Export Of Sphingosine-1-phosphate Research Articles

Short Periods of Hypoxia Upregulate Sphingosine Kinase 1 and Increase Vasodilation of Arteries to Sphingosine 1-Phosphate (S1P) via S1P3.

Sphingosine kinase [(SK), isoforms SK1 and SK2] catalyzes the formation of the bioactive lipid, sphingosine 1-phosphate (S1P). This can be exported from cells and bind to S1P receptors to modulate vascular function. We investigated the effect of short-term hypoxia on SK1 expression and the response of arteries to S1P. SK1 expression in rat aortic and coronary artery endothelial cells was studied using immunofluorescence and confocal microscopy. Responses of rat aortic rings were studied using wire myography and reversible hypoxia induced by bubbling myography chambers with 95% N2:5% CO2 Inhibitors were added 30 minutes before induction of hypoxia. S1P induced endothelium-dependent vasodilation via activation of S1P3 receptors and generation of nitric oxide. Hypoxia significantly increased relaxation to S1P and this was attenuated by (2R)-1-[[(4-[[3-methyl-5-[(phenylsulfonyl)methyl] phenoxy]methyl]phenyl]methyl]-2-pyrrolidinemethanol [(PF-543), SK1 inhibitor] but not (R)-FTY720 methyl ether [(ROMe), SK2 inhibitor]. Hypoxia also increased vessel contractility to the thromboxane mimetic, 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α, which was further increased by PF-543 and ROMe. Hypoxia upregulated SK1 expression in aortic and coronary artery endothelial cells and this was blocked by PF-543 and 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole [(SKi), SK1/2 inhibitor]. The effects of PF-543 and SKi were associated with increased proteasomal/lysosomal degradation of SK1. A short period of hypoxia increases the expression of SK1, which may generate S1P to oppose vessel contraction. Under hypoxic conditions, upregulation of SK1 is likely to lead to increased export of S1P from the cell and vasodilation via activation of endothelial S1P3 receptors. These data have significance for perfusion of tissue during episodes of ischemia.

Abstract P5-03-05: Sphingosine-1-phosphate produced by sphingosine kinase 1 and exported via ABCC1 shortens survival of mice and humans with breast cancer

Abstract Background: Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator that is generated by sphingosine kinase 1 (SphK1) when it is phosphorylated (pSphK1) inside cells, has been implicated in regulation of many process important for breast cancer progression. Previously we have shown that S1P is exported out of human breast cancer cells by ATP-binding cassette (ABC) transporter ABCC1, but not by ABCB1, both known multidrug resistance proteins that efflux chemotherapeutic agents. However, the pathological consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, we report that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients via exporting S1P. Materials and methods: Microarray based gene expression data of 2509 patients associated with their survival were obtained from METABRIC database. Single gene survival analysis based on expressin of SphK1, and dual ABCC1 or ABCB1 and SphK1 survival analyses were perfomerd. For protein analyses, tissues were obrained from 275 patients with stage 1-3 breast cancers treated in Yokohama City University Medical Center in Japan between 2006 and 2008. The expression of pSphK1 was analyzed by immunohistochemistry and investigate the relationship with clinicopathological findings. For in vitro and in vivo experiments, breast cancer cell lines were transfected by ABCB1, ABCC1 or vector transiently or stably. BALB/c nu/nu mice and BALB/c mice were used for in vivo experiments. S1P was measured by LC-ESI-MS/MS. Results: SphK1 expression significantly associate with worse overall survival (median survival of 124 months with high SphK1 expression compared to 163 months for patients with low SphK1 expression, p=0.0014). Although patients with high ABCC1 expression had only a slightly worse overall survival of 150 months, those with high levels of both SphK1 and ABCC1 had much worse prognosis with median overall survival of 114 months (p < 0.0068). Such association was not observed with ABCB1 expression. The frequency of strong pSphK1 protein expression was higher in HER2 enrhiched or TNBC than in Luminal. pSphK1 was more prevalent and increased in a larger tumors and in tumors from patients with lymph node metastases. Patients with breast cancers that express both pSphK1 and ABCC1 proteins have significantly shorter disease free survival. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 cells enhanced S1P secretion, proliferation and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary pad markedly enhanced tumor growth, angiogenesis and lymphangiogenesis with concomitant increases in lymph node and lung metastases as well as shorter survival of mice. Interestingly, S1P exported via ABCC1 from breast cancer cells upregulated transcription of SphK1 and its own formation. Conclusions: Our findings suggest that production and export of S1P via ABCC1, but not ABCB1, is associated with worse overall and disease free survival of breast cancer patients and that S1P axis play a role in aggressive biology of breast cancer progression and metastasis. Citation Format: Yamada A, Nagahashi M, Aoyagi T, Huang W-C, Lima S, Miyazaki H, Narui K, Ishikawa T, Endo I, Waters MR, Milstien S, Spiegel S, Takabe K. Sphingosine-1-phosphate produced by sphingosine kinase 1 and exported via ABCC1 shortens survival of mice and humans with breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-05.

Apolipoprotein M mediates sphingosine-1-phosphate efflux from erythrocytes

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 of ABCB1 or ATPase. Thus, ABCC1 could be involved in export of S1P from erythrocytes to apoM.

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

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

Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3-5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.

Abstract 19183: Overexpression of Apom in Liver Stimulates Formation of Larger, Apom/S1P-Enriched Plasma HDL

Apolipoprotein M (ApoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a critical signaling molecule in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulates larger nascent HDL particle formation compared to non apoM-expressing cells, but the in vivo role of apoM in HDL metabolism is poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated liver-specific apoM transgenic (apoM Tg) mice displaying ~5-fold increase in plasma apoM levels compare to wild type (WT) mice. Although plasma HDL cholesterol concentrations were similar to WT mice, apoM Tg mice had larger plasma HDL particles, enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase and S1P. Despite the presence of larger plasma HDL particles in apoM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to WT mice. ApoM Tg mice had ~5-fold increase in plasma S1P that was predominantly associated with larger plasma HDL particles. Primary hepatocytes from apoM Tg vs. WT mice generated larger nascent HDL particles, stimulated sphingolipid synthesis, and increased secretion of S1P. Chemical inhibition of ceramide synthase in hepatocytes stimulated S1P synthesis, but not secretion, suggesting that apoM is rate limiting in export of hepatocyte S1P. Our data suggest that liver-specific apoM overexpression: 1) generates large nascent HDLs and large plasma HDL particles that preferentially bind apoM and S1P, and 2) stimulates S1P synthesis for secretion into plasma. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection or other as yet unidentified functions.

Estradiol Induces Export of Sphingosine 1-Phosphate from Breast Cancer Cells via ABCC1 and ABCG2

Sphingosine 1-phosphate (S1P), a potent sphingolipid mediator produced by sphingosine kinase isoenzymes (SphK1 and SphK2), regulates diverse cellular processes important for breast cancer progression acting in an autocrine and/or paracrine manner. Here we show that SphK1, but not SphK2, increased S1P export from MCF-7 cells. Whereas for both estradiol (E(2)) and epidermal growth factor-activated SphK1 and production of S1P, only E(2) stimulated rapid release of S1P and dihydro-S1P from MCF-7 cells. E(2)-induced S1P and dihydro-S1P export required estrogen receptor-alpha, not GPR30, and was suppressed either by pharmacological inhibitors or gene silencing of ABCC1 (multidrug resistant protein 1) or ABCG2 (breast cancer resistance protein). Inhibiting these transporters also blocked E(2)-induced activation of ERK1/2, indicating that E(2) activates ERK via downstream signaling of S1P. Taken together, our findings suggest that E(2)-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E(2) important for breast cancer pathophysiology.

Estradiol Stimulates Export of Sphingosine‐1‐Phosphate From MCF7 Breast Cancer Cells By Specific ABC Transporters

Sphingosine 1‐phosphate (S1P), produced by two sphingosine kinase isoenzymes (SphK1 and SphK2), regulates many cellular processes important for breast cancer progression by binding to specific S1P receptors. SphK1 is overexpressed in breast cancer, and both estradiol (E2) and epidermal growth factor (EGF) activate SphK1. In this study, we examined how intracellularly produced S1P is secreted from breast cancer cells. Overexpression of SphK1, but not SphK2, increased S1P export from MCF7 cells, and downregulation of expression of SphK1, but not SphK2, decreased its export. Although both E2 and EGF activated SphK1 and increased intracellular S1P, only E2 significantly stimulated S1P secretion. Export of S1P was inhibited by MK571, an inhibitor of ABCC1 (multi‐drug resistant protein 1), and fumitrimorgin C, an inhibitor of ABCG2 (breast cancer resistance protein), but not by the ABCB1 inhibitor, verapamil. In addition, E2‐induced secretion of S1P was blunted by downregulation of ABCC1 or ABCG2. These findings suggest that E2‐induced export of S1P is mediated by specific ABC transporters and may play an important role in multidrug resistance in breast cancer. This work was supported by NIH grants R37 GM043880 and R01CA61774 to SS and 5K12HD055881 to KT.

The Sphingolipid Transporter Spns2 Functions in Migration of Zebrafish Myocardial Precursors

Sphingosine-1-phosphate (S1P) is a secreted lipid mediator that functions in vascular development; however, it remains unclear how S1P secretion is regulated during embryogenesis. We identified a zebrafish mutant, ko157, that displays cardia bifida (two hearts) resembling that in the S1P receptor-2 mutant. A migration defect of myocardial precursors in the ko157 mutant is due to a mutation in a multipass transmembrane protein, Spns2, and can be rescued by S1P injection. We show that the export of S1P from cells requires Spns2. spns2 is expressed in the extraembryonic tissue yolk syncytial layer (YSL), and the introduction of spns2 mRNA in the YSL restored the cardiac defect in the ko157 mutant. Thus, Spns2 in the YSL functions as a S1P transporter in S1P secretion, thereby regulating myocardial precursor migration.

Activation of sphingosine‐1‐phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration

Sphingosine-1-phosphate (S1P) acts as an extracellular ligand for a family of G-protein coupled receptors that are crucial in cell migration. S1P5 is exclusively expressed in oligodendrocytes and oligodendrocyte precursor cells (OPCs), which migrate considerable distances during brain development. The current studies suggest a physiological role for S1P and S1P5 in regulation of OPC migration. mRNA expression levels of S1P2 and S1P5 are comparable in OPCs, but S1P binding specifically to the S1P5 receptor blocked OPC migration (IC50=29 nM). Thus, knocking down S1P5 using siRNA prevented the S1P-induced decrease in OPC migration, whereas knocking down S1P2 did not have any effect. S1P-induced modulation of OPC migration was insensitive to pertussis toxin, suggesting that S1P5-initiated signaling is not mediated by the G alpha(i)-protein coupled pathway. Furthermore, S1P5 appears to engage the G alpha(12/13) protein coupled Rho/ROCK signaling pathway to impede OPC migration. To modulate OPC motility, extracellular S1P could be derived from the export of intracellular S1P generated in response to glutamate treatment of OPCs. These studies suggest that S1P could be a part of the neuron-oligodendroglial communication network regulating OPC migration and may provide directional guidance cues for migrating OPCs in the developing brain.

Toward Solving the Riddle

See related article, pages 468–476 “ It is a riddle wrapped in a mystery inside an enigma .”1 The mythical Greek Sphinx posed riddles to passers-by. Those who could not answer met their demise until Oedipus solved the riddle and the Sphinx self-destructed. Ultimately the terms sphinx and enigma became associated and in 1884 J.L.W. Thudichum, known as the “Father of Neurochemistry,” named the chemical backbone of sphingolipids for their enigmatic “Sphinx-like” properties. Recently, studies of sphingolipid actions have achieved increasing importance in understanding the pathophysiology of acute ischemia/reperfusion injury through studies of sphingosine kinase (SphK) and its end-product, sphingosine 1-phosphate (S1P). Activation of SphK is the final and rate-limiting enzymatic step in the synthesis of S1P, which is an intracellular and extracellular signaling molecule that regulates many important cellular processes including growth, survival, differentiation, cytoskeletal rearrangements, motility, angiogenesis, and calcium mobilization. In contrast, the sphingosine precursor ceramide is a growth-inhibiting lipid implicated in differentiation and apoptosis. These findings have led to the so-called “sphingolipid rheostat” hypothesis, which proposes that the relative levels of these lipids are important determinants of cell fate.2 It is now accepted that many of the actions of S1P are mediated by a family of G protein–coupled S1P receptor isoforms, termed S1P1–5. With regard to the cardiovascular system, platelets store and release S1P, which is known to regulate endothelial function and signaling. There is a substantial gradient between intracellular and extracellular concentrations of S1P. In human serum estimates of S1P concentration range from 0.5 to 1 μmol/L or more, whereas intracellular levels vary among tissues but are much lower possibly due in part to export of S1P or SphK (Figure). Also implicated in the regulation of S1P levels are degradation by both S1P lyase and dephosporylation by S1P phosphohydrolase. About 60% …