Heparin decreases serum sphingosine-1-phosphate levels in patients with vascular diseases.

BackgroundSphingosine 1‐phosphate (S1P), a bioactive lipid, has been shown to mediate cancer processes. Therefore, accurate qualitative and quantitative determination is essential. The current assay method is still cumbersome to be of practical use worldwide and the aim of this study was therefore to develop a fast, accurate, precise and efficient LC‐MS/MS method for targeted analyses of S1P in serum samples.MethodsLiquid chromatography‐tandem mass spectrometry (LC‐MS/MS) is an established method used for monitoring and analyzing S1P levels in serum. We determined the level of serum S1P in 256 patients with lung cancer and 36 healthy donors, and used Spearman';s rank correlation analysis to evaluate the difference in serum S1P levels between radiotherapy and nonradiotherapy patients.ResultsStandard curves were linear over ranges of 25–600 ng/mL for S1P with correlation coefficient (r2) greater than 0.9996. The lower limit of quantifications (LLOQs) was 25 ng/mL. The intra‐ and interbatch precisions and accuracy was less than 10% for S1P. The recoveries of the method were found to be 80%–98%. Serum S1P levels in healthy donors were different from those in patients (P < 0.001). Of 256 lung cancer patients, 124 (48.4%) received radiotherapy and were identified to have concomitant low serum S1P levels (222.13 ± 48.63), whereas 132 (51.6%) who had not received radiotherapy were identified to have high levels (315.16 ± 51.06). The serum S1P levels were therefore associated with radiotherapy (Spearman's Rho = −0.653, P < 0.001).ConclusionsOur results indicated that this new LC‐MS/MS method is rapid, sensitive, specific and reliable for the quantification of S1P levels in serum samples. The level of S1P in serum samples of patients with lung cancer who received radiotherapy was significantly lower than that in patients who did not receive radiotherapy.Key pointsAn improved method was established to quantify S1P levels in human serum by LC‐MS/MS, which enabled the change in serum S1P levels in lung cancer patients to be monitored, in combination with radiotherapy, and their clinical significance to be analyzed.

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5'-(beta,gamma-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K(m) value of 21 microm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.

To determine the changes of serum copeptin and sphingosine 1-phosphate (S1P) in patients with restenosis after stent implantation of symptomatic intracranial artery stenosis. An observational study. Changyi people's Hospital, China, from February 2016 to November 2019. A total of 76 patients with symptomatic intracranial artery stenosis and stent implantation were divided into the restenosis group (n = 16) and the non-restenosis group (n=60) according to the intracranial artery restenosis occurred after the follow-up of 1 year. Levels of serum copeptin and S1P were compared between the groups. There were significant differences in diabetes mellitus and hypertension between the two groups (p<0.001 and p = 0.017, respectively). There were no significant differences in serum copeptin and S1P levels between the two groups before and 3 days after the operation (p = 0.927, 0.792, 0.776, and 0.906, respectively). Postoperative follow-up of one year, levels of serum copeptin in the restenosis group were higher than those in the non-restenosis group (p<0.001), and levels of serum S1P in the restenosis group were lower than those in the non-restenosis group (p = 0.003). High serum copeptin level, low serum S1P level, hypertension, and diabetes mellitus are independent risk factors promoting restenosis after stent implantation in patients with symptomatic intracranial artery stenosis. Copeptin, Sphingosine 1-phosphate (S1P), Symptomatic intracranial artery stenosis, Stent implantation, Restenosis.

Sphingosine 1-phosphate (S1P) is a lipid mediator formed by the metabolism of sphingomyelin which is involved in the endothelial permeability and inflammation. Although the plasma S1P concentration is reportedly decreased in patients with cerebral malaria, the role of S1P in malaria is still unclear. The purpose of this study was to examine the impact of malaria on circulating S1P concentration and its relationship with clinical data in malaria patients. Serum S1P levels were measured in 29 patients with P. vivax, 30 patients with uncomplicated P. falciparum, and 13 patients with complicated P. falciparum malaria on admission and on day 7, compared with healthy subjects (n = 18) as control group. The lowest level of serum S1P concentration was found in the complicated P. falciparum malaria group, compared with P. vivax, uncomplicated P. falciparum patients and healthy controls (all p < 0.001). In addition, serum S1P level was positively correlated with platelet count, hemoglobin and hematocrit levels in malaria patients. In conclusions, low levels of S1P are associated with the severity of malaria, and are correlated with thrombocytopenia and anemia. These findings highlight a role of S1P in the severity of malaria and support the use of S1P and its analogue as a novel adjuvant therapy for malaria complications.

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.

Pre-infarction angina reduces myocardial infarct size by preventing the myocardium from being subjected to ischemia reperfusion (I/R) injury. Ischemic preconditioning is the proposed mechanism for this effect. Sphingosine 1 phosphate (S1P) activates ischemic preconditioning pathways and may play a role in the presence of cardioprotective effects of pre-infarction angina. Therefore, we evaluated the relationship between pre-infarction angina and serum S1P levels. Between May 2011 and January 2012, 79 patients with acute myocardial infarction were included in the study. In addition to taking routine medical histories, all of the patients were questioned as to whether or not they had pre-infarction angina. We determined patients serum levels of S1P at admission and discharge, and peak creatine kinase MB and troponin levels were also measured in the pre-infarction angina positive and negative groups. Of the 79 patients included in the study, 36 had pre-infarction angina and 43 had not. Baseline characteristics were similar between the groups. The median level of serum S1P in patients with pre-infarction angina was significantly higher than in those without pre-infarction angina both at admission and discharge [0.54 (0.14-1.35) vs. 0.26 (0.12-0.62) p = 0.014/0.51 (0.20-1.81) vs. 0.30 (0.13-0.68) p = 0.010]. Serum high sensitive troponin levels were significantly lower in patients with pre-infarction angina [0.97 (0.39-3.07) vs. 2.56 (0.9-6.51) p = 0.034]. Serum S1P levels both at admission and discharge tended to be higher in patients with more angina episodes, but the differences between these subgroups were not statistically significant. Patients who experienced pre-infarction angina had higher serum S1P levels than patients without pre-infarction angina. This study supported our hypothesis that the cardioprotective effects of pre-infarction angina may in part be mediated by S1P. Ischemic preconditioning; Pre-infarction angina; Sphingosine 1 phosphate.

INTRODUCTION: Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, regulates many cellular processes important for breast cancer progression. The aim of this study is to investigate a role for S1P produced by sphingosine kinase 1 (SphK1) in breast cancer progression and tumor-induced hemangiogenesis and lymphangiogenesis. METHODS: An isozyme-specific inhibitor of SphK1 (SK1-I) was used. Tumor burden were quantified using an in vivo imaging system (IVIS 2000). S1P and SK1-I levels were measured by LC-ESI-MS/MS. Hemangiogenesis and lymphangiogenesis are determined by morphological analysis of microvessel density as well as by flow cytometric analysis of endothelial cells. RESULTS: A significant dose dependent effect of SK1-I on inhibition of 4T1-luc2 murine breast cancer cell growth was observed. We confirmed that SK1-I decreased the enzymatic activity of SphK1 and that downregulation of SphK1 with specific siRNA also suppressed growth of these cells. We found that S1P levels increased in the tumor and in the circulation after orthotopic implantation of 4T1-luc2 cells. Similarly, serum S1P levels were significantly elevated in stage IIIA breast cancer patients who have lymph node metastases, compared to age/ethnicity-matched healthy volunteers. SK1-I blocked serum S1P increases in in vivo model and reduced lymph node and lung metastases and overall tumor burden in vivo as well. Importantly, SK1-I also decreased hem- and lymphangiogenesis not only in the primary tumor, but also in lymph nodes, the host tumor microenvironment. Whereas supernatants from 4T1-luc2 cells significantly stimulated tube formation of HUVECs and HLECs, shRNA knockdown of SphK1 markedly reduced them, indicating that S1P produced by SphK1 in 4T1-luc2 breast cancer cells is an important contributor to hem- and lymphangiogenesis. CONCLUSIONS: S1P generated by SphK1 is important for tumor progression, tumor-induced hemangiogenesis and lymphangiogenesis, and lymph node metastais. Therefore targeting SphK1 and its product S1P would be a multi-pronged attack against breast cancer. This work was supported by Sumitomo Life Social Welfare Services Foundation grant to MN, NIH (K12HD055881) and Susan G. Komen for the Cure (KG090510) to KT, and NCI (R01CA61774) to SS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4364. doi:1538-7445.AM2012-4364

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator is involved in an array of biological processes and linked to pathological manifestations. Erythrocyte is known as the major reservoir for S1P as they lack S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) and harbor sphingosine kinase-1 (SphK-1) essential for sphingosine conversion to S1P. Reduced S1P concentration in serum was correlated with disease severity in patients with Plasmodium falciparum and Plasmodium vivax infections. Herein, we aimed to identify the underlying mechanism and contribution of host erythrocytes toward depleted S1P levels in Plasmodium-infected patients vs. healthy individuals. The level and activity of SphK-1 were measured in vitro in both uninfected and cultured P. falciparum-infected erythrocytes. Infected erythrocytes demonstrated a significant decrease in SphK-1 level in a time-dependent manner. We found that 10–42 h post invasion (hpi), SphK1 level was predominantly reduced to ∼50% in rings, trophozoites, and schizonts compared to uninfected erythrocytes. We next analyzed the phosphorylation status of SphK-1, a modification responsible for its activity and S1P production, in both uninfected control and Plasmodium-infected erythrocytes. Almost ∼50% decrease in phosphorylation of SphK-1 was observed that could be corroborated with significant reduction in the production and release of S1P in infected erythrocytes. Serum S1P levels were studied in parallel in P. falciparum (N = 15), P. vivax (N = 36)-infected patients, and healthy controls (N = 6). The findings revealed that S1P concentration was significantly depleted in uncomplicated malaria cases and was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax-infected groups (∗∗p < 0.01). The lower serum S1P level could be correlated with the reduced platelet count defining the role of S1P level in platelet formation. In conclusion, erythrocyte SphK-1 and S1P levels were studied in Plasmodium-infected individuals and erythrocytes that helped in characterizing the complications associated with malaria and thrombocytopenia, providing insights into the contribution of host erythrocyte biology in malaria pathogenesis. Finally, this study proposes the use of S1P and its analog as a novel adjunct therapy for malaria complications.

Fenofibrate Increases High-Density Lipoprotein and Sphingosine 1 Phosphate Concentrations Limiting Abdominal Aortic Aneurysm Progression in a Mouse Model

Sphingosine‐1‐phosphate (S1P) regulates pathophysiological processes, including liver regeneration, vascular tone control, and immune response. In patients with liver cirrhosis, acute deterioration of liver function is associated with high mortality rates. The present study investigated whether serum S1P concentrations are associated with disease severity in patients with chronic liver disease from compensated cirrhosis (CC), acute decompensation (AD), or acute‐on‐chronic liver failure (ACLF). From August 2013 to October 2017, patients who were admitted to the University Hospital Frankfurt with CC, AD, or ACLF were enrolled in our cirrhosis cohort study. Tandem mass spectrometry was performed on serum samples of 127 patients to assess S1P concentration. Our study comprised 19 patients with CC, 55 with AD, and 51 with ACLF, aged 29 to 76 years. We observed a significant decrease of S1P according to advanced liver injury from CC and AD up to ACLF (P < 0.001). S1P levels further decreased with progression to ACLF grade 3 (P < 0.05), and S1P highly inversely correlated with the Model for End‐Stage Liver Disease score (r = −0.508; P < 0.001). In multivariate analysis, S1P remained an independent predictor of 7‐day mortality with high diagnostic accuracy (area under the curve, 0.874; P < 0.001). Conclusion: In patients with chronic liver disease, serum S1P levels dramatically decreased with advanced stages of liver disease and were predictive of early mortality. Because S1P is a potent regulator of endothelial integrity and immune response, low S1P levels may significantly influence progressive multiorgan failure. Our data justify further elucidation of the diagnostic and therapeutic role of S1P in ACLF.

Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.

Geniposide (GE) is an iridoid glycoside compound with anti-inflammatory effect. The potential of sphingosine 1-phosphate (S1P) as a plasma marker in human diseases was suggested recently in the literature, which demonstrated that, in patients with inflammatory diseases, plasma S1P was elevated. It follows that the obstructive coronary artery disease can be predicted with serum S1P. Therefore, S1P can also be potentially used as a pharmacodynamic marker to study adjuvant arthritis (AA) rats. In the current study, a UHPLC-MS/MS method combined with the microdialysis sampling technique (using FTY720 phosphate as an internal standard) was adopted and validated to measure S1P levels in the hemodialysis fluid and joint cavity dialysates of AA rats after oral administration of GE. A S1P concentration-time curve in the dialysate was established in this study. It was demonstrated that GE exerted an anti-inflammatory effect by reducing AA-induced elevated S1P levels. It is showed that changes in S1P concentrations over time can be used to monitor the pharmacodynamic effects of GE in treating AA rats in pharmacodynamic studies.

Background:Sphingosine 1-phosphate (S1P) and its receptor S1PR are involved in the interaction between the immune and bone systems. S1P-S1PR1 binding is involved in bone remodelling, allowing a balance between osteoclastogenesis...

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that regulates numerous key cardiovascular functions. High-density lipoproteins (HDLs) are the major plasma lipoprotein carriers of S1P. Fibrinolysis is a physiological process that allows fibrin clot dissolution, and decreased fibrinolytic capacity may result from increased circulating levels of plasminogen activator inhibitor-1 (PAI-1). We examined the effect of S1P associated with HDL subfractions on PAI-1 secretion from 3T3 adipocytes. S1P concentration in HDL3 averaged twice that in HDL2. Incubation of adipocytes with increasing concentrations of S1P in HDL3, but not HDL2, or with S1P complexed to albumin stimulated PAI-I secretion in a concentration-dependent manner. Quantitative RT-PCR revealed that S1P(1-3) are expressed in 3T3 adipocytes, with S1P(2) expressed in the greatest amount. Treatment of adipocytes with the S1P(1) and S1P(3) antagonist VPC23019 did not block PAI-1 secretion. Inhibiting S1P(2) with JTE-013 or reducing the expression of the gene coding for S1P(2) using silencing RNA (siRNA) technology blocked PAI-1 secretion, suggesting that the S1P(2) receptor mediates PAI-1 secretion from adipocytes exposed to HDL3 or S1P. Treatment with the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor RO-318425, or the Rho-associated protein kinase (ROCK) inhibitor Y27632 all significantly inhibited HDL3- and S1P-mediated PAI-1 release, suggesting that HDL3- and/or S1P-stimulated PAI-1 secretion from 3T3 cells is mediated by activation of multiple, downstream signaling pathways of S1P(2).

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Somatosensory neurons mediate responses to diverse mechanical stimuli, from innocuous touch to noxious pain. While recent studies have identified distinct populations of A mechanonociceptors (AMs) that are required for mechanical pain, the molecular underpinnings of mechanonociception remain unknown. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P) and S1P Receptor 3 (S1PR3) are critical regulators of acute mechanonociception. Genetic or pharmacological ablation of S1PR3, or blockade of S1P production, significantly impaired the behavioral response to noxious mechanical stimuli, with no effect on responses to innocuous touch or thermal stimuli. These effects are mediated by fast-conducting A mechanonociceptors, which displayed a significant decrease in mechanosensitivity in S1PR3 mutant mice. We show that S1PR3 signaling tunes mechanonociceptor excitability via modulation of KCNQ2/3 channels. Our findings define a new role for S1PR3 in regulating neuronal excitability and establish the importance of S1P/S1PR3 signaling in the setting of mechanical pain thresholds. https://doi.org/10.7554/eLife.33285.001 Introduction Pain is a complex sensation. It serves to protect organisms from harmful stimuli, but can also become chronic and debilitating following tissue injury and disease. Distinct cells and molecules detect noxious thermal and mechanical stimuli. Thermal pain is detected by thermosensitive TRP channels in subsets of nociceptors (Caterina et al., 2000; Vriens et al., 2011), and gentle touch is detected by Piezo2 channels in low-threshold mechanoreceptors (LTMRs) (Ranade et al., 2014; Woo et al., 2014). Aδ high-threshold mechanoreceptors (HTMRs) have been shown to play a key role in responses to painful mechanical stimuli (Arcourt et al., 2017; Ghitani et al., 2017). Recent studies have shown that there are at least two populations of HTMRs that mediate responses to noxious mechanical stimuli. The Npy2r+ subpopulation of HTMRs mediates fast paw withdrawal responses to pinprick stimulation and terminates as free nerve endings in the epidermis (Arcourt et al., 2017). The Calca+ subpopulation of circumferential-HTMRs responds to noxious force and hair pulling, and terminates as circumferential endings wrapped around guard hair follicles (Ghitani et al., 2017). Additionally, somatostatin-expressing interneurons of laminae I-III in the dorsal horn of the spinal cord receive input from nociceptors and are required for behavioral responses to painful mechanical stimuli (Duan et al., 2014). Despite these advances in defining the cells and circuits of mechanical pain, little is known about the molecular signaling pathways in mechanonociceptors. Here, we show that sphingosine 1-phosphate (S1P) is required for mechanical pain sensation. S1P is a bioactive lipid that signals via 5 G-protein coupled S1P Receptors (S1PRs 1–5). S1P signaling, mainly via S1PR1, plays a well-known role in immune cell migration and maturation (Spiegel and Milstien, 2003; Matloubian et al., 2004; Schwab et al., 2005). Additionally, recent studies have shown that S1PRs are expressed throughout the nervous system (Janes et al., 2014; Mair et al., 2011; Camprubí-Robles et al., 2013) and S1P signaling is associated with a variety of neuroinflammatory disorders, including multiple sclerosis (Brinkmann et al., 2010) and Alzheimer's disease (Couttas et al., 2014). S1P has been implicated in spontaneous pain (Camprubí-Robles et al., 2013) and thermal pain hypersensitivity (Mair et al., 2011; Finley et al., 2013; Weth et al., 2015), but due to conflicting accounts of S1P receptor expression in the CNS (Janes et al., 2014; Weth-Malsch et al., 2016) and PNS (Mair et al., 2011; Camprubí-Robles et al., 2013; Usoskin et al., 2015) as well as inconsistent reports on the effects of S1P on neuronal excitability (Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015) and pain behaviors (Mair et al., 2011; Camprubí-Robles et al., 2013; Finley et al., 2013; Weth et al., 2015), the role of S1P in somatosensation remains controversial. We found that mice lacking the S1P receptor S1PR3 display striking and selective deficits in behavioral responses to noxious mechanical stimuli. Likewise, peripheral blockade of S1PR3 signaling or S1P production impairs mechanical sensitivity. We show that S1P constitutively enhances the excitability of A mechanonociceptors (AMs) via closure of KCNQ2/3 potassium channels to tune mechanical pain sensitivity. The effects of S1P are completely dependent on S1PR3. While previous studies have shown that elevated S1P triggers acute pain and injury-evoked thermal sensitization (Mair et al., 2011; Camprubí-Robles et al., 2013), we now demonstrate that baseline levels of S1P are necessary and sufficient for setting normal mechanical pain thresholds. By contrast, elevated S1P selectively triggers thermal sensitization via activation of TRPV1+ heat nociceptors, with no effect on mechanical hypersensitivity. Our findings uncover an essential role for constitutive S1P signaling in mechanical pain. Results To identify candidate genes underlying mechanosensation, we previously performed transcriptome analysis of the sensory ganglia innervating the ultra-sensitive tactile organ (the star) of the star-nosed mole (Gerhold et al., 2013). Immunostaining revealed the tactile organ is preferentially innervated by myelinated Aδ fibers (Gerhold et al., 2013), which are primarily mechanosensitive. While our original analysis focused on ion channels enriched in the neurons of the star organ, our dataset also revealed enrichment of several components of the S1P pathway, including S1pr3. Likewise, single-cell RNA seq of mouse dorsal root ganglion (DRG) neurons revealed S1pr3 expression in a subset of myelinated mechanoreceptors (Usoskin et al., 2015) in addition to a subpopulation of peptidergic C nociceptors. S1P promotes excitability in small-diameter, capsaicin-sensitive nociceptors (Mair et al., 2011; Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015). In addition, S1PR3 has been shown to mediate spontaneous pain triggered by elevated S1P and thermal sensitization following sterile tissue injury (Camprubí-Robles et al., 2013). However, no studies have examined the role of S1PR3 in mechanosensation or in regulating somatosensory behaviors under normal conditions. Given the enrichment of S1pr3 in mechanosensory neurons of the star-nosed mole and mouse, we hypothesized that S1P signaling via S1PR3 may also play a role in mechanosensation. Thus, we set out to define the role of S1P signaling and S1PR3 in somatosensory mechanoreceptors. S1PR3 mediates acute mechanical pain We first examined a variety of somatosensory behaviors in mice lacking S1PR3 (Kono et al., 2004) (S1pr3tm1Rlp/Mmnc; referred to herein as S1PR3 KO). We initially investigated baseline responses to mechanical stimuli. S1PR3 KO mice displayed a dramatic loss of mechanical sensitivity (Figure 1A; see Figure 1—source data 1), as von Frey paw withdrawal thresholds were significantly elevated in S1PR3 KO mice relative to WT and S1PR3 HET littermates (mean thresholds: 1.737 g vs. 0.736 and 0.610 g, respectively). Moreover, S1PR3 KO mice demonstrated decreased responses to a range of noxious tactile stimuli (2–6 g; Figure 1B) and to noxious pinprick stimulation (Figure 1C), but normal responsiveness to innocuous tactile stimuli (0.6–1.4 g; Figure 1B). S1PR3 KO mice exhibited normal tape removal attempts (Ranade et al., 2014) (Figure 1D), righting reflexes (Figure 1—figure supplement 1A), radiant heat withdrawal latencies (Figure 1E), and itch-evoked scratching (Figure 1—figure supplement 1B). These results demonstrate a selective role for S1PR3 in acute mechanical pain. Figure 1 with 1 supplement see all Download asset Open asset S1PR3 mediates acute mechanical pain. (A) von Frey 50% withdrawal threshold measurements for S1pr3+/+ (WT, N = 8), S1pr3+/- (HET, N = 7) and S1pr3-/- (KO, N = 12) mice. p<0.0001 (one-way ANOVA). Tukey-Kramer post hoc comparisons for KO and HET to WT indicated on graph. (B) von Frey force-response graph for WT (N = 8) versus KO (N = 12) animals; pgenotype <0.0001 (two-way ANOVA). Tukey HSD comparisons between genotypes are indicated for given forces. (C) % withdrawal to pinprick stimulation of hindpaw for HET versus KO animals; p<0.0001 (unpaired t-test; N = 5–7 mice per group). (D) Number of attempted removal bouts in tape assay for WT (N = 2), HET (N = 2), and KO (N = 5) mice; p=0.172 (one-way ANOVA). (E) Baseline radiant heat measurements for WT (N = 8), HET (N = 3), and KO (N = 5) mice; p=0.444 (one-way ANOVA). (F) von Frey 50% withdrawal threshold measurements for mice pre- and post-injection of 500 µM TY 52156 (N = 10), 10 µM W146 (N = 6), or 1% DMSO-PBS vehicle (N = 17); p=0.016, 0.650 (two-tailed paired t-test comparing vehicle- vs. drug-injected paw). (G) von Frey force-response graph for mice injected with either 1% DMSO-PBS (N = 4) or 500 µM TY 52156 (N = 4); ptreatment <0.0001 (two-way ANOVA). Tukey HSD comparisons were made between treatment groups and significant differences at a given force are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.002 Figure 1—source data 1 S1PR3 mediates acute mechanical pain. Related to Figure 1. https://doi.org/10.7554/eLife.33285.004 Download elife-33285-fig1-data1-v2.xlsx As a complement to our analysis of somatosensation in S1PR3 KO animals, we employed a pharmacological approach, using the S1PR3-selective antagonist TY 52156 (TY) (Nussbaum et al., 2015). Similar to the phenotype of knockout animals, intradermal injection of 500 µM TY into the mouse hindpaw (the site of testing) triggered a rapid and significant elevation in von Frey paw withdrawal thresholds (Figure 1F) and decreased responsiveness to noxious (2–6 g), but not innocuous (0.6–1.4 g), tactile stimuli (Figure 1G), without affecting noxious heat sensitivity (Figure 1—figure supplement 1C). By contrast, blockade of S1PR1 with the selective antagonist W146 (Finley et al., 2013) had no effect on baseline mechanical or thermal thresholds (Figure 1F; Figure 1—figure supplement 1C). Overall, these data show that S1PR3 signaling sets mechanical pain sensitivity. Endogenous S1P mediates acute mechanical pain We next asked whether peripheral S1P was required for the S1PR3-dependent effects on mechanosensation. We decreased S1P levels via injection of the sphingosine kinase inhibitor SKI II to block local production of S1P (Chiba et al., 2010) or elevated S1P levels via intradermal injection of S1P and measured behaviors 30 min after injection. Decreasing local S1P levels with SKI II significantly reduced mechanical sensitivity (Figure 2A; see Figure 2—source data 1), comparable to the hyposensitivity phenotype observed in S1PR3 KO mice (Figure 1A). Again, similar to what was observed in S1PR3 KO animals (Figure 1E), peripheral blockade of S1P production had no effect on baseline thermal sensitivity (Figure 1—figure supplement 1C). Surprisingly, injecting exogenous S1P (10 µM; maximum solubility in saline vehicle) had no effect on mechanical sensitivity (Figure 2A–B). However, as previously reported (Mair et al., 2011; Camprubí-Robles et al., 2013), S1P injection triggered S1PR3-dependent thermal hypersensitivity and spontaneous pain (Figure 2C–D), demonstrating that the lack of effect on mechanical hypersensitivity is not due to problems with S1P delivery or degradation. Figure 2 Download asset Open asset Endogenous S1P mediates acute mechanical pain. (A) von Frey 50% withdrawal measurements for mice pre- and post-injection of 50 µM SKI II (N = 8) or 10 µM S1P (N = 7); p=0.003, 0.604 (two-tailed paired t-tests). (B) von Frey force-response graph for animals injected with 10 µM S1P or 0.1% MeOH-PBS; pgenotype >0.05 (two-way ANOVA; N = 8 mice per group). No Tukey HSD comparisons at any force between genotypes were significant. (C) Intradermal cheek injection of 10 µM S1P, 2 µM, 0.2 µM, and 20 µL 0.3% methanol PBS (vehicle), with quantification of number of forepaw wipes over the 5 min post-injection interval; p<0.0001 (one-way ANOVA; N = 3 mice per condition). Dunnett's multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (D) Radiant heat normalized paw withdrawal latencies 20–30 min post injection of 15 µL 10 µM S1P, 0.2 µM S1P. or 0.3% methanol-PBS vehicle (i.d.) into the hind paw of S1PR3 WT or KO mice; p=0.0129 (one-way ANOVA; N = 3–10 mice per condition). Dunnett's multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (E) von Frey 50% withdrawal measurements for mice pre- (baseline) and post-injection of 50 µM SKI II (N = 14) and 0 (N = 4), 10 (N = 3), 75 (N = 4), or 200 nM S1P (N = 3; one-way ANOVA; p=0.0001). Tukey Kramer comparisons are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.005 Figure 2—source data 1 Endogenous S1P mediates acute mechanical pain. Related to Figure 2. https://doi.org/10.7554/eLife.33285.006 Download elife-33285-fig2-data1-v2.xlsx These data support a model whereby S1P constitutively activates S1PR3 to set normal mechanical pain thresholds. To further test this model, we asked if the mechanical hyposensitivity elicited after endogenous S1P depletion (via SKI II) could be rescued by local injection of exogenous S1P. Indeed, we found that injection of exogenous S1P reversed SKI II-induced mechanical hyposensitivity in a dose-dependent manner, and observed a maximal effect with 200 nM S1P (Figure 2E). Although quantification of native S1P levels in skin is inaccurate owing to avid lyase activity (Shaner et al., 2009), our data establish that baseline S1P levels are sufficient to maximally exert their effect on S1PR3-dependent mechanical pain, such that increased S1P does not evoke mechanical hypersensitivity, but diminished S1P leads to mechanical hyposensitivity. These data show that constitutive activation of S1PR3 by S1P is required for normal mechanosensitivity. S1PR3 is expressed in A mechanonociceptors and thermal nociceptors Our behavioral data showing distinct roles for S1PR3 in mechanonociception and thermal hypersensitivity suggest that S1PR3 is expressed in distinct subsets of somatosensory neurons. While a previous study suggested that all somatosensory neurons express S1PR3 (Camprubí-Robles et al., 2013), single cell RNA seq data suggests S1pr3 is not expressed by all DRG neurons (Usoskin et al., 2015), and no studies have performed quantitative analysis of S1PR3 staining or co-staining to define subpopulations of S1PR3+ neurons. We thus set out to characterize the somatosensory neuron subtypes expressing S1pr3 using in situ hybridization (ISH) of wild-type somatosensory ganglia and immunohistochemistry (IHC) in an S1pr3mCherry/+ reporter mouse (Sanna et al., 2016). We first used in situ hybridization (ISH) with a specific S1pr3 probe to examine expression patterns of S1pr3 (Figure 3A–B; see Supplementary file 1). In our experiments, 43% of cells from wild-type DRG expressed S1pr3. Co-ISH revealed that one population of S1pr3+ neurons represents Aδ mechanonociceptors (AMs). These cells expressed Scn1a (39.9% of all S1pr3+), a gene that encodes the Nav1.1 sodium channel, which mediates mechanical pain in Aδ fibers (Osteen et al., 2016). S1pr3+ cells also co-expressed Npy2r (20.4% of all S1pr3+), a marker of a subset of mechanonociceptive A fibers (Arcourt et al., 2017). S1pr3 was expressed in 70.6% of Scn1a+ cells and 72% of Npy2r+ cells, comprising a majority of both of these populations. Interestingly, a subset of cells co-expressed S1pr3 and the mechanically sensitive channel Piezo2, which is expressed by Aβ, Aδ, and C fibers (Ranade et al., 2014). The remaining S1pr3+ cells were Trpv1+ and/or Trpa1+ C nociceptors (67.1% of all S1pr3+), which are reported to overlap minimally with the Scn1a+ and Npy2r+ populations (Arcourt et al., 2017; Osteen et al., 2016). Figure 3 with 1 supplement see all Download asset Open asset S1pr3 is expressed in A mechanonociceptors and C thermal nociceptors. (A) (Top) Representative co-ISH of S1pr3 (green; left) with Scn1a, Npy2r, Piezo2, and Trpv1 (magenta; center) in sectioned DRG. Right column: overlay with co-localized regions colored white (10x air objective; scale = 100 µm). (B) Bar chart showing the % of total cells expressing the indicated marker (grey) and the % of total cells co-expressing both marker and S1pr3 (green). See Table S1 for quantification. (C) Representative IHC images of sectioned DRG from S1pr3mCherry/+ animals stained with anti-DsRed (green, S1PR3) and anti-Peripherin (left, magenta) or anti-NF200 (right, magenta). Arrows indicate co-stained cells. Images were acquired using a 10x air objective (scale = 100 µm). (D) Whole-mount skin IHC confocal images with anti-DsRed antibody (S1PR3, green) and anti-NefH antibody (NF200, magenta) in an S1pr3mCherry/+ animal (20x water objective; scale = 50 µm). Arrows indicate co-positive free nerves (left image). Arrowheads indicate NF200- free nerves (left) or S1PR3- circumferential fibers (right image). (E) Sectioned skin IHC with anti-DsRed (S1PR3) and anti-NefH (NF200, left, top right) or anti-DsRed (S1PR3) and anti-beta-tubulin III (BTIII, bottom right) antibody (magenta) in S1pr3mCherry/+ skin (20x air objective; scale = 50 µm). Arrows indicate co-positive free nerve endings (left), S1PR3-negative lanceolate/circumferential hair follicle endings (top right, arrow = circumferential, arrowhead = lanceolate), or S1PR3-negative putative Merkel afferent (bottom right). (F) (Left) Quantification of sectioned DRG IHC experiments showing % of S1PR3+ cells that co-stained with indicated markers (n > 250 cells per marker). (Right) Quantification of sectioned skin IHC experiments showing % of fibers positive for indicated marker that co-stained with S1PR3 (anti-DsRed; n = 10 images per marker from two animals). https://doi.org/10.7554/eLife.33285.007 We next used an S1pr3mCherry/+ reporter mouse, which produces a functional S1PR3-mCherry fusion protein (Sanna et al., 2016), as an independent strategy to explore S1PR3 expression and localization. This strategy was used because we found that anti-S1PR3 antibodies showed broad immunoreactivity in DRG from mice lacking S1PR3, and so we instead used anti-DsRed antibodies to probe expression of the S1PR3 fusion protein (Figure 3—figure supplement 1E). We found that 42.4% of S1PR3+ cells co-stained with anti-Peripherin, demonstrating that S1PR3 is expressed in a subset of small-diameter neurons. We also observed that 69.5% of S1PR3+ cells co-stained with anti-NF200, which marks medium and large-diameter myelinated neurons. Furthermore, we observed that S1PR3+ cells were primarily of small to medium diameter (11.3–35.1 µm), whereas all cells in the DRG ranged from 11.3 to 53.9 µm. Overall, these data support the expression of S1PR3 in subsets of small-diameter thermal nociceptors and medium-diameter mechanonociceptors (Figure 3F). Additionally, no significant differences were observed between WT and S1PR3 KO DRG in number of Trpa1+, Trpv1+, Peripherin+, NF200+, or IB4+ cells (Figure 3—figure supplement 1B–C,F,G). The mean diameters of Trpv1+ neurons (Figure 3—figure supplement 1D, left), NF200+ neurons (Figure 3—figure supplement 1G), or all neurons (Figure 3—figure supplement 1D, right) in WT versus KO DRG were not significantly different, suggesting no loss of major sensory neuronal subtypes in the S1PR3 KO. We then visualized S1PR3 expression in nerve fibers that innervate the skin using anti-DsRed antibodies in whole-mount immunohistochemistry (IHC; Figure 3D). The reporter animals showed no specific antibody staining in epidermal or dermal cells (Figure 3—figure supplement 1I), and single-cell RNA seq of a diverse array of mouse epidermal and dermal cells corroborates this lack of expression (Joost et al., 2016). We observed overlap of S1PR3-expressing free nerve endings with NF200+ myelinated free nerves and NF200- putative C-fiber endings (Figure 3F), but did not observe expression of S1PR3 in NF200+ circumferential or lanceolate hair follicle receptors, or in putative Merkel afferents (Figure 3D–E). β-tubulin III, PGP9.5 (pan-neuronal markers), and NF200 staining in S1PR3 KO skin displayed patterns of epidermal and dermal innervation similar to WT skin, suggesting the phenotypes observed in the S1PR3 KO mice are not due to developmental loss of sensory neuronal innervation (pPGP9.5= 0.443 (n = 93, 38 fibers); pNefH = 0.405 (n = 61, 28 fibers); pBTIII = 0.353 (n = 104, 89 fibers); two-tailed t-tests based on average number of fibers per field of view). These results support expression of S1PR3 in subsets of myelinated A mechanonociceptors and unmyelinated C nociceptors that terminate as free nerve endings. S1P activates thermal nociceptors but not putative AMs Live imaging of cultured DRG neurons from adult reporter animals showed expression of S1PR3-mCherry fusion protein in 48.3% of neurons, mirroring our ISH and IHC results (Figure 4A). To examine the effects of S1P on A mechanonociceptors and C nociceptors, we performed ratiometric calcium imaging and electrophysiology on DRG cultures from reporter mice. Interestingly, only 56.1 ± 22.4% of mCherry-expressing neurons were activated by 1 µM S1P (Representative trace in Figure 4B; representative images in Figure 4C), which our dose-response showed to be the saturating concentration for calcium influx (Figure 4D; EC50 = 155 nM). neurons were also capsaicin-sensitive (n > sensory neurons from S1PR3 KO animals did not to S1P, as (Camprubí-Robles et al., 2013), exhibited responses that were not significantly from WT neurons (Figure 3—figure supplement The mean diameter of neurons was ± whereas the mean diameter of neurons was ± two-tailed We also performed cell experiments with studies (Mair et al., 2011; Zhang et al., 2006; Li et al., 2015), found that S1P in capsaicin-sensitive small diameter cells (Figure This that only the small-diameter, S1PR3+ putative nociceptors are by S1P. We next asked whether the S1PR3+ diameter neurons represent the mechanonociceptors observed by ISH (Figure To this we asked whether the a selective of nociceptors (Osteen et al., 2016), triggers calcium influx in S1PR3-expressing neurons. Indeed, we found that ± of neurons expressed (Figure with our staining showing expression of S1pr3 in nociceptors and the role of neurons in mechanical pain in (Osteen et al., 2016). Figure Download asset Open asset S1P activates thermal nociceptors but not mechanonociceptors. (A) (Left) Representative of in cultured adult DRG neurons from one S1pr3mCherry/+ (Right) Quantification of % of total cells expressing S1pr3 from DRG ISH and from DRG cultures (N = 2 animals (B) Representative from calcium imaging showing two neurons, one which to 1 µM S1P, 1 µM and and one which only to (C) (Left) calcium imaging (left) and after addition of 1 µM S1P in S1pr3 cultured mouse DRG neurons. Bar (Right) % of neurons that are to 1 µM S1P in ratiometric calcium imaging (n > cells from imaging from animals). (D) of mean neuronal calcium to of S1P. and (N = 2 animals). Error bars represent mean ± SD. for all S1P from which EC50 was S1P were also (E) trace of a single wild-type neuron in response to addition of 1 µM S1P and 1 µM with of to S1P and one of one also to Bar = 2 (F) (Left) calcium imaging after addition of 500 nM in S1pr3mCherry/+ neurons, which were used instead of adult DRG neurons because to without (Right) % of neurons that are (N = 1 total S1PR3 KCNQ2/3 channels to excitability We next the molecular by which S1P signaling in nociceptors may mechanical pain. We performed on the medium-diameter S1pr3mCherry/+ DRG neurons = ± which did not display calcium influx (Figure In these cells, 1 µM S1P did not (Figure supplement 1A; see Figure data or in the of injection (Figure supplement 1A; Figure However, S1P the threshold to in an S1PR3-dependent (Figure Figure supplement 1B). Figure 5 with 1 supplement see all Download asset Open asset S1PR3 KCNQ2/3 channels to experiments were performed in S1pr3mCherry/+ or DRG neurons. (A) (Left) of a single in cell and after S1P (Right) % in after S1P for S1pr3mCherry/+ (left, n = 7) and KO (right, n = 12) neurons = two-tailed paired t-tests). (B) % in input after S1P or vehicle two-tailed paired t-test; n = cells per group). (C) The is by The was by of the from the and at n = cells. Data were with a and were measured at the indicated to 20 following a was using the of the after to indicated indicated all bars represent mean ± (D) of a single neuron in cell comparing pre- and using indicated of a single neuron in cell with was in % in after indicated µM S1P, 3 µM or for S1pr3mCherry/+ medium-diameter one-way ANOVA; n = using at % in after indicated = 100 µM for S1pr3mCherry/+ medium-diameter two-tailed paired t-test; n = Figure data 1 S1PR3 KCNQ2/3 channels to Related to Figure Download We then set out to the by which S1PR3 activity neuronal excitability using studies showed that S1P capsaicin-sensitive nociceptors by sodium and potassium et al., 2006; Li et al., 2015). We found that S1P had no such effects on S1PR3+ medium-diameter cells (Figure supplement By contrast, S1P triggered a in input (Figure with the closure of potassium channels. analysis revealed that the by S1P was by potassium (Figure Additionally, S1P significantly reduced (Figure supplement 1F; Figure in an S1PR3-dependent (Figure supplement As in Aδ neurons are primarily mediated by KCNQ2/3 potassium channels et al., et al., we that S1P may modulation of these channels. Furthermore, the of the