Presence Of Sphingosine 1-phosphate Research Articles

The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts.

The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.

P-467 Optimizing in vitro growth of immature follicles from cryopreserved vs fresh human cortical ovarian tissue

Abstract Study question Can cryopreserved-thawed human ovarian cortex be cultured in vitro to produce growing follicles as efficiently as from fresh samples? Summary answer In contrast to fresh samples, cryopreserved-thawed ovarian cortex did not develop morphologically normal growing follicles, even when adding follicular growth stimulators. What is known already A multistep culture system has been shown to produce mature oocytes from immature follicles present in the human cortex, without the addition of follicular growth stimulators. However, these promising results were achieved using fresh human ovarian cortex tissue donated from cisgender women, which is not available for the purpose of fertility preservation. It remains unclear whether this multistep culture system can be used to mature follicles from cryopreserved-thawed human ovarian cortex. In addition, the applicability of the culture system to mature follicles in vitro has not been investigated using ovarian tissue from transmasculine people. Study design, size, duration Fresh and cryopreserved ovarian cortex tissue isolated from the same transmasculine people were cultured during 8 days (first step of the culture system) with or without the addition of a PI3K/Akt promotor, Sphingosine-1-Phosphate (S1P). The ovarian cortex fragments were collected a day 0 and day 8 for downstream morphological and quantitative analysis of the follicular population. This study also included data of in vitro culture of cryopreserved samples from oncological cisgender women. Participants/materials, setting, methods Ovarian cortex tissue was collected from six testosterone user transmasculine people (25,8 ± 4,9 years) and three oncological cisgender women (24,0 ± 6,0 years). Fresh cortex was either directly cultured or previously cryopreserved. All ovarian fragments were cultured in the same conditions for 8 days. After the culture period, follicular population was quantified on histological sections (haematoxylin-eosin) and compared to samples of day 0. Follicular cell types, cell proliferation and apoptosis were assessed by immunofluorescence. Main results and the role of chance Fresh ovarian cortex tissue isolated from transmasculine people showed morphologically normal primary follicles (PFs) and secondary follicles (SFs) after 8 days of culture and no antral follicles were observed. Moreover, the addition of the follicular growth stimulator, S1P, to the culture medium significantly increased the number of growing follicles (PFs, P < 0.05; SFs, P < 0.01). The addition of S1P stimulated the emergence of SFs that showed expression of anti-mullerian hormone (AMH), contained proliferative (PCNA+) granulosa cells and showed low levels of apoptotic cells (TUNEL assay). Structurally, these in vitro grown SFs showed an intact basement membrane assessed by the expression of collagen IV. By contrast, using the same culture protocol but starting from cryopreserved-thawed cortex tissue from the same patients resulted in a very low number of viable growing follicles (PFs and SFs) even in the presence of S1P. Lastly, when using the same culture protocol on cryopreserved-thawed samples from oncological cisgender women, we observed a low efficiency of follicular growth, comparable to that observed in cryopreserved ovarian tissue from (age-matched) transmasculine people. Limitations, reasons for caution Limitations of this study are: small sample size and lack of fresh cortex tissue from oncological cisgender women. Due to the high variability of follicular density among patients, the use of a larger number of samples is recommended. Wider implications of the findings Optimization of culture systems to grow and mature follicles in the ovarian cortex of cryopreserved samples (both from cis and trans persons) is urgently needed. Current culture protocols can be applied to fresh samples, but further characterization is needed to consider this as an alternative in fertility preservation treatment. Trial registration number Not applicable

The β1-integrin plays a key role in LEC invasion in an optimized 3-D collagen matrix model.

Lymphangiogenesis, or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration, and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro three-dimensional (3-D) collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), interleukin (IL)-8, and hepatocyte growth factor (HGF), were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion, and synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expressions of the integrin α5- and integrin β1-subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA-mediated knockdown of the integrin β1-subunit suppressed LEC invasion and also inhibited VEGF receptor (VEGFR)3 and ERK activation. Furthermore, exposing LECs to the inflammatory mediator lipopolysaccharide (LPS) inhibited sprouting. This optimized model for LEC invasion includes S1P, VEGF-C, and fibronectin within a 3-D collagen matrix, along with VEGF-C, VEGF-A, bFGF, and HGF in the culture medium, and provides a useful tool to investigate the functional effect of various lymphangiogenic factors and inhibitors.

Sphingosine 1-phosphate regulates proliferation, cell cycle and apoptosis of hepatocellular carcinoma cells via syndecan-1

Sphingosine 1-phosphate (S1P) plays an important role in hepatocarcinogenesis. We previously demonstrated that S1P induced epithelial–mesenchymal transition of hepatocellular carcinoma (HCC) cells via an MMP-7/Syndecan-1/TGF-β autocrine loop. In the present study, we investigated the regulative role of S1P in cell survival and progression of HCC cells, and tested whether syndecan-1 is required in the S1P action. After transfected with syndecan-1 shRNA, HepG2 and SMMC7721 cells were treated with S1P for 72 h, and then cell proliferation was detected by CCK8 assay, and cell cycle progression and cell apoptosis were detected by flow cytometry. The levels of apoptosis markers including cleaved-Caspase-3 and cleaved-PARP in SMMC7721 cells were examined by western blotting. Results showed that S1P significantly enhanced cell proliferation in HCC cells, which was significantly inhibited by syndecan-1 shRNA. S1P induced the cell proportion in S phase in HCC cells, whereas S1P decreased the proportion of cells in both early and late apoptosis. Syndecan-1 shRNA induced the G2/M arrest in the presence of S1P. In the syndecan-1 shRNA transfected HCC cells, the proportions of late and early apoptotic cells, and levels of cleaved-Caspase-3 and cleaved-PARP were significantly increased in cells with or without S1P treatment. Thus, S1P augments the proportion of cells in S phase of the cell cycle that might translate to enhance HCC cell proliferation and inhibit the cell apoptosis via syndecan-1.

Sphingosine-1-phosphate reduces adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel walls by protecting endothelial surface glycocalyx.

Sphingosine-1-phosphate (S1P) is a sphingolipid in plasma that plays a critical role in cardiovascular and immune systems. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. To test the hypothesis that S1P can reduce tumor cell adhesion to microvessel walls by protecting the ESG, we quantified the ESG and MDA-MB-231 tumor cell adhesion in the presence and absence of 1μM S1P, and in the presence of the matrix metalloproteinase (MMP) inhibitor in post-capillary venules of rat mesentery. We also measured the microvessel permeability to albumin as an indicator for the microvessel wall integrity. In the absence of S1P, ESG was ~10% of that in the presence of S1P, whereas adherent tumor cells and the permeability to albumin and were ~3.5-fold (after 30 min adhesion) and ~7.7-fold that in the presence of S1P, respectively. In the presence of the MMP inhibitor, the results are similar to those in the presence of S1P. Our results conform to the hypothesis that protecting ESG by S1P inhibits MDA-MB-231 tumor cell adhesion to the microvessel wall.

Sphingosine 1-phospate differentially modulates maturation and function of human Langerhans-like cells

BackgroundAs mediators between innate and adaptive immune responses, Langerhans cells (LCs) are in the focus of recent investigations to determine their role in allergic inflammatory diseases like allergic contact dermatitis and atopic dermatitis. Sphingosine 1-phosphate (S1P) is a crucial lipid mediator in the skin and potentially interferes with LC homeostasis but also functional properties, such as cytokine release, migration and antigen-uptake which are considered to be key events in the initiation and maintenance of pathological disorders. ObjectiveHere, we used human Langerhans-like cells to study the influence of S1P-mediated signalling on LC maturation, cytokine release, migration and endocytosis. MethodsImmature Langerhans-like cells were generated from the human acute myeloid leukaemia cell line MUTZ-3 (MUTZ-LCs) and human primary monocytes (MoLCs). S1P receptor expression was determined by quantitative RT-PCR and western blotting. Expression of maturation markers were investigated by flow cytometry. The influence of S1P signalling on cytokine release was quantified by ELISA. Migration assays and FITC-dextran uptake in the presence of S1P, specific S1 P receptor agonists and antagonists as well as fingolimod (FTY720) were analysed through fluorescence microscopy and flow cytometry. ResultsS1P receptor protein expression was confirmed for S1P1, S1P2 and S1P4 in MUTZ-LCs and S1P1 and S1P2 in MoLCs. In mature cells S1P receptors were downregulated. S1P did not induce maturation in MUTZ-LCs, whereas in MoLCs CD83 and CD86 were slightly upregulated. IL-8 release of MUTZ-LCs matured in the presence of S1P was not altered, however, reduced IL-6 and IL-12p70 levels were observed in mature MoLCs. Interestingly, immature MUTZ-LCs revealed a significantly increased S1P-dependent migratory capacity, whereas CCL20 induced migration was significantly decreased in the presence of S1P. Furthermore, migratory capacity towards CCL21 in mature MUTZ-LCs but not MoLCs was significantly lower when cells were stimulated with S1P. S1P, FTY720 and specific S1P receptor agonists did not modulate the endocytotic capacity of immature MUTZ-LCs and MoLCs. These findings were further supported by testing specific antagonists of S1P1−4 in the absence or presence of S1P. ConclusionOur data demonstrate that S1P regulates key events of human LC maturation including cytokine release and migration. These findings are of particular importance when considering the potential use of S1P in inflammatory skin disorders.

Involvement of Sphingosine 1-Phosphate in Palmitate-Induced Non-Alcoholic Fatty Liver Disease

Background/Aims: Ectopic lipid accumulation in hepatocytes has been identified as a risk factor for the progression of liver fibrosis and is strongly associated with obesity. In particular, the saturated fatty acid palmitate is involved in initiation of liver fibrosis via formation of secondary metabolites by hepatocytes that in turn activate hepatic stellate cells (HSCs) in a paracrine manner. Methods: α-smooth muscle actin-expression (α-SMA) as a marker of liver fibrosis was investigated via western blot analysis and immunofluorescence microscopy in HSCs (LX-2). Sphingolipid metabolism and the generation of the bioactive secondary metabolite sphingosine 1-phosphate (S1P) in response to palmitate were analyzed by LC-MS/MS in hepatocytes (HepG2). To identify the molecular mechanism involved in the progression of liver fibrosis real-time PCR analysis and pharmacological modulation of S1P receptors were performed. Results: Palmitate oversupply increased intra- and extracellular S1P-concentrations in hepatocytes. Conditioned medium from HepG2 cells initiated fibrosis by enhancing α-SMA-expression in LX-2 in a S1P-dependent manner. In accordance, fibrotic response in the presence of S1P was also observed in HSCs. Pharmacological inhibition of S1P receptors demonstrated that S1P₃ is the crucial receptor subtype involved in this process. Conclusion: S1P is synthesized in hepatocytes in response to palmitate and released into the extracellular environment leading to an activation of HSCs via the S1P₃ receptor.

Hypoxia augments outgrowth endothelial cell (OEC) sprouting and directed migration in response to sphingosine-1-phosphate (S1P).

Therapeutic angiogenesis provides a promising approach to treat ischemic cardiovascular diseases through the delivery of proangiogenic cells and/or molecules. Outgrowth endothelial cells (OECs) are vascular progenitor cells that are especially suited for therapeutic strategies given their ease of noninvasive isolation from umbilical cord or adult peripheral blood and their potent ability to enhance tissue neovascularization. These cells are recruited to sites of vascular injury or tissue ischemia and directly incorporate within native vascular endothelium to participate in neovessel formation. A better understanding of how OEC activity may be boosted under hypoxia with external stimulation by proangiogenic molecules remains a challenge to improving their therapeutic potential. While vascular endothelial growth factor (VEGF) is widely established as a critical factor for initiating angiogenesis, sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, has recently gained great enthusiasm as a potential mediator in neovascularization strategies. This study tests the hypothesis that hypoxia and the presence of VEGF impact the angiogenic response of OECs to S1P stimulation in vitro. We found that hypoxia altered the dynamically regulated S1P receptor 1 (S1PR1) expression on OECs in the presence of S1P (1.0 μM) and/or VEGF (1.3 nM). The combined stimuli of S1P and VEGF together promoted OEC angiogenic activity as assessed by proliferation, wound healing, 3D sprouting, and directed migration under both normoxia and hypoxia. Hypoxia substantially augmented the response to S1P alone, resulting in ~6.5-fold and ~25-fold increases in sprouting and directed migration, respectively. Overall, this report highlights the importance of establishing hypoxic conditions in vitro when studying ischemia-related angiogenic strategies employing vascular progenitor cells.

Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson’s disease and in MPP+-treated MN9D cells in vitro

Dysregulation of sphingolipid metabolism has been shown to trigger the pathophysiology of many neurodegenerative disorders. The present study focuses on the role of one of the two sphingosine kinases, Sphk2 and its metabolite sphingosine-1-phosphate (S1P) signaling in Parkinson’s disease (PD). Our study indicated a marked down regulation of Sphk2 expression in the substantia nigra region of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in the cellular PD model. Localization studies indicated that Sphk2 was predominantly present in mitochondria, proposing for its potential role in mitochondrial functions. Since mitochondrial dysfunction has been described to be the major pathological event in PD, the present study focused on the role of Sphk2/S1P signaling in promoting mitochondrial functions in the MPTP-induced mouse model of PD and in 1-methyl-4 phenylpyridinium (MPP+)-treated MN9D cells. Our study demonstrated that inhibition of Sphk2 decreased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its downstream targets nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) which are the key genes regulating mitochondrial function. In addition, there was also a significant reduction in the total cellular adenosine triphosphate (ATP) and superoxide dismutase 2 (SOD 2) with an associated increase in levels of reactive oxygen species (ROS) in the absence of Sphk2. Interestingly, it was found that treating the cells with exogenous S1P along with MPP+ exerted a neuroprotective effect by activation of p-CREB, PGC-1α and NRF-1 in the MN9D cells. Moreover, the level of ATP was unaffected in the MPP+-treated cells in the presence of S1P. It was also observed that levels of ROS were significantly decreased in the MPP+-treated cells in the presence of exogenous S1P. Our study also demonstrated that S1P exerted its protective effect through the S1P1 receptor. Taken together, these results show that Sphk2/S1P has an important role to play in the survival of the dopaminergic neurons, in the pathogenesis of PD.

Abstract 1890: Activated SphK1 and export of S1P via ABCC1 shorten disease free survival in breast cancer

Abstract ATP-binding cassette (ABC) transporters are known to be multidrug resistance proteins that efflux various compounds out of cells including chemotherapeutic agents. A number of clinical trials have been conducted targeting ABCB1: however, none showed beneficial effects. The pleiotropic bioactive lipid mediator sphingosine-1-phosphate (S1P), which is generated by sphingosine kinase 1 (SphK1) inside breast cancer cells and exerts its functions by binding to its specific cell surface G-protein coupled receptors (S1PR1-5) after being exported, is now known as a key regulatory molecule in breast cancer progression. We have previously demonstrated that ABCC1 and ABCG2, but not ABCB1 export S1P out of MCF7 human breast cancer cells. We hypothesized that ABCC1 expression in the presence of S1P produced by activated SphK1 in human breast cancer is associated with poor prognosis. We demonstrate the role of ABCC1 and SphK1/ S1P axis in breast cancer progression utilizing ABC transporter transduced breast cancer cell lines for in vitro and in vivo study. For clinical study, we analyze the expression of ABC transporters and S1P signaling factors utilizing a tissue microarray constructed from 276 breast cancer samples. Overexpression of ABCC1 in breast cancer cell lines export more S1P. ABCC1 overexpression but not ABCB1 enhance proliferation and cell migration, and promote angiogenesis (AG) and lymph angiogenesis (LAG). MK571 which is inhibitor of ABCC1 suppress them. Overexpression of ABCC1 also aggravate tumor grow and contribute poor survival in syngeneic and xenograft mice model. In the clinical study, ABCC1 expression was associated with significantly shorter disease free survival (DFS) while ABCB1 was not. pSphK1 high expression group had significantly shorter DFS. Most of the patients expressed S1PR1; however, there were no significant differences in prognosis. Patients with tumors expressing both pSphK1 and ABCC1 had significantly shorter DFS, while patients expressing both ABCB1 and pSphK1 did not. These translational findings suggest that inside-out signaling of S1P via ABCC1 may play a significant role in the course of human breast cancer progression. This work was supported by grants from the National Institute of Health (R01CA160688 to K.T and R01CA61774 to S.S). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. Citation Format: Akimitsu Yamada, Masayuki Nagahashi, Tomoyoshi Aoyagi, Wei C. Huang, Krista P. Terracina, Jeremy C. Allegood, Santiago Lima, Sheldon Milstien, Sarah Spiegel, Kumiko Kida, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. Activated SphK1 and export of S1P via ABCC1 shorten disease free survival in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1890. doi:10.1158/1538-7445.AM2014-1890

The expression of sphingosine-1 phosphate receptor-1 in chronic lymphocytic leukemia cells is impaired by tumor microenvironmental signals and enhanced by piceatannol and R406.

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.

Sphingosine-1-phosphate protects endothelial glycocalyx by inhibiting syndecan-1 shedding

Endothelial cells (ECs) are covered by a surface glycocalyx layer that forms part of the barrier and mechanosensing functions of the blood-tissue interface. Removal of albumin in bathing media induces collapse or shedding of the glycocalyx. The electrostatic interaction between arginine residues on albumin, and negatively charged glycosaminoglycans (GAGs) in the glycocalyx have been hypothesized to stabilize the glycocalyx structure. Because albumin is one of the primary carriers of the phospholipid sphingosine-1-phosphate (S1P), we evaluated the alternate hypothesis that S1P, acting via S1P1 receptors, plays the primary role in stabilizing the endothelial glycocalyx. Using confocal microscopy on rat fat-pad ECs, we demonstrated that heparan sulfate (HS), chondroitin sulfate (CS), and ectodomain of syndecan-1 were shed from the endothelial cell surface after removal of plasma protein but were retained in the presence of S1P at concentrations of >100 nM. S1P1 receptor antagonism abolished the protection of the glycocalyx by S1P and plasma proteins. S1P reduced GAGs released after removal of plasma protein. The mechanism of protection from loss of glycocalyx components by S1P-dependent pathways was shown to be suppression of metalloproteinase (MMP) activity. General inhibition of MMPs protected against loss of CS and syndecan-1. Specific inhibition of MMP-9 and MMP-13 protected against CS loss. We conclude that S1P plays a critical role in protecting the glycocalyx via S1P1 and inhibits the protease activity-dependent shedding of CS, HS, and the syndecan-1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.

Sphingosine-1-phosphate and toll-like receptor 4-cooperate to induce inflammatory and adhesive responses in human aortic valve endothelial cells

Purpose: Given the association of Toll-like receptor (TLR)-4 to calcific aortic stenosis and the relevant function of the lipid mediator sphingosine-1-phosphate (S1P) in the cardiovascular system, the putative role of TLR4 ligands and S1P in the induction of pro-inflammatory and pro-adhesive responses in endothelial cells from human aortic (AVEC) and pulmonary valves (PVEC) was investigated. Methods: AVEC and PVEC obtained from n =5 heart transplant patients with no valve disease, and AVEC derived from stenotic aortic valves collected from n = 5 patients with degenerative aortic stenosis and indication for valve replacement were studied. Reverse transcriptase PCR assays were used to evaluate the mRNA expression of TLR and S1P receptors. Cytokine secretion and adhesion molecule expression were evaluated by antibody arrays and Western blot, and adhesion of human monocytes to AVEC monolayers was assayed in vitro. Comparison between stenotic and control aortic valve and between aortic and pulmonar valve were performed. Results: Reverse transcriptase PCR assays revealed the expression of TLRs at the mRNA level in AVEC, being TLR4 the most abundant, as well as the expression of S1P receptors, the S1P1 being the most abundant subtype. As regards pro-inflammatory molecules, the exposure of control AVECs to the bacterial lipopolysaccharide (LPS), a TLR4 ligand, induced the secretion of several cytokines such as interleukin (IL)-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemotactic protein-1. Moreover, LPS cooperated with S1P to further up-regulate IL-6 and GM-CSF secretion. In addition, AVEC exposure to LPS induced the expression of the adhesion molecule ICAM-1, which was further up-regulated in the presence of S1P. Interestingly, the effect was more prominent in endothelial cells from the aortic valve than from the pulmonary valve, which rarely undergoes stenosis, and more potent in cells from stenotic valves. The cooperative effect on ICAM-1 up-regulation was inhibited by S1P1 receptor antagonists. In adhesion assays, LPS and S1P cooperated to increase the number of monocytes that adhered to the activated AVEC monolayers, and the effect was more marked in stenotic than in control AVECs. Conclusions: TLR4 and S1P1 receptor cooperate to up-regulate pro-inflammatory/pro-adhesive molecules in AVECs, what might be relevant for the pathogenesis of aortic stenosis given the stronger effect on stenotic aortic valves and the lower effect on pulmonary valves, and could open the way for new therapeutic approaches for the disease.

Plasma protein protects endothelial glycocalyx mediated by sphingosine‐1‐phosphate

It has long been known that albumin reduction in endothelial cell (EC) media induces collapse or shedding of the surface glycocalyx layer. This leads to impairment of the endothelial transport barrier and other features of vessel homeostasis. The electrostatic interaction between arginine residues on albumin and negatively charged glycosaminoglycans in the glycocalyx has been hypothesized to stabilize the glycocalyx structure. But sphingosine‐1‐phosphate (S1P), which is bound to albumin, also plays important roles in maintaining vascular barrier integrity and homeostasis. Here, the role of S1P in maintaining glycocalyx stability was investigated in this study. Using confocal microscopy on EC in vitro, we observed that heparan sulfate (HS), chondroitin sulfate (CS) and the ectodomain of syndecan‐1 were shed from the surface after removal of plasma protein, but were retained in the presence of S1P or a general MMP inhibitor. S1P receptor 1 (S1P1) antagonism abolished the protection of the glycocalyx by S1P. GAGs released to culture media and the general MMP activity increased significantly after removal of plasma protein, but could be diminished by S1P. Thus, S1P plays a critical role in protecting the glycocalyx via S1P1, and inhibits the protease activity‐dependent shedding of CS, HS and the syndecan‐1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.Grants: Supported by NIH Grant HL094889 and HL 28607.

Knockdown of the sphingosine-1-phosphate receptor S1PR1reduces pain behaviors induced by local inflammation of the rat sensory ganglion

Sphingosine 1-phosphate (S1P) is a key immune mediator regulating migration of immune cells to sites of inflammation. S1P actions are mediated by a family of five G protein-coupled receptors. Sensory neurons express many of these receptors, and in vitro S1P has excitatory effects on small-diameter sensory neurons, many mediated by the S1P receptor 1 (S1PR1). This study investigated the role of S1P in regulating the sensitivity of DRG neurons. We found that in vivo perfusion of the normal L5 DRG with S1P increased mechanical sensitivity. Microelectrode recordings in isolated whole ganglia showed that large- and medium-diameter cells, as well as small-diameter cells, increased firing in the presence of S1P. To further determine the role of S1PRs, we examined the effects of in vivo S1PR1 knockdown in the L4 and L5 sensory ganglia. Small interfering RNA directed against S1PR1 did not affect baseline mechanical sensitivity in normal animals, in which S1P levels are expected to be low. However, when the L5 ganglion was locally inflamed, a procedure that leads to rapid and sustained mechanical hypersensitivity, S1PR1 siRNA injected animals showed significantly less hypersensitivity than animals injected with scrambled siRNA. Reduced expression of S1PR1, but not S1PR2 or S1PR3, was confirmed with qPCR methods. The results indicate that the S1PR1 receptors in sensory ganglia cells may play an important role in regulating behavioral sensitivity during inflammation.

Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.

Sphingosine-1-phosphate augments agonist-mediated contraction in the bronchial smooth muscles of mice

The effects of sphingosine-1-phosphate (S1P) on bronchial smooth muscle (BSM) contractility were investigated in naive mice. S1P had no effect on the basal tone of the isolated BSM tissues. However, in the presence of S1P(10-6 M), the BSM contractions induced by acetylcholine (ACh) and endothelin-1 (ET-1) were significantly augmented: both the ACh and ET-1 concentration-response curves were significantly shifted to the left. In contrast, the pretreatment with S1P had no effect on the contractions induced by high K+ depolarization. It is thus possible that S1P augments BSM contraction induced by the activation of G protein-coupled receptors.

There is More to a Lipid than just Being a Fat: Sphingolipid-Guided Differentiation of Oligodendroglial Lineage from Embryonic Stem Cells

Dr. Robert K. Yu's research showed for the first time that the composition of glycosphingolipids is tightly regulated during embryo development. Studies in our group showed that the glycosphingolipid precursor ceramide is also critical for stem cell differentiation and apoptosis. Our new studies suggest that ceramide and its derivative, sphingosine-1-phosphate (S1P), act synergistically on embryonic stem (ES) cell differentiation. When using neural precursor cells (NPCs) derived from ES cells for transplantation, residual pluripotent stem (rPS) cells pose a significant risk of tumor formation after stem cell transplantation. We show here that rPS cells did not express the S1P receptor S1P1, which left them vulnerable to ceramide or ceramide analog (N-oleoyl serinol or S18)-induced apoptosis. In contrast, ES cell-derived NPCs expressed S1P1 and were protected in the presence of S1P or its pro-drug analog FTY720. Consistent with previous studies, FTY720-treated NPCs differentiated predominantly toward oligodendroglial lineage as tested by the expression of the oligodendrocyte precursor cell (OPC) markers Olig2 and O4. As the consequence, a combined administration of S18 and FTY720 to differentiating ES cells eliminated rPS cells and promoted oligodendroglial differentiation. In addition, we show that this combination promoted differentiation of ES cell-derived NPCs toward oligodendroglial lineage in vivo after transplantation into mouse brain.

Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation

Transient receptor potential canonical (TRPC) channels provide cation and Ca(2+) entry pathways, which have important regulatory roles in many physio-pathological processes, including muscle dystrophy. However, the mechanisms of activation of these channels remain poorly understood. Using siRNA, we provide the first experimental evidence that TRPC channel 1 (TRPC1), besides acting as a store-operated channel, represents an essential component of stretch-activated channels in C2C12 skeletal myoblasts, as assayed by whole-cell patch-clamp and atomic force microscopic pulling. The channel's activity and stretch-induced Ca(2+) influx were modulated by sphingosine 1-phosphate (S1P), a bioactive lipid involved in satellite cell biology and tissue regeneration. We also found that TRPC1 was functionally assembled in lipid rafts, as shown by the fact that cholesterol depletion resulted in the reduction of transmembrane ion current and conductance. Association between TRPC1 and lipid rafts was increased by formation of stress fibres, which was elicited by S1P and abolished by treatment with the actin-disrupting dihydrocytochalasin B, suggesting a role for cytoskeleton in TRPC1 membrane recruitment. Moreover, TRPC1 expression was significantly upregulated during myogenesis, especially in the presence of S1P, implicating a crucial role for TRPC1 in myoblast differentiation. Collectively, these findings may offer new tools for understanding the role of TRPC1 and sphingolipid signalling in skeletal muscle regeneration and provide new therapeutic approaches for skeletal muscle disorders.

The sphingosine 1-phosphate receptor 5 and sphingosine kinases 1 and 2 are localised in centrosomes: Possible role in regulating cell division

We show here that the endogenous sphingosine 1-phosphate 5 receptor (S1P 5, a G protein coupled receptor (GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 and SK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where they may participate in regulating mitosis. The centrosome is a site for active GTP–GDP cycling involving the G-protein, G i and tubulin, which are required for spindle pole organization and force generation during cell division. Therefore, the presence of S1P 5 (which normally functions as a plasma membrane guanine nucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P 5 may function as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. The addition of S1P to cells inhibits trafficking of S1P 5 to the centrosome, suggesting a dynamic shuttling endocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that the centrosomal S1P 5 receptor might function as an intracellular target of S1P linked to regulation of mitosis.