Sphingosine 1-phosphate enhances the excitability of rat sensory neurons through activation of sphingosine 1-phosphate receptors 1 and/or 3.
ABSTRACT: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1-5) and plays a key role in regulating the inflammatory response. Our previous studies demonstrated that rat sensory neurons express the mRNAs for all five S1PRs and that S1P increases neuronal excitability primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability.Isolated sensory neurons were treated with either short-interfering RNAs (siRNAs) or a variety of pharmacological agents targeted to S1PR1/R2/R3 to determine the role(s) of these receptors in regulating neuronal excitability. The excitability of isolated sensory neurons was assessed by using whole-cell patch-clamp recording to measure the capacity of these cells to fire action potentials (APs).After siRNA treatment, exposure to S1P failed to augment the excitability. Pooled siRNA targeted to S1PR1 and R3 also blocked the enhanced excitability produced by S1P. Consistent with the siRNA results, pretreatment with W146 and CAY10444, selective antagonists for S1PR1 and S1PR3, respectively, prevented the S1P-induced increase in neuronal excitability. Similarly, S1P failed to augment excitability after pretreatment with either VPC 23019, which is a S1PR1 and R3 antagonist, or VPC 44116, the phosphonate analog of VPC 23019. Acute exposure (10 to 15 min) to either of the well-established functional antagonists, FTY720 or CYM-5442, produced a significant increase in the excitability. Moreover, after a 1-h pretreatment with FTY720 (an agonist for S1PR1/R3/R4/R5), neither SEW2871 (S1PR1 selective agonist) nor S1P augmented the excitability. However, after pretreatment with CYM-5442 (selective for S1PR1), SEW2871 was ineffective, but S1P increased the excitability of some, but not all, sensory neurons.These results demonstrate that the enhanced excitability produced by S1P is mediated by activation of S1PR1 and/or S1PR3.
Project description:BACKGROUND:Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1 phosphate (S1P) following stimulation of the five plasma membrane G-protein-coupled receptors. The objective of this study is to clarify the role of S1P and its receptors (S1PRs), especially S1PR3 in airway epithelial cells. METHODS:The effects of S1P on asthma-related genes expression were examined with the human bronchial epithelial cells BEAS-2B and Calu-3 using a transcriptome analysis and siRNA of S1PRs. To clarify the role of CCL20 in the airway inflammation, BALB/c mice were immunized with ovalbumin (OVA) and subsequently challenged with an OVA-containing aerosol to induce asthma with or without intraperitoneal administration of anti-CCL20. Finally, the anti-inflammatory effect of VPC 23019, S1PR1/3 antagonist, in the OVA-induced asthma was examined. RESULTS:S1P induced the expression of some asthma-related genes, such as ADRB2, PTGER4, and CCL20, in the bronchial epithelial cells. The knock-down of SIPR3 suppressed the expression of S1P-inducing CCL20. Anti-CCL20 antibody significantly attenuated the eosinophil numbers in the bronchoalveolar lavage fluid (P<0.01). Upon OVA challenge, VPC23019 exhibited substantially attenuated eosinophilic inflammation. CONCLUSIONS:S1P/S1PR3 pathways have a role in release of proinflammatory cytokines from bronchial epithelial cells. Our results suggest that S1P/S1PR3 may be a possible candidate for the treatment of bronchial asthma.
Project description:Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P(1-5) receptors). S1P(1) and S1P(2) receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P(1)-mediated signaling by SEW2871 and FTY720, two agonists of S1P(1), significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P(1)-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P(2) signaling by JTE-013, a specific antagonist of S1P(2), S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P(1)- and S1P(2)-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P(1) and S1P(2) signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.
Project description:Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator generated when a cell membrane or its components are damaged by various factors. S1P regulates diverse cell activities via S1P receptors (S1PRs). Keratinocytes express S1PR1-5. Although it is known that S1PRs control keratinocyte differentiation, apoptosis, and wound healing, S1PR functions in keratinocyte infections have not been fully elucidated. We propose that the S1P-S1PR axis in keratinocytes works as a biosensor for bacterial invasion. Indeed, in human impetigo infection, we found high epidermal expression of S1PR1 and S1PR2 in the skin. Furthermore, in normal human epidermal keratinocytes in vitro, treatment with Staphylococcus aureus bacterial supernatant not only induced S1P production but also increased the transcription of S1PR2, confirming our in vivo observation, as well as increased the levels of TNFA, IL36G, IL6, and IL8 mRNAs. However, direct treatment of normal human epidermal keratinocytes with S1P increased the expressions of IL36G, TNFA, and IL8, but not IL6. In both S1P- and S. aureus bacterial supernatant-treated normal human epidermal keratinocytes, S1PR1 knockdown reduced IL36G, TNFA, and IL8 transcription, and the S1PR2 antagonist JTE013 blocked the secretion of these cytokines. Overall, we have proven that during infections, keratinocytes communicate damage by using S1P release and tight control of S1PR1 and 2.
Project description:Strong evidence exists for interactions of zwitterionic phosphate and amine groups in sphingosine-1 phosphate (S1P) to conserved Arg and Glu residues present at the extracellular face of the third transmembrane domain of S1P receptors. The contribution of Arg(120) and Glu(121) for high-affinity ligand-receptor interactions is essential, because single-point R(120)A or E(121)A S1P(1) mutants neither bind S1P nor transduce S1P function. Because S1P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance. Here we describe a modestly water-soluble highly selective S1P(1) agonist [2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino) ethanol (CYM-5442)] that does not require Arg(120) or Glu(121) residues for activating S1P(1)-dependent p42/p44 mitogen-activated protein kinase phosphorylation, which defines a new hydrophobic pocket in S1P(1). CYM-5442 is a full agonist in vitro for S1P(1) internalization, phosphorylation, and ubiquitination. It is noteworthy that CYM-5442 was a full agonist for induction and maintenance of S1P(1)-dependent blood lymphopenia, decreasing B lymphocytes by 65% and T lymphocytes by 85% of vehicle. Induction of CYM-5442 lymphopenia was dose- and time-dependent, requiring serum concentrations in the 50 nM range. In vitro measures of S1P(1) activation by CYM-5442 were noncompetitively inhibited by a specific S1P(1) antagonist [(R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146)], competitive for S1P, 2-amino-2-(4-octylphenethyl)propane-1,3-diol (FTY720-P), and 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2, 4-oxadiazole (SEW2871). In addition, lymphopenia induced by CYM-5442 was reversed by W146 administration or upon pharmacokinetic agonist clearance. Pharmacokinetics in mice also indicated that CYM-5442 partitions significantly in central nervous tissue. These data show that CYM-5442 activates S1P(1)-dependent pathways in vitro and to levels of full efficacy in vivo through a hydrophobic pocket separate from the orthosteric site of S1P binding that is headgroup-dependent.
Project description:Background:The apolipoprotein M (ApoM)-sphingosine-1-phosphate (S1P) axis was recently identified, and research into its function has received increasing attention. However, there are some factors which might influence the results of studies into the function of the ApoM-S1P axis using the EA.hy926 cells. This study investigated related factors, including coagulation factor VIII (FVIII), ApoM, S1P receptor subtypes (S1PRs), C-myc-tagged, and His-tagged proteins in EA.hy926 cells, as well as the effects of ApoM overexpression on S1PRs. Methods:The expression of FVIII, ApoM, S1PRs, C-myc, and His-tagged proteins in EA.hy926 cells was investigated through cellular immunofluorescence. EA.hy926 cells were infected with lentiviruses carrying (OE group) or lacking (NC group) the ApoM gene sequence. A stable cell line expressing ApoM was obtained, and the expression of ApoM mRNA was detected through single tube duplex fluorescence reverse transcription quantitative polymerase chain reaction (RT-qPCR). S1PRs expression was detected by RT-qPCR and Western blotting. Results:The results showed that EA.hy926 cells expressed FVIII, ApoM, C-myc-tagged, and His-tagged proteins. Moreover, they highly expressed S1PR1, slightly expressed S1PR3, weakly expressed S1PR2, and did not express S1PR4 and S1PR5. ApoM overexpression significantly increased S1PR1 mRNA and protein expression but did not affect the expression of S1PR3. EA.hy926 cells expressed FVIII, suggesting the cell line possesses endothelial cell characteristics and could be used for in vitro studies of the ApoM-S1P axis. Conclusions:EA.hy926 cell line is suitable for investigation of the ApoM-S1P axis in vitro. However, Since EA.hy926 cells expressed endogenous ApoM, C-myc and His tagged proteins, the exogenous recombinant ApoM should not be labeled with C-myc and His tags for distinguishing from endogenous ApoM. In addition, overexpression of ApoM should be considered to significantly increase the expression of S1PR1 when studying the APOM-S1P axis.
Project description:A bioactive lipid, sphingosine 1-phosphate (S1P), acts extracellularly as a potent mediator, and is implicated in the progression of various cancers including hepatocellular carcinoma (HCC). S1P exerts its functions by binding to five types of specific receptors, S1P receptor 1 (S1PR1), S1PR2, S1PR3, S1PR4 and S1PR5 on the plasma membrane. However, the exact roles of S1P and each S1PR in HCC cells remain to be clarified. In the present study, we investigated the effect of S1P on the hepatocyte growth factor (HGF)-induced migration of human HCC-derived HuH7 cells, and the involvement of each S1PR. S1P dose-dependently reduced the HGF-induced migration of HuH7 cells. We found that all S1PRs exist in the HuH7 cells. Among each selective agonist for five S1PRs, CYM5520, a selective S1PR2 agonist, significantly suppressed the HGF-induced HuH7 cell migration whereas selective agonists for S1PR1, S1PR3, S1PR4 or S1PR5 failed to affect the migration. The reduction of the HGF-induced migration by S1P was markedly reversed by treatment of JTE013, a selective antagonist for S1PR2, and S1PR2- siRNA. These results strongly suggest that S1P reduces the HGF-induced HCC cell migration via S1PR2. Our findings may provide a novel potential of S1PR2 to therapeutic strategy for metastasis of HCC.
Project description:Cumulating evidences suggested an important role of sphingosine-1-phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)-ANCA-positive IgG-mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO-ANCA-positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane-bound and soluble ICAM-1 and VCAM-1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO-ANCA-positive IgG-induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO-ANCA-positive IgG. S1P enhanced MPO-ANCA-positive IgG-induced membrane-bound and soluble ICAM-1/VCAM-1 up-regulation of GEnCs. Soluble ICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG increased upon pre-incubation of S1PR1 antagonist, while pre-incubation of GEnCs with the S1PR1 agonist down-regulated sICAM-1 level. Blocking S1PR2-4 reduced sICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG. Pre-incubation with S1PR5 agonist could increase sICAM-1 level in the supernatants of GEnC stimulated by S1P and MPO-ANCA-positive IgG. S1P can enhance MPO-ANCA-positive IgG-mediated GEnC activation through S1PR2-5.
Project description:Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.
Project description:Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-? and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-? gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-?, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-?, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-?-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-?, mRNA. PaCDase induced NF-?B p65 phosphorylation. The NF-?B inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-?B p65 phosphorylation and reduction in the protein level of the NF-?B inhibitor I?B?. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-? via S1P receptors, and (iii) released TNF-? stimulates the production of inflammatory mediators such as IL-8.
Project description:Sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) regulate migration of lymphocytes out of thymus to blood and lymph nodes (LNs) to efferent lymph, whereas their role in other tissue sites is not known. Here, we investigated the question of how these molecules regulate leukocyte migration from tissues through afferent lymphatics to draining LNs (dLNs). S1P, but not other chemokines, selectively enhanced human and murine CD4 T cell migration across lymphatic endothelial cells (LECs). T cell S1PR1 and S1PR4, and LEC S1PR2, were required for migration across LECs and into lymphatic vessels and dLNs. S1PR1 and S1PR4 differentially regulated T cell motility and vascular cell adhesion molecule-1 (VCAM-1) binding. S1PR2 regulated LEC layer structure, permeability, and expression of the junction molecules VE-cadherin, occludin, and zonulin-1 through the ERK pathway. S1PR2 facilitated T cell transcellular migration through VCAM-1 expression and recruitment of T cells to LEC migration sites. These results demonstrated distinct roles for S1PRs in comodulating T cell and LEC functions in migration and suggest previously unknown levels of regulation of leukocytes and endothelial cells during homeostasis and immunity.