Intracellular Sphingosine-1-phosphate Research Articles

Sphingosine 1 Phosphate Regulates Store‐Operated Calcium Entry through binding to STIM1

A growing body of evidence suggests that sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) pathway contributes to vascular dysfunction through dysregulation of Ca2+ mobilization. We demonstrated recently that intracellular S1P triggers store‐operated Ca2+ (SOC) entry and independent of G protein‐coupled S1P receptors suggesting SOC as intracellular target for S1P. However, the mechanism by which S1P modulates SOC entry is not well defined. SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. While STIM function as Ca2+ sensors within the ER, the Orai proteins constitute the pore‐forming unit of SOC in PM. We first investigated the role of SK1/S1P on STIM1/Orai1 assembly by confocal microscopy. Pretreating transiently transfected HEK293 cells with SK1 specific inhibitor inhibited angiotensin‐induced colocalization of GFP.STIM1 and mCherry Orai1. Similarly, using BRET assay, inhibition of SK1 decreased NET BRET values of STIM1‐FlAsH and Orai1‐Rluc in transiently transfected HEK293 cells stimulated with GPCR agonists. Our molecular modeling simulation predicted direct binding of S1P to EF/SAM1 domain of STIM1 molecule. This is initially assessed by in vitro binding assay revealed binding of biotinylated S1P to STIM1 in the membrane fractions of vascular smooth muscle cell lysates. We then used circular dichroism spectroscopy (CD) to measure the thermostability of the EF/SAM1 protein in the presence or absence of S1P. Addition of S1P to EF/SAM1 induced a consistent left shift indicative of lower melting temperature (Tm) compared to vehicle control. To further validate direct binding, we prepared 15N‐labeled EF/SAM1 for the NMR spectroscopy. A five point titration series with S1P caused perturbations of residue specific chemical shift due to binding interactions between EF/SAM1 and S1P using 2D TROSY (Transverse Relaxation Optimized Spectroscopy). Collectively, our data provide evidences of intracellular role for S1P in regulation of SOC through direct binding to STIM1.Support or Funding InformationAmerican Heart Association 17GRNT33700222 (HME)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Overexpression of sphingosine-1-phosphate lyase protects insulin-secreting cells against cytokine toxicity

Increasing evidence suggests a crucial role of inflammation in cytokine-mediated β-cell dysfunction and death in type 1 diabetes mellitus, although the mechanisms are incompletely understood. Sphingosine 1-phosphate (S1P) is a multifunctional bioactive sphingolipid involved in the development of many autoimmune and inflammatory diseases. Here, we investigated the role of intracellular S1P in insulin-secreting INS1E cells by genetically manipulating the S1P-metabolizing enzyme S1P lyase (SPL). The expression of spl was down-regulated by cytokines in INS1E cells and rat islets. Overexpression of SPL protected against cytokine toxicity. Interestingly, the SPL overexpression did not suppress the cytokine-induced NFκB-iNOS-NO pathway but attenuated calcium leakage from endoplasmic reticulum (ER) stores as manifested by lower cytosolic calcium levels, higher expression of the ER protein Sec61a, decreased dephosphorylation of Bcl-2-associated death promoter (Bad) protein, and weaker caspase-3 activation in cytokine-treated (IL-1β, TNFα, and IFNγ) cells. This coincided with reduced cytokine-mediated ER stress, indicated by measurements of CCAAT/enhancer-binding protein homologous protein (chop) and immunoglobulin heavy chain binding protein (bip) levels. Moreover, cytokine-treated SPL-overexpressing cells exhibited increased expression of prohibitin 2 (Phb2), involved in the regulation of mitochondrial assembly and respiration. SPL-overexpressing cells were partially protected against cytokine-mediated ATP reduction and inhibition of glucose-induced insulin secretion. siRNA-mediated spl suppression resulted in effects opposite to those observed for SPL overexpression. Knockdown of phb2 partially reversed beneficial effects of SPL overexpression. In conclusion, the relatively low endogenous Spl expression level in insulin-secreting cells contributes to their extraordinary vulnerability to proinflammatory cytokine toxicity and may therefore represent a promising target for β-cell protection in type 1 diabetes mellitus.

Evidence Suggests Sphingosine 1-Phosphate Might Be Actively Generated, Degraded, and Transported to Extracellular Spaces With Increased S1P2 and S1P3 Expression in Colon Cancer

BackgroundA pivotal role of sphingosine 1-phosphate (S1P) in cancer has been suggested based on the ceramide-S1P rheostat theory that the intracellular balance between prosurvival S1P and proapoptotic ceramide determines cell fate. Upregulation of S1P-generating sphingosine kinases (SKs) and downregulation of S1P-degrading S1P lyase (SPL) might increase intracellular S1P levels to exert a prosurvival effect in cancer in general, such as colon cancer. However, we recently observed a distinct S1P metabolism in hepatocellular carcinoma tissues that increased SPL mRNA levels with reduced S1P levels. Thus, we investigated S1P metabolism in colon cancer. Patients and MethodsWe enrolled 26 consecutive colon cancer patients, who had undergone surgical treatment. ResultsNot only SK, but also SPL, mRNA levels were increased in colon cancer tissues compared with the adjacent nontumorous tissues. Furthermore, the mRNA levels of another S1P degrading enzyme, S1P phosphatase 1, S1P transporters, spinster homolog 2, adenosine triphosphate-binding cassette subfamily C member 1, and S1P receptors, S1P2 and S1P3 were also increased, but the S1P levels were not increased in the colon cancer tissues. The reduction of SPL expression by silencing led to reduced proliferation and invasion, and overexpression of SPL caused enhanced proliferation in colon cancer cell lines. ConclusionIn human colon cancer tissues, mRNA levels of S1P-generating and S1P-degrading enzymes, transporters from inside to outside the cells, and S1P receptors, S1P2 and S1P3 were elevated, suggesting active S1P metabolism and movement. This altered S1P metabolism might play a role in colon cancer pathophysiology.

Increased sphingosine-1-phosphate production in response to osteocyte mechanotransduction

Over the past few years interest has greatly increased in how the lipid mediator sphingosine-1-phosphate (S1P) influences bone homeostasis. Recent work has postulated multiple effects of S1P on osteoblasts and osteoclasts. Based on these findings, S1P has been proposed as a potential osteoporosis treatment. However, to date, there has been only a single study investigating S1P signalling in the cells that co-ordinate bone metabolism: osteocytes. This study aimed to elucidate the role of S1P signalling in osteocyte mechanotransduction.Utilising 3D cell culture we established the expression profile of all genes related to the S1P signalling system in the Ocy454 osteocyte cell line. Exposure to mechanical loading resulted in a downregulation in Sost, Spns2, the S1P transporter, Sgpl1 and Sgppl1 the enzymes responsible for degradation and dephosphorylation of S1P. These findings, in conjunction with fluid-flow induced upregulation of Sphk1, the kinase responsible for phosphorylation of sphingosine, suggest that mechanical stimulation of osteocytes leads to an increase in intracellular S1P. This was confirmed with mechanical loading of Ocy454 cells rapidly increasing S1P production in conditioned media and protein lysates. These findings strongly suggest an important role for S1P in the response to mechanical loading of bone.

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

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, S1P1, 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 S1P1, but not S1P2 or S1P3, with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P1 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 S1P1.

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

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.

Targeting sphingosine-1-phosphate signaling in lung diseases.

Sphingosine-1-phosphate (S1P), a simple, bioactive sphingolipid metabolite, plays a key role, both intracellularly and extracellularly, in various cellular processes such as proliferation, survival, migration, inflammation, angiogenesis, and endothelial barrier integrity. The cellular S1P level is low and is tightly regulated by its synthesis and degradation. Sphingosine Kinases (SphKs) 1 and 2, catalyze the ATP-dependent phosphorylation of sphingosine to S1P, while the degradation is mediated by the reversible dephosphorylation catalyzed by the S1P phosphatases and lipid phosphate phosphatases and the irreversible degradation to hexadecenal and ethanolamine phosphate by sphingosine-1-phosphate lyase (S1PL). As a ligand for specific G-protein-coupled receptors, S1P1-5, which are differentially expressed in different cell types, S1P generates downstream signals that play crucial role in developmental and disease related pathologies. In addition to acting extracellularly on receptors located on the plasma membrane, S1P can also act intracellularly, independently of S1P1-5, affecting calcium homeostasis and cell proliferation. The SphKs /S1P /S1PL metabolic pathway is implicated in numerous human pathologies including respiratory disorders, thereby raising the possibility that manipulating intracellular S1P levels could offer therapeutic potential in ameliorating lung diseases. This review focuses on the prospects of targeting S1P signaling and S1P metabolizing enzymes using small molecule inhibitors, receptor agonists, and antagonists in the treatment of lung diseases.

Regulation of Store‐Operated Calcium Channels by Sphingosine 1 Phosphate

Several studies have implicated sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) axis in mobilization of intracellular Ca2+ and defective vascular tone. We and other independent investigators demonstrated recently that SK1/S1P mediates transmembrane Ca2+ flux through store‐operated Ca2+ channel (SOC). SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. STIM function as Ca2+ sensors within the ER, and Orai proteins at PM. Depletion of Ca2+ from ER leads to oligomerization and translocation of STIM to bind Orai at the PM to activate Ca2+ flux. We found that S1P regulates SOC through yet undefined intracellular mechanism that does not involve activation of G protein‐coupled S1P receptors. Here, we have studied the mechanism by which SK/S1P mediates activation of SOC. Molecular modeling predicted the binding of S1P with STIM1 of SOC in monomer and dimer forms. Unlike biotin ceramide and biotin controls, only biotinylated S1P bound to endogenous STIM1 in the membrane fraction of cell lysates that was inhibited with competitive cold S1P suggesting specificity of S1P binding to STIM1. Intriguingly, direct mutation of the predicted S1P binding sites to STIM1 markedly inhibited angiotensinII (AngII) and intracellular S1P‐dependent Ca2+ flux without affecting Ca2+ release from intracellular stores. To examine the effect of SK1/S1P on STIM1/Orai1 assembly by BRET assay, we generated STIM1‐FlAsH and Orai1‐Rluc plasmids. We found that NET BRET values of AngII‐dependent stimulated HEK293 cells transiently transfected with STIM1‐FlAsH and Orai1‐Rluc were significantly inhibited by SK1 inhibitor. Functionally, transfecting STIM1 mutants in HEK293 cells inhibited cell proliferation compared to vehicle control. These findings suggest the requirement of intracellular S1P for activation of SOC via modulation of STIM1 and provide novel mechanistic and functional insights into pathophysiology of vascular dysfunction.

ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex

We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

Sphingosine Kinases (SK): Key Molecules Associated with the Activation, Proliferation and Ibrutinib-Induced Cell Death of Chronic Lympocytic Leukemia Cells

Leukemic cells from CLL patients can survive and proliferate within lymphoid tissues where the supportive microenvironment favors their accumulation. We have previously reported that the activation of CLL cells reduces the expression of the main receptor for sphingosine 1-phosphate (S1P) (Borge M, J Immunol, 2014), a bioactive phospholipid that participates in lymphocyte egress from lymphoid tissues. S1P also mediates several biological functions, including cell growth stimulation and protection of apoptosis, through receptor independent intracellular mechanisms. S1P is generated by two isoforms of sphingosine kinases (SK1/2) and its levels are tightly controlled via degradation by intracellular S1P lyases (S1PL). Several studies have implicated the SK/S1P/S1PL pathway as an essential regulator of cell proliferation and survival in different cancer cells. The aims of our study were: a) to evaluate the expression of SK and S1PL in CLL cells, b) to assess whether key microenvironment signals are able to modulate this expression and c) to evaluate the effect of SK inhibitors on the activation and survival of the leukemic clone.We measured the basal expression of SK and S1PL by qRT-PCR on purified B cells from CLL patients (n=22) and aged-matched healthy donors (n=9), and found that CLL cells express high levels of SK1, favoring an increased SK1/S1PL ratio in the malignant clone compared to healthy B lymphocytes ( p<0.05). Similar results were obtained when SK2 was evaluated. The in vitro activation of CLL cells with anti-IgM+CD40L increased SK1/S1PL ratio (n=10, p<0.01) and the expression of the activation marker CD69. To evaluate the expression of SK1/S1PL within in vivo activated CLL cell subpopulations, we segregated the proliferative fraction (PF) of circulating CLL cells from the quiescent fraction (QF) in the same sample. As it was previously described, this PF is characterized by the presence of an active class-switch recombination process and a high expression of proliferation-related genes, such as Ki-67, c-myc, CD49d, and p27-Kip1 (Palacios F, Blood, 2010). Interestingly, SK1/S1PL ratio was increased in the PF compared with the QF (n=3). Additionally, bone marrow leukemic cells expressing high levels of CD38, which defines a subpopulation of activated lymphocytes, showed a higher S1P/S1PL ratio compared to CD38 low or negative counterparts (n=3),showing that the in vivo activated CLL cells expressed higher ratios of SK1/S1PL compared with the rest of the leukemic clone. Finally, we wondered whether the inhibition of SK impairs the survival, activation and proliferation of the leukemic clone. To this aim we employed a commercial selective SK1 and SK2 inhibitor (SKI-II, 5 and 15μM), which did not affect cell viability (n=10, evaluated at 24, 48, 72 and 96hs). However, it was able to impair the expected upregulation of CD69 induced by IgM+CD40L at 24hs (n=10, p<0.001) and the leukemic cell proliferation evaluated by CFSE dilution assay at 96hs (n=10 , p<0.001). Moreover, while SKI-II did not increase the sensitivity of CLL cells to Fludarabine or Bendamustine, it was able to enhance the cell death induced by Ibrutinib (0.3 and 3μM) (n=5, p<0.05 and p<0.01 respectively).Taking together, our results suggest that SK/S1P/S1PL axis might participate in the accumulation of the malignant clone in CLL patients and the disruption of this pathway might be a potentially effective treatment option in the future. DisclosuresBezares:Janssen: Honoraria. Gamberale:Janssen: Honoraria.

Resveratrol exerts a biphasic effect on apolipoprotein M

Resveratrol exerts a range of beneficial actions in several areas of pathophysiology, including vascular biology. Here, we have investigated the effects of resveratrol on apolipoprotein M (apoM), a carrier and modulator of sphingosine 1-phosphate (S1P), a vasoactive lipid mediator. We used a hepatoma cell line (HepG2), human primary hepatocytes and C57BL/6 mice. We measured apoM, S1P and related enzymes, LDL receptors and sirtuin1 activity, using Western blotting, RT-PCR and enzyme assays. We also used si-RNA to knock-down sirtuin1 in HepG2 cells. In cultures of HepG2 cells, resveratrol (1-10 μM) increased intracellular apoM and S1P. High concentrations of resveratrol (100 μM) decreased extracellular (in the culture medium) apoM, whereas moderate concentrations of resveratrol (1-10 μM) increased extracellular apoM. High concentrations of resveratrol also increased LDL receptor expression, while all concentrations of resveratrol activated the histone deacetylase sirtuin1. In cultures of human primary hepatocytes, resveratrol, at all concentrations, increased both intra- and extracellular apoM. When wild-type mice were fed a resveratrol-containing chow (0.3% w/w) for 2 weeks, both the plasma and hepatic apoM and S1P levels were increased. However, the resveratrol diet did not affect hepatic LDL receptor levels in this in vivo study. Resveratrol increased intra- and extracellular levels of apoM, along with intracellular S1P levels, while a high concentration of resveratrol reduced extracellular apoM. The present findings suggest that resveratrol has novel effects on the metabolic kinetics of S1P, a multi-functional bioactive phospholipid.

A systematic methodology for large scale compound screening: A case study on the discovery of novel S1PL inhibitors

Decrease in sphingosine 1-phosphate (S1P) concentration induces migration of pathogenic T cells to the blood stream, disrupts the CNS and it is implicated in multiple sclerosis (MS), a progressive inflammatory disorder of the central nervous system (CNS), and Alzheimer’s disease (AD). A promising treatment alternative for MS and AD is inhibition of the activity of the microsomal enzyme sphingosine 1-phosphate lyase (S1PL), which degrades intracellular S1P. This report describes an integrated systematic approach comprising virtual screening, molecular docking, substructure search and molecular dynamics simulation to discover novel S1PL inhibitors. Virtual screening of the ZINC database via ligand-based and structure-based pharmacophore models yielded 10000 hits. After molecular docking, common substructures of the top ranking hits were identified. The ligand binding poses were optimized by induced fit docking. MD simulations were performed on the complex structures to determine the stability of the S1PL-ligand complex and to calculate the binding free energy. Selectivity of the selected molecules was examined by docking them to hERG and cytochrome P450 receptors. As a final outcome, 15 compounds from different chemotypes were proposed as potential S1PL inhibitors. These molecules may guide future medicinal chemistry efforts in the discovery of new compounds against the destructive action of pathogenic T cells.

Inhibition of Sphingosine Kinase 1 Ameliorates Angiotensin II-Induced Hypertension and Inhibits Transmembrane Calcium Entry via Store-Operated Calcium Channel.

Angiotensin II (AngII) plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca(2+) mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1-phosphate (S1P) in the mobilization of intracellular Ca(2+) through a yet-undefined mechanism. Here we demonstrate that AngII-induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris-phosphate-dependent release of intracellular calcium, followed by a sustained transmembrane Ca(2+) influx through store-operated calcium channels (SOCs). Inhibition of SK1 attenuates the second phase of transmembrane Ca(2+) influx, suggesting a role for SK1 in AngII-dependent activation of SOC. Intracellular S1P triggers SOC-dependent Ca(2+) influx independent of S1P receptors, whereas external application of S1P stimulated S1P receptor-dependent Ca(2+) influx that is insensitive to inhibitors of SOCs, suggesting that the SK1/S1P axis regulates store-operated calcium entry via intracellular rather than extracellular actions. Genetic deletion of SK1 significantly inhibits both the acute hypertensive response to AngII in anaesthetized SK1 knockout mice and the sustained hypertensive response to continuous infusion of AngII in conscious animals. Collectively these data implicate SK1 as the missing link that connects the angiotensin AT1A receptor to transmembrane Ca(2+) influx and identify SOCs as a potential intracellular target for SK1.

Sphingosine Kinase 1 Mediates Transmembrane Calcium Entry via Store‐Operated Calcium Channel

Angiotensin II plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca2+ mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1‐phosphate (S1P) in the mobilization of intracellular Ca2+ through a yet undefined mechanism. Here we demonstrate that angiotensin II induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris‐phosphate‐dependent release of intracellular calcium, followed by a sustained transmembrane Ca2+ influx through store operated calcium channels (SOC). Inhibition of SK1 attenuates the second phase of transmembrane Ca2+ influx, suggesting a role for SK1 in angiotensin II‐dependent activation of SOC. Intracellular S1P triggers SOC‐dependent Ca2+ influx independent of S1P receptors, while external application of S1P stimulated S1P receptor‐dependent Ca2+ influx that is insensitive to inhibitors of SOC, suggesting that the SK1/S1P axis regulates SOC entry via intracellular rather than extracellular actions. Collectively, these data implicate SK1 as the missing link that connects the angiotensin AT1A receptor to transmembrane Ca2+ influx and identify SOC as a potential intracellular target for SK1/S1P axis.

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

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.

SKIP Is Underexpressed in AML Leading to Sphingosine Kinase Hypofunction

Background: Sphingosine kinase interacting protein (SKIP) has been shown to be mostly silenced by hypermethylation in AML [1]. SKIP interacts with and regulates the function of sphingosine kinase (SK) enzyme. SK activity results in phosphorylation of sphingosine (SPH) to form sphingosine 1 phosphate (S1P), which promotes cell survival and resistance to apoptosis. On the other hand, S1P precursors ceramide (CER) and SPH mediate antiproliferative and apoptotic responses. SKIP has been reported to negatively regulate SK1 activity in fibroblasts. Therefore, we investigated the consequences of SKIP silencing in primary AML cells. In addition, we studied the effects of SKIP re-expression in leukemic cell lines.Methods: CTS and K562 cells were transfected with SKIP gene using standard techniques. SKIP is normally silenced in both cell lines. Intracellular and extracellular S1P, SPH and CER were measured by UPLC-MS/MS. In addition, intracellular SK activity was determined based on C17 S1P production from C17 SPH substrate. Chemosensitivity to doxorubicin, Imatinib and Ara-C in transfected cells was also studied.Results: In Primary AML cells, intracellular S1P and CER concentrations were reduced compared to G-CSF-mobilized peripheral blood mononuclear cells. In addition, SK activity was found to be downregulated in AML primary cells. When we transfected leukemic cell lines with SKIP gene, S1P and CER showed at least 2 fold increase in intracellular and extracellular basal levels compared to vector alone control (Figure 1). Further studies confirmed a significant increase in intracellular SK activity in SKIP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase in S1P and CER was associated with increasing apoptotic signals as evidenced by Annexin V staining and cleaved PARP expression. Moreover, chemosensitivity to Imatinib and AraC was significantly increased in SKIP transfected cell lines. These experiments confirm the regulation of SK1 function by SKIP.Conclusion: These data indicate that SKIP is downregulated in AML leading to reduced SK activity, which ultimately inhibits the apoptosis response. [Display omitted] 1. Saied, M.H., et al., Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats. PLoS One, 2012. 7(3): p. e33213.EAG and PS contributed equallyJG and DT contributed equally DisclosuresNo relevant conflicts of interest to declare.

The Role of Sphingosine-1-Phosphate (S1P) in Thrombopoiesis

Human megakaryocytes (MKs) release trillions of platelets each day into the circulation to maintain normal homeostatic platelet levels. However, the signals that control platelet biogenesis in vivo remain incompletely understood. We have recently identified that extracellular sphingosine 1-phosphate (S1P) plays a key role in thrombopoiesis. Using conditional mutants and intravital multiphoton microscopy, we demonstrate that the lipid mediator S1P serves as a critical directional cue guiding the elongation of megakaryocytic proplatelet (PP) extensions from the interstitium into bone marrow sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. In addition to its role as an extracellular mediator, S1P can also function as a second messenger within the intracellular compartment. Correspondingly, we have demonstrated that MKs express the S1P-generating enzyme sphingosine kinase 2 (Sphk2). Sphk2 predominantly localizes to the nucleus and is the major source of intracellular S1P in MKs. Loss of Sphk2 significantly reduced intracellular S1P in MKs and downregulated the expression and activity of Src family kinases (SFKs). At the same time, loss of Sphk2 and inhibition of SFK activity resulted in defective intravascular PP shedding, the final stage of thrombopoiesis. Correspondingly, mice lacking Sphk2 in the hematopoietic system display thrombocytopenia. Collectively, our findings uncover a novel function of S1P as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia. DisclosuresNo relevant conflicts of interest to declare.

Intracellular Sphingosine 1-Phosphate Contributes to Collagen Expression of Hepatic Myofibroblasts in Human Liver Fibrosis Independent of Its Receptors

Sphingosine 1-phosphate (S1P) is involved in multiple pathological processes, including fibrogenesis. S1P participates in mouse liver fibrogenesis via a paracrine manner. Herein, we investigated the involvement of S1P in human liver fibrosis. Human fibrotic samples were obtained from livers of patients undergoing liver transplantation. Expression of sphingosine kinase (SphK1), collagen (Col) α1(I), Col α1(III), α-smooth muscle actin, and p-Smad2/3 was characterized by immunofluorescence, real-time RT-PCR, high-content analysis, or Western blot analysis in the fibrotic liver, human bone marrow-derived mesenchymal stem cells, and human hepatogenic profibrotic cells. The effect of SphK1 was assessed using siSphK1 or SphK-specific inhibitor. SphK1, which was expressed in human fibrotic liver myofibroblasts, could be detected in human bone marrow-derived mesenchymal stem cells or human hepatogenic profibrotic cells activated by transforming growth factor β1 (TGF-β1). TGF-β1 evoked the activation of SphK1, increased intracellular S1P, and up-regulated expression of SphK1, Col α1(I), and Col α1(III) in a TGF-β receptor-dependent manner. TGF-β1 induced expression of Col α1(I) and Col α1(III) via SphK1, which was mediated by intracellular S1P, independent of S1P receptors. TGF-β1 evoked nuclear translocation of p-Smad2 and p-Smad3 in TGF-β receptor-dependent, but SphK1-independent, manner. In conclusion, intracellular S1P plays a crucial role in the TGF-β1-induced expression of Col α1(I) and Col α1(III), which is required for human fibrosis development. S1P exerts its effects in S1P receptor-independent manner.

Critical role of Spns2, a sphingosine-1-phosphate transporter, in lung cancer cell survival and migration.

The sphingosine-1-phosphate (S1P) transporter Spns2 regulates myocardial precursor migration in zebrafish and lymphocyte trafficking in mice. However, its function in cancer has not been investigated. We show here that ectopic Spns2 expression induced apoptosis and its knockdown enhanced cell migration in non-small cell lung cancer (NSCLC) cells. Metabolically, Spns2 expression increased the extracellular S1P level while its knockdown the intracellular. Pharmacological inhibition of S1P synthesis abolished the augmented cell migration mediated by Spns2 knockdown, indicating that intracellular S1P plays a key role in this process. Cell signaling studies indicated that Spns2 expression impaired GSK-3β and Stat3 mediated pro-survival pathways. Conversely, these pathways were activated by Spns2 knockdown, which explains the increased cell migration since they are also crucial for migration. Alterations of Spns2 were found to affect several enzymes involved in S1P metabolism, including sphingosine kinases, S1P phosphatases, and S1P lyase 1. Genetically, Spns2 mRNA level was found to be reduced in advanced lung cancer (LC) patients as quantified by using a small scale qPCR array. These data show for the first time that Spns2 plays key roles in regulating the cellular functions in NSCLC cells, and that its down-regulation is a potential risk factor for LC.

Export of sphingosine-1-phosphate and cancer progression

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that promotes cell survival, proliferation, migration, angiogenesis, lymphangiogenesis, and immune response; all are critical processes of cancer progression. Although some important roles of intracellular S1P have recently been uncovered, the majority of its biological effects are known to be mediated via activation of five specific G protein-coupled receptors [S1P receptor (S1PR)1-S1PR5] located on the cell surface. Secretion of S1P produced inside cells by sphingosine kinases can then signal through these receptors in autocrine, paracrine, and/or endocrine manners, coined "inside-out" signaling of S1P. Numerous studies suggest that secreted S1P plays important roles in cancer progression; thus, understanding the mechanism by which S1P is exported out of cells, particularly cancer cells, is both interesting and important. Here we will review the current understanding of the transport of S1P out of cancer cells and its potential roles in the tumor microenvironment.