed CCI-induced mechanical allodynia without tolerance development, and concomitantly resulted in a dose-dependent reduction of G-protein activation by the S1PR1-selective agonist SEW2871 in the lumbar spinal cord and brain. These findings indicate FTY720-induced desensitization of S1PR1 signaling coincides with its anti-allodynic effects. Consistent with this finding, a single injection of FTY720 reversed mechanical allodynia while concomitantly producing partial desensitization of S1PR1-stimulated G-protein activation in the CNS. However, mechanical allodynia returned 24-hr post injection, despite S1PR1 desensitization at that time, demonstrating a dissociation between these measures. Furthermore, CCI surgery led to elevations of sphingolipid metabolites, including S1P, which were unaffected by daily FTY720 administration, suggesting FTY720 reversed mechanical allodynia by targeting S1PR1 rather than sphingolipid metabolism. Supporting this hypothesis, acute administration of the S1PR1-selective agonist CYM-5442 mimicked the anti-allodynic effect of FTY720. In contrast, the S1PR1-selective antagonist NIBR-0213 prevented the anti-allodynic effect of FTY720, but NIBR-0213 given alone did not affect nociception. These results indicate that FTY720 alleviates CCI-induced allodynia through a mechanism distinct from functional antagonism.

Extracellular α-synuclein impairs sphingosine 1-phosphate receptor type 3 (S1PR3)-regulated lysosomal delivery of cathepsin D in HeLa cells.

α-Synuclein (α-Syn)-positive intracellular fibrillar protein deposits, known as Lewy bodies, are thought to be involved in the pathogenesis of Parkinson's disease (PD). Although recent lines of evidence suggested that extracellular α-Syn secreted from pathogenic neurons contributes to the propagation of PD pathology, the precise mechanism of action remains unclear. We have reported that extracellular α-Syn caused sphingosine 1-phosphate (S1P) receptor type 1 (S1PR1) uncoupled from Gi and inhibited downstream G-protein signaling in SH-SY5Y cells, although its patho/physiological role remains to be clarified. Here we show that extracellular α-Syn caused S1P receptor type 3 (S1PR3) uncoupled from G protein in HeLa cells. Further studies indicated that α-Syn treatment reduced cathepsin D activity while enhancing the secretion of immature pro-cathepsin D into cell culture medium, suggesting that lysosomal delivery of cathepsin D was disturbed. Actually, extracellular α-Syn attenuated the retrograde trafficking of insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptor, which is under the regulation of S1PR3. These findings shed light on the understanding of dissemination of the PD pathology, that is, the mechanism underlying how extracellular α-Syn secreted from pathogenic cells causes lysosomal dysfunction of the neighboring healthy cells, leading to propagation of the disease.

Apolipoprotein M and Sphingosine-1-Phosphate Receptor 1 Promote the Transendothelial Transport of High-Density Lipoprotein.

Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.

Sphingosine 1-phosphate receptor type 2 positively regulates interleukin (IL)-4/IL-13-induced STAT6 phosphorylation

Previous reports have demonstrated that sphingosine 1-phosphate receptor type 2 (S1P2) is involved in the activation of signal transducer and activator of transcription (STAT) 6. Additionally, the major signaling pathway of S1P2 is the Rho-Rho kinase pathway. In this study, we examined the role of S1P2 in STAT6 activation in a macrophage (Mφ) model using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA). We established S1P2knockout THP-1 cells using the CRISPR-Cas9 gene editing system. The PMA-treated S1P2knockout THP-1 Mφs showed decreases in IL-4/IL-13-induced phosphorylation of Janus-activated kinase (JAK) 1, JAK2, and STAT6 as well as mRNA expression of the M2 marker ARG1 compared with wild-type THP-1 Mφs. Pretreatment of PMA-treated THP-1 Mφs with the S1P2 antagonist JTE-013, the Rho inhibitor Rhosin or the Rho kinase inhibitor Y27632 inhibited the IL-4/IL-13-induced increase in STAT6 phosphorylation. The expressions of suppressor of cytokine signaling 3 in the S1P2knockout THP-1 Mφs were higher than those in wild-type THP-1 Mφs. In addition, the protein tyrosine phosphatase inhibitor vanadate enhanced IL-4-induced STAT6 phosphorylation in the S1P2knockout THP-1 Mφs, suggesting that S1P2-Rho-Rho kinase inhibited the negative regulation of STAT6. These results suggest that the S1P2-Rho-Rho kinase pathway is necessary for full activation of STAT6 by IL-4/IL-13 in Mφs.

Targeted Deletion of Sphingosine‐1‐Phosphate Receptor Type 1 (S1PR1) Reveals a Predominant Functional Localization of S1PR1 in Astrocytes in the Spinal Cord

Sphingosine-1-phosphate (S1P) is a tightly regulated bioactive lysolipid that acts as the endogenous ligand for G-protein coupled S1P receptors (S1PR) types 1-5. The S1PR system has been targeted for immune modulation, but research indicates its potential as a target for pain relief. FTY720 is a S1PR immunomodulatory prodrug that is phosphorylated in vivo to its active form, FTY720-phosphate (FTY-P), which activates S1PR 1, 3, 4, and 5. FTY720 is currently FDA approved for the treatment of Multiple Sclerosis, but preclinical evidence has indicated both S1P and FTY720 produce antinociception in models of acute and neuropathic pain. Results with S1PR1 selective agonists and antagonists suggest S1PR ligands most likely act to alleviate pain specifically through S1PR1. Here we used conditional gene deletion to determine the cell type(s) mediating S1P-stimulated G-protein activation in the spinal cord, measured using agonist-stimulated [35S]GTPγS binding. Deletion of S1PR1 in S1PR1-floxed mice expressing cre recombinase under control of the nestin promoter, which deletes S1PR1 from neurons, astrocytes, and oligodendroglia, showed a profound decrease in G-protein activation stimulated by S1P (by ~85%) or the S1PR1 agonist SEW2871 (>95%) in the spinal cord, suggesting the majority of S1PR activity in spinal cord is mediated by S1PR1. We next explored the extent to which S1PR1-mediated G-protein activation in the lumbar spinal cord of male and female mice is specifically localized in astrocytes. The lumbar spinal cord is thought to be a major point of transduction of pain signals from nerve-injury models of neuropathic pain in rodents. S1PR1-floxed mice were crossed with cre recombinase under the control of a glial fibrillary acidic protein (GFAP)-promotor. In this model, there was a ~75% loss in G-protein activation by S1P in the astrocyte specific conditional knock out (cKO) compared to the control mice, whereas there was a near complete (>95%) loss of SEW2871-stimulated G-protein activation in these cKO mice. No sex differences in S1PR-stimulated G-protein activation or the effect of S1PR1 deletion was observed. This loss of S1PR1-stimulated G-protein activation in the lumbar spinal cord upon astrocyte specific deletion of S1PR1 suggests S1PR1 activity is mainly localized in astrocytes within the lumbar spinal cord. These results aid in the understanding of S1PR1 localization and supports recently reported evidence for a role for S1PR1 in astrocytes in FTY720-responsive neuropathic pain models. Studies are also ongoing to determine the functional localization of S1PR1 in astrocytes in brain regions associated with pain perception and regulation.

In Vitro Acute Ischemic Injury Altered Human Brain Endothelial MMP‐9 Activity and Associated Cell Surface Adhesion Protein Expression in Part by S1PR Type 1 Activation

Cerebrovascular endothelial cells are among the first cells to be impacted by acute ischemic stroke (AIS) which poses them as important therapeutic targets pivotal to the maintenance of blood brain barrier (BBB) integrity. Following AIS, matrix metalloproteinase 9 (MMP-9) proteolytic activity contributes to the loss in BBB integrity, increased hemorrhagic risk, and worse outcomes. We have previously shown that sphingosine-1-phosphate receptor (S1PR) ligands attenuated hypoxia plus glucose deprivation (HGD)-induced decreases in brain microvascular endothelial trans-endothelial resistance (TEER), suggesting a potential role for S1PR ligands on barrier function during AIS. Endothelial vascular cell adhesion molecule 1 (VCAM-1) is an inflammatory mediator and adhesion molecule that contributes to the loss of endothelial barrier integrity by serving as a scaffold for immune cell extravasation as well as a mediator for intracellular signals involved in MMP-9 activation. Cell surface adhesion receptor, CD44, likewise plays a key role in facilitating MMP-9 activity via co-localization at the endothelial surface. Thusly, we sought to investigate protein expression levels of VCAM-1, MMP-9, tight junctional proteins, (ZO-1 and claudin-5), as well as to characterize CD44 protein expression in cerebrovascular endothelial cells following HGD. Pediatric male human brain microvascular endothelial cells (HBMECs) were treated with ozanimod (selective S1PR type 1 ligand; 0.5 nM) at P7 and immediately exposed to normoxia (21% O2) or HGD (1% O2) for 3h, a timepoint determined from our previous TEER studies which revealed deterioration of the endothelial barrier as early as 3h. To verify S1PR type 1 dependency, selective blockade with antagonist W146 was applied to select experiments. We observed through standard immunoblotting that HGD induced a simultaneous decrease in ZO-1 and claudin-5 in parallel to increased expression of VCAM-1. Additionally, intracellular pro-MMP-9 (inactive enzymatic form) levels decreased following HGD and the ratio of active MMP-9 to pro-MMP-9 was increased. Ozanimod did not alter these responses. Using immunocytochemistry, anti-CD44 fluorescence was increased following HGD exposure, and ozanimod attenuated this response. S1PR type 1 blockade demonstrated receptor dependence. In conclusion, HGD induced endothelial activation characterized by increased adhesion molecule levels and involvement of MMP-9 activity potentially contributing to tight junction protein loss. Furthermore, S1PR type 1 ligands may in part attenuate HGD-induced BBB integrity loss by modulating the bioavailability of MMP-9 to disrupt endothelial barrier integrity. Mechanism(s) by which S1PR type 1 ligands regulate MMP-9 activity and its associated cell surface adhesion proteins (e.g. CD44 and VCAM-1) following ischemic-like injury warrants further investigation.

Design, synthesis and biological evaluation as immunosuppressant of amino alcohol derivatives containing thioether

To develop a safer immunosuppressant for organ transplantation and autoimmune disease treatment, in this study, several of novel amino alcohol derivatives containing thioether moiety were synthesized with 7-bromo-tetralin-2-one as starting material, and Suzuki coupling reaction and Bucherer-Bergs reaction as key steps. Their activity as sphingosine 1-phosphate receptor type 1 (S1P1) agonists were evaluated by [γ-35S] GTP binding assay. Among the thioether substituted compounds, compound10showed good activity as S1P1agonist at low micromolar concentration (EC50 = 0.698 μmol/L). The result suggested that it has potential activity against autoimmune diseases and immunosuppressant of organ transplantations.

The role of sphingosine 1‐phosphate type 2 receptor on IL‐4/IL‐13‐stimulated STAT6 phosphorylation in phorbol12‐myristate13‐acetate‐treated THP‐1 cells

Background/Purpose We previously observed reduced activation of STAT6 in broncho-alveolar lavage fluids (BALF) cells from bleomycin-administered sphingosine 1-phosphate receptor type 2 (S1P2) knockout mice compared to BALF cells from bleomycin-administered wild-type mice (PLoS One, 2018). In this study, we examined the role of S1P2 on STAT6 activation using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) as a model for macrophages (MΦs). Methods THP-1 cells were cultured in RPMI-1640 containing 10% fetal bovine serum. THP-1 cells were treated with PMA to differentiate into adherent MΦs. S1P2knockout THP-1 cells were established using CRISPR-Cas9 gene editing system. Quantification of mRNA expression was performed by quantitative RT-PCR using specific primers. Jak/STAT6 activation was performed by Western blot analysis using antibodies against tyrosine-phosphorylated Jak1, Jak2 and STAT6. Results Quantitative PCR analysis showed that THP-1 cells expresses S1P2. We examined the responses of PMA-treated THP-1 MΦs to stimulation with IL-4/IL-13 in the presence of S1P. IL-4/IL-13 stimulation induced a strong increase in STAT6 phosphorylation in PMA-treated THP-1 MΦs. The major signaling pathway of S1P2 is a Rho–Rho kinase pathway. Pretreatment of PMA-treated THP-1 MΦs with the S1P2 inhibitor JTE-013 and the Rho kinase inhibitor Y27632 inhibited IL-4/IL-13–induced increase in STAT6 phosphorylation. PMA-treated S1P2knockout THP-1 MΦs also showed a lower extent of STAT6 phosphorylation in response to IL-4/IL-13 stimulation than PMA-treated wild-type THP-1 MΦs. mRNA expression of IL-4/IL-13 receptors was not different between wild-type and S1P2knockout THP-1 MΦs. No difference in mRNA and protein expression of STAT6 was observed between wild-type and S1P2knockout THP-1 MΦs. The phosphorylation of Jak1 and Jak2 in S1P2knockout THP-1 MΦs was diminished compared with wild-type THP-1 MΦs. Finally, S1P2 knockout in THP-1 MΦs attenuated IL-4/IL-13-induced increase in the mRNA expression of the M2 marker arginase 1. Conclusion These results suggested that S1P2/Rho kinase pathway is necessary for full activation of STAT6 by IL-4 or IL-13 in MΦs and could be a drug target for Th2-associated diseases.

Apoptotic cell extrusion depends on single-cell synthesis of sphingosine-1-phosphate by sphingosine kinase 2

Collecting duct cells are physiologically subject to the hypertonic environment of the kidney. This condition is necessary for kidney maturation and function but represents a stress condition that requires active strategies to ensure epithelial integrity. Madin-Darby Canine Kidney (MDCK) cells develop the differentiated phenotype of collecting duct cells when subject to hypertonicity, serving as a model to study epithelial preservation and homeostasis in this particular environment. The integrity of epithelia is essential to achieve the required functional barrier. One of the mechanisms that ensure integrity is cell extrusion, a process initiated by sphingosine-1-phosphate (S1P) to remove dying or surplus cells while maintaining the epithelium barrier. Both types start with the activation of S1P receptor type 2, located in neighboring cells. In this work, we studied the effect of cell differentiation induced by hypertonicity on cell extrusion in MDCK cells, and we provide new insights into the associated molecular mechanism. We found that the different stages of differentiation influence the rate of apoptotic cell extrusion. Besides, we used a novel methodology to demonstrate that S1P increase in extruding cells of differentiated monolayers. These results show for first time that cell extrusion is triggered by the single-cell synthesis of S1P by sphingosine kinase 2 (SphK2), but not SphK1, of the extruding cell itself. Moreover, the inhibition or knockdown of SphK2 prevents cell extrusion and cell-cell junction protein degradation, but not apoptotic nuclear fragmentation. Thus, we propose SphK2 as the biochemical key to ensure the preservation of the epithelial barrier under hypertonic stress.

Validation of a monoclonal antibody directed against the human sphingosine 1-phosphate receptor type 1

The sphingosine 1-phosphate receptor type 1 (S1PR1) has several important functions, including stabilizing endothelial barrier and maintaining lymphocyte circulation. These functions are critically dependent on the regulation of S1PR1 cell surface expression. Currently available antibodies against human S1PR1 are not able to pick up cell surface expression on living cells by flow cytometry due to intracellular epitopes or unspecific binding. Here we describe the generation of a mouse monoclonal antibody specific for the N-terminal region of human S1PR1. It has an immunoglobulin M (IgM) kappa isotype and detects cell surface expression of recombinant human S1PR1 on overexpressing cells. Due to unspecific intracellular cell staining, it cannot be used for staining of dead cells and tissue slides or in microscopic analyses. It is also not suitable for Western blot analysis and immunoprecipitation. However, the antibody can stain for endogenous S1PR1 on human endothelial cell lines and primary human umbilical vein endothelial cells (HUVEC). Incubation of these cells with various S1PR1 agonists revealed potent S1PR1 internalization, which was not the case with the specific antagonist W146. Surprisingly, human T and B cells isolated from blood and palatine tonsils did not show specific staining, demonstrating significantly lower endogenous S1PR1 surface expression on lymphocytes than on endothelial cells.

Sphingosine 1-phosphate (S1P) receptor type 1 signaling induces an anti-atherogenic phenotype in macrophages and attenuates atherosclerosis in LDL-receptor-deficient mice

Background and Aims: Sphingosine 1-phosphate (S1P) accounts for antiatherogenic properties of high-density lipoproteins, but the S1P receptor subtype mediating the atheroprotective effects of S1P and the underlying molecular mechanisms remain enigmatic. Experimental models engineered to amplify the signaling of endogenous S1P over its specific receptors may help in clarifying this issue. To this purpose, we generated a mouse model overexpressing S1P receptor type 1 (S1P1) specifically in the myeloid compartment and studied the effect on atherosclerosis development.

Blockage of sphingosine-1-phosphate receptor 2 attenuates 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice.

Sphingosine-1-phosphate (S1P) and its receptors have been implicated in functions of Langerhans cells and atopic dermatitis. In this study, we investigated the roles of S1P receptor type 2 (S1P2) in a mouse model of atopic dermatitis, which was induced by topical application of 2,4-dinitrochlorobenzene (DNCB) on ventral skin on D0, followed by repeated DNCB challenge on both ears from D7 to D49. Wild-type mice with atopic dermatitis displayed severe inflammation and mast cell accumulation in ear tissues and elevated IgE levels in serum. Furthermore, the mice showed significantly increased sizes of draining lymph nodes, high levels of inflammatory cytokines (IL-4, IL-13, IL-17, and IFN-γ) in the ears and lymph nodes and high levels of chemokines CCL17 and CCL22 in ears. Administration of JTE-013, a selective antagonist of S1P2 (3 mg/kg, i.p, from D19 to D49) before DNCB challenge significantly suppressed DNCB-induced atopic responses in ears and lymph nodes. JTE-013 administration also significantly decreased the lymph nodes sizes, the levels of inflammatory cytokines (IL-4, IL-13, IL-17, and IFN-γ) in the ears and lymph nodes, and the levels of chemokines CCL17 and CCL22 in ears. Furthermore, the inflammatory responses of atopic dermatitis were greatly ameliorated in S1pr2 gene-deficient mice. As CCL17 and CCL22 are CCR4 ligands, acting as Th2-attracting chemokines, we investigated CCL17 and CCL22 expression in bone marrow-derived dendritic cells (BMDCs) from wild-type and S1pr2 gene-deficient mice. Addition of IL-4 (10 ng/mL) markedly increased the levels of CCL17 and CCL22, but IL-4-induced CCL17 and CCL22 expression was significantly blunted in BMDCs from S1pr2 gene-deficient mice. Furthermore, pretreatment with JTE-013 (1-30 μM) dose-dependently suppressed this induction in BMDCs from wild-type mice. Our results demonstrate that blockage of S1P2 ameliorates not only DNCB-induced atopic dermatitis symptoms but also Th2 cell-attracting capacity of dendritic cells, suggesting S1P2 as a potential therapeutic target for atopic dermatitis.

Inflammatory Conditions Disrupt Constitutive Endothelial Cell Barrier Stabilization by Alleviating Autonomous Secretion of Sphingosine 1-Phosphate.

The breakdown of the endothelial cell (EC) barrier contributes significantly to sepsis mortality. Sphingosine 1-phosphate (S1P) is one of the most effective EC barrier-stabilizing signaling molecules. Stabilization is mainly transduced via the S1P receptor type 1 (S1PR1). Here, we demonstrate that S1P was autonomously produced by ECs. S1P secretion was significantly higher in primary human umbilical vein endothelial cells (HUVEC) compared to the endothelial cell line EA.hy926. Constitutive barrier stability of HUVEC, but not EA.hy926, was significantly compromised by the S1PR1 antagonist W146 and by the anti-S1P antibody Sphingomab. HUVEC and EA.hy926 differed in the expression of the S1P-transporter Spns2, which allowed HUVEC, but not EA.hy926, to secrete S1P into the extracellular space. Spns2 deficient mice showed increased serum albumin leakage in bronchoalveolar lavage fluid (BALF). Lung ECs isolated from Spns2 deficient mice revealed increased leakage of fluorescein isothiocyanate (FITC) labeled dextran and decreased resistance in electric cell-substrate impedance sensing (ECIS) measurements. Spns2 was down-regulated in HUVEC after stimulation with pro-inflammatory cytokines and lipopolysaccharides (LPS), which contributed to destabilization of the EC barrier. Our work suggests a new mechanism for barrier integrity maintenance. Secretion of S1P by EC via Spns2 contributed to constitutive EC barrier maintenance, which was disrupted under inflammatory conditions via the down-regulation of the S1P-transporter Spns2.

Sphingosine 1 phosphate receptor-1 (S1PR1) signaling protects cardiac function by inhibiting cardiomyocyte autophagy.

ObjectiveTo investigate the role of sphingosine-1-phosphate (S1P) and its receptors in cardiomyocyte autophagy, cardiomyocyte hypertrophy and cardiac function.MethodsCardiomyocytes were isolated from neonatal Vista rats. Autophagy and hypertrophy of cardiomyocytes were induced via starvation culture and phenylephrine (PE), respectively, and S1P was used to treat the cardiomyocytes. The effect of S1P on cardiomyocyte autophagy was evaluated by the number of autophagosomes, the expression of autophagy-related proteins and autophagic marker genes in cardiomyocytes. The effect of S1P on cardiomyocyte hypertrophy was evaluated by examining the surface area of cardiomyocytes and the expression of hypertrophic genes. Subsequently, different small interfering RNAs (siRNAs) were used to knockdown the expression of the three types of S1P receptors on cardiomyocytes and to analyze the type of receptor that mediates S1P signaling in cardiomyocytes. Finally, sphingosine 1 phosphate receptor-1 (S1PR1) was knockout in the mouse cardiomyocytes using the Cas9 technique. The effect of S1PR1 on cardiac autophagy and cardiac hypertrophy was examined by assessing cardiomyocyte autophagy, cardiomyocyte hypertrophy and cardiac function.ResultsStarvation-induced cardiomyocyte autophagy and PE-induced cardiomyocyte hypertrophy were significantly attenuated by S1P. The results showed that the formation of autophagosomes was decreased, the autophagy-associated protein LC3II/I and the expression of autophagic marker genes Atg5, Atg12, Beclin1 and LC3B decreased after S1P treatment. The surface area of the cardiomyocytes was decreased, and the expression of hypertrophic genes, including atrial natriuretic factor (ANF), skeletal muscle and cardiac actin (SKA), myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP) were all decreased after S1P treatment. The autophagy and hypertrophy of cardiomyocytes in the S1PR1 knocked-down group were significantly increased compared to those in the control group, the S1PR2 and the S1PR3 knocked-down groups. In vivo, the knockout of S1PR1 in cardiomyocytes exacerbated stress-induced cardiac autophagy, cardiac hypertrophy and the impairment of cardiac function.ConclusionS1P could inhibit cardiomyocyte autophagy, thereby inhibiting cardiomyocyte hypertrophy and protecting cardiac function by activating S1PR1 in pressure-overloaded cardiomyocytes in mice.

Role of Sphingosine ‐1‐Phosphate on Expression of MAPK and Akt Signaling Pathways in Hypoxic Human Extravillous Trophoblasts

Preeclampsia (PE), is a serious pregnancy disorder characterized by placental ischemia, hypertension, and proteinuria. A successful pregnancy requires the migration and invasion of extravillous trophoblasts (EVTs), from the villous tips of the placenta into the maternal decidua and subsequently the spiral arteries. Failure of EVTs to adequately migrate and invade into the spiral arteries leads to insufficient blood flow to the developing placenta commonly found in PE. Sphingosine‐1‐phosphate (S1P) functions as a key regulator of cellular homeostasis in angiogenesis, inflammation, and endothelium permeability. S1P acts on its receptor to control cell migration through the PI3‐Akt and p38 MAPK signaling pathways. We have previously reported that expression of placenta ceramidase, sphingosine kinases 1, and sphingosine kinases 2 were altered in reduced uterine perfusion (RUP) mouse model. In the present study, we tested the hypothesis that serine 473 phosphorylated Akt (pAkt) and phosphorylated p38 MAPK expressions are decreased in hypoxic condition and treatment with S1PR 1 agonist and antagonist alter these expression levels.MethodsThe human extravillous trophoblasts (EVTs) cell line SGHPL‐4 was cultured in either normoxia 21% oxygen or hypoxia 2% oxygen conditions for 24 hours. The cultures were treated for one hour with vehicle control or with S1PR1 receptor agonists (SEW2871, 1uM) or S1PR antagonist (W146, 18 nM).ResultsBoth pAkt and p38 MAPK were significantly decreased in hypoxia EVTs compared to normoxia EVTs (p38 MAPK: 50% and pAkt: 25% decrease). SEW2871 increased p38 MAPK in hypoxia EVTs to a level similar to normaxia EVTs. Inhibition of S1PR1 by W146 decreased p38 MAPK in both hypoxia and normoxia. SEW2871 normalized the differences of pAkt expression between normoxia and hypoxia EVTs. While W146 did not change the pAkt in normoxia, it significantly increased pAkt in hypoxia EVTs cells. This data suggests that activation of S1P receptor type 1 increases the expression of angiogenic pMAPK in hypoxic SGHPL‐4 trophoblast cells.Support or Funding InformationDr. Intapad was supported by funding from the AHA 16SDG27770041, ASN Kidney Research Career Development grant, and Start‐up funds from Tulane University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS-induced BBB disruption.

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine-1-phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context-dependent manner. We utilized lipopolysaccharide (LPS)-injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood-brain barrier (BBB) breakdown invivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMVs), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMVs and brain tissue. Primary mouse brain microvascular endothelial cells (MBMECs) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER) as a measure of permeability invitro. We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMVs, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP1) and S1P phosphatase 1 on the other hand, as well as a down-regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMECs did not alter TEER, but serum from LPS-treated mice lead to a breakdown of the barrier compared to serum from vehicle-treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis. Read the Editorial Highlight for this article on page 115. Cover Image for this Issue: doi. 10.1111/jnc.14161.

Synthesis and biological evaluation of amino alcohol containing 1,3,4-oxadiazole

A series of amino alcohol derivatives containing 1,3,4-oxadiazole moieties was synthesized with 7-bromo-2-tetralone as starting materials, 2,2-dimethyl-1,3-oxazolidine as intermediates and Strecker reaction and cyclization with POCl3 as key steps. The structures of the key intermediate and target compounds were confirmed by 1H NMR, 13C NMR and HRMS. Some compounds have resulted in the generation of highly potent sphingosine 1-phosphate receptor type 1(S1P1) agonists.

Abstract 130: Sphingosine-1-Phosphate Signaling Plays a Role in High Blood Pressure Programmed by Intrauterine Growth Restriction in Mouse

Intrauterine growth restriction (IUGR) is a risk factor for hypertension and cardiovascular (CV) disease in later life, but the underlying mechanisms remain unclear. The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) is critically involved in CV development in the fetus, and plays a significant role in the regulation of CV health in adulthood. S1P receptor (S1PR) type 1, 2 and 3 are widely expressed in CV system which S1PR1 has a protective role against kidney injury, while S1PR3 is involved in controlling BP. Yet, the contribution of S1P on BP in IUGR is unknown. In the present studies, we tested the hypothesis that IUGR alters renal S1P receptors expression during- and post-nephrogenesis, which contributes to high BP in male IUGR mouse. C57bl/6J mice underwent sham or reduced uterine perfusion (RUP) at day 13 of gestation with delivery at full term. IUGR offspring (from RUP dam) had a lower birth weight than control (p<0.05). Kidneys were isolated from 2 day old male pups or adult 24 week old male control and IUGR. S1PR3 protein expression was increased in 2 day old IUGR kidneys (2.4 fold vs control, N=3, p< 0.01). At 24 weeks of age, S1PR3 mRNA levels were increased (1.2 fold vs control, N=4, p< 0.05) whereas S1PR3 protein levels were decreased (0.75 fold vs control, N=4, p< 0.05) in IUGR kidneys. mRNA and protein expression levels of S1PR1 and S1PR2 were not different between control and IUGR kidneys. Finally, we assessed the role of S1PRs agonist on BP of IUGR. Male IUGR offspring had a significantly higher BP compared to male control via carotid catheter in the conscious state (control: 112.1±2.1, IUGR: 125.0±3.7 mmHg; N=7, P <0.05). Acute administration of FTY720 (1 mg/kgBW i.p, Fingomod), a S1P receptor type 1, 3 agonist did not significantly alter BP in control (106.0 ± 5.7 mmHg) but significantly decreased BP in IUGR (105.7±2.3 mmHg, p< 0.05). A dose response to FTY720 (10 mg/kgBW) decreased BP in both control (94.0±2.0 mmHg, p< 0.05) and IUGR (99.3±2.3 mmHg, p< 0.05). Together our data suggest that IUGR programs an alteration of renal S1PR3 expression in both during- and post-nephrogenesis thereby contributing to an increase in sensitivity to S1PRs agonist. Thus, S1P signaling is a putative mechanism underlying the hypertension of IUGR offspring.

Improving heart function by modulating myocardiocyte autophagy: a possible novel mechanism for cardiovascular protection of high-density lipoprotein.

BackgroundHigh-density lipoprotein (HDL) has been shown to confer cardiovascular protection in clinical and epidemiologic studies. Emerging evidence suggests that many of the cardioprotective functions of HDL may be due to the phospholipid sphingosine-1-phosphate (S1P).Presentation of the hypothesisHDL-S1P binds to S1P receptors in the heart, activating PI3K/Akt signaling and myocyte survival. PI3K/Akt is a classic signaling modulator of autophagy. Excessive autophagy due to cell death and cardiomyocyte loss may contribute to impaired heart function during pressure overload-induced heart failure. Therefore, we hypothesize that HDL-S1P may suppress excessive autophagy of cardiomyocytes through activation of PI3K/Akt signaling. Further, reconstituted HDL (including S1P) may protect heart function during pressure overload-induced heart failure.Testing the hypothesisWe will design the following experiments to test this hypothesis. (1) We will treat cells and mice with PI-3 kinase inhibitors to examine if HDL-S1P downregulates expression of Autophagy-related genes (ATGs) and proteins via activation of PI3K/Akt signaling. (2) We will use siRNA against S1P receptors or inhibitors of S1P receptors to determine which types of S1P receptors participate in this mechanism. (3) We will also examine if reconstituted HDL (including S1P) improves heart function during pressure overload-induced heart failure by suppressing excessive autophagy of cardiomyocytes through activation of PI3K/Akt signaling.Implications of the hypothesisUnderstanding the autophagy signaling pathway modulated by HDL-S1P will make a major contribution to the field by identifying a novel mechanism for cardiovascular protection of high-density lipoprotein. Further, using reconstituted HDL to improve heart function would provide a novel therapeutic approach for pressure overload–induced heart failure.