Sphingosine-1-Phosphate Signaling in Inflammatory Bowel Disease

DEFF Research Database (Denmark)

Nielsen, Ole Haagen; Li, Yuan; Lindbom, Bengt Johansson

2017-01-01

primary non-responders or lose responsiveness during maintenance treatment. A new class of small molecules, sphingosine-1-phosphate (S1P) receptor modulators, has recently shown efficacy in IBD. Here we provide an overview of the mechanism of action of this novel treatment principle in the context...... of intestinal inflammation. The remarkable impact of therapeutic modulation of the S1P/S1P receptor axis reflects the complexity of the pathogenesis of IBD and the fact that S1P receptor modulation may be a logical therapeutic approach for the future management of IBD....

Sphingosine 1-phosphate as a novel immune regulator of dendritic ...

Indian Academy of Sciences (India)

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to be involved in several physiological and pathological functions as an extracellular mediator. S1P receptors are widely expressed and thought to regulate important functions in cell signalling.

Role of sphingosine 1-phosphate (S1P and effects of fingolimod, an S1P receptor 1 functional antagonist in lymphocyte circulation and autoimmune diseases

Directory of Open Access Journals (Sweden)

Kenji Chiba

2014-11-01

Full Text Available Sphingosine 1-phosphate (S1P, a multi-functional phospholipid mediator, is generated from sphingosine by sphingosine kinases and binds to five known G protein-coupled S1P receptors (S1P1, S1P2, S1P3, S1P4, and S1P5. It is widely accepted that S1P receptor 1 (S1P1 plays an essential role in lymphocyte egress from the secondary lymphoid organs (SLO and thymus, because lymphocyte egress from these organs to periphery is at extremely low levels in mice lacking lymphocytic S1P1. Fingolimod hydrochloride (FTY720 is a first-in-class, orally active S1P1 functional antagonist which was discovered by chemical modification of a natural product, myriocin. Since FTY720 has a structure closely related to sphingosine, the phosphorylated FTY720 (FTY720-P is converted by sphingosine kinases and binds 4 types of S1P receptors. FTY720-P strongly induces down-regulation of S1P1 by internalization and degradation of this receptor and acts as a functional antagonist at S1P1. Consequently, FTY720 inhibits S1P1-dependent lymphocyte egress from the SLO and thymus to reduce circulating lymphocytes including autoreactive Th17 cells, and is highly effective in experimental autoimmune encephalomyelitis (EAE, an animal model of multiple sclerosis (MS. In relapsing remitting MS patients, oral FTY720 shows a superior efficacy when compared to intramuscular interferon-β-1a. Based on these data, it is presumed that modulation of the S1P-S1P1 axis provides an effective therapy for autoimmune diseases including MS.

Sphingosine kinase 1/sphingosine-1-phosphate (S1P)/S1P receptor axis is involved in ovarian cancer angiogenesis.

Science.gov (United States)

Dai, Lan; Liu, Yixuan; Xie, Lei; Wu, Xia; Qiu, Lihua; Di, Wen

2017-09-26

Sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) signaling pathway has been implicated in a variety of pathological processes of ovarian cancer. However, the function of this axis in ovarian cancer angiogenesis remains incompletely defined. Here we provided the first evidence that SphK1/S1P/S1PR 1/3 pathway played key roles in ovarian cancer angiogenesis. The expression level of SphK1, but not SphK2, was closely correlated with the microvascular density (MVD) of ovarian cancer tissue. In vitro , the angiogenic potential and angiogenic factor secretion of ovarian cancer cells could be attenuated by SphK1, but not SphK2, blockage and were restored by the addition of S1P. Moreover, in these cells, we found S1P stimulation induced the angiogenic factor secretion via S1PR 1 and S1PR 3 , but not S1PR 2 . Furthermore, inhibition of S1PR 1/3 , but not S1PR 2 , attenuated the angiogenic potential and angiogenic factor secretion of the cells. in vivo , blockage of SphK or S1PR 1/3 could attenuate ovarian cancer angiogenesis and inhibit angiogenic factor expression in mouse models. Collectively, the current study showed a novel role of SphK1/S1P/S1PR 1/3 axis within the ovarian cancer, suggesting a new target to block ovarian cancer angiogenesis.

Sphingosine-1-Phosphate and Its Receptors: A Mutual Link between Blood Coagulation and Inflammation

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Shailaja Mahajan-Thakur

2015-01-01

Full Text Available Sphingosine-1-phosphate (S1P is a versatile lipid signaling molecule and key regulator in vascular inflammation. S1P is secreted by platelets, monocytes, and vascular endothelial and smooth muscle cells. It binds specifically to a family of G-protein-coupled receptors, S1P receptors 1 to 5, resulting in downstream signaling and numerous cellular effects. S1P modulates cell proliferation and migration, and mediates proinflammatory responses and apoptosis. In the vascular barrier, S1P regulates permeability and endothelial reactions and recruitment of monocytes and may modulate atherosclerosis. Only recently has S1P emerged as a critical mediator which directly links the coagulation factor system to vascular inflammation. The multifunctional proteases thrombin and FXa regulate local S1P availability and interact with S1P signaling at multiple levels in various vascular cell types. Differential expression patterns and intracellular signaling pathways of each receptor enable S1P to exert its widespread functions. Although a vast amount of information is available about the functions of S1P and its receptors in the regulation of physiological and pathophysiological conditions, S1P-mediated mechanisms in the vasculature remain to be elucidated. This review summarizes recent findings regarding the role of S1P and its receptors in vascular wall and blood cells, which link the coagulation system to inflammatory responses in the vasculature.

Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration.

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Koch, Alexander; Jäger, Manuel; Völzke, Anja; Grammatikos, Georgios; Zu Heringdorf, Dagmar Meyer; Huwiler, Andrea; Pfeilschifter, Josef

2015-06-01

Sphingosine 1-phosphate (S1P) is generated by sphingosine kinase (SK)-1 and -2 and acts mainly as an extracellular ligand at five specific receptors, denoted S1P1-5. After activation, S1P receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration and survival. Previously, we showed that dexamethasone enhances SK-1 activity and S1P formation, which protected mesangial cells from stress-induced apoptosis. Here we demonstrate that dexamethasone treatment lowered S1P1 mRNA and protein expression levels in rat mesangial cells. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU-486. In addition, in vivo studies showed that dexamethasone downregulated S1P1 expression in glomeruli isolated from mice treated with dexamethasone (10 mg/kg body weight). Functionally, we identified S1P1 as a key player mediating S1P-induced mesangial cell migration. We show that dexamethasone treatment significantly lowered S1P-induced migration of mesangial cells, which was again reversed in the presence of RU-486. In summary, we suggest that dexamethasone inhibits S1P-induced mesangial cell migration via downregulation of S1P1. Overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells.

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

Energy Technology Data Exchange (ETDEWEB)

Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

2014-08-01

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

International Nuclear Information System (INIS)

Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

2014-01-01

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling.

Science.gov (United States)

Gatfield, John; Monnier, Lucile; Studer, Rolf; Bolli, Martin H; Steiner, Beat; Nayler, Oliver

2014-07-01

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase - by facilitating S1P1 receptor recycling - is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates. Copyright © 2014 Elsevier Inc. All rights reserved.

Cardiovascular effects of sphingosine-1-phosphate and other sphingomyelin metabolites

NARCIS (Netherlands)

Alewijnse, Astrid E.; Peters, Stephan L. M.; Michel, Martin C.

2004-01-01

1 Upon various stimuli, cells metabolize sphingomyelin from the cellular plasma membrane to form sphingosylphosphorylcholine (SPC) or ceramide. The latter can be further metabolized to sphingosine and then sphingosine-1-phosphate (S1P). Apart from local formation, S1P and SPC are major constituents

Sphingosine 1-Phosphate as a Link between Blood Coagulation and Inflammation

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Bernhard Hermann Rauch

2014-06-01

Full Text Available Sphingosine 1-phosphate (S1P is a multifunctional signaling lipid generated from sphingosine by sphingosine kinases. S1P formation has been shown in numerous cells in the circulation, including platelets, vascular endothelial and smooth muscle cells and monocytes. S1P also exerts multiple effects on these cells, i.e. cell proliferation and migration, activation of proinflammatory signaling pathways and release of additional inflammatory mediators. Similar activities and targets have also been identified for activated clotting factors such as thrombin or the activated factor-X (FXa, suggesting a possible involvement of S1P in thrombus-associated cellular signaling and thrombin-induced inflammatory reactions. Several levels of S1P-mediated, thrombin /FXa-induced signaling have already been identified: regulation of sphingosine kinase expression and activity, stimulation of S1P release from platelets and other cells and, possibly regulation of S1P-receptors on target cells. This review summarizes the current state of knowledge about S1P as a clotting factor-regulated molecular link between blood coagulation and inflammation. It is concluded that S1P might represent an until now underestimated lipid mediator of inflammatory reactions following activation of the clotting system and, in this context, also involved in the development and progression of atherosclerosis.

Impaired endothelial barrier function in apolipoprotein M-deficient mice is dependent on sphingosine-1-phosphate receptor 1

DEFF Research Database (Denmark)

Christensen, Pernille M; Liu, Catherine H; Swendeman, Steven L

2016-01-01

Apolipoprotein M (ApoM) transports sphingosine-1-phosphate (S1P) in plasma, and ApoM-deficient mice (Apom(-/-)) have ∼50% reduced plasma S1P levels. There are 5 known S1P receptors, and S1P induces adherens junction formation between endothelial cells through the S1P1 receptor, which in turn...... suppresses vascular leak. Increased vascular permeability is a hallmark of inflammation. The purpose of this study was to explore the relationships between vascular leakage in ApoM deficiency and S1P1 function in normal physiology and in inflammation. Vascular permeability in the lungs was assessed...... by accumulation of dextran molecules (70 kDa) and was increased ∼40% in Apom(-/-) mice compared to WT (C57Bl6/j) mice. Reconstitution of plasma ApoM/S1P or treatment with an S1P1 receptor agonist (SEW2871) rapidly reversed the vascular leakage to a level similar to that in WT mice, suggesting that it is caused...

Sphingosine 1-Phosphate and Cancer: Lessons from Thyroid Cancer Cells

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Kid Törnquist

2013-05-01

Full Text Available Sphingomyelin is found in the cell membrane of all eukaryotic cells, and was for a long time considered merely as a structural component. However, during the last two decades, metabolites of sphingomyelin, especially sphingosine 1-phosphate (S1P, have proven to be physiologically significant regulators of cell function. Through its five different G protein-coupled receptors, S1P regulates a wide array of cellular processes, ranging from stimulating cellular proliferation and migration, to the inhibition of apoptosis and induction of angiogenesis and modulation of cellular calcium homeostasis. Many of the processes regulated by S1P are important for normal cell physiology, but may also induce severe pathological conditions, especially in malignancies like cancer. Thus, understanding S1P signaling mechanisms has been the aim of a multitude of investigations. Great interest has also been shown in understanding the action of sphingosine kinase (SphK, i.e., the kinase phosphorylating sphingosine to S1P, and the interactions between S1P and growth factor signaling. In the present review, we will discuss recent findings regarding the possible importance of S1P and SphK in the etiology of thyroid cancer. Although clinical data is still scarce, our in vitro findings suggest that S1P may function as a “double-edged sword”, as the receptor profile of thyroid cancer cells largely determines whether S1P stimulates or blocks cellular migration. We will also discuss the interactions between S1P- and VEGF-evoked signaling, and the importance of a S1P1-VEGF receptor 2 complex in thyroid cancer cells.

A sphingosine 1-phosphate receptor agonist ameliorates animal model of vasculitis.

Science.gov (United States)

Miyabe, Chie; Miyabe, Yoshishige; Komiya, Takaki; Shioya, Hiroki; Miura, Noriko N; Takahashi, Kei; Ohno, Naohito; Tsuboi, Ryoji; Luster, Andrew D; Kawai, Shinichi; Miyasaka, Nobuyuki; Nanki, Toshihiro

2017-04-01

Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to cell surface receptors (S1P 1-5 ). In this study, we examined the effect of S1P 1 agonist, ONO-W061, on murine Candida albicans water-soluble fraction (CAWS)-induced vasculitis. Mice were administered ONO-W061, and the number of peripheral blood cells was counted. Vasculitis was induced by an intraperitoneal injection of CAWS. Expression of S1P receptors and CXCL1 was analyzed by quantitative RT-PCR. ONO-W061 was orally administered, and vasculitis was evaluated histologically. Number of neutrophils, macrophages and T cells in the vasculitis tissue was counted using flow cytometry. Production of chemokines from S1P-stimulated human umbilical vein endothelial cells (HUVECs) was measured by ELISA. Number of peripheral blood lymphocytes was decreased by ONO-W061. Expression of CXCL1 and S1P 1 was enhanced in CAWS-induced vasculitis tissue. Vasculitis score, CXCL1 and number of neutrophils in the vasculitis tissue were lower in ONO-W061-treated mice. Treatment of HUVECs with S1P upregulated the production of CXCL1 and IL-8 in vitro, and this was inhibited by ONO-W061. ONO-W061 significantly improved CAWS-induced vasculitis. This effect may be partly exerted through the inhibited production of chemokines by endothelial cells, which in turn could induce neutrophil recruitment into inflamed vessels.

The Role of Sphingosine-1-Phosphate and Ceramide-1-Phosphate in Inflammation and Cancer

Directory of Open Access Journals (Sweden)

Nitai C. Hait

2017-01-01

Full Text Available Inflammation is part of our body’s response to tissue injury and pathogens. It helps to recruit various immune cells to the site of inflammation and activates the production of mediators to mobilize systemic protective processes. However, chronic inflammation can increase the risk of diseases like cancer. Apart from cytokines and chemokines, lipid mediators, particularly sphingosine-1-phosphate (S1P and ceramide-1-phosphate (C1P, contribute to inflammation and cancer. S1P is an important player in inflammation-associated colon cancer progression. On the other hand, C1P has been recognized to be involved in cancer cell growth, migration, survival, and inflammation. However, whether C1P is involved in inflammation-associated cancer is not yet established. In contrast, few studies have also suggested that S1P and C1P are involved in anti-inflammatory pathways regulated in certain cell types. Ceramide is the substrate for ceramide kinase (CERK to yield C1P, and sphingosine is phosphorylated to S1P by sphingosine kinases (SphKs. Biological functions of sphingolipid metabolites have been studied extensively. Ceramide is associated with cell growth inhibition and enhancement of apoptosis while S1P and C1P are associated with enhancement of cell growth and survival. Altogether, S1P and C1P are important regulators of ceramide level and cell fate. This review focuses on S1P and C1P involvement in inflammation and cancer with emphasis on recent progress in the field.

Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

International Nuclear Information System (INIS)

Sato, Chieri; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime

2012-01-01

Highlights: ► We investigated the role of S1P signaling for osteoblast differentiation. ► Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. ► S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. ► MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.

Selectivity and specificity of sphingosine-1-phosphate receptor ligands: caveats and critical thinking in characterizing receptor-mediated effects.

Science.gov (United States)

Salomone, Salvatore; Waeber, Christian

2011-01-01

Receptors for sphingosine-1-phosphate (S1P) have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444), used extensively as specific S1P(2) and S1P(3) receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P(2) receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca(2+) concentration via P(2) receptor or α(1A)-adrenoceptor stimulation and α(1A)-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P(3)-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P(1/3) receptor antagonist, VPC23019, does not inhibit S1P(3)-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes.

Selectivity and specificity of sphingosine-1-phosphate receptor ligands: caveats and critical thinking in characterizing receptor-mediated effects

Directory of Open Access Journals (Sweden)

Christian eWaeber

2011-02-01

Full Text Available Receptors for sphingosine-1-phosphate (S1P have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444, used extensively as specific S1P2 and S1P3 receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P2 receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca2+ concentration via P2 receptor or α1A-adrenoceptor stimulation and α1A-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P3-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P1/3 receptor antagonist, VPC23019, does not inhibit S1P3-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes.

Sphingosine-1-phosphate signalling induces the production of Lcn-2 by macrophages to promote kidney regeneration

DEFF Research Database (Denmark)

Sola, Anna; Weigert, Andreas; Jung, Michaela

2011-01-01

the kidney. The present study describes a mechanism for renal tissue regeneration after ischaemia/reperfusion injury. Following injury, apoptotic cell-derived sphingosine-1-phosphate (S1P) or exogenously administered sphingosine analogue FTY720 activates macrophages to support the proliferation and healing...... of renal epithelium, once inflammatory conditions are terminated. Both suppression of inflammation and renal regeneration might require S1P receptor 3 (S1P3) signalling and downstream release of neutrophil gelatinase-associated lipocalin (NGAL/Lcn-2) from macrophages. Overall, our data point...

Hematopoietic Sphingosine 1-Phosphate Lyase Deficiency Decreases Atherosclerotic Lesion Development in LDL-Receptor Deficient Mice

NARCIS (Netherlands)

Bot, Martine; Van Veldhoven, Paul P.; de Jager, Saskia C. A.; Johnson, Jason; Nijstad, Niels; Van Santbrink, Peter J.; Westra, Marijke M.; Van Der Hoeven, Gerd; Gijbels, Marion J.; Mueller-Tidow, Carsten; Varga, Georg; Tietge, Uwe J. F.; Kuiper, Johan; Van Berkel, Theo J. C.; Nofer, Jerzy-Roch; Bot, Ilze; Biessen, Erik A. L.

2013-01-01

Aims: Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets

Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice

NARCIS (Netherlands)

Bot, Martine; van Veldhoven, Paul P.; de Jager, Saskia C. A.; Johnson, Jason; Nijstad, Niels; van Santbrink, Peter J.; Westra, Marijke M.; van der Hoeven, Gerd; Gijbels, Marion J.; Müller-Tidow, Carsten; Varga, Georg; Tietge, Uwe J. F.; Kuiper, Johan; van Berkel, Theo J. C.; Nofer, Jerzy-Roch; Bot, Ilze; Biessen, Erik A. L.

2013-01-01

Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets relevant to

Hematopoietic Sphingosine 1-Phosphate Lyase Deficiency Decreases Atherosclerotic Lesion Development in LDL-Receptor Deficient Mice

NARCIS (Netherlands)

Bot, M.; Veldhoven, van P.P.; Jager, de S.C.; Johnson, J.; Nijstad, N.; van, Santbrink P.J.; Westra, M.M.; Hoeven, van der G.; Gijbels, M.J.; Muller-Tidow, C.; Varga, G.; Tietge, U.J.; Kuiper, J.; Berkel, van T.J.; Nofer, J.R.; Bot, I.; Biessen, E.A.

2013-01-01

Abstract Aims Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1−/−) deficiency on leukocyte

Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

Energy Technology Data Exchange (ETDEWEB)

Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

2012-06-22

Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

Sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis is involved in liver fibrosis-associated angiogenesis.

Science.gov (United States)

Yang, Le; Yue, Shi; Yang, Lin; Liu, Xin; Han, Zhen; Zhang, Yuanyuan; Li, Liying

2013-07-01

Sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) axis is involved in multiple biological processes, including liver fibrosis. Angiogenesis is an important pathophysiological process closely associated with liver fibrosis; however, the functional role of SphK/S1P/S1PR in this process remains incompletely defined. Bile duct ligation or carbon tetrachloride was used to induce liver fibrosis in mice. Human fibrotic samples were obtained from livers of patients undergoing liver transplantation. S1P levels in the liver were examined by HPLC. Expression of angiogenic markers, including angiopoietin 1, CD31, vascular cell adhesion molecule-1, and von Willebrand factor, was characterized by immunofluorescence, real-time RT-PCR, and Western blot in the fibrotic liver and primary mouse hepatic stellate cells (HSCs). SphK inhibitor (SKI) or S1PR antagonists were administered intraperitoneally in mice. S1P levels in the liver were closely correlated with mRNA expression of angiogenic markers. Ang1 is expressed in activated HSCs of the fibrotic liver and in primary HSCs. In HSCs, by using specific antagonists or siRNAs, we demonstrated S1P stimulation induced Ang1 expression via S1PR1 and S1PR3. In vivo, S1P reduction by SKI inhibited angiogenesis in fibrotic mice. Furthermore, S1PR1/3 antagonist significantly blocked upregulation of angiogenic markers in the injured liver, and attenuated the extent of liver fibrosis, while S1PR2 antagonist had no effect on angiogenesis, supporting the key role of S1PR1 and S1PR3 in angiogenesis underlying liver fibrosis process. SphK1/S1P/S1PR1/3 axis plays a crucial role in the angiogenic process required for fibrosis development, which may represent an effective therapeutic strategy for liver fibrosis. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Sphingosine-1-Phosphate Evokes Unique Segment-Specific Vasoconstriction of the Renal Microvasculature

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Singletary, Sean T.; Cook, Anthony K.; Hobbs, Janet L.; Pollock, Jennifer S.; Inscho, Edward W.

2014-01-01

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, has been implicated in regulating vascular tone and participating in chronic and acute kidney injury. However, little is known about the role of S1P in the renal microcirculation. Here, we directly assessed the vasoresponsiveness of preglomerular and postglomerular microvascular segments to exogenous S1P using the in vitro blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (0.001–10 μM) evoked concentration-dependent vasoconstriction in preglomerular microvessels, predominantly afferent arterioles. After administration of 10 μM S1P, the diameter of afferent arterioles decreased to 35%±5% of the control diameter, whereas the diameters of interlobular and arcuate arteries declined to 50%±12% and 68%±6% of the control diameter, respectively. Notably, efferent arterioles did not respond to S1P. The S1P receptor agonists FTY720 and FTY720-phosphate and the specific S1P1 receptor agonist SEW2871 each evoked modest afferent arteriolar vasoconstriction. Conversely, S1P2 receptor inhibition with JTE-013 significantly attenuated S1P-mediated afferent arteriolar vasoconstriction. Moreover, blockade of L-type voltage-dependent calcium channels with diltiazem or nifedipine attenuated S1P-mediated vasoconstriction. Intravenous injection of S1P in anesthetized rats reduced renal blood flow dose dependently. Western blotting and immunofluorescence revealed S1P1 and S1P2 receptor expression in isolated preglomerular microvessels and microvascular smooth muscle cells. These data demonstrate that S1P evokes segmentally distinct preglomerular vasoconstriction via activation of S1P1 and/or S1P2 receptors, partially via L-type voltage-dependent calcium channels. Accordingly, S1P may have a novel function in regulating afferent arteriolar resistance under physiologic conditions. PMID:24578134

Sphingosine-1-Phosphate Signaling in Immune Cells and Inflammation: Roles and Therapeutic Potential

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Masayo Aoki

2016-01-01

Full Text Available Sphingosine-1-phosphate (S1P is a bioactive sphingolipid metabolite involved in many critical cell processes. It is produced by the phosphorylation of sphingosine by sphingosine kinases (SphKs and exported out of cells via transporters such as spinster homolog 2 (Spns2. S1P regulates diverse physiological processes by binding to specific G protein-binding receptors, S1P receptors (S1PRs 1–5, through a process coined as “inside-out signaling.” The S1P concentration gradient between various tissues promotes S1PR1-dependent migration of T cells from secondary lymphoid organs into the lymphatic and blood circulation. S1P suppresses T cell egress from and promotes retention in inflamed peripheral tissues. S1PR1 in T and B cells as well as Spns2 in endothelial cells contributes to lymphocyte trafficking. FTY720 (Fingolimod is a functional antagonist of S1PRs that induces systemic lymphopenia by suppression of lymphocyte egress from lymphoid organs. In this review, we summarize previous findings and new discoveries about the importance of S1P and S1PR signaling in the recruitment of immune cells and lymphocyte retention in inflamed tissues. We also discuss the role of S1P-S1PR1 axis in inflammatory diseases and wound healing.

Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

International Nuclear Information System (INIS)

Kim, Mi-Kyoung; Lee, Ha Young; Kwak, Jong-Young; Park, Joo-In; Yun, Jeanho; Bae, Yoe-Sik

2006-01-01

Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P 2 , S1P 3 , S1P 4 , but not S1P 1 . When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P 1 - and S1P 4 -selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G i protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process

sphingosine-1-phosphate transport and its role in immunology

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Reitsema, V.; Bouma, Hjalmar; Kok, Jan

2014-01-01

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite with many important functions in cellular and systemic physiology, including the immune system. As it cannot traverse the membrane, it is exported from cells by transporters. Several members of the ATP-binding cassette (ABC) transporter

Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis Can Promote Mouse and Human Primary Mast Cell Angiogenic Potential through Upregulation of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-2

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Alena Chumanevich

2016-01-01

Full Text Available Mast cells (MC are present in most vascularized tissues around the vasculature likely exerting immunomodulatory functions. Endowed with diverse mediators, resident MC represent first-line fine-tuners of local microenvironment. Sphingosine-1-phosphate (S1P functions as a pluripotent signaling sphingolipid metabolite in health and disease. S1P formation occurs at low levels in resting MC and is upregulated upon activation. Its export can result in type 2 S1P receptor- (S1PR2- mediated stimulation of MC, further fueling inflammation. However, the role of S1PR2 ligation in proangiogenic vascular endothelial growth factor- (VEGF- A and matrix metalloproteinase- (MMP- 2 release from MC is unknown. Using a preclinical MC-dependent model of acute allergic responses and in vitro stimulated primary mouse bone marrow-derived MC (BMMC or human primary skin MC, we report that S1P signaling resulted in substantial amount of VEGF-A release. Similar experiments using S1pr2-deficient mice or BMMC or selective S1P receptor agonists or antagonists demonstrated that S1P/S1PR2 ligation on MC is important for VEGF-A secretion. Further, we show that S1P stimulation triggered transcriptional upregulation of VEGF-A and MMP-2 mRNA in human but not in mouse MC. S1P exposure also triggered MMP-2 secretion from human MC. These studies identify a novel proangiogenic axis encompassing MC/S1P/S1PR2 likely relevant to inflammation.

Analysis of Onset Mechanisms of a Sphingosine 1-Phosphate Receptor Modulator Fingolimod-Induced Atrioventricular Conduction Block and QT-Interval Prolongation

Energy Technology Data Exchange (ETDEWEB)

Yagi, Yukihiro [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama, Kanagawa 222–8567 (Japan); Nakamura, Yuji [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Kitahara, Ken [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Division of Cardiovascular Medicine, Department of Internal Medicine, Faculty of Medicine, Toho University, 6-11-1 Omori-nishi, Ota-ku, Tokyo 143–8541 (Japan); Harada, Takuma [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Kato, Kazuhiko; Ninomiya, Tomohisa [Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama, Kanagawa 222–8567 (Japan); Cao, Xin [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Ohara, Hiroshi [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Division of Cardiovascular Medicine, Department of Internal Medicine, Faculty of Medicine, Toho University, 6-11-1 Omori-nishi, Ota-ku, Tokyo 143–8541 (Japan); Izumi-Nakaseko, Hiroko [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Suzuki, Kokichi [Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama, Kanagawa 222–8567 (Japan); Ando, Kentaro [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); and others

2014-11-15

Fingolimod, a sphingosine 1-phosphate (S1P) receptor subtype 1, 3, 4 and 5 modulator, has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular conduction block and/or QT-interval prolongation have been reported in some patients after the first dose. In this study, we directly compared the electropharmacological profiles of fingolimod with those of siponimod, a modulator of sphingosine 1-phosphate receptor subtype 1 and 5, using in vivo guinea-pig model and in vitro human ether-a-go-go-related gene (hERG) assay to better understand the onset mechanisms of the clinically observed adverse events. Fingolimod (0.01 and 0.1 mg/kg) or siponimod (0.001 and 0.01 mg/kg) was intravenously infused over 10 min to the halothane-anaesthetized guinea pigs (n = 4), whereas the effects of fingolimod (1 μmol/L) and siponimod (1 μmol/L) on hERG current were examined (n = 3). The high doses of fingolimod and siponimod induced atrioventricular conduction block, whereas the low dose of siponimod prolonged PR interval, which was not observed by that of fingolimod. The high dose of fingolimod prolonged QT interval, which was not observed by either dose of siponimod. Meanwhile, fingolimod significantly inhibited hERG current, which was not observed by siponimod. These results suggest that S1P receptor subtype 1 in the heart could be one of the candidates for fingolimod- and siponimod-induced atrioventricular conduction block since S1P receptor subtype 5 is localized at the brain, and that direct I{sub Kr} inhibition may play a key role in fingolimod-induced QT-interval prolongation. - Highlights: • Fingolimod and siponimod are S1P{sub 1,3,4,5} and S1P{sub 1,5} receptor modulators, respectively. • Fingolimod and siponimod induced AV block in the halothane-anesthetized guinea pigs. • S1P{sub 1} in the hearts may be the target of fingolimod- and siponimod-induced AV block. • Fingolimod directly inhibited hERG current, which was not

Analysis of Onset Mechanisms of a Sphingosine 1-Phosphate Receptor Modulator Fingolimod-Induced Atrioventricular Conduction Block and QT-Interval Prolongation

International Nuclear Information System (INIS)

Yagi, Yukihiro; Nakamura, Yuji; Kitahara, Ken; Harada, Takuma; Kato, Kazuhiko; Ninomiya, Tomohisa; Cao, Xin; Ohara, Hiroshi; Izumi-Nakaseko, Hiroko; Suzuki, Kokichi; Ando, Kentaro

2014-01-01

Fingolimod, a sphingosine 1-phosphate (S1P) receptor subtype 1, 3, 4 and 5 modulator, has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular conduction block and/or QT-interval prolongation have been reported in some patients after the first dose. In this study, we directly compared the electropharmacological profiles of fingolimod with those of siponimod, a modulator of sphingosine 1-phosphate receptor subtype 1 and 5, using in vivo guinea-pig model and in vitro human ether-a-go-go-related gene (hERG) assay to better understand the onset mechanisms of the clinically observed adverse events. Fingolimod (0.01 and 0.1 mg/kg) or siponimod (0.001 and 0.01 mg/kg) was intravenously infused over 10 min to the halothane-anaesthetized guinea pigs (n = 4), whereas the effects of fingolimod (1 μmol/L) and siponimod (1 μmol/L) on hERG current were examined (n = 3). The high doses of fingolimod and siponimod induced atrioventricular conduction block, whereas the low dose of siponimod prolonged PR interval, which was not observed by that of fingolimod. The high dose of fingolimod prolonged QT interval, which was not observed by either dose of siponimod. Meanwhile, fingolimod significantly inhibited hERG current, which was not observed by siponimod. These results suggest that S1P receptor subtype 1 in the heart could be one of the candidates for fingolimod- and siponimod-induced atrioventricular conduction block since S1P receptor subtype 5 is localized at the brain, and that direct I Kr inhibition may play a key role in fingolimod-induced QT-interval prolongation. - Highlights: • Fingolimod and siponimod are S1P 1,3,4,5 and S1P 1,5 receptor modulators, respectively. • Fingolimod and siponimod induced AV block in the halothane-anesthetized guinea pigs. • S1P 1 in the hearts may be the target of fingolimod- and siponimod-induced AV block. • Fingolimod directly inhibited hERG current, which was not observed by

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

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Mathias J Gerl

Full Text Available Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1 HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation

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Bernacchioni Caterina

2012-07-01

Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-1 is the most important physiological regulator of skeletal muscle progenitor cells, which are responsible for adult skeletal muscle regeneration. The ability of IGF-1 to affect multiple aspects of skeletal muscle cell biology such as proliferation, differentiation, survival and motility is well recognized, although the molecular mechanisms implicated in its complex biological action are not fully defined. Since sphingosine 1-phosphate (S1P has recently emerged as a key player in skeletal muscle regeneration, we investigated the possible involvement of the sphingosine kinase (SK/S1P receptor axis on the biological effects of IGF-1 in murine myoblasts. Methods RNA interference, chemical inhibition and immunofluorescence approaches were used to assess the role of the SK/S1P axis on the myogenic and mitogenic effects of IGF-1 in C2C12 myoblasts. Results We show that IGF-1 increases SK activity in mouse myoblasts. The effect of the growth factor does not involve transcriptional regulation of SK1 or SK2, since the protein content of both isoforms is not affected; rather, IGF-1 enhances the fraction of the active form of SK. Moreover, transactivation of the S1P2 receptor induced by IGF-1 via SK activation appears to be involved in the myogenic effect of the growth factor. Indeed, the pro-differentiating effect of IGF-1 in myoblasts is impaired when SK activity is pharmacologically inhibited, or SK1 or SK2 are specifically silenced, or the S1P2 receptor is downregulated. Furthermore, in this study we show that IGF-1 transactivates S1P1/S1P3 receptors via SK activation and that this molecular event negatively regulates the mitogenic effect elicited by the growth factor, since the specific silencing of S1P1 or S1P3 receptors increases cell proliferation induced by IGF-1. Conclusions We demonstrate a dual role of the SK/S1P axis in response to myoblast challenge with IGF-1, that likely is important to

Expansion of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor Function in Normal and Cancer Cells: From Membrane Restructuring to Mediation of Estrogen Signaling and Stem Cell Programming

Science.gov (United States)

2018-01-01

Sphingolipids, sphingolipid metabolizing enzymes, and their receptors network are being recognized as part of the signaling mechanisms, which govern breast cancer cell growth, migration, and survival during chemotherapy treatment. Approximately 70% of breast cancers are estrogen receptor (ER) positive and, thus, rely on estrogen signaling. Estrogen activates an intracellular network composed of many cytoplasmic and nuclear mediators. Some estrogen effects can be mediated by sphingolipids. Estrogen activates sphingosine kinase 1 (SphK1) and amplifies the intracellular concentration of sphingosine-1-phosphate (S1P) in breast cancer cells during stimulation of proliferation and survival. Specifically, Estrogen activates S1P receptors (S1PR) and induces growth factor receptor transactivation. SphK, S1P, and S1PR expression are causally associated with endocrine resistance and progression to advanced tumor stages in ER-positive breast cancers in vivo. Recently, the network of SphK/S1PR was shown to promote the development of ER-negative cancers and breast cancer stem cells, as well as stimulating angiogenesis. Novel findings confirm and broaden our knowledge about the cross-talk between sphingolipids and estrogen network in normal and malignant cells. Current S1PRs therapeutic inhibition was indicated as a promising chemotherapy approach in non-responsive and advanced malignancies. Considering that sphingolipid signaling has a prominent role in terminally differentiated cells, the impact should be considered when designing specific SphK/S1PR inhibitors. This study analyzes the dynamic of the transformation of sphingolipid axis during a transition from normal to pathological condition on the level of the whole organism. The sphingolipid-based mediation and facilitation of global effects of estrogen were critically accented as a bridging mechanism that should be explored in cancer prevention. PMID:29385066

Expansion of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor Function in Normal and Cancer Cells: From Membrane Restructuring to Mediation of Estrogen Signaling and Stem Cell Programming

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Olga A. Sukocheva

2018-01-01

Full Text Available Sphingolipids, sphingolipid metabolizing enzymes, and their receptors network are being recognized as part of the signaling mechanisms, which govern breast cancer cell growth, migration, and survival during chemotherapy treatment. Approximately 70% of breast cancers are estrogen receptor (ER positive and, thus, rely on estrogen signaling. Estrogen activates an intracellular network composed of many cytoplasmic and nuclear mediators. Some estrogen effects can be mediated by sphingolipids. Estrogen activates sphingosine kinase 1 (SphK1 and amplifies the intracellular concentration of sphingosine-1-phosphate (S1P in breast cancer cells during stimulation of proliferation and survival. Specifically, Estrogen activates S1P receptors (S1PR and induces growth factor receptor transactivation. SphK, S1P, and S1PR expression are causally associated with endocrine resistance and progression to advanced tumor stages in ER-positive breast cancers in vivo. Recently, the network of SphK/S1PR was shown to promote the development of ER-negative cancers and breast cancer stem cells, as well as stimulating angiogenesis. Novel findings confirm and broaden our knowledge about the cross-talk between sphingolipids and estrogen network in normal and malignant cells. Current S1PRs therapeutic inhibition was indicated as a promising chemotherapy approach in non-responsive and advanced malignancies. Considering that sphingolipid signaling has a prominent role in terminally differentiated cells, the impact should be considered when designing specific SphK/S1PR inhibitors. This study analyzes the dynamic of the transformation of sphingolipid axis during a transition from normal to pathological condition on the level of the whole organism. The sphingolipid-based mediation and facilitation of global effects of estrogen were critically accented as a bridging mechanism that should be explored in cancer prevention.

Sphingosine 1-phosphate mediates hyperalgesia via a neutrophil-dependent mechanism.

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Amanda Finley

Full Text Available Novel classes of pain-relieving molecules are needed to fill the void between non-steroidal anti-inflammatory agents and narcotics. We have recently shown that intraplantar administration of sphingosine 1-phosphate (S1P in rats causes peripheral sensitization and hyperalgesia through the S1P(1 receptor subtype (S1PR(1: the mechanism(s involved are largely unknown and were thus explored in the present study. Intraplantar injection of carrageenan in rats led to a time-dependent development of thermal hyperalgesia that was associated with pronounced edema and infiltration of neutrophils in paw tissues. Inhibition of 1 S1P formation with SK-I, a sphingosine kinase inhibitor, 2 S1P bioavailability with the S1P blocking antibody Sphingomab, LT1002 (but not its negative control, LT1017 or 3 S1P actions through S1PR(1 with the selective S1PR(1 antagonist, W146 (but not its inactive enantiomer, W140 blocked thermal hyperalgesia and infiltration of neutrophils. Taken together, these findings identify S1P as an important contributor to inflammatory pain acting through S1PR(1 to elicit hyperalgesia in a neutrophil-dependant manner. In addition and in further support, we demonstrate that the development of thermal hyperalgesia following intraplantar injection of S1P or SEW2871 (an S1PR(1 agonist was also associated with neutrophilic infiltration in paw tissues as these events were attenuated by fucoidan, an inhibitor of neutrophilic infiltration. Importantly, FTY720, an FDA-approved S1P receptor modulator known to block S1P-S1PR(1 signaling, attenuated carrageenan-induced thermal hyperalgesia and associated neutrophil infiltration. Targeting the S1P/S1PR(1 axis opens a therapeutic strategy for the development of novel non-narcotic anti-hyperalgesic agents.

Sphingosine 1-Phosphate and Atherosclerosis

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Yatomi, Yutaka

2018-01-01

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that works on five kinds of S1P receptors located on the cell membrane. In the circulation, S1P is distributed to HDL, followed by albumin. Since S1P and HDL share several bioactivities, S1P is believed to be responsible for the pleiotropic effects of HDL. Plasma S1P levels are reportedly lower in subjects with coronary artery disease, suggesting that S1P might be deeply involved in the pathogenesis of atherosclerosis. In basic experiments, however, S1P appears to possess both pro-atherosclerotic and anti-atherosclerotic properties; for example, S1P possesses anti-apoptosis, anti-inflammation, and vaso-relaxation properties and maintains the barrier function of endothelial cells, while S1P also promotes the egress and activation of lymphocytes and exhibits pro-thrombotic properties. Recently, the mechanism for the biased distribution of S1P on HDL has been elucidated; apolipoprotein M (apoM) carries S1P on HDL. ApoM is also a modulator of S1P, and the metabolism of apoM-containing lipoproteins largely affects the plasma S1P level. Moreover, apoM modulates the biological properties of S1P. S1P bound to albumin exerts both beneficial and harmful effects in the pathogenesis of atherosclerosis, while S1P bound to apoM strengthens anti-atherosclerotic properties and might weaken the pro-atherosclerotic properties of S1P. Although the detailed mechanisms remain to be elucidated, apoM and S1P might be novel targets for the alleviation of atherosclerotic diseases in the future. PMID:28724841

HDL activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) promotes regeneration and suppresses fibrosis in the liver.

Science.gov (United States)

Ding, Bi-Sen; Liu, Catherine H; Sun, Yue; Chen, Yutian; Swendeman, Steven L; Jung, Bongnam; Chavez, Deebly; Cao, Zhongwei; Christoffersen, Christina; Nielsen, Lars Bo; Schwab, Susan R; Rafii, Shahin; Hla, Timothy

2016-12-22

Regeneration of hepatic sinusoidal vasculature is essential for non-fibrotic liver regrowth and restoration of its metabolic capacity. However, little is known about how this specialized vascular niche is regenerated. Here we show that activation of endothelial sphingosine-1-phosphate receptor-1 (S1P 1 ) by its natural ligand bound to HDL (HDL-S1P) induces liver regeneration and curtails fibrosis. In mice lacking HDL-S1P, liver regeneration after partial hepatectomy was impeded and associated with aberrant vascular remodeling, thrombosis and peri-sinusoidal fibrosis. Notably, this "maladaptive repair" phenotype was recapitulated in mice that lack S1P 1 in the endothelium. Reciprocally, enhanced plasma levels of HDL-S1P or administration of SEW2871, a pharmacological agonist specific for S1P 1 enhanced regeneration of metabolically functional vasculature and alleviated fibrosis in mouse chronic injury and cholestasis models. This study shows that natural and pharmacological ligands modulate endothelial S1P 1 to stimulate liver regeneration and inhibit fibrosis, suggesting that activation of this pathway may be a novel therapeutic strategy for liver fibrosis.

Indomethacin differentiates the renal effects of sphingosine-1-phosphate and sphingosylphosphorylcholine

NARCIS (Netherlands)

Czyborra, Claudia; Bischoff, Angela; Michel, Martin C.

2006-01-01

The sphingomyelin breakdown products sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) constrict intrarenal microvessels in vitro in a pertussis toxin (PTX) sensitive manner, and S1P also reduces renal blood flow in vivo. Nevertheless, both S1P and SPC have been reported to enhance

The signaling lipid sphingosine 1-phosphate regulates mechanical pain

Science.gov (United States)

Hill, Rose Z; Hoffman, Benjamin U; Morita, Takeshi; Campos, Stephanie M; Lumpkin, Ellen A; Brem, Rachel B

2018-01-01

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. PMID:29561262

ASP4058, a novel agonist for sphingosine 1-phosphate receptors 1 and 5, ameliorates rodent experimental autoimmune encephalomyelitis with a favorable safety profile.

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Rie Yamamoto

Full Text Available Sphingosine-1-phosphate (S1P is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P1-S1P5. S1P1 is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P1. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl-4-{[(2S-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058, a novel next-generation S1P receptor agonist selective for S1P1 and S1P5. ASP4058 preferentially activates S1P1 and S1P5 compared with S1P2, 3, 4 in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.

Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice.

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Martine Bot

Full Text Available AIMS: Altered sphingosine 1-phosphate (S1P homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/- deficiency on leukocyte subsets relevant to atherosclerosis. METHODS AND RESULTS: LDL receptor deficient mice that were transplanted with Sgpl1(-/- bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/- chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. CONCLUSIONS: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

Apolipoprotein M mediates sphingosine-1-phosphate efflux from erythrocytes

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Christensen, Pernille M.; Bosteen, Markus H.; Hajny, Stefan

2017-01-01

Sphingosine-1-phosphate (S1P) is a bioactive lipid implicated in e.g. angiogenesis, lymphocyte trafficking, and endothelial barrier function. Erythrocytes are a main source of plasma S1P together with platelets and endothelial cells. Apolipoprotein M (apoM) in HDL carries 70% of plasma S1P, whereas...... 30% is carried by albumin. The current aim was to investigate the role of apoM in export of S1P from human erythrocytes. Erythrocytes exported S1P more efficiently to HDL than to albumin, particularly when apoM was present in HDL. In contrast, export of sphingosine to HDL was unaffected...... by the presence of apoM. The specific ability of apoM to promote export of S1P was independent of apoM being bound in HDL particles. Treatment with MK-571, an inhibitor of the ABCC1 transporter, effectively reduced export of S1P from human erythrocytes to apoM, whereas the export was unaffected by inhibitors...

Sphingosine 1-phosphate (S1P) signaling in glioblastoma multiforme-A systematic review.

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Mahajan-Thakur, Shailaja; Bien-Möller, Sandra; Marx, Sascha; Schroeder, Henry; Rauch, Bernhard H

2017-11-17

The multifunctional sphingosine-1-phosphate (S1P) is a lipid signaling molecule and central regulator in the development of several cancer types. In recent years, intriguing information has become available regarding the role of S1P in the progression of Glioblastoma multiforme (GBM), the most aggressive and common brain tumor in adults. S1P modulates numerous cellular processes in GBM, such as oncogenesis, proliferation and survival, invasion, migration, metastasis and stem cell behavior. These processes are regulated via a family of five G-protein-coupled S1P receptors (S1PR1-5) and may involve mainly unknown intracellular targets. Distinct expression patterns and multiple intracellular signaling pathways of each S1PR subtype enable S1P to exert its pleiotropic cellular actions. Several studies have demonstrated alterations in S1P levels, the involvement of S1PRs and S1P metabolizing enzymes in GBM pathophysiology. While the tumorigenic actions of S1P involve the activation of several kinases and transcription factors, the specific G-protein (Gi, Gq, and G12/13)-coupled signaling pathways and downstream mediated effects in GBM remain to be elucidated in detail. This review summarizes the recent findings concerning the role of S1P and its receptors in GBM. We further highlight the current insights into the signaling pathways considered fundamental for regulating the cellular processes in GMB and ultimately patient prognosis.

Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration.

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Polzin, Amin; Knoop, Betül; Böhm, Andreas; Dannenberg, Lisa; Zurek, Mark; Zeus, Tobias; Kelm, Malte; Levkau, Bodo; Rauch, Bernhard H

2018-01-01

Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies. © 2017 S. Karger AG, Basel.

Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness

International Nuclear Information System (INIS)

Young, Nicholas; Van Brocklyn, James R.

2007-01-01

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P 1-5 . S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P 1 , S1P 2 and S1P 3 all contribute positively to S1P-stimulated glioma cell proliferation, with S1P 1 being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P 5 blocks glioma cell proliferation, and inhibits ERK activation. S1P 1 and S1P 3 enhance glioma cell migration and invasion. S1P 2 inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P 2 also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P 2 -stimulated glioma invasion. Thus, while S1P 2 decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix

Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M

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Christoffersen, Christina; Obinata, Hideru; Kumaraswamy, Sunil B

2011-01-01

Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM(+) HDL contained S1P, whereas ApoM(-) HDL did...... not. Moreover, HDL in Apom(-/-) mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM......(+) HDL induced S1P(1) receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM(-) HDL did not. Importantly, lack of S1P in the HDL fraction of Apom(-/-) mice decreased basal endothelial barrier function in lung...

Regulation of cellular sphingosine-1-phosphate by sphingosine kinase 1 and sphingosine-1-phopshate lyase determines chemotherapy resistance in gastroesophageal cancer

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Matula, Kasia; Collie-Duguid, Elaina; Murray, Graeme; Parikh, Khyati; Grabsch, Heike; Tan, Patrick; Lalwani, Salina; Garau, Roberta; Ong, Yuhan; Bain, Gillian; Smith, Asa-Dahle; Urquhart, Gordon; Bielawski, Jacek; Finnegan, Michael; Petty, Russell

2015-01-01

Resistance to chemotherapy is common in gastroesophageal cancer. Mechanisms of resistance are incompletely characterised and there are no predictive biomarkers in clinical practice for cytotoxic drugs. We used new cell line models to characterise novel chemotherapy resistance mechanisms and validated them in tumour specimens to identify new targets and biomarkers for gastroesophageal cancer. Cell lines were selected for resistance to oxaliplatin, cisplatin and docetaxel and gene expression examined using Affymetrix Exon 1.0 ST arrays. Leads were validated by qRT-PCR and HPLC of tumour metabolites. Protein expression and pharmacological inhibition of lead target SPHK1 was evaluated in independent cell lines, and by immunohistochemistry in gastroesophageal cancer patients. Genes with differential expression in drug resistant cell lines compared to the parental cell line they were derived from, were identified for each drug resistant cell line. Biological pathway analysis of these gene lists, identified over-represented pathways, and only 3 pathways - lysosome, sphingolipid metabolism and p53 signalling- were identified as over-represented in these lists for all three cytotoxic drugs investigated. The majority of genes differentially expressed in chemoresistant cell lines from these pathways, were involved in metabolism of glycosphingolipids and sphingolipids in lysosomal compartments suggesting that sphingolipids might be important mediators of cytotoxic drug resistance in gastroeosphageal cancers . On further investigation, we found that drug resistance (IC50) was correlated with increased sphingosine kinase 1(SPHK1) mRNA and also with decreased sphingosine-1-phosphate lysase 1(SGPL1) mRNA. SPHK1 and SGPL1 gene expression were inversely correlated. SPHK1:SGPL1 ratio correlated with increased cellular sphingosine-1-phosphate (S1P), and S1P correlated with drug resistance (IC50). High SPHK1 protein correlated with resistance to cisplatin (IC50) in an independent

Exosomes from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells (hiPSC-MSCs) Protect Liver against Hepatic Ischemia/ Reperfusion Injury via Activating Sphingosine Kinase and Sphingosine-1-Phosphate Signaling Pathway.

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Du, Yingdong; Li, Dawei; Han, Conghui; Wu, Haoyu; Xu, Longmei; Zhang, Ming; Zhang, Jianjun; Chen, Xiaosong

2017-01-01

This study aimed to evaluate the effects of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury, as well as the underlying mechanisms. Exosomes derived from hiPSC-MSCs were isolated and characterized both biochemically and biophysically. hiPSC-MSCs-Exo were injected systemically into a murine ischemia/reperfusion injury model via the inferior vena cava, and then the therapeutic effects were evaluated. The serum levels of transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as histological changes were examined. Primary hepatocytes and human hepatocyte cell line HL7702 were used to test whether exosomes could induce hepatocytes proliferation in vitro. In addition, the expression levels of proliferation markers (proliferation cell nuclear antigen, PCNA; Phosphohistone-H3, PHH3) were measured by immunohistochemistry and Western blot. Moreover, SK inhibitor (SKI-II) and S1P1 receptor antagonist (VPC23019) were used to investigate the role of sphingosine kinase and sphingosine-1-phosphate-dependent pathway in the effects of hiPSC-MSCs-Exo on hepatocytes. hiPSCs were efficiently induced into hiPSC-MSCs that had typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 100 to 200 nm and expressed exosome markers (Alix, CD63 and CD81). After hiPSC-MSCs-Exo administration, hepatocyte necrosis and sinusoidal congestion were markedly suppressed in the ischemia/reperfusion injury model, with lower histopathological scores. The levels of hepatocyte injury markers AST and ALT were significantly lower in the treatment group compared to control, and the expression levels of proliferation markers (PCNA and PHH3) were greatly induced after hiPSC-MSCs-Exo administration. Moreover, hiPSC-MSCs-Exo also induced primary hepatocytes and HL7702 cells proliferation in vitro in a dose-dependent manner. We found that hiPSC-MSCs-Exo could

Advance in the Study of the Mechanisms Regulated by Sphingosine-1-Phosphate

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Ye, Fei; Kong, Xiangqian; Luo, Cheng

2010-09-01

Sphingosine-1-phosphate (S1P) is a bioactive lipid messenger in the cells that regulate gene expression and NF-KB signal pathway through unknown mechanisms. Recently, Cheng Luo, associate professor of DDDC in Shanghai Institute of Materia Medica, whose project was funded by the National Natural Science Foundation of China, joined in a research team led by Professor Sarah Spiegel of Virginia Commonwealth University. The team continuously made significant breakthroughs in understanding the regulation mechanism of Sphingosine-1-Phosphate. In September 2009, in a paper published on SCIENCE magazine (Science 2009, 325: 1254-7), they firstly demonstrated that S1P is a physiologically important regulator of histone deacetylases (HDACs), HDACs are direct intracellular targets of S1P. Furthermore, they identified the mechanism that S1P regulates gene expression through regulating the activity of HDACs. In June 24th, 2010, in another paper to be published on NATURE magazine (Nature 2010, June 24th, advance online publication) which reports the regulation of NF-KB signaling pathway by S1P. They demonstrate that S1P is the missing cofactor for TRAF2 (tumour-necrosis factor receptor-associated factor 2) and indicate a new paradigm for the regulation of lysine-63-linked poly-ubiquitination. The study also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-KB activation pathway, and then play crucial role in inflammatory, antiapoptotic and immune processes. The identification of new mechanisms by which S1P regulates gene expression and TNF and NF-KB signaling pathway will light up the road to develop novel inhibitors that might be useful for treatment of cancer and inflammatory diseases.

Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation

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Quarta, Serena; Camprubí-Robles, Maria; Schweigreiter, Rüdiger; Matusica, Dusan; Haberberger, Rainer V.; Proia, Richard L.; Bandtlow, Christine E.; Ferrer-Montiel, Antonio; Kress, Michaela

2017-01-01

The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P1-deficient neurons. Depletion of S1P3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P1 receptors to stimulate elongation or S1P3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons. PMID:29066950

Role of Sphingosine-1-Phosphate in Mast Cell Functions and Asthma and Its Regulation by Non-Coding RNA

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Rohit Saluja

2017-05-01

Full Text Available Sphingolipid metabolites are emerging as important signaling molecules in allergic diseases specifically asthma. One of the sphingolipid metabolite, sphingosine-1-phosphate (S1P, is involved in cell differentiation, proliferation, survival, migration, and angiogenesis. In the allergic diseases, alteration of S1P levels influences the differentiation and responsiveness of mast cells (MCs. S1P is synthesized by two sphingosine kinases (SphKs, sphingosine kinase 1, and sphingosine kinase 2. Engagement of IgE to the FcεRI receptor induces the activation of both the SphKs and generates S1P. Furthermore, SphKs are also essential to FcεRI-mediated MC activation. Activated MCs export S1P into the extracellular space and causes inflammatory response and tissue remodeling. S1P signaling has dual role in allergic responses. Activation of SphKs and secretion of S1P are required for MC activation; however, S1P signaling plays a vital role in the recovery from anaphylaxis. Several non-coding RNAs have been shown to play a crucial role in controlling the MC-associated inflammatory and allergic responses. Thus, S1P signaling pathway and its regulation by non-coding RNA could be explored as an exciting potential therapeutic target for asthma and other MC-associated diseases.

Plasma levels of sphingosine-1-phosphate and apolipoprotein M in patients with monogenic disorders of HDL metabolism

NARCIS (Netherlands)

Karuna, Ratna; Park, Rebekka; Othman, Alaa; Holleboom, Adriaan G.; Motazacker, Mohammad Mahdi; Sutter, Iryna; Kuivenhoven, Jan Albert; Rohrer, Lucia; Matile, Hugues; Hornemann, Thorsten; Stoffel, Markus; Rentsch, Katharina M.; von Eckardstein, Arnold

2011-01-01

Apolipoprotein M (apoM) has been identified as a specific sphingosine-1-phosphate (S1P) binding protein of HDL. To investigate the in vivo effects of disturbed apoM or HDL metabolism we quantified S1P and apoM in plasmas of wild-type, apoM-knock-out, and apoM transgenic mice as well as 50 patients

Controlled release of sphingosine-1-phosphate agonist with gelatin hydrogels for macrophage recruitment.

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Murakami, Masahiro; Saito, Takashi; Tabata, Yasuhiko

2014-11-01

The objective of this study is to design a drug delivery system (DDS) for the in vivo promotion of macrophage recruitment. As the drug, a water-insoluble agonist of sphingosine-1-phosphate type 1 receptor (SEW2871) was selected. SEW2871 (SEW) was water-solubilized by micelle formation with gelatin grafted by L-lactic acid oligomer. SEW micelles were mixed with gelatin, followed by dehydrothermal crosslinking of gelatin to obtain gelatin hydrogels incorporating SEW micelles. SEW was released from the hydrogels incorporating SEW micelles in vitro and in vivo. The water-solubilized SEW showed in vitro macrophage migration activity. When implanted into the back subcutis or the skin wound defect of mice, the hydrogel incorporating SEW micelles promoted macrophage migration toward the tissue around the implanted site to a significantly great extent compared with SEW-free hydrogel and that mixed with SEW micelles. The hydrogel is a promising DDS to enhance macrophage recruitment in vivo. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Critical role of sphingosine-1-phosphate receptor 2 (S1PR2) in acute vascular inflammation.

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Zhang, Guoqi; Yang, Li; Kim, Gab Seok; Ryan, Kieran; Lu, Shulin; O'Donnell, Rebekah K; Spokes, Katherine; Shapiro, Nathan; Aird, William C; Kluk, Michael J; Yano, Kiichiro; Sanchez, Teresa

2013-07-18

The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S1PRs). Here, we studied the role of S1PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S1PR2-specific antagonist (JTE013), we found that S1PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotoxemia. Experiments with bone marrow chimeras (S1pr2(+/+) → S1pr2(+/+), S1pr2(+/+) → S1pr2(-/-), and S1pr2(-/-) → S1pr2(+/+)) indicate the critical role of S1PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE013 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S1PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S1PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S1PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S1PR2 as a novel therapeutic target for vascular disorders.

In vitro and in vivo study of endothelial cells radio-induced death modulation by Sphingosine-1-Phosphate; Etude in vitro et in vivo de la regulation de la mort radioinduite des cellules endotheliales par la Sphingosine-1-Phosphate

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Bonnaud, St

2007-01-15

Protecting the vasculature from radiation-induced death is a major concern in tissue radioprotection. Developing a model of endothelial cells radiosensitivity, we proved that HMEC-1 undergo 2 waves of death after exposure to 15 Gy: an early pre mitotic apoptosis dependent of ceramide generation and a delayed DNA damage-induced mitotic death. Sphingosine-1-Phosphate (S1P), a ceramide antagonist, protects HMEC-1 only from early apoptosis, but not from mitotic death. We confirmed in vivo the S1P radioprotection from ceramide-mediated radio-induced apoptosis, and that S1P radioprotection is partially mediated by S1Ps receptors. Segregation between these 2 types of death may give the opportunity to define a new class of radioprotectors for normal tissue where quiescent endothelium represent the most sensitive target, while excluding malignant tumor containing pro-proliferating angiogenic endothelial cells, sensitive to mitotic death. (author)

Sphingosine-1-Phosphate (S1P)-Related Response of Human Conjunctival Fibroblasts After Filtration Surgery for Glaucoma.

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Aoyama-Araki, Yuka; Honjo, Megumi; Uchida, Takatoshi; Yamagishi, Reiko; Kano, Kuniyuki; Aoki, Junken; Aihara, Makoto

2017-04-01

To investigate levels of sphingosine-1-phosphate (S1P) in aqueous fluid samples taken before and after filtration surgery and S1P-induced human conjunctival fibroblast (HCF) responses. Levels of S1P and its related sphingophospholipids in aqueous fluid obtained immediately before and after filtration surgery were determined by liquid chromatography-tandem mass spectrometry. HCFs were used for all in vitro experiments. The expression of five S1P receptor subtypes in HCFs was examined by quantitative real-time PCR. The effect of S1P and receptor-specific antagonists on HCF viability and cell migration was assessed by WST-1 assay and scratch migration assay, respectively. Differentiation to myofibroblasts and extracellular matrix production was evaluated by examining changes in F-actin, α-smooth muscle actin (αSMA), and collagen expression with immunocytochemistry, Western blotting, and collagen accumulation assay, respectively. No significant S1P levels in the aqueous fluid samples were detectable immediately before surgery, but postoperative levels of several lysophospholipids, including S1P, dehydro-S1P, and sphingosine, were significantly increased to bioactive concentrations in aqueous fluid in the blebs (P S1P receptor subtypes was detected in HCFs. Although S1P levels did not influence HCF proliferation, S1P enhanced cell migration, which could be inhibited by the S1P2 antagonist JTE 013. F-actin, αSMA, and collagen expression was significantly increased by S1P stimulation and was reduced by JTE 013. Bioactive S1P concentrations were present in the aqueous fluid at the end of filtration surgery. S1P activated HCFs via S1P2 receptors. These results revealed the potential of S1P2 antagonists in preventing scarring after glaucoma filtration surgery.

Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier

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van Doorn Ruben

2012-06-01

Full Text Available Abstract Background The sphingosine 1-phosphate (S1P receptor modulator FTY720P (Gilenya® potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood–brain barrier (BBB functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function. Methods We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. Results We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. Conclusion Our

In vitro and in vivo study of endothelial cells radio-induced death modulation by Sphingosine-1-Phosphate

International Nuclear Information System (INIS)

Bonnaud, St.

2007-01-01

Protecting the vasculature from radiation-induced death is a major concern in tissue radioprotection. Developing a model of endothelial cells radiosensitivity, we proved that HMEC-1 undergo 2 waves of death after exposure to 15 Gy: an early pre mitotic apoptosis dependent of ceramide generation and a delayed DNA damage-induced mitotic death. Sphingosine-1-Phosphate (S1P), a ceramide antagonist, protects HMEC-1 only from early apoptosis, but not from mitotic death. We confirmed in vivo the S1P radioprotection from ceramide-mediated radio-induced apoptosis, and that S1P radioprotection is partially mediated by S1Ps receptors. Segregation between these 2 types of death may give the opportunity to define a new class of radioprotectors for normal tissue where quiescent endothelium represent the most sensitive target, while excluding malignant tumor containing pro-proliferating angiogenic endothelial cells, sensitive to mitotic death. (author)

Cellular function and signaling pathways of vascular smooth muscle cells modulated by sphingosine 1-phosphate

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Takuji Machida

2016-12-01

Full Text Available Sphingosine 1-phosphate (S1P plays important roles in cardiovascular pathophysiology. S1P1 and/or S1P3, rather than S1P2 receptors, seem to be predominantly expressed in vascular endothelial cells, while S1P2 and/or S1P3, rather than S1P1 receptors, seem to be predominantly expressed in vascular smooth muscle cells (VSMCs. S1P has multiple actions, such as proliferation, inhibition or stimulation of migration, and vasoconstriction or release of vasoactive mediators. S1P induces an increase of the intracellular Ca2+ concentration in many cell types, including VSMCs. Activation of S1P3 seems to play an important role in Ca2+ mobilization. S1P induces cyclooxygenase-2 expression in VSMCs via both S1P2 and S1P3 receptors. S1P2 receptor activation in VSMCs inhibits inducible nitric oxide synthase (iNOS expression. At the local site of vascular injury, vasoactive mediators such as prostaglandins and NO produced by VSMCs are considered primarily as a defensive and compensatory mechanism for the lack of endothelial function to prevent further pathology. Therefore, selective S1P2 receptor antagonists may have the potential to be therapeutic agents, in view of their antagonism of iNOS inhibition by S1P. Further progress in studies of the precise mechanisms of S1P may provide useful knowledge for the development of new S1P-related drugs for the treatment of cardiovascular diseases.

Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs

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Mathew, Biji; Jacobson, Jeffrey R.; Berdyshev, Evgeny; Huang, Yong; Sun, Xiaoguang; Zhao, Yutong; Gerhold, Lynnette M.; Siegler, Jessica; Evenoski, Carrie; Wang, Ting; Zhou, Tong; Zaidi, Rafe; Moreno-Vinasco, Liliana; Bittman, Robert; Chen, Chin Tu; LaRiviere, Patrick J.; Sammani, Saad; Lussier, Yves A.; Dudek, Steven M.; Natarajan, Viswanathan; Weichselbaum, Ralph R.; Garcia, Joe G. N.

2011-01-01

Clinically significant radiation-induced lung injury (RILI) is a common toxicity in patients administered thoracic radiotherapy. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung gene mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We hypothesized that sphingolipid signaling components serve as modulators and novel therapeutic targets of RILI. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to sphingosine 1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage fluid, and lung tissue. Mice with a targeted deletion of SphK1 (SphK1−/−) or with reduced expression of S1P receptors (S1PR1+/−, S1PR2−/−, and S1PR3−/−) exhibited marked RILI susceptibility. Finally, studies of 3 potent vascular barrier-protective S1P analogs, FTY720, (S)-FTY720-phosphonate (fTyS), and SEW-2871, identified significant RILI attenuation and radiation-induced gene dysregulation by the phosphonate analog, fTyS (0.1 and 1 mg/kg i.p., 2×/wk) and to a lesser degree by SEW-2871 (1 mg/kg i.p., 2×/wk), compared with those in controls. These results support the targeting of S1P signaling as a novel therapeutic strategy in RILI.—Mathew, B., Jacobson, J. R., Berdyshev, E., Huang, Y., Sun, X., Zhao, Y., Gerhold, L. M., Siegler, J., Evenoski, C., Wang, T., Zhou, T., Zaidi, R., Moreno-Vinasco, L., Bittman, R., Chen, C. T., LaRiviere, P. J., Sammani, S., Lussier, Y. A., Dudek, S. M., Natarajan, V., Weichselbaum, R. R., Garcia, J. G. N. Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs. PMID:21712494

Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

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Ami Oizumi

Full Text Available 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-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes", which form a stratum corneum. PaCDase alone did not affect TNF-α 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-α, 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-α, 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-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. 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

Enhancement of Human Endothelial Cell Adhesion to Type I Collagen by Lysophosphatidic Acid (LPA and Sphingosine-1-Phosphate (S1P

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Hsinyu Lee

2004-06-01

Full Text Available The diverse cellular effects of lysophosphatidic acid (LPA and sphingosine-1-phosphate (S1P are transduced by two structurally homologous subfamilies of G protein-coupled receptors, which are encoded by endothelial differentiation genes (Edg Rs. Human umbilical cord vein endothelial cells (HUVECs express Edg Rs for LPA (Edg2 and S1P (Edg1 and 3, which transduce signals for migration of HUVECs through micropore filters coated with type I collagen. Since activation of integrins is essential for optimal migration of endothelial cells, we now examine the capacity of LPA and S1P to augment integrin mediation of endothelial cell binding to type I collagen. Lysophospholipid enhancement of HUVEC adhesion to type I collagen is detectable within 20 minutes. Enhancement of adhesion by both LPA and S1P is significant at 50 nM and optimal at 5µM. Pertussis toxin (PTx, a specific inhibitor of Gi, and C3 exotoxin, a specific inhibitor of Rho, both suppress LPA and S1P enhancement of HUVEC adhesion. In contrast, PD98059, which blocks MAP kinase kinase (MEK, and wortmannin, which inhibits phosphatidylinositol 3-kinase (PI3K, had no effect on LPA- or S1P-enhancement of HUVEC adhesion. Neutralizing monoclonal antibodies specific for α2 and β1 integrin chains, concomitantly decrease LPA and S1P enhancement of HUVEC adhesion to type I collagen. LPA and S1P thus promote type I collagen-dependent adhesion and migration of HUVECs by recruiting α2 and β1 integrin through both Gi and Rho pathways. Integrin α2/β1 therefore appears to be critical on the effects of LPA and S1P on endothelial cell physiology.

Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a S1PR2/STAT3 signaling pathway.

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Kenneth C Loh

Full Text Available Sphingosine-1-phosphate (S1P activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD, were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3, a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

Epigenetic regulation of pro-inflammatory cytokine secretion by sphingosine 1-phosphate (S1P) in acute lung injury: Role of S1P lyase.

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Ebenezer, David L; Fu, Panfeng; Suryadevara, Vidyani; Zhao, Yutong; Natarajan, Viswanathan

2017-01-01

Cellular level of sphingosine-1-phosphate (S1P), the simplest bioactive sphingolipid, is tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and degradation mediated by S1P phosphatases, lipid phosphate phosphatases, and S1P lyase. The pleotropic actions of S1P are attributed to its unique inside-out (extracellular) signaling via G-protein-coupled S1P1-5 receptors, and intracellular receptor independent signaling. Additionally, S1P generated in the nucleus by nuclear SphK2 modulates HDAC1/2 activity, regulates histone acetylation, and transcription of pro-inflammatory genes. Here, we present data on the role of S1P lyase mediated S1P signaling in regulating LPS-induced inflammation in lung endothelium. Blocking S1P lyase expression or activity attenuated LPS-induced histone acetylation and secretion of pro-inflammatory cytokines. Degradation of S1P by S1P lyase generates Δ2-hexadecenal and ethanolamine phosphate and the long-chain fatty aldehyde produced in the cytoplasmic compartment of the endothelial cell seems to modulate histone acetylation pattern, which is different from the nuclear SphK2/S1P signaling and inhibition of HDAC1/2. These in vitro studies suggest that S1P derived long-chain fatty aldehyde may be an epigenetic regulator of pro-inflammatory genes in sepsis-induced lung inflammation. Trapping fatty aldehydes and other short chain aldehydes such as 4-hydroxynonenal derived from S1P degradation and lipid peroxidation, respectively by cell permeable agents such as phloretin or other aldehyde trapping agents may be useful in treating sepsis-induced lung inflammation via modulation of histone acetylation. . Copyright © 2016 Elsevier Ltd. All rights reserved.

The role of sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) in the trafficking of normal and malignant cells

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Ratajczak, Mariusz Z.; Suszynska, Malwina; Borkowska, Sylwia; Ratajczak, Janina; Schneider, Gabriela

2014-01-01

Introduction A common feature of many types of cells is their responsiveness to chemotactic gradients of factors for which they express the corresponding receptors. The most studied chemoattractants so far are peptide-based growth factors and a family of cytokines endowed with strong chemotactic properties, called chemokines. However, additional evidence has accumulated that, in addition to these peptide-based chemoattractants, an important role in cell migration is played by bioactive lipids. Areas covered Solid evidence has accumulated that two bioactive phosphorylated sphingolipids that are derivatives of sphingolipid metabolism, namely sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are potent chemoattractants for a variety of cells. In this review, we will discuss the effect of these two phosphorylated sphingolipids on the trafficking of normal and malignant cells, and, in particular, we will focus on their role in trafficking of normal hematopoietic stem/progenitor cells. Unlike other mediators, S1P under steady state conditions maintain a steep gradient between interstitial fluid and peripheral blood and lymph across the endothelial barrier, which is important in the egress of cells from bone marrow. Both S1P and C1P may be upregulated in damaged tissues, which may result in reversal of this gradient. Expert opinion S1P and C1P are important regulators of the trafficking of normal and malignant cells, and modification of their biological effects will have important applications in optimizing stem cell mobilization and homing, tissue organ/regeneration, and preventing cancer metastasis. PMID:24188167

Sphingosine 1-Phosphate (S1P) Receptors 1 and 2 Coordinately Induce Mesenchymal Cell Migration through S1P Activation of Complementary Kinase Pathways*

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Quint, Patrick; Ruan, Ming; Pederson, Larry; Kassem, Moustapha; Westendorf, Jennifer J.; Khosla, Sundeep; Oursler, Merry Jo

2013-01-01

Normal bone turnover requires tight coupling of bone resorption and bone formation to preserve bone quantity and structure. With aging and during several pathological conditions, this coupling breaks down, leading to either net bone loss or excess bone formation. To preserve or restore normal bone metabolism, it is crucial to determine the mechanisms by which osteoclasts and osteoblast precursors interact and contribute to coupling. We showed that osteoclasts produce the chemokine sphingosine 1-phosphate (S1P), which stimulates osteoblast migration. Thus, osteoclast-derived S1P may recruit osteoblasts to sites of bone resorption as an initial step in replacing lost bone. In this study we investigated the mechanisms by which S1P stimulates mesenchymal (skeletal) cell chemotaxis. S1P treatment of mesenchymal (skeletal) cells activated RhoA GTPase, but this small G protein did not contribute to migration. Rather, two S1P receptors, S1PR1 and S1PR2, coordinately promoted migration through activation of the JAK/STAT3 and FAK/PI3K/AKT signaling pathways, respectively. These data demonstrate that the chemokine S1P couples bone formation to bone resorption through activation of kinase signaling pathways. PMID:23300082

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis

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Takeshita, Harunori; Kitano, Masayasu; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto; Miyazawa, Keiji; Hla, Timothy; Sano, Hajime

2012-01-01

Highlights: ► MH7A cells and CD4 + T cells expressed S1P1 and RANKL. ► S1P increased RANKL expression in MH7A cells and CD4 + T cells. ► The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4 + T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4 + T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4 + T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4 + T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4 + T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

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Takeshita, Harunori [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Kitano, Masayasu, E-mail: mkitano6@hyo-med.ac.jp [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi [Department of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima Kobe, Hyogo 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Miyazawa, Keiji [Discovery Research III, Research and Development, Kissei Pharmaceutical Company, 4365-1 Hodakakashiwara, Azumino, Nagano 399-8304 (Japan); Hla, Timothy [Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 1300 York Avenue, Box 69, NY 10065 (United States); Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

2012-03-09

Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration.

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Westwood, Melissa; Al-Saghir, Khiria; Finn-Sell, Sarah; Tan, Cherlyn; Cowley, Elizabeth; Berneau, Stéphane; Adlam, Daman; Johnstone, Edward D

2017-12-01

Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function. A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH) 2 D 3 . QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression. EVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH) 2 D 3 for 48 or 72 h reduces S1PR2 (4-fold; S1P did not inhibit the migration of cells exposed to 1,25(OH) 2 D 3 (p S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα 12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Co-ordinate regulation of growth factor receptors and lipid phosphate phosphatase-1 controls cell activation by exogenous lysophosphatidate.

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Pilquil, C; Ling, Z C; Singh, I; Buri, K; Zhang, Q X; Brindley, D N

2001-11-01

The serum-derived lipid growth factors, lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P), activate cells selectively through different members of a family of endothelial differentiation gene (EDG) receptors. Activation of EDG receptors by LPA and S1P provides a variety of signalling cascades depending upon the G-protein coupling of the different EDG receptors. This leads to chemotactic and mitogenic responses, which are important in wound healing. For example, LPA stimulates fibroblast division and S1P stimulates the chemotaxis and division of endothelial cells leading to angiogenesis. Counteracting these effects of LPA and S1P, are the actions of lipid phosphate phosphatases (LPP, or phosphatidate phosphohydrolases, Type 2). The isoform LPP-1 is expressed in the plasma membrane with its active site outside the cell. This enzyme is responsible for 'ecto-phosphatase' activity leading to the degradation of exogenous lipid phosphate mediators, particularly LPA. Expression of LPP-1 decreases cell activation by exogenous LPA. The mechanism for this is controversial and several mechanisms have been proposed. Evidence will be presented that the LPPs cross-talk with EDG and other growth factor receptors, thus, regulating the responses of the cells to lipid phosphate mediators of signal transduction.

Sphingosine 1-phosphate (S1P) signalling: Role in bone biology and potential therapeutic target for bone repair.

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Sartawi, Ziad; Schipani, Ernestina; Ryan, Katie B; Waeber, Christian

2017-11-01

The lipid mediator sphingosine 1-phosphate (S1P) affects cellular functions in most systems. Interest in its therapeutic potential has increased following the discovery of its G protein-coupled receptors and the recent availability of agents that can be safely administered in humans. Although the role of S1P in bone biology has been the focus of much less research than its role in the nervous, cardiovascular and immune systems, it is becoming clear that this lipid influences many of the functions, pathways and cell types that play a key role in bone maintenance and repair. Indeed, S1P is implicated in many osteogenesis-related processes including stem cell recruitment and subsequent differentiation, differentiation and survival of osteoblasts, and coupling of the latter cell type with osteoclasts. In addition, S1P's role in promoting angiogenesis is well-established. The pleiotropic effects of S1P on bone and blood vessels have significant potential therapeutic implications, as current therapeutic approaches for critical bone defects show significant limitations. Because of the complex effects of S1P on bone, the pharmacology of S1P-like agents and their physico-chemical properties, it is likely that therapeutic delivery of S1P agents will offer significant advantages compared to larger molecular weight factors. Hence, it is important to explore novel methods of utilizing S1P agents therapeutically, and improve our understanding of how S1P and its receptors modulate bone physiology and repair. Copyright © 2017 Elsevier Ltd. All rights reserved.

PPARγ agonists upregulate sphingosine 1-phosphate (S1P) receptor 1 expression, which in turn reduces S1P-induced [Ca(2+)]i increases in renal mesangial cells.

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Koch, Alexander; Völzke, Anja; Puff, Bianca; Blankenbach, Kira; Meyer Zu Heringdorf, Dagmar; Huwiler, Andrea; Pfeilschifter, Josef

2013-11-01

We previously identified peroxisome proliferator-activated receptor gamma (PPARγ) agonists (thiazolidinediones, TZDs) as modulators of the sphingolipid metabolism in renal mesangial cells. TZDs upregulated sphingosine kinase 1 (SK-1) and increased the formation of intracellular sphingosine 1-phosphate (S1P), which in turn reduced the expression of pro-fibrotic connective tissue growth factor. Since S1P also acts as extracellular ligand at specific S1P receptors (S1PR, S1P1-5), we investigated here the effect of TZDs on S1PR expression in mesangial cells and evaluated the functional consequences by measuring S1P-induced increases in intracellular free Ca(2+) concentration ([Ca(2+)]i). Treatment with two different TZDs, troglitazone and rosiglitazone, enhanced S1P1 mRNA and protein expression in rat mesangial cells, whereas S1P2-5 expression levels were not altered. Upregulation of S1P1 mRNA upon TZD treatment was also detected in human mesangial cells and mouse glomeruli. PPARγ antagonism and promoter studies revealed that the TZD-dependent S1P1 mRNA induction involved a functional PPAR response element in the S1P1 promoter. Pharmacological approaches disclosed that S1P-induced [Ca(2+)]i increases in rat mesangial cells were predominantly mediated by S1P2 and S1P3. Interestingly, the transcriptional upregulation of S1P1 by TZDs resulted in a reduction of S1P-induced [Ca(2+)]i increases, which was reversed by the S1P1/3 antagonist VPC-23019, the protein kinase C (PKC) inhibitor PKC-412, and by S1P1 siRNA. These data suggest that PPARγ-dependent upregulation of S1P1 leads to an inhibition of S1P-induced Ca(2+) signaling in a PKC-dependent manner. Overall, these results reveal that TZDs not only modulate intracellular S1P levels but also regulate S1PR signaling by increasing S1P1 expression in mesangial cells. © 2013.

Induction of antiproliferative connective tissue growth factor expression in Wilms' tumor cells by sphingosine-1-phosphate receptor 2.

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Li, Mei-Hong; Sanchez, Teresa; Pappalardo, Anna; Lynch, Kevin R; Hla, Timothy; Ferrer, Fernando

2008-10-01

Connective tissue growth factor (CTGF), a member of the CCN family of secreted matricellular proteins, regulates fibrosis, angiogenesis, cell proliferation, apoptosis, tumor growth, and metastasis. However, the role of CTGF and its regulation mechanism in Wilms' tumor remains largely unknown. We found that the bioactive lipid sphingosine-1-phosphate (S1P) induced CTGF expression in a concentration- and time-dependent manner in a Wilms' tumor cell line (WiT49), whereas FTY720-phosphate, an S1P analogue that binds all S1P receptors except S1P2, did not. Further, the specific S1P2 antagonist JTE-013 completely inhibited S1P-induced CTGF expression, whereas the S1P1 antagonist VPC44116 did not, indicating that this effect was mediated by S1P2. This was confirmed by adenoviral transduction of S1P2 in WiT49 cells, which showed that overexpression of S1P2 increased the expression of CTGF. Induction of CTGF by S1P was sensitive to ROCK inhibitor Y-27632 and c-Jun NH2-terminal kinase inhibitor SP600125, suggesting the requirement of RhoA/ROCK and c-Jun NH2-terminal kinase pathways for S1P-induced CTGF expression. Interestingly, the expression levels of CTGF were decreased in 8 of 10 Wilms' tumor tissues compared with matched normal tissues by quantitative real-time PCR and Western blot analysis. In vitro, human recombinant CTGF significantly inhibited the proliferation of WiT49 cells. In addition, overexpression of CTGF resulted in significant inhibition of WiT49 cell growth. Taken together, these data suggest that CTGF protein induced by S1P2 might act as a growth inhibitor in Wilms' tumor.

DEVELOPMENT OF A METHOD FOR QUANTITATING SPHINGOID BASE 1-PHOSPHATES IN BLOOD SPOTS

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Red blood cells (RBC) accumulate, store and release sphingoid base 1-phosphates,important ligands for the extracellular receptors S1P1-5. The ability of RBC to accumulate these bioactive lipids is because, with the exception of sphingosine kinase, the enzymes responsible for metabolizing sphingosine...

Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy

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Diem-Hang Nguyen-Tran

2014-01-01

Full Text Available Duchenne muscular dystrophy (DMD is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI, an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx, upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor.

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Onuma, Takashi; Tanabe, Kumiko; Kito, Yuko; Tsujimoto, Masanori; Uematsu, Kodai; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Doi, Tomoaki; Nagase, Kiyoshi; Akamatsu, Shigeru; Tokuda, Haruhiko; Ogura, Shinji; Iwama, Toru; Kozawa, Osamu; Iida, Hiroki

2017-08-01

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR Activity.

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Firhan A Malik

Full Text Available The cystic fibrosis transmembrane conductance regulator (CFTR attenuates sphingosine-1-phosphate (S1P signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK, establishing a potential feedback link. In Baby Hamster Kidney (BHK cells expressing wild-type human CFTR, S1P (1μmol/L attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds, transient and correlates with CFTR serine residue 737 (S737 phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019 and AMPK inhibition (80μmol/L Compound C or AMPK siRNA attenuate S1P-stimluated (i AMPK phosphorylation, (ii CFTR S737 phosphorylation and (iii CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508, the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure. S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody.

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Camaré, Caroline; Trayssac, Magali; Garmy-Susini, Barbara; Mucher, Elodie; Sabbadini, Roger; Salvayre, Robert; Negre-Salvayre, Anne

2015-01-01

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect. Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice. Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody. These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL. © 2014 The British Pharmacological Society.

Decreased plasma levels of the endothelial protective sphingosine-1-phosphate are associated with dengue-induced plasma leakage

NARCIS (Netherlands)

Michels, M.; Japtok, L.; Alisjahbana, B.; Wisaksana, R.; Sumardi, U.; Puspita, M.; Kleuser, B.; Mast, Q. de; Ven, A.J.A.M. van der

2015-01-01

BACKGROUND: A transient endothelial hyperpermeability is a hallmark of severe dengue infections. Sphingosine-1-phosphate (S1P) maintains vascular integrity and protects against plasma leakage. We related plasma S1P levels to dengue-induced plasma leakage and studied mechanisms that may underlie the

Emerging Role of Sphingosine-1-phosphate in Inflammation, Cancer, and Lymphangiogenesis

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Kazuaki Takabe

2013-07-01

Full Text Available The main function of the lymphatic system is to control and maintain fluid homeostasis, lipid transport, and immune cell trafficking. In recent years, the pathological roles of lymphangiogenesis, the generation of new lymphatic vessels from preexisting ones, in inflammatory diseases and cancer progression are beginning to be elucidated. Sphingosine-1-phosphate (S1P, a bioactive lipid, mediates multiple cellular events, such as cell proliferation, differentiation, and trafficking, and is now known as an important mediator of inflammation and cancer. In this review, we will discuss recent findings showing the emerging role of S1P in lymphangiogenesis, in inflammation, and in cancer.

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

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Benamer, Najate [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Fares, Nassim [Laboratoire de Physiologie, Faculte de Medecine, Universite Saint Joseph, Beyrouth (Lebanon); Bois, Patrick [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Faivre, Jean-Francois, E-mail: Jean-Francois.Faivre@univ-poitiers.fr [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France)

2011-04-29

Highlights: {yields} In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. {yields} S1P increases cell proliferation through SUR2/Kir6.1 activation. {yields} S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. {yields} S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

International Nuclear Information System (INIS)

Benamer, Najate; Fares, Nassim; Bois, Patrick; Faivre, Jean-Francois

2011-01-01

Highlights: → In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. → S1P increases cell proliferation through SUR2/Kir6.1 activation. → S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. → S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.

Sphingosine kinase 1 is regulated by peroxisome proliferator-activated receptor α in response to free fatty acids and is essential for skeletal muscle interleukin-6 production and signaling in diet-induced obesity.

Science.gov (United States)

Ross, Jessica S; Hu, Wei; Rosen, Bess; Snider, Ashley J; Obeid, Lina M; Cowart, L Ashley

2013-08-02

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3). Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1(-/-) mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1(-/-) mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.

Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells

NARCIS (Netherlands)

Hendriks-Balk, Mariëlle C.; Hajji, Najat; van Loenen, Pieter B.; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

2009-01-01

Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations

Extracellular sphingosine-1-phosphate: a novel actor in human glioblastoma stem cell survival.

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Elena Riccitelli

Full Text Available Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs, a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ, and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source

High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation

International Nuclear Information System (INIS)

Akao, Yukihiro; Banno, Yoshiko; Nakagawa, Yoshihito; Hasegawa, Nobuko; Kim, Tack-Joong; Murate, Takashi; Igarashi, Yasuyuki; Nozawa, Yoshinori

2006-01-01

Although most of pharmacological therapies for cancer utilize the apoptotic machinery of the cells, the available anti-cancer drugs are limited due to the ability of prostate cancer cells to escape from the anti-cancer drug-induced apoptosis. A human prostate cancer cell line PC3 is resistant to camptothecin (CPT). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of S1P receptors, S1P 1 and S1P 3 , as compared with those of LNCaP cells. The knockdown of SPHK1 by small interfering RNA and inhibition of S1P receptor signaling by pertussis toxin in PC3 cells induced significant inhibition of cell growth, suggesting implication of SPHK1 and S1P receptors in cell proliferation in PC3 cells. Furthermore, the treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1P 1 /S1P 3 receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT

Inflammatory lipid sphingosine-1-phosphate upregulates C-reactive protein via C/EBPβ and potentiates breast cancer progression

NARCIS (Netherlands)

Kim, E.S.; Cha, Y.; Ham, M.; Jung, J.; Kim, S.G.; Hwang, S.; Kleemann, R.; Moon, A.

2014-01-01

A crucial role of the inflammatory lipid sphingosine-1-phosphate (S1P) in breast cancer aggressiveness has been reported. Recent clinical studies have suggested that C-reactive protein (CRP) has a role in breast cancer development. However, limited information is available on the molecular basis for

Sphingosine 1-Phosphate Activation of EGFR As a Novel Target for Meningitic Escherichia coli Penetration of the Blood-Brain Barrier

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Wang, Xiangru; Maruvada, Ravi; Morris, Andrew J.; Liu, Jun O.; Baek, Dong Jae; Kim, Kwang Sik

2016-01-01

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood–brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics. PMID:27711202

Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling

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Serafimidis, Ioannis; Rodriguez-Aznar, Eva; Lesche, Mathias; Yoshioka, Kazuaki; Takuwa, Yoh; Dahl, Andreas; Pan, Duojia; Gavalas, Anthony

2017-01-01

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches. PMID:28248965

Ablation of sphingosine 1-phosphate receptor subtype 3 impairs hippocampal neuron excitability in vitro and spatial working memory in vivo

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Daniela Weth-Malsch

2016-11-01

Full Text Available Understanding the role of the bioactive lipid mediator sphingosine 1-phosphate (S1P within the central nervous system has recently gained more and more attention, as it has been connected to major diseases such as multiple sclerosis and Alzheimer's disease. Even though much data about the functions of the five S1P receptors has been collected for other organ systems, we still lack a complete understanding for their specific roles, in particular within the brain. Therefore, it was the aim of this study to further elucidate the role of S1P receptor subtype 3 (S1P3 in vivo and in vitro with a special focus on the hippocampus. Using an S1P3 knock-out mouse model we applied a range of behavioral tests, performed expression studies and whole cell patch clamp recordings in acute hippocampal slices. We were able to show that S1P3 deficient mice display a significant spatial working memory deficit within the T-maze test, but not in anxiety related tests. Furthermore, S1p3 mRNA was expressed throughout the hippocampal formation. Principal neurons in area CA3 lacking S1P3 showed significantly increased interspike intervals and a significantly decreased input resistance. Upon stimulation with S1P CA3 principal neurons from both wildtype and S1P3-/- mice displayed significantly increased evoked EPSC amplitudes and decay times, whereas rise times remained unchanged. These results suggest a specific involvement of S1P3 for the establishment of spatial working memory and neuronal excitability within the hippocampus.

S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells

NARCIS (Netherlands)

Hendriks-Balk, Mariëlle C.; van Loenen, Pieter B.; Hajji, Najat; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

2009-01-01

Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells

Smad3 deficiency leads to mandibular condyle degradation via the sphingosine 1-phosphate (S1P)/S1P3 signaling axis.

Science.gov (United States)

Mori, Hiroki; Izawa, Takashi; Tanaka, Eiji

2015-10-01

Temporomandibular joint osteoarthritis is a degenerative disease that is characterized by permanent cartilage destruction. Transforming growth factor (TGF)-β is one of the most abundant cytokines in the bone matrix and is shown to regulate the migration of osteoprogenitor cells. It is hypothesized that TGF-β/Smad3 signaling affects cartilage homeostasis by influencing sphingosine 1-phosphate (S1P)/S1P receptor signaling and chondrocyte migration. We therefore investigated the molecular mechanisms by which crosstalk may occur between TGF-β/Smad3 and S1P/S1P receptor signaling to maintain condylar cartilage and to prevent temporomandibular joint osteoarthritis. Abnormalities in the condylar subchondral bone, including dynamic changes in bone mineral density and microstructure, were observed in Smad3(-/-) mice by microcomputed tomography. Cell-free regions and proteoglycan loss characterized the cartilage degradation present, and increased numbers of apoptotic chondrocytes and matrix metalloproteinase 13(+) chondrocytes were also detected. Furthermore, expression of S1P receptor 3 (S1P3), but not S1P1 or S1P2, was significantly down-regulated in the condylar cartilage of Smad3(-/-) mice. By using RNA interference technology and pharmacologic tools, S1P was found to transactivate Smad3 in an S1P3/TGF-β type II receptor-dependent manner, and S1P3 was found to be required for TGF-β-induced migration of chondrocyte cells and downstream signal transduction via Rac1, RhoA, and Cdc42. Taken together, these results indicate that the Smad3/S1P3 signaling pathway plays an important role in the pathogenesis of temporomandibular joint osteoarthritis. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Sphingosine-1-Phosphate Signaling Regulates Myogenic Responsiveness in Human Resistance Arteries.

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Sonya Hui

Full Text Available We recently identified sphingosine-1-phosphate (S1P signaling and the cystic fibrosis transmembrane conductance regulator (CFTR as prominent regulators of myogenic responsiveness in rodent resistance arteries. However, since rodent models frequently exhibit limitations with respect to human applicability, translation is necessary to validate the relevance of this signaling network for clinical application. We therefore investigated the significance of these regulatory elements in human mesenteric and skeletal muscle resistance arteries. Mesenteric and skeletal muscle resistance arteries were isolated from patient tissue specimens collected during colonic or cardiac bypass surgery. Pressure myography assessments confirmed endothelial integrity, as well as stable phenylephrine and myogenic responses. Both human mesenteric and skeletal muscle resistance arteries (i express critical S1P signaling elements, (ii constrict in response to S1P and (iii lose myogenic responsiveness following S1P receptor antagonism (JTE013. However, while human mesenteric arteries express CFTR, human skeletal muscle resistance arteries do not express detectable levels of CFTR protein. Consequently, modulating CFTR activity enhances myogenic responsiveness only in human mesenteric resistance arteries. We conclude that human mesenteric and skeletal muscle resistance arteries are a reliable and consistent model for translational studies. We demonstrate that the core elements of an S1P-dependent signaling network translate to human mesenteric resistance arteries. Clear species and vascular bed variations are evident, reinforcing the critical need for further translational study.

High density lipoprotein (HDL)-associated sphingosine 1-phosphate (S1P) inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression

DEFF Research Database (Denmark)

Feuerborn, Renata; Becker, Susen; Potì, Francesco

2017-01-01

BACKGROUND AND AIMS: Macrophage apoptosis is critically involved in atherosclerosis. We here examined the effect of anti-atherogenic high density lipoprotein (HDL) and its component sphingosine-1-phosphate (S1P) on apoptosis in RAW264.7 murine macrophages. METHODS: Mitochondrial or endoplasmic re...

Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo

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Daniela eWeth

2015-04-01

Full Text Available At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P. It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (105/µl, 106/µl, 107/µl and assessed in mice with different genetic backgrounds (WT, S1P1fl/fl, SNS-S1P1-/-, S1P3-/-. Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralisation of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P.

Bile Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Promotes Neuroinflammation during Hepatic Encephalopathy in Mice

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Matthew McMillin

2017-07-01

Full Text Available Hepatic encephalopathy (HE is a neuropsychiatric complication that occurs due to deteriorating hepatic function and this syndrome influences patient quality of life, clinical management strategies and survival. During acute liver failure, circulating bile acids increase due to a disruption of the enterohepatic circulation. We previously identified that bile acid-mediated signaling occurs in the brain during HE and contributes to cognitive impairment. However, the influences of bile acids and their downstream signaling pathways on HE-induced neuroinflammation have not been assessed. Conjugated bile acids, such as taurocholic acid (TCA, can activate sphingosine-1-phosphate receptor 2 (S1PR2, which has been shown to promote immune cell infiltration and inflammation in other models. The current study aimed to assess the role of bile-acid mediated S1PR2 signaling in neuroinflammation and disease progression during azoxymethane (AOM-induced HE in mice. Our findings demonstrate a temporal increase of bile acids in the cortex during AOM-induced HE and identified that cortical bile acids were elevated as an early event in this model. In order to classify the specific bile acids that were elevated during HE, a metabolic screen was performed and this assay identified that TCA was increased in the serum and cortex during AOM-induced HE. To reduce bile acid concentrations in the brain, mice were fed a diet supplemented with cholestyramine, which alleviated neuroinflammation by reducing proinflammatory cytokine expression in the cortex compared to the control diet-fed AOM-treated mice. S1PR2 was expressed primarily in neurons and TCA treatment increased chemokine ligand 2 mRNA expression in these cells. The infusion of JTE-013, a S1PR2 antagonist, into the lateral ventricle prior to AOM injection protected against neurological decline and reduced neuroinflammation compared to DMSO-infused AOM-treated mice. Together, this identifies that reducing bile acid

A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).

Science.gov (United States)

Yamamoto, Sayaka; Yako, Yuta; Fujioka, Yoichiro; Kajita, Mihoko; Kameyama, Takeshi; Kon, Shunsuke; Ishikawa, Susumu; Ohba, Yusuke; Ohno, Yusuke; Kihara, Akio; Fujita, Yasuyuki

2016-02-01

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells. © 2016 Yamamoto, Yako, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Causes Increased Cardiac S1P Levels and Bradycardia in Rats.

Science.gov (United States)

Harris, Christopher M; Mittelstadt, Scott; Banfor, Patricia; Bousquet, Peter; Duignan, David B; Gintant, Gary; Hart, Michelle; Kim, Youngjae; Segreti, Jason

2016-10-01

Inhibition of the sphingosine-1-phosphate (S1P)-catabolizing enzyme S1P lyase (S1PL) elevates the native ligand of S1P receptors and provides an alternative mechanism for immune suppression to synthetic S1P receptor agonists. S1PL inhibition is reported to preferentially elevate S1P in lymphoid organs. Tissue selectivity could potentially differentiate S1PL inhibitors from S1P receptor agonists, the use of which also results in bradycardia, atrioventricular block, and hypertension. But it is unknown if S1PL inhibition would also modulate cardiac S1P levels or cardiovascular function. The S1PL inhibitor 6-[(2R)-4-(4-benzyl-7-chlorophthalazin-1-yl)-2-methylpiperazin-1-yl]pyridine-3-carbonitrile was used to determine the relationship in rats between drug concentration, S1P levels in select tissues, and circulating lymphocytes. Repeated oral doses of the S1PL inhibitor fully depleted circulating lymphocytes after 3 to 4 days of treatment in rats. Full lymphopenia corresponded to increased levels of S1P of 100- to 1000-fold in lymph nodes, 3-fold in blood (but with no change in plasma), and 9-fold in cardiac tissue. Repeated oral dosing of the S1PL inhibitor in telemeterized, conscious rats resulted in significant bradycardia within 48 hours of drug treatment, comparable in magnitude to the bradycardia induced by 3 mg/kg fingolimod. These results suggest that S1PL inhibition modulates cardiac function and does not provide immune suppression with an improved cardiovascular safety profile over fingolimod in rats. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Vascular and Immunobiology of the Circulatory Sphingosine 1-Phosphate Gradient

Science.gov (United States)

Yanagida, Keisuke; Hla, Timothy

2017-01-01

Vertebrates are endowed with a closed circulatory system, the evolution of which required novel structural and regulatory changes. Furthermore, immune cell trafficking paradigms adapted to the barriers imposed by the closed circulatory system. How did such changes occur mechanistically? We propose that spatial compartmentalization of the lipid mediator sphingosine 1-phosphate (S1P) may be one such mechanism. In vertebrates, S1P is spatially compartmentalized in the blood and lymphatic circulation, thus comprising a sharp S1P gradient across the endothelial barrier. Circulatory S1P has critical roles in maturation and homeostasis of the vascular system as well as in immune cell trafficking. Physiological functions of S1P are tightly linked to shear stress, the key biophysical stimulus from blood flow. Thus, circulatory S1P confinement could be a primordial strategy of vertebrates in the development of a closed circulatory system. This review discusses the cellular and molecular basis of the S1P gradients and aims to interpret its physiological significance as a key feature of the closed circulatory system. PMID:27813829

Data on subgroup specific baseline characteristics and serum sphingosine-1-phosphate concentrations in the Study of Health in Pomerania

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Eileen Moritz

2017-06-01

Full Text Available In this data article, we provide subgroup specific baseline characteristics and serum sphingosine-1-phosphate (S1P concentrations for healthy individuals within the Study of Health in Pomerania (SHIP-TREND cohort. After exclusion of subjects with cardiovascular disease, diabetes mellitus, hypertension, metabolic syndrome, elevated liver enzymes and/or chronic kidney disease stadium III or IV, four subgroups were defined according to different limits for body mass index (BMI, alterations in blood lipid levels and smoking status. Tables show respective clinical and laboratory parameters stratified by gender. Serum S1P concentrations are also stratified by age groups. The data presented herein is related to the research article entitled “Reference intervals for serum sphingosine-1-phosphate in the population-based Study of Health in Pomerania” (E. Moritz, D. Wegner, S. Groß, M. Bahls, M. Dörr, S.B. Felix, T. Ittermann, S. Oswald, M. Nauck, N. Friedrich, R.H. Böger, G. Daum, E. Schwedhelm, B.H. Rauch, Clin Chim Acta. 468 (2017 25–31 [1].

Vehicle-dependent Effects of Sphingosine 1-phosphate on Plasminogen Activator Inhibitor-1 Expression

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Takahashi, Chiharu; Kurano, Makoto; Nishikawa, Masako; Kano, Kuniyuki; Dohi, Tomotaka; Miyauchi, Katsumi; Daida, Hiroyuki; Shimizu, Tomo; Aoki, Junken

2017-01-01

Aim: Sphingosine 1-phosphate (S1P) has been suggested to be a positive regulator of plasminogen activator inhibitor 1 (PAI-1) in adipocytes, while some studies are not consistent with this prothrombotic property of S1P. Since S1P is bound to apolipoprotein M (apoM) on HDL or to albumin in plasma, we compared the properties of these two forms on the PAI-1 induction. Methods: We investigated the associations of S1P, apoM, and PAI-1 concentrations in the plasma of normal coronary artery (NCA), stable angina pectoris (SAP), and acute coronary syndrome (ACS) subjects (n = 32, 71, and 38, respectively). Then, we compared the effects of S1P with various vehicles on the PAI-1 expression in 3T3L1 adipocytes. We also investigated the modulation of the PAI-1 levels in mice infected with adenovirus coding apoM. Results: Among ACS subjects, the PAI-1 level was positively correlated with the S1P level, but not the apoM level. In adipocytes, S1P bound to an apoM-rich vehicle induced PAI-1 expression to a lesser extent than the control vehicle, while S1P bound to an apoM-depleted vehicle induced PAI-1 expression to a greater extent than the control vehicle in 3T3L1 adipocytes. Additionally, apoM overexpression in mice failed to modulate the plasma PAI-1 level and the adipose PAI-1 expression level. S1P bound to albumin increased PAI-1 expression through the S1P receptor 2-Rho/ROCK-NFκB pathway. Conclusion: S1P bound to albumin, but not to apoM, induces PAI-1 expression in adipocytes, indicating that S1P can exert different properties on the pathogenesis of vascular diseases, depending on its vehicle. PMID:28321011

Sphingosine-1-Phosphate (S1P) and S1P Signaling Pathway: Therapeutic Targets in Autoimmunity and Inflammation.

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Tsai, Hsing-Chuan; Han, May H

2016-07-01

Sphingosine-1-phosphate (S1P) and S1P receptors (S1PR) are ubiquitously expressed. S1P-S1PR signaling has been well characterized in immune trafficking and activation in innate and adaptive immune systems. However, the full extent of its involvement in the pathogenesis of autoimmune diseases is not well understood. FTY720 (fingolimod), a non-selective S1PR modulator, significantly decreased annualized relapse rates in relapsing-remitting multiple sclerosis (MS). FTY720, which primarily targets S1P receptor 1 as a functional antagonist, arrests lymphocyte egress from secondary lymphoid tissues and reduces neuroinflammation in the central nervous system (CNS). Recent studies suggest that FTY720 also decreases astrogliosis and promotes oligodendrocyte differentiation within the CNS and may have therapeutic benefit to prevent brain atrophy. Since S1P signaling is involved in multiple immune functions, therapies targeting S1P axis may be applicable to treat autoimmune diseases other than MS. Currently, over a dozen selective S1PR and S1P pathway modulators with potentially superior therapeutic efficacy and better side-effect profiles are in the pipeline of drug development. Furthermore, newly characterized molecules such as apolipoprotein M (ApoM) (S1P chaperon) and SPNS2 (S1P transporter) are also potential targets for treatment of autoimmune diseases. Finally, the application of therapies targeting S1P and S1P signaling pathways may be expanded to treat several other immune-mediated disorders (such as post-infectious diseases, post-stroke and post-stroke dementia) and inflammatory conditions beyond their application in primary autoimmune diseases.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2012-02-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2011-05-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Sphingosine 1-Phosphate (S1P) Carrier-dependent Regulation of Endothelial Barrier

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Wilkerson, Brent A.; Grass, G. Daniel; Wing, Shane B.; Argraves, W. Scott; Argraves, Kelley M.

2012-01-01

Sphingosine 1-phosphate (S1P) is a blood-borne lysosphingolipid that acts to promote endothelial cell (EC) barrier function. In plasma, S1P is associated with both high density lipoproteins (HDL) and albumin, but it is not known whether the carriers impart different effects on S1P signaling. Here we establish that HDL-S1P sustains EC barrier longer than albumin-S1P. We showed that the sustained barrier effects of HDL-S1P are dependent on signaling by the S1P receptor, S1P1, and involve persistent activation of Akt and endothelial NOS (eNOS), as well as activity of the downstream NO target, soluble guanylate cyclase (sGC). Total S1P1 protein levels were found to be higher in response to HDL-S1P treatment as compared with albumin-S1P, and this effect was not associated with increased S1P1 mRNA or dependent on de novo protein synthesis. Several pieces of evidence indicate that long term EC barrier enhancement activity of HDL-S1P is due to specific effects on S1P1 trafficking. First, the rate of S1P1 degradation, which is proteasome-mediated, was slower in HDL-S1P-treated cells as compared with cells treated with albumin-S1P. Second, the long term barrier-promoting effects of HDL-S1P were abrogated by treatment with the recycling blocker, monensin. Finally, cell surface levels of S1P1 and levels of S1P1 in caveolin-enriched microdomains were higher after treatment with HDL-S1P as compared with albumin-S1P. Together, the findings reveal S1P carrier-specific effects on S1P1 and point to HDL as the physiological mediator of sustained S1P1-PI3K-Akt-eNOS-sGC-dependent EC barrier function. PMID:23135269

Crystal Structure of a Lipid G Protein-Coupled Receptor

Energy Technology Data Exchange (ETDEWEB)

Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C [Scripps; (Receptos)

2012-03-01

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P₁-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P₁, resulting in the modulation of immune and stromal cell responses.

Sphingosine-1-Phosphate (S1P) Impacts Presynaptic Functions by Regulating Synapsin I Localization in the Presynaptic Compartment.

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Riganti, Loredana; Antonucci, Flavia; Gabrielli, Martina; Prada, Ilaria; Giussani, Paola; Viani, Paola; Valtorta, Flavia; Menna, Elisabetta; Matteoli, Michela; Verderio, Claudia

2016-04-20

Growing evidence indicates that sphingosine-1-P (S1P) upregulates glutamate secretion in hippocampal neurons. However, the molecular mechanisms through which S1P enhances excitatory activity remain largely undefined. The aim of this study was to identify presynaptic targets of S1P action controlling exocytosis. Confocal analysis of rat hippocampal neurons showed that S1P applied at nanomolar concentration alters the distribution of Synapsin I (SynI), a presynaptic phosphoprotein that controls the availability of synaptic vesicles for exocytosis. S1P induced SynI relocation to extrasynaptic regions of mature neurons, as well as SynI dispersion from synaptic vesicle clusters present at axonal growth cones of developing neurons. S1P-induced SynI relocation occurred in a Ca(2+)-independent but ERK-dependent manner, likely through the activation of S1P3 receptors, as it was prevented by the S1P3 receptor selective antagonist CAY1044 and in neurons in which S1P3 receptor was silenced. Our recent evidence indicates that microvesicles (MVs) released by microglia enhance the metabolism of endogenous sphingolipids in neurons and stimulate excitatory transmission. We therefore investigated whether MVs affect SynI distribution and whether endogenous S1P could be involved in the process. Analysis of SynI immunoreactivity showed that exposure to microglial MVs induces SynI mobilization at presynaptic sites and growth cones, whereas the use of inhibitors of sphingolipid cascade identified S1P as the sphingolipid mediating SynI redistribution. Our data represent the first demonstration that S1P induces SynI mobilization from synapses, thereby indicating the phosphoprotein as a novel target through which S1P controls exocytosis. Growing evidence indicates that the bioactive lipid sphingosine and its metabolite sphingosine-1-P (S1P) stimulate excitatory transmission. While it has been recently clarified that sphingosine influences directly the exocytotic machinery by activating the

Reduction in serum sphingosine 1-phosphate concentration in malaria.

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Chuchard Punsawad

Full Text Available 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.

Binding Characteristics of Sphingosine-1-Phosphate to ApoM hints to Assisted Release Mechanism via the ApoM Calyx-Opening

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Zhang, Hansi; Pluhackova, Kristyna; Jiang, Zhenyan; Böckmann, Rainer A.

2016-08-01

Sphingosine-1-phosphate (S1P) is a lysophospholipid mediator carried by the HDL-associated apoM protein in blood, regulating many physiological processes by activating the G protein-coupled S1P receptor in mammals. Despite the solved crystal structure of the apoM-S1P complex, the mechanism of S1P release from apoM as a part of the S1P pathway is unknown. Here, the dynamics of the wild type apoM-S1P complex as well as of mutants were investigated by means of atomistic molecular dynamics simulations. The potential of mean force for S1P unbinding from apoM reflected a large binding strength of more than 60 kJ/mol. This high unbinding free energy for S1P underlines the observed specificity of the physiological effects of S1P as it suggests that the spontaneous release of S1P from apoM is unlikely. Instead, S1P release and thus the control of this bioactive lipid probably requires the tight interaction with other molecules, e.g. with the S1P receptor. Mutations of specific S1P anchoring residues of apoM decreased the energetic barrier by up to 20 kJ/mol. Moreover, the ligand-free apoM protein is shown to adopt a more open upper hydrophilic binding pocket and to result in complete closure of the lower hydrophobic cavity, suggesting a mechanism for adjusting the gate for ligand access.

Sphingosine-1-Phosphate Receptor-1 Selective Agonist Enhances Collateral Growth and Protects against Subsequent Stroke.

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Masahiko Ichijo

Full Text Available Collateral growth after acute occlusion of an intracranial artery is triggered by increasing shear stress in preexisting collateral pathways. Recently, sphingosine-1-phosphate receptor-1 (S1PR1 on endothelial cells was reported to be essential in sensing fluid shear stress. Here, we evaluated the expression of S1PR1 in the hypoperfused mouse brain and investigated the effect of a selective S1PR1 agonist on leptomeningeal collateral growth and subsequent ischemic damage after focal ischemia.In C57Bl/6 mice (n = 133 subjected to unilateral common carotid occlusion (CCAO and sham surgery. The first series examined the time course of collateral growth, cell proliferation, and S1PR1 expression in the leptomeningeal arteries after CCAO. The second series examined the relationship between pharmacological regulation of S1PR1 and collateral growth of leptomeningeal anastomoses. Animals were randomly assigned to one of the following groups: LtCCAO and daily intraperitoneal (i.p. injection for 7 days of an S1PR1 selective agonist (SEW2871, 5 mg/kg/day; sham surgery and daily i.p. injection for 7 days of SEW2871 after surgery; LtCCAO and daily i.p. injection for 7 days of SEW2871 and an S1PR1 inverse agonist (VPC23019, 0.5 mg/kg; LtCCAO and daily i.p. injection of DMSO for 7 days after surgery; and sham surgery and daily i.p. injection of DMSO for 7 days. Leptomeningeal anastomoses were visualized 14 days after LtCCAO by latex perfusion method, and a set of animals underwent subsequent permanent middle cerebral artery occlusion (pMCAO 7 days after the treatment termination. Neurological functions 1 hour, 1, 4, and 7 days and infarction volume 7 days after pMCAO were evaluated.In parallel with the increase in S1PR1 mRNA levels, S1PR1 expression colocalized with endothelial cell markers in the leptomeningeal arteries, increased markedly on the side of the CCAO, and peaked 7 days after CCAO. Mitotic cell numbers in the leptomeningeal arteries increased after

Chemical Hypoxia Brings to Light Altered Autocrine Sphingosine-1-Phosphate Signalling in Rheumatoid Arthritis Synovial Fibroblasts

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Chenqi Zhao

2015-01-01

Full Text Available Emerging evidence suggests a role for sphingosine-1-phosphate (S1P in various aspects of rheumatoid arthritis (RA pathogenesis. In this study we compared the effect of chemical hypoxia induced by cobalt chloride (CoCl2 on the expression of S1P metabolic enzymes and cytokine/chemokine secretion in normal fibroblast-like synoviocytes (FLS and RAFLS. RAFLS incubated with CoCl2, but not S1P, produced less IL-8 and MCP-1 than normal FLS. Furthermore, incubation with the S1P2 and S1P3 receptor antagonists, JTE-013 and CAY10444, reduced CoCl2-mediated chemokine production in normal FLS but not in RAFLS. RAFLS showed lower levels of intracellular S1P and enhanced mRNA expression of S1P phosphatase 1 (SGPP1 and S1P lyase (SPL, the enzymes that are involved in intracellular S1P degradation, when compared to normal FLS. Incubation with CoCl2 decreased SGPP1 mRNA and protein and SPL mRNA as well. Inhibition of SPL enhanced CoCl2-mediated cytokine/chemokine release and restored autocrine activation of S1P2 and S1P3 receptors in RAFLS. The results suggest that the sphingolipid pathway regulating the intracellular levels of S1P is dysregulated in RAFLS and has a significant impact on cell autocrine activation by S1P. Altered sphingolipid metabolism in FLS from patients with advanced RA raises the issue of synovial cell burnout due to chronic inflammation.

Regulation of S1P receptors and sphingosine kinases expression in acute pulmonary endothelial cell injury

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Huiying Liu

2016-12-01

Full Text Available Background Acute lung injury and acute respiratory distress syndrome (ALI/ARDS is a severe clinical syndrome with mortality rate as high as 30–40%. There is no treatment yet to improve pulmonary endothelial barrier function in patients with severe pulmonary edema. Developing therapies to protect endothelial barrier integrity and stabilizing gas exchange is getting more and more attention. Sphingosine-1-phosphate (S1P is able to enhance the resistance of endothelial cell barrier. S1P at physiological concentrations plays an important role in maintaining endothelial barrier function. Proliferation, regeneration and anti-inflammatory activity that mesenchymal stem cells (MSCs exhibit make it possible to regulate the homeostatic control of S1P. Methods By building a pulmonary endothelial cell model of acute injury, we investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs during the treatment of acute lung injury using RT-PCR, and investigated the HPAECs Micro-electronics impedance using Real Time Cellular Analysis. Results It was found that the down-regulation of TNF-α expression was more significant when MSC was used in combination with S1P. The combination effection mainly worked on S1PR2, S1PR3 and SphK2. The results show that when MSCs were used in combination with S1P, the selectivity of S1P receptors was increased and the homeostatic control of S1P concentration was improved through regulation of expression of S1P metabolic enzymes. Discussions The study found that, as a potential treatment, MSCs could work on multiple S1P related genes simultaneously. When it was used in combination with S1P, the expression regulation result of related genes was not simply the superposition of each other, but more significant outcome was obtained. This study establishes the experimental basis for further exploring the efficacy of improving endothelial barrier function in acute lung injury, using MSCs in combination with S1

Regulation of S1P receptors and sphingosine kinases expression in acute pulmonary endothelial cell injury.

Science.gov (United States)

Liu, Huiying; Zhang, Zili; Li, Puyuan; Yuan, Xin; Zheng, Jing; Liu, Jinwen; Bai, Changqing; Niu, Wenkai

2016-01-01

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is a severe clinical syndrome with mortality rate as high as 30-40%. There is no treatment yet to improve pulmonary endothelial barrier function in patients with severe pulmonary edema. Developing therapies to protect endothelial barrier integrity and stabilizing gas exchange is getting more and more attention. Sphingosine-1-phosphate (S1P) is able to enhance the resistance of endothelial cell barrier. S1P at physiological concentrations plays an important role in maintaining endothelial barrier function. Proliferation, regeneration and anti-inflammatory activity that mesenchymal stem cells (MSCs) exhibit make it possible to regulate the homeostatic control of S1P. By building a pulmonary endothelial cell model of acute injury, we investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs during the treatment of acute lung injury using RT-PCR, and investigated the HPAECs Micro-electronics impedance using Real Time Cellular Analysis. It was found that the down-regulation of TNF- α expression was more significant when MSC was used in combination with S1P. The combination effection mainly worked on S1PR2, S1PR3 and SphK2. The results show that when MSCs were used in combination with S1P, the selectivity of S1P receptors was increased and the homeostatic control of S1P concentration was improved through regulation of expression of S1P metabolic enzymes. The study found that, as a potential treatment, MSCs could work on multiple S1P related genes simultaneously. When it was used in combination with S1P, the expression regulation result of related genes was not simply the superposition of each other, but more significant outcome was obtained. This study establishes the experimental basis for further exploring the efficacy of improving endothelial barrier function in acute lung injury, using MSCs in combination with S1P and their possible synergistic mechanism.

Sphingosine signaling and atherogenesis

DEFF Research Database (Denmark)

Xu, Cang-bao; Hansen-Schwartz, Jacob; Edvinsson, Lars

2004-01-01

Sphingosine-1-phosphate (S1P) has diverse biological functions acting inside cells as a second messenger to regulate cell proliferation and survival, and extracellularly, as a ligand for a group of G protein-coupled receptors (GPCRs) named the endothelial differentiation gene (EDG) family. Five...... closely related GPCRs of EDG family (EDG1, EDG3, EDG5, EDG6, and EDG8) have recently been identified as high-affinity S1P receptors. These receptors are coupled via Gi, Gq, G12/13, and Rho. The signaling pathways are linked to vascular cell migration, proliferation, apoptosis, intracellular Ca2......+ mobilization, and expression of adhesion molecules. The formation of an atherosclerotic lesion occurs through activation of cellular events that include monocyte adhesion to the endothelium and vascular smooth muscle cell (VSMC) migration and proliferation. Thus, S1P signaling may play an important role...

Sphingosine-1-Phosphate Induces Dose-Dependent Chemotaxis or Fugetaxis of T-ALL Blasts through S1P1 Activation

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Messias, Carolina V.; Santana-Van-Vliet, Eliane; Lemos, Julia P.; Moreira, Otacilio C.; Cotta-de-Almeida, Vinicius; Savino, Wilson; Mendes-da-Cruz, Daniella Arêas

2016-01-01

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5). S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL) did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM) did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000–10000 nM) through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts. PMID:26824863

Sphingosine-1-Phosphate Induces Dose-Dependent Chemotaxis or Fugetaxis of T-ALL Blasts through S1P1 Activation.

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Carolina V Messias

Full Text Available Sphingosine-1-phosphate (S1P is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5. S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000-10000 nM through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts.

Circulating levels of sphingosine-1-phosphate are elevated in severe, but not mild psoriasis and are unresponsive to anti-TNF-α treatment

Science.gov (United States)

Checa, Antonio; Xu, Ning; Sar, Daniel G.; Haeggström, Jesper Z.; Ståhle, Mona; Wheelock, Craig E.

2015-07-01

Sphingolipids are bioactive molecules with a putative role in inflammation. Alterations in sphingolipids, in particular ceramides, have been consistently observed in psoriatic skin. Herein, we quantified the circulating sphingolipid profile in individuals with mild or severe psoriasis as well as healthy controls. In addition, the effects of anti-TNF-α treatment were determined. Levels of sphingoid bases, including sphingosine-1-phosphate (S1P), increased in severe (P S1P response to treatment may have pathobiological implications due to its close relation to the vascular and immune systems. In particular, increased levels of sphingolipids and especially S1P in severe psoriasis patients requiring biological treatment may potentially be associated with cardiovascular comorbidities. The fact that shifts in S1P levels were not ameliorated by anti-TNF-α treatment, despite improvements in the skin lesions, further supports targeting S1P receptors as therapy for severe psoriasis.

A rapid and validated HPLC method to quantify sphingosine 1-phosphate in human plasma using solid-phase extraction followed by derivatization with fluorescence detection

NARCIS (Netherlands)

Butter, Jan J.; Koopmans, Richard P.; Michel, Martin C.

2005-01-01

We describe the development and validation of analytical methodology for the determination of sphingosine 1-phosphate (S1P) in plasma. It uses solid-phase extraction (SPE) followed by an automated reversed-phase gradient HPLC column-switching system with a pre-column derivatization with

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells.

Science.gov (United States)

Völzke, Anja; Koch, Alexander; Meyer Zu Heringdorf, Dagmar; Huwiler, Andrea; Pfeilschifter, Josef

2014-01-01

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE2 formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β). Treatment of rat and human mesangial cells with S1P led to concentration-dependent enhanced expression of COX-2. Pharmacological and molecular biology approaches revealed that the S1P-dependent increase of COX-2 mRNA and protein expression was mediated via activation of S1P receptor 2 (S1P2). Further, inhibition of Gi and p42/p44 MAPK signaling, both downstream of S1P2, abolished the S1P-induced COX-2 expression. In addition, S1P/S1P2-dependent upregulation of COX-2 led to significantly elevated PGE2 levels, which were further potentiated in the presence of Ang II and IL-1β. A functional consequence downstream of S1P/S1P2 signaling is mesangial cell migration that is stimulated by S1P. Interestingly, inhibition of COX-2 by celecoxib and SC-236 completely abolished the migratory response. Overall, our results demonstrate that extracellular S1P induces COX-2 expression via activation of S1P2 and subsequent Gi and p42/p44 MAPK-dependent signaling in renal mesangial cells leading to enhanced PGE2 formation and cell migration that essentially requires COX-2. Thus, targeting S1P/S1P2 signaling pathways might be a novel strategy to treat renal inflammatory diseases. © 2013.

SPHINGOSINE-1 PHOSPHATE: A NEW MODULATOR OF IMMUNE PLASTICITY IN THE TUMOR MICROENVIRONMENT

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Yamila I Rodriguez

2016-10-01

Full Text Available In the last 15 years, increasing evidences demonstrate a strong link between sphingosine-1-phosphate (S1P in both normal physiology and progression of different diseases, including cancer and inflammation. Indeed, numerous studies show that tissue levels of this sphingolipid metabolite are augmented in many cancers, affecting survival, proliferation, angiogenesis and metastatic spread. Recent insights into the possible role of S1P as a therapeutic target has attracted enormous attention and opened new opportunities in this evolving field. In this review we will focus on the role of S1P in cancer with particular emphasis in new developments that highlight the many functions of this sphingolipid in the tumor microenvironment. We will discuss how S1P modulates phenotypic plasticity of macrophages and mast cells, tumor-induced immune evasion, differentiation and survival of immune cells in the tumor milieu, interaction between cancer and stromal cells and hypoxic response.

Sphingosine-1-Phosphate Metabolism and Its Role in the Development of Inflammatory Bowel Disease

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Tomasz Wollny

2017-03-01

Full Text Available Beyond their role as structural molecules, sphingolipids are involved in many important cellular processes including cell proliferation, apoptosis, inflammation, and migration. Altered sphingolipid metabolism is observed in many pathological conditions including gastrointestinal diseases. Inflammatory bowel disease (IBD represents a state of complex, unpredictable, and destructive inflammation of unknown origin within the gastrointestinal tract. The mechanisms explaining the pathophysiology of IBD involve signal transduction pathways regulating gastro-intestinal system’s immunity. Progressive intestinal tissue destruction observed in chronic inflammation may be associated with an increased risk of colon cancer. Sphingosine-1-phosphate (S1P, a sphingolipid metabolite, functions as a cofactor in inflammatory signaling and becomes a target in the treatment of IBD, which might prevent its conversion to cancer. This paper summarizes new findings indicating the impact of (S1P on IBD development and IBD-associated carcinogenesis.

Sustained release of sphingosine 1-phosphate for therapeutic arteriogenesis and bone tissue engineering.

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Sefcik, Lauren S; Petrie Aronin, Caren E; Wieghaus, Kristen A; Botchwey, Edward A

2008-07-01

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that impacts migration, proliferation, and survival in diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. In this study, we investigated the effects of sustained release of S1P on microvascular remodeling and associated bone defect healing in vivo. The murine dorsal skinfold window chamber model was used to evaluate the structural remodeling response of the microvasculature. Our results demonstrated that 1:400 (w/w) loading and subsequent sustained release of S1P from poly(lactic-co-glycolic acid) (PLAGA) significantly enhanced lumenal diameter expansion of arterioles and venules after 3 and 7 days. Incorporation of 5-bromo-2-deoxyuridine (BrdU) at day 7 revealed significant increases in mural cell proliferation in response to S1P delivery. Additionally, three-dimensional (3D) scaffolds loaded with S1P (1:400) were implanted into critical-size rat calvarial defects, and healing of bony defects was assessed by radiograph X-ray, microcomputed tomography (muCT), and histology. Sustained release of S1P significantly increased the formation of new bone after 2 and 6 weeks of healing and histological results suggest increased numbers of blood vessels in the defect site. Taken together, these experiments support the use of S1P delivery for promoting microvessel diameter expansion and improving the healing outcomes of tissue-engineered therapies.

Paradoxical Association of Postoperative Plasma Sphingosine-1-Phosphate with Breast Cancer Aggressiveness and Chemotherapy

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Rajesh Ramanathan

2017-01-01

Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid mediator that has been shown to serve an important regulatory function in breast cancer progression. This study analyzes plasma S1P levels in breast cancer patients undergoing adjuvant therapy as compared to healthy control volunteers. 452 plasma S1P samples among 158 breast cancer patients, along with 20 healthy control volunteers, were analyzed. Mean S1P levels did not significantly differ between cancer patients and controls. Smoking was associated with higher S1P levels in cancer patients. Baseline S1P levels had weak inverse correlation with levels of the inflammatory mediator interleukin- (IL- 17 and CCL-2 and positive correlation with tumor necrosis factor alpha (TNF-α. Midpoint S1P levels during adjuvant therapy were lower than baseline, with near return to baseline after completion, indicating a relationship between chemotherapy and circulating S1P. While stage of disease did not correlate with plasma S1P levels, they were lower among patients with Her2-enriched and triple-negative breast cancer as compared to luminal-type breast cancer. Plasma S1P levels are paradoxically suppressed in aggressive breast cancer and during adjuvant chemotherapy, which raises the possibility that postoperative plasma S1P levels do not reflect S1P secretion from resected breast cancer.

Sphingosine kinase 2-deficiency mediated changes in spinal pain processing

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Jastrow eCanlas

2015-08-01

Full Text Available Chronic pain is one of the most burdensome health issues facing the planet (as costly as diabetes and cancer combined, and in desperate need for new diagnostic targets leading to better therapies. The bioactive lipid sphingosine 1-phosphate (S1P and its receptors have recently been shown to modulate nociceptive signalling at the level of peripheral nociceptors and central neurons. However, the exact role of S1P generating enzymes, in particular sphingosine kinase 2 (Sphk2, in nociception remains unknown. We found that both sphingosine kinases, Sphk1 and Sphk2, were expressed in spinal cord with higher levels of Sphk2 mRNA compared to Sphk1. All three Sphk2 mRNA-isoforms were present with the Sphk2.1 mRNA showing the highest relative expression. Mice deficient in Sphk2 (Sphk2-/- showed in contrast to mice deficient in Sphk1 (Sphk1-/- substantially lower spinal S1P levels compared to wild-type C57BL/6 mice. In the formalin model of acute peripheral inflammatory pain, Sphk2-/- mice showed facilitation of nociceptive transmission during the late response, whereas responses to early acute pain, and the number of c-Fos immunoreactive dorsal horn neurons were not different between Sphk2-/- and wild-type mice. Chronic peripheral inflammation (CPI caused a bilateral increase in mechanical sensitivity in Sphk2-/- mice. Additionally, CPI increased the relative mRNA expression of P2X4 receptor, brain-derived neurotrophic factor and inducible nitric oxide synthase in the ipsilateral spinal cord of wild-type but not Sphk2-/- mice. Similarly, Sphk2-/- mice showed in contrast to wild-type no CPI-dependent increase in areas of the dorsal horn immunoreactive for the microglia marker Iba-1 and the astrocyte marker GFAP. Our results suggest that the tightly regulated cell signalling enzyme Sphk2 may be a key component for facilitation of nociceptive circuits in the CNS leading to central sensitization and pain memory formation.

Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

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Tani, Alessia; Anderloni, Giulia; Pierucci, Federica; Matteini, Francesca; Chellini, Flaminia; Zecchi Orlandini, Sandra; Meacci, Elisabetta

2014-01-01

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration. PMID:25264785

TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

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Ito, Shiori; Iwaki, Soichiro; Kondo, Rie; Satoh, Masashi; Iwabuchi, Kazuya; Ohkawa, Ryunosuke; Mishima, Yuko; Yatomi, Yutaka; Furumoto, Tomoo; Tsutsui, Hiroyuki; Fujii, Satoshi

2014-06-01

Natural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-α, in NKT cell hybridomas and mouse NKT cells. NKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and α-galactosylceramide (α-GalCer), the major ligand to produce cytokines in NKT cells. TNF-α mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. Hybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and α-GalCer increased TNF-α mRNA expression and protein production. S1P enhanced TNF-α induction by α-GalCer. S1P receptor antagonists decreased the TNF-α mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-α mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-α mRNA expression in mouse NKT cells. Plasma TNF-α levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (NKT cells and enhances TNF-α production. TNF-α overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

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Yu-Ting Huang

2007-12-01

Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

Native and reconstituted HDL activate Stat3 in ventricular cardiomyocytes via ERK1/2: role of sphingosine-1-phosphate.

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Frias, Miguel A; James, Richard W; Gerber-Wicht, Christine; Lang, Ursula

2009-05-01

High-density lipoprotein (HDL) has been reported to have cardioprotective properties independent from its cholesterol transport activity. The influence of native HDL and reconstituted HDL (rHDL) on Stat3, the transcription factor playing an important role in myocardium adaptation to stress, was analysed in neonatal rat ventricular cardiomyocytes. We have investigated modulating the composition of rHDL as a means of expanding its function and potential cardioprotective effects. Stat3 phosphorylation and activation were determined by western blotting and electrophoretic mobility shift assay (EMSA). In ventricular cardiomyocytes, HDL and the HDL constituent sphingosine-1-phosphate (S1P) induce a concentration- and time-dependent increase in Stat3 activation. They also enhance extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. U0126, a specific inhibitor of MEK1/2, the upstream activator of ERK1/2, abolishes HDL- and S1P-induced Stat3 activation, whereas the p38 MAPK blocker SB203580 has no significant effect. Inhibition of the tyrosine kinase family Src (Src) caused a significant reduction of Stat3 activation, whereas inhibition of phosphatidylinositol 3-kinase (PI3K) had no effect. S1P and rHDL containing S1P have a similar strong stimulatory action on Stat3, ERK1/2, and p38 MAPK comparable to native HDL. S1P-free rHDL has a much weaker effect. Experiments with agonists and antagonists of the S1P receptor subtypes indicate that HDL and S1P activate Stat3 mainly through the S1P2 receptor. In ventricular cardiomyocytes, addition of S1P to rHDL enhances its therapeutic potential by improving its capacity to activate Stat3. Activation of Stat3 occurs mainly via the S1P constituent and the lipid receptor S1P2 requiring stimulation of ERK1/2 and Src but not p38 MAPK or PI3K. The study underlines the therapeutic potential of tailoring rHDL to confront particular clinical situations.

Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

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Asghar, Muhammad Yasir; Bergelin, Nina; Jaakkola, Panu; Törnquist, Kid

2013-01-01

Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1. PMID:23824493

Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells.

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Veronica Kalhori

Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1 is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3, Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.

Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro

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J.E. Nóbrega Jr.

2014-08-01

Full Text Available This study describes the effect of sphingosine 1-phosphate (S1P for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL. All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05 in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC and Control Culture (CC, which was followed by an increase of activated follicles (intermediary, primary and secondary. In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability.

Intracellular sphingosine releases calcium from lysosomes.

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Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

2015-11-27

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard

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Mirzaian, Mina; Wisse, Patrick; Ferraz, Maria J.; Marques, André R. A.; Gabriel, Tanit L.; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; van Eijk, Marco; Codée, Jeroen D. C.; van der Marel, Gijsbert A.; Overkleeft, Herman S.; Aerts, Johannes M.

2016-01-01

We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of

Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease.

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Sun, Kaiqi; D'Alessandro, Angelo; Ahmed, Mostafa H; Zhang, Yujin; Song, Anren; Ko, Tzu-Ping; Nemkov, Travis; Reisz, Julie A; Wu, Hongyu; Adebiyi, Morayo; Peng, Zhangzhe; Gong, Jing; Liu, Hong; Huang, Aji; Wen, Yuan Edward; Wen, Alexander Q; Berka, Vladimir; Bogdanov, Mikhail V; Abdulmalik, Osheiza; Han, Leng; Tsai, Ah-Lim; Idowu, Modupe; Juneja, Harinder S; Kellems, Rodney E; Dowhan, William; Hansen, Kirk C; Safo, Martin K; Xia, Yang

2017-11-10

Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression. Functional studies revealed that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure at 1.9 Å resolution deciphered that S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to α1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with α2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying S1P-mediated pathogenic metabolic reprogramming in SCD and novel therapeutic avenues.

Uncleaved ApoM signal peptide is required for formation of large ApoM/sphingosine 1-phosphate (S1P)-enriched HDL particles.

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Liu, Mingxia; Allegood, Jeremy; Zhu, Xuewei; Seo, Jeongmin; Gebre, Abraham K; Boudyguina, Elena; Cheng, Dongmei; Chuang, Chia-Chi; Shelness, Gregory S; Spiegel, Sarah; Parks, John S

2015-03-20

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM(Q22A)) introduces a functional signal peptidase cleavage site. Expression of apoM(Q22A) in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM(WT)). When apoM(Q22A) was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM(WT). Hepatocytes isolated from both apoM(WT)- and apoM(Q22A)-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM(WT), apoM(Q22A) hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM(Q22A) bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM(WT) and apoM(Q22A) hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Activation-induced cell death of dendritic cells is dependent on sphingosine kinase 1

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Anja eSchwiebs

2016-04-01

Full Text Available Sphingosine 1-phosphate (S1P is an immune modulatory lipid mediator and has been implicated in numerous pathophysiological processes. S1P is produced by sphingosine kinase 1 (Sphk1 and Sphk2. Dendritic cells (DCs are central for the direction of immune responses and crucially involved in autoimmunity and cancerogenesis. In this study we examined the function and survival of bone marrow-derived DCs under long-term inflammatory stimulation. We observed that differentiated cells undergo activation-induced cell death upon LPS stimulation with an increased metabolic activity shortly after stimulation, followed by a rapid activation of caspase 3 and subsequent augmented apoptosis. Importantly, we highlight a profound role of Sphk1 in secretion of inflammatory cytokines and survival of dendritic cells that might be mediated by a change in sphingolipid levels as well as by a change in STAT3 expression. Cell growth during differentiation of Sphk1-deficient cells treated with the functional S1P receptor antagonist FTYP was reduced. Importantly, in dendritic cells we did not observe a compensatory regulation of Sphk2 mRNA in Sphk1-deficient cells. Instead, we discovered a massive increase in Sphk1 mRNA concentration upon long-term stimulation with LPS in wild type cells that might function as an attempt to rescue from inflammation-caused cell death. Taken together, in this investigation we describe details of a crucial involvement of sphingolipids and Sphk1 in activation-induced cell death during long-term immunogenic activity of DCs that might play an important role in autoimmunity and might explain the differences in immune response observed in in vivo studies of Sphk1 modulation.

The Transporter Spns2 Is Required for Secretion of Lymph but Not Plasma Sphingosine-1-Phosphate

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Alejandra Mendoza

2012-11-01

Full Text Available Plasma sphingosine-1-phosphate (S1P regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how S1P is delivered into circulatory fluids. Here, we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As a differential requirement for lymph and blood S1P, Spns2 may be an attractive target for immune suppressive drugs.

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

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Rodriguez, A M; Graef, A J; LeVine, D N; Cohen, I R; Modiano, J F; Kim, J-H

2015-01-01

Sphingosine-1-phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs. S1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA). Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used. This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT-PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high-performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca(2+) mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining. Canine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1 , decreased S1P1 protein expression and induced apoptosis of HSA cells. S1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Sphingosine 1-Phosphate-Induced ICAM-1 Expression via NADPH Oxidase/ROS-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells

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Lin, Chih-Chung; Yang, Chien-Chung; Cho, Rou-Ling; Wang, Chen-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

2016-01-01

The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47phox, and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with

Sphingosine 1-phosphate induces neutrophil chemoattractant IL-8: repression by steroids.

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Md Mostafizur Rahman

Full Text Available The bioactive sphingolipid sphingosine 1-phosphate (S1P is found in increased amounts in the airways of asthmatics. S1P can regulate airway smooth muscle functions associated with asthmatic inflammation and remodeling, including cytokine secretion. To date however, whether S1P induces secretion of an important chemokine responsible for neutrophilia in airway inflammation--IL-8--was unexplored. The aim of this study was to investigate whether S1P induces IL-8 gene expression and secretion to enhance neutrophil chemotaxis in vitro, as well as examine the molecular mechanisms responsible for repression by the corticosteroid dexamethasone. We show that S1P upregulates IL-8 secretion from ASM cells and enhance neutrophil chemotaxis in vitro. The corticosteroid dexamethasone significantly represses IL-8 mRNA expression and protein secretion in a concentration- and time-dependent manner. Additionally, we reveal that S1P-induced IL-8 secretion is p38 MAPK and ERK-dependent and that these key phosphoproteins act on the downstream effector mitogen- and stress-activated kinase 1 (MSK1 to control secretion of the neutrophil chemoattractant cytokine IL-8. The functional relevance of this in vitro data was demonstrated by neutrophil chemotaxis assays where S1P-induced effects can be significantly attenuated by pretreatment with dexamethasone, pharmacological inhibition of p38 MAPK- or ERK-mediated pathways, or by knocking down MSK-1 with siRNA. Taken together, our study reveals the molecular pathways responsible for IL-8 secretion from ASM cells in response to S1P and indicates ways in which the impact on IL-8-driven neutrophilia may be lessened.

Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

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Sun, Hui-Yan, E-mail: shy35309@sohu.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Wei, Shu-Ping, E-mail: weishuping_83@163.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Xu, Rui-Cheng, E-mail: xu_rc@sohu.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Xu, Peng-Xiao, E-mail: xupengxiao1228@sina.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Zhang, Wen-Cheng, E-mail: wenchengzhang@yahoo.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China)

2010-05-07

Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

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Sun, Hui-Yan; Wei, Shu-Ping; Xu, Rui-Cheng; Xu, Peng-Xiao; Zhang, Wen-Cheng

2010-01-01

Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

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Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M; Zhou, Xinchun

2014-10-01

Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft

Evaluation of plasma sphingosine 1-phosphate, hepcidin and cardiovascular damage biomarkers (cardiac troponin I and homocysteine) in rats infected with brucellosis and vaccinated (Rev-1, RB-51).

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Azimzadeh, Kaveh; Nasrollahi Nargesabad, Reza; Vousooghi, Nasim

2017-08-01

Brucellosis is known as one of important zoonosis. Studying the histological and biochemical effects of the disease could help to increase our knowledge about it. The aim of the present study was to evaluate changes of plasma parameters after intraperitoneal injection of two species of Brucella (Brucella melitensis and Brucella abortus) and two vaccines (Rev-1, RB-51) in the rat. Forty male rats were divided into five groups (n = 8 in each group). Two groups received suspensions of Brucella abortus and Brucella melitensis and two other groups were injected intraperitoneally with two mentioned vaccines and the last group received only distilled water. The results showed a significant increase in sphingosine 1-phosphate, Malondialdehyde, hepcidin, homocysteine, cardiac troponin I and copper levels and a considerable decrease in the levels of iron and zinc (P ≤ 0.01) in infected groups compared to the control animals. In vaccinated groups, hepcidin was increased but other parameters were not changed in comparison to the control group. It can be concluded that increase of homocysteine and cardiac troponin I in brucellosis could be a warning for cardiac adverse effects. Besides, increase of sphingosine 1-phosphate probably indicates its stimulant and modulatory effects in anti- Brucellosis biochemical pathways of the host. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sphingosine-1-phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti-myeloperoxidase antibody-positive IgG.

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Sun, Xiao-Jing; Chen, Min; Zhao, Ming-Hui

2018-03-01

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. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Early double-negative thymocyte export in Trypanosoma cruzi infection is restricted by sphingosine receptors and associated with human chagas disease.

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Ailin Lepletier

2014-10-01

Full Text Available The protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironmental and lymphoid compartments. Acute infection results in severe atrophy of the organ and early release of immature thymocytes into the periphery. To date, the pathophysiological effects of thymic changes promoted by parasite-inducing premature release of thymocytes to the periphery has remained elusive. Herein, we show that sphingosine-1-phosphate (S1P, a potent mediator of T cell chemotaxis, plays a role in the exit of immature double-negative thymocytes in experimental Chagas disease. In thymuses from T. cruzi-infected mice we detected reduced transcription of the S1P kinase 1 and 2 genes related to S1P biosynthesis, together with increased transcription of the SGPL1 sphingosine-1-lyase gene, whose product inactivates S1P. These changes were associated with reduced intrathymic levels of S1P kinase activity. Interestingly, double-negative thymocytes from infected animals expressed high levels of the S1P receptor during infection, and migrated to lower levels of S1P. Moreover, during T. cruzi infection, this thymocyte subset expresses high levels of IL-17 and TNF-α cytokines upon polyclonal stimulation. In vivo treatment with the S1P receptor antagonist FTY720 resulted in recovery the numbers of double-negative thymocytes in infected thymuses to physiological levels. Finally, we showed increased numbers of double-negative T cells in the peripheral blood in severe cardiac forms of human Chagas disease.

Exogenous sphingosine-1-phosphate boosts acclimatization in rats exposed to acute hypobaric hypoxia: assessment of haematological and metabolic effects.

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Sonam Chawla

Full Text Available The physiological challenges posed by hypobaric hypoxia warrant exploration of pharmacological entities to improve acclimatization to hypoxia. The present study investigates the preclinical efficacy of sphingosine-1-phosphate (S1P to improve acclimatization to simulated hypobaric hypoxia.Efficacy of intravenously administered S1P in improving haematological and metabolic acclimatization was evaluated in rats exposed to simulated acute hypobaric hypoxia (7620 m for 6 hours following S1P pre-treatment for three days.Altitude exposure of the control rats caused systemic hypoxia, hypocapnia (plausible sign of hyperventilation and respiratory alkalosis due to suboptimal renal compensation indicated by an overt alkaline pH of the mixed venous blood. This was associated with pronounced energy deficit in the hepatic tissue along with systemic oxidative stress and inflammation. S1P pre-treatment improved blood oxygen-carrying-capacity by increasing haemoglobin, haematocrit, and RBC count, probably as an outcome of hypoxia inducible factor-1α mediated erythropoiesis and renal S1P receptor 1 mediated haemoconcentation. The improved partial pressure of oxygen in the blood could further restore aerobic respiration and increase ATP content in the hepatic tissue of S1P treated animals. S1P could also protect the animals from hypoxia mediated oxidative stress and inflammation.The study findings highlight S1P's merits as a preconditioning agent for improving acclimatization to acute hypobaric hypoxia exposure. The results may have long term clinical application for improving physiological acclimatization of subjects venturing into high altitude for occupational or recreational purposes.

Sphingosine 1-phosphate-induced ICAM-1 expression via NADPH oxidase/ROS-dependent NF-kappaB cascade on human pulmonary alveolar epithelial cells

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Chin-Chung eLin

2016-03-01

Full Text Available The intercellular adhesion molecule-1 (ICAM-1 expression is frequently correlated with the lung inflammation. A bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P, was involved in inflammation through the adhesion molecules induction, and then caused lung injury. However, the transduction mechanisms of the S1P stimulation to induce ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs remain unclear. Here, we demonstrated that exposure of HPAEpiCs to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCdelta, PF431396 (PYK2, diphenyleneiodonium chloride (DPI, apocynin (NADPH oxidase, Edaravone (ROS, and Bay11-7082 (NF-kappaB. Consistently, knockdown with siRNA transfection of PKCdelta, PYK2, p47phox, and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A and Gi/o-coupled receptor antagonist (GPA2 also blocked S1P-induced ICAM-1 protein and mRNA expression. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCdelta-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-kappaB p65 phosphorylation and translocation from the cytosol to the nucleus in HPAEpiCs, which was inhibited by Rottlerin, PF431396, APO, DPI, or Edaravone. In the in vitro study, we established that S1P induced monocyte adhesion via an ICAM-1-dependent pathway. In the in vivo study, we found that S1P induced ICAM-1 protein and mRNA levels in the lung fractions, pulmonary hematoma, and leukocyte (mainly eosinophils and neutrophils count in bronchoalveolar lavage (BAL fluid in mice via a PKCdelta/PYK2/NADPH oxidase/ROS/NF-kappaB signaling pathway. We concluded that S1P may induce lung

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes[S

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Kobayashi, Naoki; Otsuka, Masato; Yamaguchi, Akihito; Nishi, Tsuyoshi

2016-01-01

Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers. PMID:27655910

Sphingosine-1-phosphate prevents chemotherapy-induced human primordial follicle death.

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Li, Fang; Turan, Volkan; Lierman, Sylvie; Cuvelier, Claude; De Sutter, Petra; Oktay, Kutluk

2014-01-01

Can Sphingosine-1-phosphate (S1P), a ceramide-induced death pathway inhibitor, prevent cyclophosphamide (Cy) or doxorubicin (Doxo) induced apoptotic follicle death in human ovarian xenografts? S1P can block human apoptotic follicle death induced by both drugs, which have differing mechanisms of cytotoxicity. S1P has been shown to decrease the impact of chemotherapy and radiation on germinal vesicle oocytes in animal studies but no human translational data exist. Experimental human ovarian xenografting to test the in vivo protective effect of S1P on primordial follicle survival in the chemotherapy setting. The data were validated by assessing the same protective effect in the ovaries of xenografted mice in parallel. Xenografted mice were treated with Cy (75 mg/kg), Cy+S1P (200 μM), Doxo (10 mg/kg), Doxo+S1P or vehicle only (Control). S1P was administered via continuous infusion using a mini-osmotic pump beginning 24 h prior to and ending 72 h post-chemotherapy. Grafts were then recovered and stained with anti-caspase 3 antibody for the detection of apoptosis in primordial follicles. The percentage of apoptotic to total primordial follicles was calculated in each group. Both Cy and Doxo resulted in a significant increase in apoptotic follicle death in human ovarian xenografts compared with controls (62.0 ± 3.9% versus 25.7 ± 7.4%, P 0.05). The findings from the ovaries of the severe combined immunodeficient mice mirrored the findings with human tissue. The functionality of the rescued human ovarian follicles needs to be evaluated in future studies though the studies in rodents showed that rescued oocytes can result in healthy offspring. In addition, the impact of S1P on cancer cells should be further studied. S1P and its future analogs hold promise for preserving fertility by pharmacological means for patients undergoing chemotherapy. This research is supported by NIH's NICHD and NCI (5R01HD053112-06 and 5R21HD061259-02) and the Flemish Foundation for Scientific

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

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Weske, Sarah; Vaidya, Mithila; Reese, Alina; von Wnuck Lipinski, Karin; Keul, Petra; Bayer, Julia K; Fischer, Jens W; Flögel, Ulrich; Nelsen, Jens; Epple, Matthias; Scatena, Marta; Schwedhelm, Edzard; Dörr, Marcus; Völzke, Henry; Moritz, Eileen; Hannemann, Anke; Rauch, Bernhard H; Gräler, Markus H; Heusch, Gerd; Levkau, Bodo

2018-05-01

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P 2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3β-β-catenin and WNT5A-LRP5 pathways. Accordingly, S1P 2 -deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis.

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Andreas Billich

Full Text Available BACKGROUND: Sphingosine-1-phosphate (S1P regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1. Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. METHODOLOGY: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. PRINCIPAL FINDINGS: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE. T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. SIGNIFICANCE: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.

Loss of Sphingosine Kinase Alters Life History Traits and Locomotor Function in Caenorhabditis elegans

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Jason P. Chan

2017-09-01

Full Text Available Sphingolipid metabolism is important to balance the abundance of bioactive lipid molecules involved in cell signaling, neuronal function, and survival. Specifically, the sphingolipid sphingosine mediates cell death signaling, whereas its phosphorylated form, sphingosine-1-phosphate (S1P, mediates cell survival signaling. The enzyme sphingosine kinase produces S1P, and the activity of sphingosine kinase impacts the ability of cells to survive under stress and challenges. To examine the influence of sphingolipid metabolism, particularly enzymes regulating sphingosine and S1P, in mediating aging, neuronal function and stress response, we examined life history traits, locomotor capacities and heat stress responses of young and old animals using the model organism Caenorhabditis elegans. We found that C. elegans sphk-1 mutants, which lack sphingosine kinase, had shorter lifespans, reduced brood sizes, and smaller body sizes compared to wild type animals. By analyzing a panel of young and old animals with genetic mutations in the sphingolipid signaling pathway, we showed that aged sphk-1 mutants exhibited a greater decline in neuromuscular function and locomotor behavior. In addition, aged animals lacking sphk-1 were more susceptible to death induced by acute and prolonged heat exposure. On the other hand, older animals with loss of function mutations in ceramide synthase (hyl-1, which converts sphingosine to ceramide, showed improved neuromuscular function and stress response with age. This phenotype was dependent on sphk-1. Together, our data show that loss of sphingosine kinase contributes to poor animal health span, suggesting that sphingolipid signaling may be important for healthy neuronal function and animal stress response during aging.

Sphingosine kinase-1, S1P transporter spinster homolog 2 and S1P2 mRNA expressions are increased in liver with advanced fibrosis in human.

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Sato, Masaya; Ikeda, Hitoshi; Uranbileg, Baasanjav; Kurano, Makoto; Saigusa, Daisuke; Aoki, Junken; Maki, Harufumi; Kudo, Hiroki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

2016-08-26

The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3-4 compared to those with 0-2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.

High density lipoprotein (HDL)-associated sphingosine 1-phosphate (S1P) inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression.

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Feuerborn, Renata; Becker, Susen; Potì, Francesco; Nagel, Petra; Brodde, Martin; Schmidt, Harmut; Christoffersen, Christina; Ceglarek, Uta; Burkhardt, Ralph; Nofer, Jerzy-Roch

2017-02-01

Macrophage apoptosis is critically involved in atherosclerosis. We here examined the effect of anti-atherogenic high density lipoprotein (HDL) and its component sphingosine-1-phosphate (S1P) on apoptosis in RAW264.7 murine macrophages. Mitochondrial or endoplasmic reticulum-dependent apoptosis was induced by exposure of macrophages to etoposide or thapsigargin/fukoidan, respectively. Cell death induced by these compounds was inhibited by S1P as inferred from reduced annexin V binding, TUNEL staining, and caspase 3, 9 and 12 activities. S1P induced expression of the inhibitor of apoptosis protein (IAP) family proteins cIAP1, cIAP2 and survivin, but only the inhibitor of survivin expression YM155 and not the cIAP1/2 blocker GDC0152 reversed the inhibitory effect of S1P on apoptosis. Moreover, S1P activated signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) and the stimulatory effect of S1P on survivin expression and inhibitory effects on apoptosis were attenuated by STAT3 or JAK2 inhibitors, S3I-201 or AG490, respectively. The effects of S1P on STAT3 activation, survivin expression and macrophage apoptosis were emulated by HDL, HDL lipids, and apolipoprotein (apo) M-containing HDL, but not by apoA-I or HDL deprived of S1P or apoM. In addition, JTE013 and CAY10444, S1P receptor 2 and 3 antagonists, respectively, compromised the S1P and HDL capacities to stimulate STAT3 activation and survivin expression, and to inhibit apoptosis. HDL-associated S1P inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression. The suppression of macrophage apoptosis may represent a novel mechanism utilized by HDL to exert its anti-atherogenic effects. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Modulation of sphingosine 1-phosphate (S1P) attenuates spatial learning and memory impairments in the valproic acid rat model of autism.

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Wu, Hongmei; Zhang, Quanzhi; Gao, Jingquan; Sun, Caihong; Wang, Jia; Xia, Wei; Cao, Yonggang; Hao, Yanqiu; Wu, Lijie

2018-03-01

Autism spectrum disorders (ASD) are a set of pervasive neurodevelopmental disorders that manifest in early childhood, and it is growing up to be a major cause of disability in children. However, the etiology and treatment of ASD are not well understood. In our previous study, we found that serum levels of sphingosine 1-phosphate (S1P) were increased significantly in children with autism, indicating that S1P levels may be involved in ASD. The objective of this study was to identify a link between increased levels of S1P and neurobehavioral changes in autism. We utilized a valproic acid (VPA) -induced rat model of autism to evaluate the levels of S1P and the expression of sphingosine kinase (SphK), a key enzyme for S1P production, in serum and hippocampal tissue. Furthermore, we assessed cognitive functional changes and histopathological and neurochemical alterations in VPA-exposed rats after SphK blockade to explore the possible link between increased levels of S1P and neurobehavioral changes in autism. We found that SphK2 and S1P are upregulated in hippocampal tissue from VPA-exposed rats, while pharmacological inhibition of SphK reduced S1P levels, attenuated spatial learning and memory impairments, increased the expression of phosphorylated CaMKII and CREB and autophagy-related proteins, inhibited cytochrome c release, decreased the expression of apoptosis related proteins, and protected against neuronal loss in the hippocampus. We have demonstrated that an increased level of SphK2/S1P is involved in the spatial learning and memory impairments of autism, and this signaling pathway represents a novel therapeutic target and direction for future studies.

Sphingosine-1-phosphate suppresses chondrosarcoma metastasis by upregulation of tissue inhibitor of metalloproteinase 3 through suppressing miR-101 expression.

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Tsai, Chun-Hao; Yang, Dong-Ying; Lin, Chih-Yang; Chen, Tsung-Ming; Tang, Chih-Hsin; Huang, Yuan-Li

2017-10-01

Chondrosarcoma is the second most common primary malignancy form of bone cancer, exhibiting resistance to chemotherapy and radiation therapy as well as developing high metastasis ability in late-stage tumors. Thus, understanding the metastatic processes of chondrosarcoma is considered a strategy for the treatment of this disease. Sphingosine 1-phosphate (S1P), a bioactive sphingolipid, is produced intracellularly by sphingosine kinase (SphK) and is regarded as a second signaling molecule that regulates inflammation, proliferation, angiogenesis, and metastasis. However, the effect of S1P on chondrosarcoma remains uncertain. As demonstrated by the transwell, immunoblotting, and real-time PCR analyses, we found that S1P inhibited cell migration and MMP-2 expression through the upregulation of the tissue inhibitor of metalloproteinase-3 (TIMP-3) expression in human chondrosarcoma cells. Additionally, we also showed that microRNA (miRNA)-101, which targets the 3' untranslated region (3'UTR) of TIMP-3, decreased significantly following S1P treatment. After transfection with miR-101 mimics, the S1P-regulated cell migration and TIMP-3 expression were both reversed. Furthermore, we also showed that the S1P-inhibited cell migration is mediated through the c-Src/MEK/ERK signaling axis. Meanwhile, the in vivo study indicated that overexpression of SphK1 decreases chondrosarcoma metastasis to the lungs. Our results illustrate the clinical significance between SphK1, TIMP-3, and miR-101 in human chondrosarcoma patients. Taken together, our results suggest that S1P and miR-101 may prove to be potential therapeutic targets for future chondrosarcoma treatment. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Serum-Sphingosine-1-Phosphate Concentrations Are Inversely Associated with Atherosclerotic Diseases in Humans.

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Irina Soltau

Full Text Available Atherosclerotic changes of arteries are the leading cause for deaths in cardiovascular disease and greatly impair patient's quality of life. Sphingosine-1-phosphate (S1P is a signaling sphingolipid that regulates potentially pro-as well as anti-atherogenic processes. Here, we investigate whether serum-S1P concentrations are associated with peripheral artery disease (PAD and carotid stenosis (CS.Serum was sampled from blood donors (controls, N = 174 and from atherosclerotic patients (N = 132 who presented to the hospital with either clinically relevant PAD (N = 102 or CS (N = 30. From all subjects, serum-S1P was measured by mass spectrometry and blood parameters were determined by routine laboratory assays. When compared to controls, atherosclerotic patients before invasive treatment to restore blood flow showed significantly lower serum-S1P levels. This difference cannot be explained by risk factors for atherosclerosis (old age, male gender, hypertension, hypercholesteremia, obesity, diabetes or smoking or comorbidities (Chronic obstructive pulmonary disease, kidney insufficiency or arrhythmia. Receiver operating characteristic curves suggest that S1P has more power to indicate atherosclerosis (PAD and CS than high density lipoprotein-cholesterol (HDL-C. In 35 patients, serum-S1P was measured again between one and six months after treatment. In this group, serum-S1P concentrations rose after treatment independent of whether patients had PAD or CS, or whether they underwent open or endovascular surgery. Post-treatment S1P levels were highly associated to platelet numbers measured pre-treatment.Our study shows that PAD and CS in humans is associated with decreased serum-S1P concentrations and that S1P may possess higher accuracy to indicate these diseases than HDL-C.

Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

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Smirnov, Anna; Pohlmann, Stephanie; Nehring, Melanie; Ali, Shafaqat; Mann-Nüttel, Ritu; Scheu, Stefanie; Antoni, Anne-Charlotte; Hansen, Wiebke; Büettner, Manuela; Gardiasch, Miriam J.; Westendorf, Astrid M.; Wirsdörfer, Florian; Pastille, Eva; Dudda, Marcel; Flohé, Stefanie B.

2017-01-01

Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs) secrete enhanced levels of interleukin (IL) 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP), a model for human polymicrobial sepsis. Bone marrow cells (BMC) were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs) were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR) 2, the receptor for C-C chemokine ligand (CCL) 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs) that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are activated

Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

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Anna Smirnov

2017-11-01

Full Text Available Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs secrete enhanced levels of interleukin (IL 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP, a model for human polymicrobial sepsis. Bone marrow cells (BMC were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR 2, the receptor for C-C chemokine ligand (CCL 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are

Individual variation of human S1P₁ coding sequence leads to heterogeneity in receptor function and drug interactions.

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Obinata, Hideru; Gutkind, Sarah; Stitham, Jeremiah; Okuno, Toshiaki; Yokomizo, Takehiko; Hwa, John; Hla, Timothy

2014-12-01

Sphingosine 1-phosphate receptor 1 (S1P₁), an abundantly-expressed G protein-coupled receptor which regulates key vascular and immune responses, is a therapeutic target in autoimmune diseases. Fingolimod/Gilenya (FTY720), an oral medication for relapsing-remitting multiple sclerosis, targets S1P₁ receptors on immune and neural cells to suppress neuroinflammation. However, suppression of endothelial S1P₁ receptors is associated with cardiac and vascular adverse effects. Here we report the genetic variations of the S1P₁ coding region from exon sequencing of >12,000 individuals and their functional consequences. We conducted functional analyses of 14 nonsynonymous single nucleotide polymorphisms (SNPs) of the S1PR1 gene. One SNP mutant (Arg¹²⁰ to Pro) failed to transmit sphingosine 1-phosphate (S1P)-induced intracellular signals such as calcium increase and activation of p44/42 MAPK and Akt. Two other mutants (Ile⁴⁵ to Thr and Gly³⁰⁵ to Cys) showed normal intracellular signals but impaired S1P-induced endocytosis, which made the receptor resistant to FTY720-induced degradation. Another SNP mutant (Arg¹³ to Gly) demonstrated protection from coronary artery disease in a high cardiovascular risk population. Individuals with this mutation showed a significantly lower percentage of multi-vessel coronary obstruction in a risk factor-matched case-control study. This study suggests that individual genetic variations of S1P₁ can influence receptor function and, therefore, infer differential disease risks and interaction with S1P₁-targeted therapeutics. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Sphingosine kinase 1 deficiency confers protection against hyperoxia-induced bronchopulmonary dysplasia in a murine model: role of S1P signaling and Nox proteins.

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Harijith, Anantha; Pendyala, Srikanth; Reddy, Narsa M; Bai, Tao; Usatyuk, Peter V; Berdyshev, Evgeny; Gorshkova, Irina; Huang, Long Shuang; Mohan, Vijay; Garzon, Steve; Kanteti, Prasad; Reddy, Sekhar P; Raj, J Usha; Natarajan, Viswanathan

2013-10-01

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Berberine reduces fibronectin expression by suppressing the S1P-S1P2 receptor pathway in experimental diabetic nephropathy models.

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Kaipeng Huang

Full Text Available The accumulation of glomerular extracellular matrix (ECM is one of the critical pathological characteristics of diabetic renal fibrosis. Fibronectin (FN is an important constituent of ECM. Our previous studies indicate that the activation of the sphingosine kinase 1 (SphK1-sphingosine 1- phosphate (S1P signaling pathway plays a key regulatory role in FN production in glomerular mesangial cells (GMCs under diabetic condition. Among the five S1P receptors, the activation of S1P2 receptor is the most abundant. Berberine (BBR treatment also effectively inhibits SphK1 activity and S1P production in the kidneys of diabetic models, thus improving renal injury. Based on these data, we further explored whether BBR could prevent FN production in GMCs under diabetic condition via the S1P2 receptor. Here, we showed that BBR significantly down-regulated the expression of S1P2 receptor in diabetic rat kidneys and GMCs exposed to high glucose (HG and simultaneously inhibited S1P2 receptor-mediated FN overproduction. Further, BBR also obviously suppressed the activation of NF-κB induced by HG, which was accompanied by reduced S1P2 receptor and FN expression. Taken together, our findings suggest that BBR reduces FN expression by acting on the S1P2 receptor in the mesangium under diabetic condition. The role of BBR in S1P2 receptor expression regulation could closely associate with its inhibitory effect on NF-κB activation.

Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling.

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Okajima, F; Tomura, H; Sho, K; Kimura, T; Sato, K; Im, D S; Akbar, M; Kondo, Y

1997-01-01

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(L-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTP gamma S-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.

Sphingosine-1-phosphate (S1P) activates STAT3 to protect against de novo acute heart failure (AHF).

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Deshpande, Gaurang P; Imamdin, Aqeela; Lecour, Sandrine; Opie, Lionel H

2018-03-01

Acute heart failure (AHF) is a burden disease, with high mortality and re-hospitalisations. Using an ex-vivo model of AHF, we have previously reported that sphingosine-1-phosphate (S1P) confers cardioprotection. However, the mechanisms remain to be elucidated. In the present study, we aimed to examine the role of the cardioprotective signal transducer and activator of transcription 3 (STAT3) in S1P mediated improved functional recovery in AHF. Isolated hearts from male Long-Evans rats were subjected to hypotensive AHF for 35 min followed by a recovery phase of 30 min (n ≥ 4/group). S1P (10 nM) was given during either the hypotensive or the recovery phase with/without an inhibitor of STAT3, AG490. Functional parameters were recorded throughout the experiment. Following an AHF insult, S1P, given during the recovery phase, improved the heart rate (HR) compared to the control (175.2 ± 30.7 vs. 71.6 ± 27.4 beats per minute (BPM); p S1P abolished the cardioprotective effect of S1P (42.3 ± 17.1 vs. 148.8 ± 26.4 BPM for S1P; p S1P protects in an ex-vivo rat heart model of AHF by activation of STAT3 and provide further evidence for the usage of S1P as a potential therapy in patients suffering from AHF. Copyright © 2018 Elsevier Inc. All rights reserved.

Fenretinide causes emphysema, which is prevented by sphingosine 1-phoshate.

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Masanori Yasuo

Full Text Available Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW, a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P (100 µg/kg BW. Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α, vascular endothelial growth factor (VEGF and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2, and nuclear factor (erythroid-derived 2-like 2 (Nrf2 protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of

Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter-endothelial communication.

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Solomon A Mensah

Full Text Available Vasculoprotective endothelium glycocalyx (GCX shedding plays a critical role in vascular disease. Previous work demonstrated that GCX degradation disrupts endothelial cell (EC gap junction connexin (Cx proteins, likely blocking interendothelial molecular transport that maintains EC and vascular tissue homeostasis to resist disease. Here, we focused on GCX regeneration and tested the hypothesis that vasculoprotective EC function can be stimulated via replacement of GCX when it is shed. We used EC with [i] intact heparan sulfate (HS, the most abundant GCX component; [ii] degraded HS; or [iii] HS that was restored after enzyme degradation, by cellular self-recovery or artificially. Artificial HS restoration was achieved via treatment with exogenous HS, with or without the GCX regenerator and protector sphingosine 1- phosphate (S1P. In these cells we immunocytochemically examined expression of Cx isotype 43 (Cx43 at EC borders and characterized Cx-containing gap junction activity by measuring interendothelial spread of gap junction permeable Lucifer Yellow dye. With intact HS, 60% of EC borders expressed Cx43 and dye spread to 2.88 ± 0.09 neighboring cells. HS degradation decreased Cx43 expression to 30% and reduced dye spread to 1.87± 0.06 cells. Cellular self-recovery of HS restored baseline levels of Cx43 and dye transfer. Artificial HS recovery with exogenous HS partially restored Cx43 expression to 46% and yielded dye spread to only 1.03 ± 0.07 cells. Treatment with both HS and S1P, recovered HS and restored Cx43 to 56% with significant dye transfer to 3.96 ± 0.23 cells. This is the first evidence of GCX regeneration in a manner that effectively restores vasculoprotective EC communication.

New extracellular factors in glioblastoma multiforme development: neurotensin, growth differentiation factor-15, sphingosine-1-phosphate and cytomegalovirus infection

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Korbecki, Jan; Gutowska, Izabela; Kojder, Ireneusz; Jeżewski, Dariusz; Goschorska, Marta; Łukomska, Agnieszka; Lubkowska, Anna; Chlubek, Dariusz; Baranowska-Bosiacka, Irena

2018-01-01

Recent years have seen considerable progress in understanding the biochemistry of cancer. For example, more significance is now assigned to the tumor microenvironment, especially with regard to intercellular signaling in the tumor niche which depends on many factors secreted by tumor cells. In addition, great progress has been made in understanding the influence of factors such as neurotensin, growth differentiation factor-15 (GDF-15), sphingosine-1-phosphate (S1P), and infection with cytomegalovirus (CMV) on the ‘hallmarks of cancer’ in glioblastoma multiforme. Therefore, in the present work we describe the influence of these factors on the proliferation and apoptosis of neoplastic cells, cancer stem cells, angiogenesis, migration and invasion, and cancer immune evasion in a glioblastoma multiforme tumor. In particular, we discuss the effect of neurotensin, GDF-15, S1P (including the drug FTY720), and infection with CMV on tumor-associated macrophages (TAM), microglial cells, neutrophil and regulatory T cells (Treg), on the tumor microenvironment. In order to better understand the role of the aforementioned factors in tumoral processes, we outline the latest models of intratumoral heterogeneity in glioblastoma multiforme. Based on the most recent reports, we discuss the problems of multi-drug therapy in treating glioblastoma multiforme. PMID:29467963

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

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2013-01-01

Background Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice. Methods We treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles. Results Short-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function. Conclusions These data show that S1P is

Combination of ciclopirox olamine and sphingosine-1-phosphate as granulation enhancer in diabetic wounds.

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Lim, Natalie Sheng Jie; Sham, Adeline; Chee, Stella Min Ling; Chan, Casey; Raghunath, Michael

2016-09-01

Granulation tissue formation requires a robust angiogenic response. As granulation tissue develops, collagen fibers are deposited and compacted. Forces generated in the wake of this process drive wound contraction to reduce the wound area. In diabetics, both angiogenesis and wound contraction are diminished leading to impaired wound healing. To emulate this pathology and to address it pharmacologically, we developed a wound healing model in the diabetic Zucker fatty rat and tested a topical proangiogenic strategy combining antifungal agent ciclopirox olamine (CPX) and lysophospholipid sphingosine-1-phosphate (S1P) to promote diabetic wound closure. In vitro, we demonstrated that CPX + S1P up-regulates a crucial driver of angiogenesis, hypoxia-inducible factor-1, in endothelial cells. Injection of CPX + S1P into subcutaneously implanted sponges in experimental rats showed, in an additive manner, a fivefold increased endothelial infiltration and lectin-perfused vessel length. We developed a splinted diabetic rodent model to achieve low wound contraction rates that are characteristic for the healing mode of diabetic ulcers in humans. We discovered specific dorsal sites that allowed for incremental full-thickness excisional wound depths from 1 mm (superficial) to 3 mm (deep). This enabled us to bring down wound contraction from 51% in superficial wounds to 8% in deep wounds. While the effects of topical gel treatment of CPX + S1P were masked by the rodent-characteristic dominant contraction in superficial wounds, they became clearly evident in deep diabetic wounds. Here, a fivefold increase of functional large vessels resulted in accelerated granulation tissue formulation, accompanied by a 40% increase of compacted thick collagen fibers. This was associated with substantially reduced matrix metalloproteinase-3 and -13 expression. These findings translated into a fivefold increase in granulation-driven contraction, promoting diabetic wound closure. With CPX

Effects of inhibition of serine palmitoyltransferase (SPT and sphingosine kinase 1 (SphK1 on palmitate induced insulin resistance in L6 myotubes.

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Agnieszka Mikłosz

Full Text Available BACKGROUND: The objective of this study was to examine the effects of short (2 h and prolonged (18 h inhibition of serine palmitoyltransferase (SPT and sphingosine kinase 1 (SphK1 on palmitate (PA induced insulin resistance in L6 myotubes. METHODS: L6 myotubes were treated simultaneously with either PA and myriocin (SPT inhibitor or PA and Ski II (SphK1inhibitor for different time periods (2 h and 18 h. Insulin stimulated glucose uptake was measured using radioactive isotope. Expression of insulin signaling proteins was determined using Western blot analyses. Intracellular sphingolipids content [sphinganine (SFA, ceramide (CER, sphingosine (SFO, sphingosine-1-phosphate (S1P] were estimated by HPLC. RESULTS: Our results revealed that both short and prolonged time of inhibition of SPT by myriocin was sufficient to prevent ceramide accumulation and simultaneously reverse palmitate induced inhibition of insulin-stimulated glucose transport. In contrast, prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3β/GSK3β ratio in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes. CONCLUSION: Myriocin is more effective in restoration of palmitate induced insulin resistance in L6 myocytes, despite of the time of SPT inhibition, comparing to SKII (a specific SphK1 inhibitor. Observed changes in insulin signaling proteins were related to the content of specific sphingolipids, namely to the reduction of ceramide. Interestingly, inactivation of SphK1 augmented the effect of PA induced insulin resistance in L6 myotubes, which was associated with further inhibition of insulin stimulated PKB and GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.

Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

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Berdyshev, Evgeny V.; Gorshkova, Irina; Usatyuk, Peter; Kalari, Satish; Zhao, Yutong; Pyne, Nigel J.; Pyne, Susan; Sabbadini, Roger A.; Garcia, Joe G. N.; Natarajan, Viswanathan

2011-01-01

Background Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility. Methodology/Principal Findings Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1Pint, and attenuated S1Pext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1Pint and potentiated motility of HPAECs to S1Pext or serum. S1Pext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1Pext or 4-deoxypyridoxine-dependent endothelial cell motility. Conclusions/Significance These results suggest S1Pext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL. PMID:21304987

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase.

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Evgeny V Berdyshev

Full Text Available BACKGROUND: Earlier we have shown that extracellular sphingosine-1-phosphate (S1P induces migration of human pulmonary artery endothelial cells (HPAECs through the activation of S1P(1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs and S1P lyase (S1PL, that regulate intracellular S1P accumulation, in HPAEC motility. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino-4-(p-Chlorophenyl Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P(int, and attenuated S1P(ext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P(int and potentiated motility of HPAECs to S1P(ext or serum. S1P(ext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting "inside-out" signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs. Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P(ext or 4-deoxypyridoxine-dependent endothelial cell motility. CONCLUSIONS/SIGNIFICANCE: These results suggest S1P(ext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.

Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

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Martin, Claire [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); Lafosse, Jean-Michel [CHU Toulouse, Hopital Rangueil, Service d' orthopedie et Traumatologie, Toulouse F-31000 (France); Malavaud, Bernard [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); CHU Toulouse, Hopital Rangueil, Service d' Urologie et de Transplantation Renale, Toulouse F-31000 (France); Cuvillier, Olivier, E-mail: olivier.cuvillier@ipbs.fr [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France)

2010-01-01

Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

A lipid binding domain in sphingosine kinase 2

International Nuclear Information System (INIS)

Don, Anthony S.; Rosen, Hugh

2009-01-01

The lipid second messenger sphingosine 1-phosphate (S1P) is a critical mediator of cellular proliferation and survival signals, and is essential for vasculogenesis and neurogenesis. S1P formation is catalysed by sphingosine kinases 1 and 2 (Sphk1 and Sphk2). We have found that the endogenous glycolipid sulfatide (3-O-sulfogalactosylceramide) binds to and inhibits the activity of Sphk2 and the closely related ceramide kinase (Cerk), but not Sphk1. Using sulfatide as a probe, we mapped the lipid binding domain to the N-terminus of Sphk2 (residues 1-175), a region of sequence that is absent in Sphk1, but aligns with a pleckstrin homology domain in Cerk. Accordingly, Sphk2 bound to phosphatidylinositol monophosphates but not to abundant cellular phospholipids. Deleting the N-terminal domain reduced Sphk2 membrane localisation in cells. We have therefore identified a lipid binding domain in Sphk2 that is important for the enzyme's sub-cellular localisation.

The relationship between the high-density lipoprotein (HDL)-associated sphingosine-1-phosphate (S1P) and coronary in-stent restenosis.

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Jing, Xiao-Dong; Wei, Xiao-Ming; Deng, Song-Bai; Du, Jian-Lin; Liu, Ya-Jie; She, Qiang

2015-06-15

High-density lipoprotein (HDL)-associated sphingosine-1-phosphate (S1P) contributed to several beneficial effects in the cardiovascular system. We explored the relationship between the HDL-S1P concentrations and coronary in-stent restenosis (ISR). Fifty consecutive patients with ISR and 50 normal control subjects were included. The serum S1P, HDL-S1P and clinical data were collected to explore the relationships between these parameters and ISR. The patients with ISR had significantly lower concentrations of serum S1P (96.10 ± 26.33 vs. 113.40 ± 32.72; P = 0.004) and HDL-S1P (32.81 ± 10.02 vs. 42.72 ± 11.75; P S1P: Quartile 1 (18.63-28.51 ng/ml), Quartile 2 (28.62-37.28 ng/ml), Quartile 3 (37.35-45.27 ng/ml), and Quartile 4 (45.59-79.36 ng/ml). The rates of ISR were 84%, 48%, 40% and 28%, respectively. The patients in Quartile 1 exhibited significantly higher rates of ISR compared with the other groups (P = 0.001). A multivariate stepwise logistic regression analysis indicated that HDL-S1P (OR = 0.846, 95% CI = 0.767-0.932, P = 0.001) was an independent predictor of ISR. An ROC analysis indicated that HDL-S1P = 30.37 ng/ml and had a 90% sensitivity and a 52% specificity in predicting ISR. HDL-S1P is an independent predictor of ISR, and patients with higher concentrations of HDL-S1P have a low risk of ISR. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of Sphingosine-1-Phosphate in Human Plasma Using Liquid Chromatography Coupled with Q-Tof Mass Spectrometry

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Egom, Emmanuel E.; Fitzgerald, Ross; Canning, Rebecca; Pharithi, Rebabonye B.; Murphy, Colin; Maher, Vincent

2017-01-01

Evidence suggests that high-density lipoprotein (HDL) components distinct from cholesterol, such as sphingosine-1-phosphate (S1P), may account for the anti-atherothrombotic effects attributed to this lipoprotein. The current method for the determination of plasma levels of S1P as well as levels associated with HDL particles is still cumbersome an assay method to be worldwide practical. Recently, a simplified protocol based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific quantification of plasma levels of S1P with good accuracy has been reported. This work utilized a triple quadrupole (QqQ)-based LC-MS/MS system. Here we adapt that method for the determination of plasma levels of S1P using a quadrupole time of flight (Q-Tof) based LC-MS system. Calibration curves were linear in the range of 0.05 to 2 µM. The lower limit of quantification (LOQ) was 0.05 µM. The concentration of S1P in human plasma was determined to be 1 ± 0.09 µM (n = 6). The average accuracy over the stated range of the method was found to be 100 ± 5.9% with precision at the LOQ better than 10% when predicting the calibration standards. The concentration of plasma S1P in the prepared samples was stable for 24 h at room temperature. We have demonstrated the quantification of plasma S1P using Q-Tof based LC-MS with very good sensitivity, accuracy, and precision that can used for future studies in this field. PMID:28820460

Fingolimod modulates microglial activation to augment markers of remyelination

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Baker David

2011-07-01

Full Text Available Abstract Introduction Microglial activation in multiple sclerosis has been postulated to contribute to long-term neurodegeneration during disease. Fingolimod has been shown to impact on the relapsing remitting phase of disease by modulating autoreactive T-cell egress from lymph organs. In addition, it is brain penetrant and has been shown to exert multiple effects on nervous system cells. Methods In this study, the impact of fingolimod and other sphingosine-1-phosphate receptor active molecules following lysophosphotidyl choline-induced demyelination was examined in the rat telencephalon reaggregate, spheroid cell culture system. The lack of immune system components allowed elucidation of the direct effects of fingolimod on CNS cell types in an organotypic situation. Results Following demyelination, fingolimod significantly augmented expression of myelin basic protein in the remyelination phase. This increase was not associated with changes in neurofilament levels, indicating de novo myelin protein expression not associated with axonal branching. Myelin wrapping was confirmed morphologically using confocal and electron microscopy. Increased remyelination was associated with down-regulation of microglial ferritin, tumor necrosis factor alpha and interleukin 1 during demyelination when fingolimod was present. In addition, nitric oxide metabolites and apoptotic effectors caspase 3 and caspase 7 were reduced during demyelination in the presence of fingolimod. The sphingosine-1-phosphate receptor 1 and 5 agonist BAF312 also increased myelin basic protein levels, whereas the sphingosine-1-phosphate receptor 1 agonist AUY954 failed to replicate this effect on remyelination. Conclusions The results presented indicate that modulation of S1P receptors can ameliorate pathological effectors associated with microglial activation leading to a subsequent increase in protein and morphological markers of remyelination. In addition, sphingosine-1-phosphate

Sphingosine-1-phosphate receptors: Zooming in on ligand-induced intracellular trafficking and its functional implications

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Verzijl, Dennis; Peters, Stephan L. M.; Alewijnse, Astrid E.

2010-01-01

Regulatory processes including receptor phosphorylation and intracellular trafficking, also referred to as receptor internalization, are important processes to terminate G protein-coupled receptor (GPCR) signaling. Compelling evidence now indicates that internalization of a receptor is not

Sphingosine kinase activity is not required for tumor cell viability.

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Karen Rex

Full Text Available Sphingosine kinases (SPHKs are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P. In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

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Schwalm, Stephanie; Doell, Frauke; Roemer, Isolde; Bubnova, Svetlana; Pfeilschifter, Josef; Huwiler, Andrea

2008-01-01

Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1α and HIF-2α, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1α or HIF-2α by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression

Modeling Tolerance Development for the Effect on Heart Rate of the Selective S1P1 Receptor Modulator Ponesimod.

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Lott, Dominik; Lehr, Thorsten; Dingemanse, Jasper; Krause, Andreas

2017-09-15

Ponesimod is a selective sphingosine-1-phosphate-1 (S1P 1 ) receptor modulator currently under investigation for the treatment of multiple sclerosis. S1P receptor modulators reduce heart rate following treatment initiation. This effect disappears with repeated dosing, enabling development of innovative uptitration regimens to optimize patient safety. There are currently no published pharmacokinetic/pharmacodynamic models describing the heart rate reduction of S1P receptor modulators in humans. The model developed here provides quantification of this effect for ponesimod. A direct-effect I max model with estimated maximum reduction of 45%, tolerance development, and circadian variation best described this effect. The pooled data from nine clinical studies enabled characterization of interindividual variability. The model was used to simulate different treatment regimens to compare the effect of high initial doses vs. gradual uptitration with respect to the occurrence of bradycardia. The results indicate a better safety profile when using gradual uptitration. The model allows studying dosing regimens not clinically tested in silico. © 2017 American Society for Clinical Pharmacology and Therapeutics.

Genetic evidence for involvement of neuronally expressed S1P₁ receptor in nociceptor sensitization and inflammatory pain.

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Norbert Mair

2011-02-01

Full Text Available Sphingosine-1-phosphate (S1P is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P₁ receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P₁ receptor. Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium.

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Fu, Panfeng; Ebenezer, David L; Berdyshev, Evgeny V; Bronova, Irina A; Shaaya, Mark; Harijith, Anantha; Natarajan, Viswanathan

2016-12-30

Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P 1 , in lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P 1 , but not S1P 2 or S1P 3 , with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P 1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2 . Taken together, these results suggest that HGF/c-Met-mediated lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P 1 . © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Selective coupling of the S1P3 receptor subtype to S1P-mediated RhoA activation and cardioprotection.

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Yung, Bryan S; Brand, Cameron S; Xiang, Sunny Y; Gray, Charles B B; Means, Christopher K; Rosen, Hugh; Chun, Jerold; Purcell, Nicole H; Brown, Joan Heller; Miyamoto, Shigeki

2017-02-01

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P 1 , S1P 2 , and S1P 3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to G q . We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα 13 but not Gα 12 , Gα q , or Gα i . Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P 3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P 3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P 3 KO mouse heart. CYM-51736, an S1P 3 -specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P 3 receptor- and Gα 13 -mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P 3 receptors could provide therapeutic benefits in ischemic heart disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation.

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Lavieu, Grégory; Scarlatti, Francesca; Sala, Giusy; Carpentier, Stéphane; Levade, Thierry; Ghidoni, Riccardo; Botti, Joëlle; Codogno, Patrice

2006-03-31

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1(wt)-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.

Manipulating Endothelial Progenitor Cell Homing with Sphingosine-1-Phosphate for Terapeutic Angiogenesis

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Williams, Priscilla Anne

Ischemic vascular diseases are the main cause of mortality worldwide and yet current therapies only delay disease progression and improve quality of life without addressing the fundamental problem of tissue loss. Within the field of tissue engineering, therapeutic angiogenesis provides a promising approach to alternatively provide new blood vessel formation via spatiotemporally controlled delivery of proangiogenic agents. Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid that is upregulated under ischemic conditions, has recently gained great enthusiasm as a potential mediator in neovascularization strategies given its essential roles in promoting both neovessel formation and stabilization, and cellular trafficking along highly regulated endogenous gradients. Herein, the governing hypothesis guiding this dissertation is that local biomaterial-controlled delivery of S1P may be used to enhance migration and recruitment of vascular progenitor cells for enhanced therapeutic angiogenesis within ischemic tissue. The initial work in this dissertation investigated the effect of hypoxia on the angiogenic response of both mature and progenitor endothelial cells to S1P stimulation in vitro. Outgrowth endothelial cells (OECs) were isolated from human umbilical cord blood to provide a clinically relevant source of vascular progenitor cells for the studies conducted within this dissertation. S1P stimulation promoted angiogenic activity of both ECs and OECs under both ambient and hypoxic (1%) oxygen tensions. Furthermore, dual therapy with the combination of S1P and vascular endothelial growth factor (VEGF) further enhanced cellular responses. Interestingly, hypoxia substantially augmented the functional response of OECs to S1P, resulting in 25-fold and 6.5-fold increases in directed migration and sprouting, respectively. Thus, these studies highlighted the potential for S1P as a therapeutic agent for treatment of ischemic diseases. An injectable biomaterial system

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons

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Qi, Yanmei; Mair, Norbert; Kummer, Kai K.; Leitner, Michael G.; Camprubí-Robles, María; Langeslag, Michiel; Kress, Michaela

2018-01-01

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl− current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl− current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl− current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl− currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception. PMID:29479306

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons

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Yanmei Qi

2018-02-01

Full Text Available Sphingosine-1-phosphate (S1P is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl− current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl− current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl− current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl− currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

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Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

2015-01-01

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

Plasma sphingosine-1-phosphate is elevated in obesity.

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Greg M Kowalski

Full Text Available BACKGROUND: Dysfunctional lipid metabolism is a hallmark of obesity and insulin resistance and a risk factor for various cardiovascular and metabolic complications. In addition to the well known increase in plasma triglycerides and free fatty acids, recent work in humans and rodents has shown that obesity is associated with elevations in the bioactive class of sphingolipids known as ceramides. However, in obesity little is known about the plasma concentrations of sphinogsine-1-phosphate (S1P, the breakdown product of ceramide, which is an important signaling molecule in mammalian biology. Therefore, the purpose of this study was to examine the impact of obesity on circulating S1P concentration and its relationship with markers of glucose metabolism and insulin sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: Plasma S1P levels were determined in high-fat diet (HFD-induced and genetically obese (ob/ob mice along with obese humans. Circulating S1P was elevated in both obese mouse models and in obese humans compared with lean healthy controls. Furthermore, in humans, plasma S1P positively correlated with total body fat percentage, body mass index (BMI, waist circumference, fasting insulin, HOMA-IR, HbA1c (%, total and LDL cholesterol. In addition, fasting increased plasma S1P levels in lean healthy mice. CONCLUSION: We show that elevations in plasma S1P are a feature of both human and rodent obesity and correlate with metabolic abnormalities such as adiposity and insulin resistance.

Exercise increases sphingoid base-1-phosphate levels in human blood and skeletal muscle in a time- and intensity-dependent manner

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Baranowski, Marcin; Błachnio-Zabielska, Agnieszka U; Charmas, Małgorzata

2015-01-01

PURPOSE: Sphingosine-1-phosphate (S1P) regulates cardiovascular function and plays an important role in muscle biology. We have previously reported that cycling exercise increased plasma S1P. Here, we investigated the effect of exercise duration and intensity on plasma and skeletal muscle S1P lev...

Sphingosine-1-phosphate promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes under the designated culturing conditions

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Zhang Henggui

2011-06-01

Full Text Available Abstract Background It is of growing interest to develop novel approaches to initiate differentiation of mesenchymal stem cells (MSCs into cardiomyocytes. The purpose of this investigation was to determine if Sphingosine-1-phosphate (S1P, a native circulating bioactive lipid metabolite, plays a role in differentiation of human umbilical cord mesenchymal stem cells (HUMSCs into cardiomyocytes. We also developed an engineered cell sheet from these HUMSCs derived cardiomyocytes by using a temperature-responsive polymer, poly(N-isopropylacrylamide (PIPAAm cell sheet technology. Methods Cardiomyogenic differentiation of HUMSCs was performed by culturing these cells with either designated cardiomyocytes conditioned medium (CMCM alone, or with 1 μM S1P; or DMEM with 10% FBS + 1 μM S1P. Cardiomyogenic differentiation was determined by immunocytochemical analysis of expression of cardiomyocyte markers and patch clamping recording of the action potential. Results A cardiomyocyte-like morphology and the expression of α-actinin and myosin heavy chain (MHC proteins can be observed in both CMCM culturing or CMCM+S1P culturing groups after 5 days' culturing, however, only the cells in CMCM+S1P culture condition present cardiomyocyte-like action potential and voltage gated currents. A new approach was used to form PIPAAm based temperature-responsive culture surfaces and this successfully produced cell sheets from HUMSCs derived cardiomyocytes. Conclusions This study for the first time demonstrates that S1P potentiates differentiation of HUMSCs towards functional cardiomyocytes under the designated culture conditions. Our engineered cell sheets may provide a potential for clinically applicable myocardial tissues should promote cardiac tissue engineering research.

Crosstalk between phospholipase D and sphingosine kinase in plant stress signaling

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Xuemin eWang

2012-03-01

Full Text Available The activation of phospholipase D (PLD produces phosphatidic acid (PA, whereas sphingosine kinase (SPHK phosphorylates long-chain bases (LCBs to generate LCB-1-phosphates (LCBPs such as phytosphingosine-1-phosphate (phyto-S1P. PA and phyto-S1P have been identified as lipid messengers. Recent studies have shown that PA interacts directly with SPHKs in Arabidopsis, and that the interaction promotes SPHK activity. However, SPHK and phyto-S1P act upstream of PLDα1 and PA in the stomatal response to abscisic acid (ABA. These findings indicate that SPHK/phyto-S1P and PLD/PA are co-dependent in the amplification of lipid messengers, and that crosstalk between the sphingolipid- and phospholipid-mediated signaling pathways may play important roles in plant stress signaling.

Differential Effects of Long Term FTY720 Treatment on Endothelial versus Smooth Muscle Cell Signaling to S1P in Rat Mesenteric Arteries

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Shishavan, Mahdi Hamidi; Bidadkosh, Arash; Yazdani, Saleh; Lambooy, Sebastiaan; van den Born, Jacob; Buikema, Hendrik; Henning, Robert H.; Deelman, Leo E.

2016-01-01

The sphingosine-1-phosphate (S1P) analog FTY720 exerts pleiotropic effects on the cardiovascular system and causes down-regulation of S1P receptors. Myogenic constriction is an important mechanism regulating resistance vessel function and is known to be modulated by S1P. Here we investigated

The effect of S1P receptor signaling pathway on the survival and drug resistance in multiple myeloma cells.

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Fu, Di; Li, Yingchun; Li, Jia; Shi, Xiaoyan; Yang, Ronghui; Zhong, Yuan; Wang, Huihan; Liao, Aijun

2017-01-01

Multiple myeloma (MM) remains incurable by conventional chemotherapy. Sphingosine-1-phosphate (S1P) receptor-mediated signaling has been recently demonstrated to have critical roles in cell survival and drug resistance in a number of hematological malignancies. To dissect the roles of S1P receptor pathway in MM, we systematically examined cell viability and protein expression associated with cell survival and drug resistance in MM cell lines upon treatment with either pathway activator (S1P) or inhibitor (FTY720). Our results reveal that FTY720 inhibits cell proliferation by downregulating expression of target genes, while S1P has an opposite effect. Knocking down of S1P receptor S1P5R results in a reduction of cell survival-related gene expression; however, it does not have impacts on expression of drug resistance genes. These results suggest that S1P signaling plays a role in cell proliferation and drug resistance in MM, and targeting this pathway will provide a new therapeutic direction for MM management.

Blocking S1P interaction with S1P1 receptor by a novel competitive S1P1-selective antagonist inhibits angiogenesis

International Nuclear Information System (INIS)

Fujii, Yasuyuki; Ueda, Yasuji; Ohtake, Hidenori; Ono, Naoya; Takayama, Tetsuo; Nakazawa, Kiyoshi; Igarashi, Yasuyuki; Goitsuka, Ryo

2012-01-01

Highlights: ► The effect of a newly developed S1P 1 -selective antagonist on angiogenic responses. ► S1P 1 is a critical component of VEGF-related angiogenic responses. ► S1P 1 -selective antagonist showed in vitro activity to inhibit angiogenesis. ► S1P 1 -selective antagonist showed in vivo activity to inhibit angiogenesis. ► The efficacy of S1P 1 -selective antagonist for anti-cancer therapies. -- Abstract: Sphingosine 1-phosphate receptor type 1 (S1P 1 ) was shown to be essential for vascular maturation during embryonic development and it has been demonstrated that substantial crosstalk exists between S1P 1 and other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. We developed a novel S1P 1 -selective antagonist, TASP0277308, which is structurally unrelated to S1P as well as previously described S1P 1 antagonists. TASP0277308 inhibited S1P- as well as VEGF-induced cellular responses, including migration and proliferation of human umbilical vein endothelial cells. Furthermore, TASP0277308 effectively blocked a VEGF-induced tube formation in vitro and significantly suppressed tumor cell-induced angiogenesis in vivo. These findings revealed that S1P 1 is a critical component of VEGF-related angiogenic responses and also provide evidence for the efficacy of TASP0277308 for anti-cancer therapies.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.

Science.gov (United States)

He, Xiaoli; H'ng, Shiau-Chen; Leong, David T; Hutmacher, Dietmar W; Melendez, Alirio J

2010-08-01

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Fingolimod phosphate attenuates oligomeric amyloid β-induced neurotoxicity via increased brain-derived neurotrophic factor expression in neurons.

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Yukiko Doi

Full Text Available The neurodegenerative processes that underlie Alzheimer's disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P receptor (S1PR agonist fingolimod phosphate (FTY720-P-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimer's disease.

Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

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Lin, Chih-Chung; Lee, I-Ta; Hsu, Chun-Hao; Hsu, Chih-Kai; Chi, Pei-Ling; Hsiao, Li-Der; Yang, Chuen-Mao

2015-01-01

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation. PMID:25734900

Sphingosine Kinase 2 and Ceramide Transport as Key Targets of the Natural Flavonoid Luteolin to Induce Apoptosis in Colon Cancer Cells.

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Loubna Abdel Hadi

Full Text Available The plant flavonoid luteolin exhibits different biological effects, including anticancer properties. Little is known on the molecular mechanisms underlying its actions in colorectal cancer (CRC. Here we investigated the effects of luteolin on colon cancer cells, focusing on the balance between ceramide and sphingosine-1-phosphate (S1P, two sphingoid mediators with opposite roles on cell fate. Using cultured cells, we found that physiological concentrations of luteolin induce the elevation of ceramide, followed by apoptotic death of colon cancer cells, but not of differentiated enterocytes. Pulse studies revealed that luteolin inhibits ceramide anabolism to complex sphingolipids. Further experiments led us to demonstrate that luteolin induces an alteration of the endoplasmic reticulum (ER-Golgi flow of ceramide, pivotal to its metabolic processing to complex sphingolipids. We report that luteolin exerts its action by inhibiting both Akt activation, and sphingosine kinase (SphK 2, with the consequent reduction of S1P, an Akt stimulator. S1P administration protected colon cancer cells from luteolin-induced apoptosis, most likely by an intracellular, receptor-independent mechanism. Overall this study reveals for the first time that the dietary flavonoid luteolin exerts toxic effects on colon cancer cells by inhibiting both S1P biosynthesis and ceramide traffic, suggesting its dietary introduction/supplementation as a potential strategy to improve existing treatments in CRC.

Oncogenic S1P signalling in EBV-associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3.

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Lee, Hui Min; Lo, Kwok-Wai; Wei, Wenbin; Tsao, Sai Wah; Chung, Grace Tin Yun; Ibrahim, Maha Hafez; Dawson, Christopher W; Murray, Paul G; Paterson, Ian C; Yap, Lee Fah

2017-05-01

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1, and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transforming growth factor-β-sphingosine kinase 1/S1P signaling upregulates microRNA-21 to promote fibrosis in renal tubular epithelial cells.

Science.gov (United States)

Liu, Xiujuan; Hong, Quan; Wang, Zhen; Yu, Yanyan; Zou, Xin; Xu, Lihong

2016-02-01

Renal fibrosis is a progressive pathological change characterized by tubular cell apoptosis, tubulointerstitial fibroblast proliferation, and excessive deposition of extracellular matrix (ECM). miR-21 has been implicated in transforming growth factor-β (TGF-β)-stimulated tissue fibrosis. Recent studies showed that sphingosine kinase/sphingosine-1-phosphate (SphK/S1P) are also critical for TGF-β-stimulated tissue fibrosis; however, it is not clear whether SphK/S1P interacts with miR-21 or not. In this study, we hypothesized that SphK/S1P signaling is linked to upregulation of miR-21 by TGF-β. To verify this hypothesis, we first determined that miR-21 was highly expressed in renal tubular epithelial cells (TECs) stimulated with TGF-β by using qRT-PCR and Northern blotting. Simultaneously, inhibition of miR-21, mediated by the corresponding antimir, markedly decreased the expression and deposition of type I collagen, fibronectin (Fn), cysteine-rich protein 61 (CCN1), α-smooth muscle actin, and fibroblast-specific protein1 in TGF-β-treated TECs. ELISA and qRT-PCR were used to measure the S1P and SphK1 levels in TECs. S1P production was induced by TGF-β through activation of SphK1. Furthermore, it was observed that TGF-β-stimulated upregulation of miR-21 was abolished by SphK1 siRNA and was restored by the addition of exogenous S1P. Blocking S1PR2 also inhibited upregulation of miR-21. Additionally, miR-21 overexpression attenuated the repression of TGF-β-stimulated ECM deposition and epithelial-mesenchymal transition by SphK1 and S1PR2 siRNA. In summary, our study demonstrates a link between SphK1/S1P and TGF-β-induced miR-21 in renal TECs and may represent a novel therapeutic target in renal fibrosis. © 2015 by the Society for Experimental Biology and Medicine.

Expression of S1P metabolizing enzymes and receptors correlate with survival time and regulate cell migration in glioblastoma multiforme.

Science.gov (United States)

Bien-Möller, Sandra; Lange, Sandra; Holm, Tobias; Böhm, Andreas; Paland, Heiko; Küpper, Johannes; Herzog, Susann; Weitmann, Kerstin; Havemann, Christoph; Vogelgesang, Silke; Marx, Sascha; Hoffmann, Wolfgang; Schroeder, Henry W S; Rauch, Bernhard H

2016-03-15

A signaling molecule which is involved in proliferation and migration of malignant cells is the lipid mediator sphingosine-1-phosphate (S1P). There are hints for a potential role of S1P signaling in malignant brain tumors such as glioblastoma multiforme (GBM) which is characterized by a poor prognosis. Therefore, a comprehensive expression analysis of S1P receptors (S1P1-S1P5) and S1P metabolizing enzymes in human GBM (n = 117) compared to healthy brain (n = 10) was performed to evaluate their role for patient´s survival. Furthermore, influence of S1P receptor inhibition on proliferation and migration were studied in LN18 GBM cells. Compared to control brain, mRNA levels of S1P1, S1P2, S1P3 and S1P generating sphingosine kinase-1 were elevated in GBM. Kaplan-Meier analyses demonstrated an association between S1P1 and S1P2 with patient´s survival times. In vitro, an inhibitory effect of the SphK inhibitor SKI-II on viability of LN18 cells was shown. S1P itself had no effect on viability but stimulated LN18 migration which was blocked by inhibition of S1P1 and S1P2. The participation of S1P1 and S1P2 in LN18 migration was further supported by siRNA-mediated silencing of these receptors. Immunoblots and inhibition experiments suggest an involvement of the PI3-kinase/AKT1 pathway in the chemotactic effect of S1P in LN18 cells.In summary, our data argue for a role of S1P signaling in proliferation and migration of GBM cells. Individual components of the S1P pathway represent prognostic factors for patients with GBM. Perspectively, a selective modulation of S1P receptor subtypes could represent a therapeutic approach for GBM patients and requires further evaluation.

Lysophospholipid Receptors Are Differentially Expressed in Rat Terminal Schwann Cells, As Revealed by a Single Cell RT-PCR and In Situ Hybridization

International Nuclear Information System (INIS)

Kobashi, Hiroaki; Yaoi, Takeshi; Oda, Ryo; Okajima, Seiichiro; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Fushiki, Shinji

2006-01-01

Terminal Schwann cells (TSCs) that cover motor neuron terminals, are known to play an important role in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, the molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. By using our previously reported method of selectively and efficiently collecting TSCs, we have analyzed the difference in expression patterns of lysophospholipid (LPL) receptor genes (LPA 1 , LPA 2 , LPA 3 , S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 ) between TSCs and myelinating Schwann cells (MSCs). LPL, which includes lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), is the bioactive lipid that induces a myriad of cellular responses through specific members of G-protein coupled receptors for LPA. It turned out that LPA 3 was expressed only in TSCs, whereas S1P 1 was expressed in TSCs and skeletal muscle, but not in MSCs. Other types of LPL receptor genes, including LPA 1 , S1P 2 , S1P 3 , S1P 4 , were expressed in both types of Schwann cells. None of the LPL receptor gene family showed MSCs-specific expression

Sphingosine kinase-2 maintains viral latency and survival for KSHV-infected endothelial cells.

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Lu Dai

Full Text Available Phosphorylation of sphingosine by sphingosine kinases (SphK1 and SphK2 generates sphingosine-1-phosphate (S1P, a bioactive sphingolipid which promotes cancer cell survival and tumor progression in vivo. We have recently reported that targeting SphK2 induces apoptosis for human primary effusion lymphoma (PEL cell lines infected by the Kaposi's sarcoma-associated herpesvirus (KSHV, and this occurs in part through inhibition of canonical NF-κB activation. In contrast, pharmacologic inhibition of SphK2 has minimal impact for uninfected B-cell lines or circulating human B cells from healthy donors. Therefore, we designed additional studies employing primary human endothelial cells to explore mechanisms responsible for the selective death observed for KSHV-infected cells during SphK2 targeting. Using RNA interference and a clinically relevant pharmacologic approach, we have found that targeting SphK2 induces apoptosis selectively for KSHV-infected endothelial cells through induction of viral lytic gene expression. Moreover, this effect occurs through repression of KSHV-microRNAs regulating viral latency and signal transduction, including miR-K12-1 which targets IκBα to facilitate activation of NF-κB, and ectopic expression of miR-K12-1 restores NF-κB activation and viability for KSHV-infected endothelial cells during SphK2 inhibition. These data illuminate a novel survival mechanism and potential therapeutic target for KSHV-infected endothelial cells: SphK2-associated maintenance of viral latency.

Estrogen-Modulated Response of Breast Cancer To Vitamin D and Its Analogs: Role of IGF

National Research Council Canada - National Science Library

Dolezalova, Hana

1999-01-01

... (LPA) and sphingosine 1-phosphate (S1P). Estrogen receptor positive and estrogen receptor negative cells express predominantly Edg-2 and Edg-4 Rs for LPA and Edg-3 for Sip, which transduce proliferative responses by direct nuclear signaling...

Differential S1P Receptor Profiles on M1- and M2-Polarized Macrophages Affect Macrophage Cytokine Production and Migration.

Science.gov (United States)

Müller, Jan; von Bernstorff, Wolfram; Heidecke, Claus-Dieter; Schulze, Tobias

2017-01-01

Introduction . Macrophages are key players in complex biological processes. In response to environmental signals, macrophages undergo polarization towards a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that acts via 5 G-protein coupled receptors (S1P 1-5 ) in order to influence a broad spectrum of biological processes. This study assesses S1P receptor expression on macrophages before and after M1 and M2 polarization and performs a comparative analysis of S1P signalling in the two activational states of macrophages. Methods . Bone marrow derived macrophages (BMDM) from C57 BL/6 mice were cultured under either M1- or M2-polarizing conditions. S1P-receptor expression was determined by quantitative RT-PCR. Influence of S1P on macrophage activation, migration, phagocytosis, and cytokine secretion was assessed in vitro. Results . All 5 S1P receptor subclasses were expressed in macrophages. Culture under both M1- and M2-polarizing conditions led to significant downregulation of S1P 1 . In contrast, M1-polarized macrophages significantly downregulated S1P 4 . The expression of the remaining three S1P receptors did not change. S1P increased expression of iNOS under M2-polarizing conditions. Furthermore, S1P induced chemotaxis in M1 macrophages and changed cytokine production in M2 macrophages. Phagocytosis was not affected by S1P-signalling. Discussion . The expression of different specific S1P receptor profiles may provide a possibility to selectively influence M1- or M2-polarized macrophages.

Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard.

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Mirzaian, Mina; Wisse, Patrick; Ferraz, Maria J; Marques, André R A; Gabriel, Tanit L; van Roomen, Cindy P A A; Ottenhoff, Roelof; van Eijk, Marco; Codée, Jeroen D C; van der Marel, Gijsbert A; Overkleeft, Herman S; Aerts, Johannes M

2016-08-01

We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels. Earlier it was reported that S1P is elevated in plasma of Fabry disease patients. We investigated this with the improved quantification. No clear conclusion could be drawn for patient plasma samples given the lack of uniformity of blood collection and plasma preparation. To still obtain insight, plasma and tissues were identically collected from α-galactosidase A deficient Fabry mice and matched control animals. No significant difference was observed in plasma S1P levels. A significant 2.3 fold increase was observed in kidney of Fabry mice, but not in liver and heart. Comparative analysis of S1P in cultured fibroblasts from normal subjects and classically affected Fabry disease males revealed no significant difference. In conclusion, accurate quantification of S1P in biological materials is feasible by mass spectrometry using the internal standards (13)C5 C18-S1P or C17-S1P. Significant local increases of S1P in the kidney might occur in Fabry disease as suggested by the mouse model. Copyright © 2016 Elsevier B.V. All rights reserved.

Sphingosine-1-Phosphate (S1P) Is a Feasible Biomarker in Predicting the Efficacy of Polymyxin B-Immobilized Fiber Direct Hemoperfusion (PMX-DHP) in Patients with Septic Shock.

Science.gov (United States)

Inoue, Satoshi; Sakamoto, Yuichiro; Koami, Hiroyuki; Yamada C, Kosuke; Nagashima, Futoshi; Miike, Toru; Iwamura, Takashi; Obata, Toru

2018-01-01

The aim of this study was to identify a useful biomarker to predict the efficacy of polymyxin B-immobilized fiber direct hemoperfusion (PMX-DHP) in patients with septic shock. The 44 patients included in this study were divided into two groups. Group A had an increase in systolic blood pressure (SBP) over 30 mmHg after PMX-DHP treatment. Group B had an increase in SBP less than 30 mmHg after PMX-DHP treatment. We evaluated the clinical characteristics and demographics of both groups. We also assessed whether the cause of sepsis affected the efficacy of PMX-DHP and compared the prognosis of both groups. Finally, we investigated whether there were any significant differences in the levels of sepsis-related biomarkers, including sphingosine-1-phosphate (S1P), between both groups before PMX-DHP in an effort to identify a biomarker that could predict the efficacy of PMX-DHP. PMX-DHP significantly increased SBP regardless of the cause of sepsis. Although there was some tendency, PMX-DHP did not significantly improve the prognosis of effective cases in comparison with non-effective cases, probably because of the limited number of patients included. Among the sepsis-related biomarkers, only S1P values were significantly different between the two groups before PMX-DHP, and S1P levels were significantly increased after treatment in the effective cases. S1P levels prior to PMX-DHP can be used to predict its efficacy. In addition, continuous monitoring of S1P levels can indicate the effectiveness of PMX-DHP in patients with septic shock.

Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

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Cliona M Stapleton

2011-04-01

Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

Calcium Overload Accelerates Phosphate-Induced Vascular Calcification Via Pit-1, but not the Calcium-Sensing Receptor.

Science.gov (United States)

Masumoto, Asuka; Sonou, Tomohiro; Ohya, Masaki; Yashiro, Mitsuru; Nakashima, Yuri; Okuda, Kouji; Iwashita, Yuko; Mima, Toru; Negi, Shigeo; Shigematsu, Takashi

2017-07-01

Vascular calcification (VC) is a risk factor of cardiovascular and all-cause mortality in patients with chronic kidney disease (CKD). CKD-mineral and bone metabolism disorder is an important problem in patients with renal failure. Abnormal levels of serum phosphate and calcium affect CKD-mineral and bone metabolism disorder and contribute to bone disease, VC, and cardiovascular disease. Hypercalcemia is a contributing factor in progression of VC in patients with CKD. However, the mechanisms of how calcium promotes intracellular calcification are still unclear. This study aimed to examine the mechanisms underlying calcium-induced calcification in a rat aortic tissue culture model. Aortic segments from 7-week-old male Sprague-Dawley rats were cultured in serum-supplemented medium for 10 days. We added high calcium (HiCa; calcium 3.0 mM) to high phosphate (HPi; phosphate 3.8 mM) medium to accelerate phosphate and calcium-induced VC. We used phosphonoformic acid and the calcimimetic R-568 to determine whether the mechanism of calcification involves Pit-1 or the calcium-sensing receptor. Medial VC was significantly augmented by HPi+HiCa medium compared with HPi alone (300%, p＜0.05), and was associated with upregulation of Pit-1 protein. Pit-1 protein concentrations in HPi+HiCa medium were greater than those in HPi medium. Phosphonoformic acid completely negated the augmentation of medial VC induced by HPi+HiCa. R-568 had no additive direct effect on medial VC. These results indicated that exposure to HPi+HiCa accelerates medial VC, and this is mediated through Pit-1, not the calcium-sensing receptor.

Divergent Synthesis of Diastereomeric Sphingosines from a Chiral Aziridine

Energy Technology Data Exchange (ETDEWEB)

Kang, On-Yu; Shin, Mi-Ri; Kang, Han-Young [Chungbuk National University, Gongju (Korea, Republic of)

2016-07-15

All four stereoisomers of sphingosines were synthesized starting from a single intermediate, chiral aziridine (2), which was efficiently prepared by enzymatic desymmetrization in an enatiopure form. Aziridine (2) was converted to 3, which was used for the synthesis of 4. Both the advanced key intermediates, vinylaziridines 3 and 4, were successfully converted to threo-sphingosines 1a and 1b, respectively. Ring-closing metathesis (RCM) using the Grubbs II catalyst was the key reaction in the synthesis. Two erythro-sphingosines 1c and 1d were synthesized by the ring-expansion reactions of vinylaziridines 3 and 4, followed by RCM reactions. The successful divergent synthesis confirmed that chiral vinylaziridine 2 can be used as a key intermediate for the synthesis of sphingosine-related natural products.

Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence.

Science.gov (United States)

Uranbileg, Baasanjav; Ikeda, Hitoshi; Kurano, Makoto; Enooku, Kenichiro; Sato, Masaya; Saigusa, Daisuke; Aoki, Junken; Ishizawa, Takeaki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

2016-01-01

Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.

Blocking S1P interaction with S1P{sub 1} receptor by a novel competitive S1P{sub 1}-selective antagonist inhibits angiogenesis

Energy Technology Data Exchange (ETDEWEB)

Fujii, Yasuyuki, E-mail: y.fujii@po.rd.taisho.co.jp [Department of Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co. Ltd., 1-403 Saitama, Saitama 331-9530 (Japan); Ueda, Yasuji; Ohtake, Hidenori; Ono, Naoya; Takayama, Tetsuo; Nakazawa, Kiyoshi [Department of Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co. Ltd., 1-403 Saitama, Saitama 331-9530 (Japan); Igarashi, Yasuyuki [Laboratory of Biomembrane and Biofunctional Chemistry, Hokkaido University, Sapporo, Hokkaido 060-0812 (Japan); Goitsuka, Ryo [Division of Development and Aging, Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022 (Japan)

2012-03-23

Highlights: Black-Right-Pointing-Pointer The effect of a newly developed S1P{sub 1}-selective antagonist on angiogenic responses. Black-Right-Pointing-Pointer S1P{sub 1} is a critical component of VEGF-related angiogenic responses. Black-Right-Pointing-Pointer S1P{sub 1}-selective antagonist showed in vitro activity to inhibit angiogenesis. Black-Right-Pointing-Pointer S1P{sub 1}-selective antagonist showed in vivo activity to inhibit angiogenesis. Black-Right-Pointing-Pointer The efficacy of S1P{sub 1}-selective antagonist for anti-cancer therapies. -- Abstract: Sphingosine 1-phosphate receptor type 1 (S1P{sub 1}) was shown to be essential for vascular maturation during embryonic development and it has been demonstrated that substantial crosstalk exists between S1P{sub 1} and other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. We developed a novel S1P{sub 1}-selective antagonist, TASP0277308, which is structurally unrelated to S1P as well as previously described S1P{sub 1} antagonists. TASP0277308 inhibited S1P- as well as VEGF-induced cellular responses, including migration and proliferation of human umbilical vein endothelial cells. Furthermore, TASP0277308 effectively blocked a VEGF-induced tube formation in vitro and significantly suppressed tumor cell-induced angiogenesis in vivo. These findings revealed that S1P{sub 1} is a critical component of VEGF-related angiogenic responses and also provide evidence for the efficacy of TASP0277308 for anti-cancer therapies.

FTY720 on the way from the base camp to the summit of the mountain: relevance for remyelination

NARCIS (Netherlands)

Kipp, M.; Amor, S.

2012-01-01

FTY720 (fingolimod; Gilenya®), a sphingosine 1-phosphate (S1P) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. FTY720 is rapidly converted in vivo to the active S-fingolimod-phosphate, which binds to S1P

Metastatic triple-negative breast cancer is dependent on SphKs/S1P signaling for growth and survival.

Science.gov (United States)

Maiti, Aparna; Takabe, Kazuaki; Hait, Nitai C

2017-04-01

About 40,000 American women die from metastatic breast cancer each year despite advancements in treatment. Approximately, 15% of breast cancers are triple-negative for estrogen receptor, progesterone receptor, and HER2. Triple-negative cancer is characterized by more aggressive, harder to treat with conventional approaches and having a greater possibility of recurrence. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid signaling mediator has emerged as a key regulatory molecule in breast cancer progression. Therefore, we investigated whether cytosolic sphingosine kinase type 1 (SphK1) and nuclear sphingosine kinase type 2 (SphK2), the enzymes that make S1P are critical for growth and PI3K/AKT, ERK-MAP kinase mediated survival signaling of lung metastatic variant LM2-4 breast cancer cells, generated from the parental triple-negative MDA-MB-231 human breast cancer cell line. Similar with previous report, SphKs/S1P signaling is critical for the growth and survival of estrogen receptor positive MCF-7 human breast cancer cells, was used as our study control. MDA-MB-231 did not show a significant effect of SphKs/S1P signaling on AKT, ERK, and p38 pathways. In contrast, LM2-4 cells that gained lung metastatic phenotype from primary MDA-MB-231 cells show a significant effect of SphKs/S1P signaling requirement on cell growth, survival, and cell motility. PF-543, a selective potent inhibitor of SphK1, attenuated epidermal growth factor (EGF)-mediated cell growth and survival signaling through inhibition of AKT, ERK, and p38 MAP kinase pathways mainly in LM2-4 cells but not in parental MDA-MB-231 human breast cancer cells. Moreover, K-145, a selective inhibitor of SphK2, markedly attenuated EGF-mediated cell growth and survival of LM2-4 cells. We believe this study highlights the importance of SphKs/S1P signaling in metastatic triple-negative breast cancers and targeted therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Different pathways of [3H]inositol phosphate formation mediated by α 1a- and α 1b-adrenergic receptors

International Nuclear Information System (INIS)

Wilson, K.M.; Minneman, K.P.

1990-01-01

The types of inositol phosphates (InsPs) formed in response to activation of alpha 1-adrenergic receptor subtypes were determined in collagenase-dispersed renal cells and hepatocytes by high pressure liquid chromatography separation. In hepatocytes, which contain only the alpha 1b subtype, norepinephrine stimulated rapid (10-s) formation of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 and slower (5-min) formation of Ins(1,4)P2 and Ins(1)P. Selective inactivation of alpha 1b receptors by chloroethylclonidine almost completely blocked the effects of norepinephrine in hepatocytes. In renal cells, which contain both alpha 1a and alpha 1b receptors in a 60:40 ratio, norepinephrine did not significantly increase the size of any peaks until 5 min after agonist activation. At this time, only a peak eluting with Ins(1)P and one eluting shortly after Ins(1,4)P2 were significantly elevated. Incubation with norepinephrine for 2 h caused small but significant increases in peaks co-eluting with Ins(1)P and Ins(1,4,5)P3 in renal cells; however, only the increase in Ins(1)P was inhibited by chloroethylclonidine pretreatment. Extraction under neutral conditions suggested that cyclic InsPs may be the primary compounds formed in response to norepinephrine in renal cells. Removal of extracellular Ca2+ caused a 60% reduction in the InsP response to norepinephrine in renal cells but had no effect in hepatocytes. These results suggest that activation of alpha 1a and alpha 1b receptor subtypes results in formation of different InsPs and that the response to alpha 1a activation may require influx of extracellular Ca2+

Sphingosine in plants--more riddles from the Sphinx?

Science.gov (United States)

Islam, M Nurul; Jacquemot, Marie-Pierre; Coursol, Sylvie; Ng, Carl K-Y

2012-01-01

• Sphingolipids are emerging as important mediators of cellular and developmental processes in plants, and advances in lipidomics have yielded a wealth of information on the composition of plant sphingolipidomes. Studies using Arabidopsis thaliana showed that the dihydroxy long-chain base (LCB) is desaturated at carbon position 8 (d18:1(Δ8)). This raised important questions on the role(s) of sphingosine (d18:1(Δ4)) and sphingosine-1-phosphate (d18:1(Δ4)-P) in plants, as these LCBs appear to be absent in A. thaliana. • Here, we surveyed 21 species from various phylogenetic groups to ascertain the position of desaturation of the d18:1 LCB, in order to gain further insights into the prevalence of d18:1(Δ4) and d18:1(Δ8) in plants. • Our results showed that d18:1(Δ8) is common in gymnosperms, whereas d18:1(Δ4) is widespread within nonseed land plants and the Poales, suggesting that d18:1(Δ4) is evolutionarily more ancient than d18:1(Δ8) in Viridiplantae. Additionally, phylogenetic analysis indicated that the sphingolipid Δ4-desaturases from Viridiplantae form a monophyletic group, with Angiosperm sequences falling into two distinct clades, the Eudicots and the Poales. • We propose that efforts to elucidate the role(s) of d18:1(Δ4) and d18:1(Δ4)-P should focus on genetically tractable Viridiplantae species where the d18:1 LCB is desaturated at carbon position 4. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

Science.gov (United States)

McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

2016-03-29

Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles.

Science.gov (United States)

Hernández-Coronado, C G; Guzmán, A; Espinosa-Cervantes, R; Romano, M C; Verde-Calvo, J R; Rosales-Torres, A M

2015-02-01

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In

The promotion of mandibular defect healing by the targeting of S1P receptors and the recruitment of alternatively activated macrophages.

Science.gov (United States)

Das, Anusuya; Segar, Claire E; Hughley, Brian B; Bowers, Daniel T; Botchwey, Edward A

2013-12-01

Endogenous signals originating at the site of injury are involved in the paracrine recruitment, proliferation, and differentiation of circulating progenitor and diverse inflammatory cell types. Here, we investigate a strategy to exploit endogenous cell recruitment mechanisms to regenerate injured bone by local targeting and activation of sphingosine-1-phosphate (S1P) receptors. A mandibular defect model was selected for evaluating regeneration of bone following trauma or congenital disease. The particular challenges of mandibular reconstruction are inherent in the complex anatomy and function of the bone given that the area is highly vascularized and in close proximity to muscle. Nanofibers composed of poly(DL-lactide-co-glycolide) (PLAGA) and polycaprolactone (PCL) were used to delivery FTY720, a targeted agonist of S1P receptors 1 and 3. In vitro culture of bone progenitor cells on drug-loaded constructs significantly enhanced SDF1α mediated chemotaxis of bone marrow mononuclear cells. In vivo results show that local delivery of FTY720 from composite nanofibers enhanced blood vessel ingrowth and increased recruitment of M2 alternatively activated macrophages, leading to significant osseous tissue ingrowth into critical sized defects after 12 weeks of treatment. These results demonstrate that local activation of S1P receptors is a regenerative cue resulting in recruitment of wound healing or anti-inflammatory macrophages and bone healing. Use of such small molecule therapy can provide an alternative to biological factors for the clinical treatment of critical size craniofacial defects. Copyright © 2013 Elsevier Ltd. All rights reserved.

Targeting the S1P Axis and Development of a Novel Therapy for Obesity Related Triple Negative Breast Cancer

Science.gov (United States)

2017-09-01

with the options ‘intersection-nonempty’ mode, non- stranded , minimum quality 10, and ‘exon’ was used as the feature type, with ‘gene_id’ as the GTF...Keywords: sphingosine-1-phosphate, sphingosine kinase, estradiol, breast cancer, FTY720/fingolimod. Abbreviations: BCSCs, breast cancer stem cells; ,ER...pathway along with p38MAPK in breast cancer stem cells (BCSCs) leading to proliferation and tumorigenicity (Hirata et al., 2014). BCSCs can also be