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Plasma phospholipid transfer protein (PLTP) is a risk factor for cardiovascular diseases. Sphingosine-1-phosphate (S1P), carried by high-density lipoprotein (HDL), is a potent lipid mediator and is also associated with cardiovascular diseases. We found that germline Pltp gene knockout (KO) mice have decreased circulating S1P without influencing apoM, a major S1P carrier on HDL. We then hypothesized that, like apoM, PLTP is another S1P carrier. We established inducible Pltp- KO, Apom-KO, and Pltp/Apom double KO mice and measured plasma lipoprotein and S1P levels under different diets. We found that PLTP deficiency, and the double deficiency have a similar effect on HDL reduction. Importantly, we found that all mice have about 50% reduction in plasma S1P levels, compared to WT mice, and PLTP deficiency significantly reduces apoM levels (about 40%), while apoM deficiency has no effect on PLTP activity, indicating that PLTP depletion reduces S1P through HDL reduction. To further evaluate this HDL reduction--mediated effect, we overexpressed PLTP which also caused a reduction of HDL. We found that the overexpression reduces S1P and apoM as well as apoA-I, a major apolipoprotein on HDL. Furthermore, we found that albumin (another reported S1P carrier) deficiency in mice has no effect on plasma S1P. We also found that the influence of PLTP on HDL may not require its direct binding to the particle. In conclusion, PLTP is not a direct S1P carrier. PLTP depletion or overexpression in adulthood dramatically reduces plasma S1P through HDL reduction. ApoM, but not albumin, deficiency reduces plasma S1P levels. [ABSTRACT FROM AUTHOR]

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1- phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide–treated GCs and a 3-nitropropionic acid–induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress–induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress–induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide–KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid–induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback. [ABSTRACT FROM AUTHOR]

The vascular and lymphatic systems both comprise a series of structurally distinct vessels lined with an inner layer of endothelial cells that function to provide a semipermeable barrier to blood and lymph. Regulation of the endothelial barrier is critical for maintaining vascular and lymphatic barrier homeostasis. One of the regulators of endothelial barrier function and integrity is sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite secreted into the blood by erythrocytes, platelets, and endothelial cells and into the lymph by lymph endothelial cells. Binding of S1P to its G protein-coupled receptors, known as S1PR1-5, regulates its pleiotropic functions. This review outlines the structural and functional differences between vascular and lymphatic endothelium and describes current understanding of the importance of S1P/S1PR signaling in regulation of barrier functions. Most studies thus far have been primarily focused on the role of the S1P/S1PR1 axis in vasculature and have been summarized in several excellent reviews, and thus, we will only discuss new perspectives on the molecular mechanisms of action of S1P and its receptors. Much less is known about the responses of the lymphatic endothelium to S1P and the functions of S1PRs in lymph endothelial cells, and this is the major focus of this review. We also discuss current knowledge related to signaling pathways and factors regulated by the S1P/S1PR axis that control lymphatic endothelial cell junctional integrity. Gaps and limitations in current knowledge are highlighted together with the need to further understand the role of S1P receptors in the lymphatic system. [ABSTRACT FROM AUTHOR]

The translocation of sphingosine kinase 1 (SK1) to the plasma membrane (PM) is crucial in promoting oncogenesis. We have previously proposed that SK1 exists as both a monomer and dimer in equilibrium, although it is unclear whether these species translocate to the PM via the same or different mechanisms. We therefore investigated the structural determinants involved to better understand how translocation might potentially be targeted for therapeutic intervention. We report here that monomeric WT mouse SK1 (GFP-mSK1) translocates to the PM of MCF-7L cells stimulated with carbachol or phorbol 12-myristate 13-acetate, whereas the dimer translocates to the PM in response to sphingosine-1-phosphate; thus, the equilibrium between the monomer and dimer is sensitive to cellular stimulus. In addition, carbachol and phorbol 12-myristate 13-acetate induced translocation of monomeric GFP-mSK1 to lamellipodia, whereas sphingosine-1-phosphate induced translocation of dimeric GFP-mSK1 to filopodia, suggesting that SK1 regulates different cell biological processes dependent on dimerization. GFP-mSK1 mutants designed to modulate dimerization confirmed this difference in localization. Regulation by the C-terminal tail of SK1 was investigated using GFP-mSK1 truncations. Removal of the last five amino acids (PPEEP) prevented translocation of the enzyme to the PM, whereas removal of the last ten amino acids restored translocation. This suggests that the penultimate five amino acids (SRRGP) function as a translocation brake, which can be released by sequestration of the PPEEP sequence. We propose that these determinants alter the arrangement of N-terminal and C-terminal domains in SK1, leading to unique surfaces that promote differential translocation to the PM. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple KO (TKO) HAP1 cells that lacked all PLPPs (PLPP1-3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared with the WT cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPPindependent S1P uptake pathway. Tracer experiments using [32P]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of sphingolipid transporter 2 (SPNS2) or of major facilitator superfamily domain containing 2B (MFSD2B), both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas KO of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and capable of not only exporting but also importing S1P. Furthermore, we observed that erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration. [ABSTRACT FROM AUTHOR]

It is a great honor to be asked to write a "Reflections" article by one of the true icons of biochemistry, Herb Tabor. I felt humbled, especially since it follows many written by biochemists I admire and whose contributions have shaped major advances in biochemistry and molecular biology in the last century. Here I present my personal reflections on my adventure with the bioactive sphingolipid metabolite sphingosine-1-phosphate intertwined with those of my family life as a wife, mother, and grandmother. These reflections brought back many memories of events in my early career that played significant roles in determining the path I have taken for more than 40 years and that brought much fun and satisfaction into my life. It has been an exciting journey so far, with many surprises along the way, that still continues. [ABSTRACT FROM AUTHOR]

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression. [ABSTRACT FROM AUTHOR]

α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein-protein interaction analysis, we demonstrate that extracellularα-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1P1R) from the lipid raft fractions. S1P1R regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1P1R became refractory to S1P stimulation required for activating inhibitory G-protein (G1) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1P1R on internal vesicles, resulting in the reduced amount ofCD63molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn-treated cells. Furthermore, cholesterol-depleting agent-induced S1P1R expulsion from the rafts also resulted in S1P1R uncoupling. Taken together, these results suggest that extracellular α-Syn-induced expulsion of S1P1R from lipid rafts promotes the uncoupling of S1P1R from G1, thereby blocking subsequent G1 signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis. [ABSTRACT FROM AUTHOR]

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gβγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gβγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests a crucial role of inflammation in cytokine-mediatedβ-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-mediatedATPreduction 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. [ABSTRACT FROM AUTHOR]

Previously, we showed that levels of sphingosine-1 phosphate receptor 3 (S1PR3) are increased in a panel of cultured human lung adenocarcinoma cell lines, and that S1PR3-mediated signaling pathways regulate proliferation, soft agar growth, and invasion of human lung adenocarcinoma cells in vitro. In the present study, we examine S1PR3 levels in human lung adenocarcinoma specimens. cDNA array and tumor microarray analysis shows that mRNA and protein levels of S1PR3 are significantly increased in human lung adenocarcinomas when compared with normal lung epithelial cells. Promoter analysis shows 16 candidate SMAD3 binding sites in the promoter region of S1PR3. ChIP indicates that TGF-β treatment stimulates the binding of SMAD3 to the promoter region of S1PR3. Luciferase reporter assay demonstrates that SMAD3 transactivates S1PR3 promoter. TGF-β stimulation or ectopic expression of TGF-β up-regulates S1PR3 levels in vitro and ex vivo. Pharmacologic inhibition of TGF-β receptor or SMAD3 abrogates the TGF-β-stimulated S1PR3 up-regulation. Moreover, S1PR3 knockdown dramatically inhibits tumor growth and lung metastasis, whereas ectopic expression of S1PR3 promotes the growth of human lung adenocarcinoma cells in animals. Pharmacological inhibition of S1PR3 profoundly inhibits the growth of lung carcinoma in mice. Our studies suggest that levels of S1PR3 are up-regulated in human lung adenocarcinomas, at least in part due to the TGF-β/SMAD3 signaling axis. Furthermore, S1PR3 activity promotes the progression of human lung adenocarcinomas. Therefore,S1PR3mayrepresentanoveltherapeutictargetfor the treatment of deadly lung adenocarcinomas. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1-/- mice, Sgpp2-/- mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2-/- mice had normal pancreatic islet size; however, they exhibited defective adaptive β-cell proliferation that was demonstrated after treatment with either a high-fat diet or the β-cell-specific toxin, streptozotocin. Importantly, β-cells from untreated Sgpp2-/- mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with β-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes β-cell endoplasmic reticulum stress, which is a known cause of β-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is a rare, but highly malignant primary hepatobiliary cancer with a very poor prognosis and limited treatment options. Our recent studies reported that conjugated bile acids (CBAs) promote the invasive growth of CCA via activation of sphingosine 1-phosphate receptor 2 (S1PR2). Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is the most abundant prostaglandin in various human malignancies including CCA. Previous studies have indicated that COX-2 was highly expressed in CCA tissues, and the survival rate of CCA patients was negatively associated with high COX-2 expression levels. It has also been reported that CBAs induce COX-2 expression, whereas free bile acids inhibit COX-2 expression in CCA mouse models. However, the underlying cellular mechanisms and connection between S1PR2 and COX-2 expression in CCA cells have still not been fully elucidated. In the current study, we examined the role of S1PR2 in conjugated bile acid (taurocholate, (TCA))-induced COX-2 expression in a human HuCCT1 CCA cell line and further identified the potential underlying cellular mechanisms. The results indicated that TCA-induced invasive growth of human CCA cells was correlated with S1PR2-medated up-regulation of COX-2 expression and PGE2 production. Inhibition of S1PR2 activation with chemical antagonist (JTE-013) or down-regulation of S1PR2 expression with gene-specific shRNA not only reduced COX-2 expression, but also inhibited TCA-induced activation of EGFR and the ERK1/2/Akt-NF-κB signaling cascade. In conclusion, S1PR2 plays a critical role in TCA-induced COX-2 expression and CCA growth and may represent a novel therapeutic target for CCA. [ABSTRACT FROM AUTHOR]

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FNintegrin- dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) is synthesized from sphingosine by sphingosine kinases (SPHK1 and SPHK2) in invertebrates and vertebrates, whereas specific receptors for S1P (S1PRs) selectively appear in vertebrates, suggesting that S1P acquires novel functions in vertebrates. Because the developmental functions of SPHK1 and SPHK2 remain obscure in vertebrates, we generated sphk1 or sphk2 gene-disrupted zebrafish by introducing premature stop codons in their coding regions using transcription activator-like effector nucleases. Both zygotic sphk1 and sphk2 zebrafish mutants exhibited no obvious developmental defects and grew to adults. The maternal-zygotic sphk2 mutant (MZsphk2), but not the maternal-zygotic sphk1 mutant and maternal sphk2 mutant, had a defect in the cardiac progenitor migration and a concomitant decrease in S1P level, leading to a two-heart phenotype (cardia bifida). Cardia bifida in MZsphk2, which was rescued by injecting sphk2 mRNA, was a phenotype identical to that of zygotic mutants of the S1P transporter spns2 and S1P receptor s1pr2, indicating that the Sphk2-Spns2-S1pr2 axis regulates the cardiac progenitor migration in zebrafish. The contribution of maternally supplied lipid mediators during vertebrate organogenesis presents as a requirement for maternal-zygotic Sphk2. [ABSTRACT FROM AUTHOR]

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys63-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response. [ABSTRACT FROM AUTHOR]

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 hepatocyte and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoMQ22A) introduces a functional signal peptidase cleavage site. Expression of apoMQ22A in ABCA1-expressing HEK293 cells resulted in formation of smaller nascent HDL particles compared to wild type apoM (apoMWT). When apoMQ22A was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoMWT. Hepatocytes isolated from both apoMWT- and apoMQ22A-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoMWT, apoMQ22A hepatocytes displayed more rapid apoM and S1P secretion but minimal apoMQ22A bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not media S1P in both apoMWT and apoMQ22Ahepatocytes. 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 likely precursors of larger apoM/S1P-enriched plasma HDL. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) is a vasoactive lipid mediator that is speculated to be involved in various aspects of atherosclerosis. About 70% of circulating plasma S1P is carried on HDL, and several pleiotropic properties of HDL have been ascribed to S1P. In the previous study with human subjects, however, LDL cholesterol or apoB, but not HDL cholesterol or apoA-I, had a significant positive correlation with the plasma S1P level, suggesting that the metabolic pathway for LDL might have some roles in the metabolism of S1P. In this study, we analyzed the association between LDL receptor, an important protein in the clearance of LDL, and circulating S1P. We observed that in LDL receptor-overexpressing mice, the plasma S1P levels as well as apolipoprotein M (apoM), a carrier of S1P, were decreased and that exogenously administered C17S1P bound to apoM-containing lipoproteins was cleared more rapidly. Unlike the situation in wild-type mice, LDL receptor overexpression in apoE-deficient mice did not reduce the plasma S1 P or apoM levels, suggesting that apoE might be a ligand for the LDL receptor during the clearance of these factors. The present findings clarify the novel roles of the LDL receptor and apoE in the clearance of S1P, a multifunctional bioactive phospholipid. [ABSTRACT FROM AUTHOR]

The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P. [ABSTRACT FROM AUTHOR]

The ceramide-sphingosine 1-phosphate (S1P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S1P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy-induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents. We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S1P formation in the spinal dorsal horn that corresponded with the engagement of S1P receptor subtype 1 (S1PR1)-dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-1β). Intrathecal delivery of the S1PR1 antagonist W146 reduced these neuroinflammatory processes but increased IL-10 and IL-4, potent anti-inflammatory/neuroprotective cytokines. Additionally, spinal W146 reversed established neuropathic pain. Noteworthy, systemic administration of the S1PR1 modulator FTY720 (Food and Drug Administration-approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S1PR1 modulators (ponesimod and CYM-5442) and S1PR1 antagonists (NIBR-14/15) but not S1PR1 agonists (SEW2871). Our findings identify for the first time the S1P/S1PR1 axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients. [ABSTRACT FROM AUTHOR]

ApolipoproteinM(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 FROM AUTHOR]

Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P3), thus providing a tractable in vitro system to characterize molecular mechanism(s) underlying the S1P3 receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P3/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P3 or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P3/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, i.e., ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P3-regulated chemotactic response. [ABSTRACT FROM AUTHOR]

Sphingosine kinases (Sphks), which catalyze the formation of sphingosine 1-phosphate (S1P) from sphingosine, have been implicated as essential intracellular messengers in inflammatory responses. Specifically, intracellular Sphk1-derived S1P was reported to be required for NFB induction during inflammatory cytokine action. To examine the role of intracellular S1P in the inflammatory response of innate immune cells, we derived murine macrophages that lack both Sphk1 and Sphk2 (MΦ Sphk dKO). Compared with WT counterparts, MΦ Sphk dKO cells showed marked suppression of intracellular S1P levels whereas sphingosine and ceramide levels were strongly upregulated. Cellular proliferation and apoptosis were similar in MΦ Sphk dKO cells compared with WT counterparts. Treatment of WT and MSphk dKO with inflammatory mediators TNF or Escherichia coli LPS resulted in similar NFB activation and cytokine expression. Furthermore, LPS-induced inflammatory responses, mortality, and thioglycolate-induced macrophage recruitment to the peritoneum were indistinguishable between MΦ Sphk dKO and littermate control mice. Interestingly, autophagic markers were constitutively induced in bone marrow-derived macrophages from Sphk dKO mice. Treatment with exogenous sphingosine further enhanced intracellular sphingolipid levels and autophagosomes. Inhibition of autophagy resulted in caspase-dependent cell death. Together, these data suggest that attenuation of Sphk activity, particularly Sphk2, leads to increased intracellular sphingolipids and autophagy in macrophages. [ABSTRACT FROM AUTHOR]

The fetal development of the mammalian eyelid involves the expansion of the epithelium over the developing cornea, fusion into a continuous sheet covering the eye, and a splitting event several weeks later that results in the formation of the upper and lower eyelids. Recent studies have revealed a significant number of molecular signaling components that are essential mediators of eyelid development. Receptor-mediated sphingosine 1-phosphate (S1P) signaling is known to influence diverse biological processes, but its involvement in eyelid development has not been reported. Here, we show that two S1P receptors, S1P2 and S1P3, are collectively essential mediators of eyelid closure during murine development. Homozygous deletion of the gene encoding either receptor has no apparent effect on eyelid development, but double-null embryos are born with an "eyes open at birth" defect due to a delay in epithelial sheet extension. Both receptors are expressed in the advancing epithelial sheet during the critical period of extension. Fibroblasts derived from double-null embryos have a deficient response to epidermal growth factor, suggesting that S1P2 and S1P3 modulate this essential signaling pathway during eyelid closure. [ABSTRACT FROM AUTHOR]

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 dietinduced 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). Dietinduced 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. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1Pspecific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1-/- mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1-/- pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1-/- mice. Keratinocytes isolated from the skin of Sgpp1-/- pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) binds G-protein-coupled receptors (S1P1-5) to regulate a multitude of physiological effects, especially those in the vascular and immune systems. S1P receptors in the vascular system have been characterized primarily in mammals. Here, we report that the S1P receptors and metabolic enzymes are conserved in the genome of zebrafish Danio rerio. Bioinformatic analysis identified seven S1P receptor-like sequences in the zebrafish genome, including duplicated orthologs of receptors 3 and 5. Sphingolipidomic analysis detected erythrocyte and plasma S1P as well as high plasma ceramides and sphingosine. Morpholino-mediated knockdown of s1pr1 causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels, decreased proliferation of intersegmental and axial vessels, and hypersprouting in the caudal vein plexus. The s1pr2 gene was previously characterized as a regulator of cell migration and heart development, but its role in angiogenesis is not known. However, when expression of both s1pr1 and s1pr2 is suppressed, severely reduced vascular development of the intersegmental vessels was observed with doses of the s1pr1 morpholino that alone did not cause any discernible vascular defects, suggesting that s1pr1 and s1pr2 function cooperatively to regulate vascular development in zebrafish. Similarly, the S1P transporter, spns2, also cooperated with s1pr1. We propose that extracellular S1P acts through vascular S1P receptors to regulate vascular development. [ABSTRACT FROM AUTHOR]

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3'- phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110β, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2β, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P1-3.Westudied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P1 in ECs. Knockdown of either PI3K-C2α or class I p110β markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110β was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110β were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110β markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110β suppressed S1P-induced S1P1 internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P1 internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P1 internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) is a blood-borne lysosphingo-lipid 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. [ABSTRACT FROM AUTHOR]

Sphingosine kinase 1 (SK1) is an important enzyme involved in the production of the bioactive lipid sphingosine 1-phosphate (S1P). SK1 is overexpressed in many forms of cancer, however, the contribution of SK1 to cancer progression is still unclear. One of the best characterized mutations found in several forms of human cancer is an activating point mutation in the Ras oncogene, which disrupts its GTPase activity and leads to stimulation of the MEK/ERK pathway. Because SK1 activity and subcellular localization have been shown to be regulated by ERK, we wished to investigate the effect of oncogenic Ras, a potent activator of the Raf/MEK/ERK pathway, on the activity of SK1 and sphingolipid metabolism. Using HEK293T cells transiently transfected with the K-RasG12V oncogene and both wild type and Sphk1-/- mouse embryonic fibroblasts stably infected with retroviral K-RasG12V, we found that K-RasG12V increases the production of S1P and decreases the production of ceramide in a SK1- dependent manner. In addition, we found that expression of the K-RasG12V oncogene leads to plasma membrane localization of SK1 and a reduction in cytosolic levels of SK1. This effect is likely mediated by the Raf/MEK/ERK pathway as constitutively active B-Raf orMEK1are able to activate SK1, but constitutively active Akt1 is not.Webelieve this research has important implications for how sphingolipids may be contributing to oncogenic transformation and provide some of the first evidence for oncogenes inducing specific changes in sphingolipid metabolism through SK1 regulation. [ABSTRACT FROM AUTHOR]

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate lyase (S1P lyase) irreversibly cleaves sphingosine 1-phosphate (S1P) in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphate are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. Indeed, we have previously reported that S1P lyase deficiency causes neurodegeneration and other adverse symptoms. We next asked the question whether and how S1P lyase deficiency affects the metabolism of (glyco)sphingolipids and cholesterol, two lipid classes that might be involved in the neurodegenerative processes observed in S1P lyase-deficient mice. As predicted, there was a considerable increase in free and phosphorylated sphingoid bases upon elimination of S1P lyase, but to our surprise, rather than increasing, the mass of (glyco)sphingolipids persisted at wild type levels. This was discovered to be due to reduced de novo sphingoid base biosynthesis and a corresponding increase in the recycling of the backbones via the salvage pathway. There was also a considerable increase in cholesterol esters, although free cholesterol persisted at wild type levels, which might be secondary to the shifts in sphingolipid metabolism. All in all, these findings show that accumulation of free and phosphorylated sphingoid bases by loss of S1P lyase causes an interesting readjustment of the balance between de novo biosynthesis and recycling to maintain (glyco)sphingolipid homeostasis. These changes, and their impact on the metabolism of other cellular lipids, should be explored as possible contributors to the neurodegeneration in S1P lyase deficiency. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) and ceramide have been implicated in both autophagy and apoptosis. However, the roles of these sphingolipid metabolites in the links between these two processes are not completely understood. Depletion of S1P phosphohydrolase-1 (SPP1), which degrades intracellular S1P, induces the unfolded protein response and endoplasmic reticulum stress-induced autophagy (Lépine, S., Allegood, J. C., Park, M., Dent, P., Milstien, S., and Spiegel, S. (2011) Cell Death Differ. 18, 350-361). Surprisingly, however, treatment with doxorubicin, which by itself also induced autophagy, markedly reduced the extent of autophagy mediated by depletion of SPP1. Concomitantly, doxorubicin-induced apoptosis was greatly enhanced by down-regulation of SPP1. Autophagy and apoptosis seemed to be sequentially linked because inhibiting autophagy with 3-methyladenine also markedly attenuated apoptosis. Moreover, silencing Atg5 or the three sensors of the unfolded protein response, IRE1α, ATF6, and PKR-like eIF2α kinase (PERK), significantly decreased both autophagy and apoptosis. Doxorubicin stimulated calpain activity and Atg5 cleavage, which were significantly enhanced in SPP1-depleted cells. Inhibition or depletion of calpain not only suppressed Atg5 cleavage, it also markedly decreased the robust apoptosis induced by doxorubicin in SPP1-deficient cells. Importantly, doxorubicin also increased de novo synthesis of the pro-apoptotic sphingolipid metabolite ceramide. Elevation of ceramide in turn stimulated calpain; conversely, inhibiting ceramide formation suppressed Atg5 cleavage and apoptosis. Hence, doxorubicin switches protective autophagy in SPP1-depleted cells to apoptosis by calpain-mediated Atg5 cleavage. [ABSTRACT FROM AUTHOR]

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) β-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein-coupled receptors, designated as S1P1-5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple KO (TKO) HAP1 cells that lacked all PLPPs (PLPP1-3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared with the WT cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPP-independent S1P uptake pathway. Tracer experiments using [ 32 P]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of sphingolipid transporter 2 (SPNS2) or of major facilitator superfamily domain containing 2B (MFSD2B), both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas KO of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and capable of not only exporting but also importing S1P. Furthermore, we observed that erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sphingosine-1-phosphate (S1P), a natural multifunctional phospholipid, is highly increased in plasma from patients with pulmonary arterial hypertension and mediates proliferation of pulmonary artery smooth muscle cells (PASMCs) by activating the Notch3 signaling pathway. However, the mechanisms underpinning S1P-mediated induction of PASMCs proliferation remain unclear. In this study, using biochemical and molecular biology approaches, RNA interference and gene expression analyses, 5'-ethynyl-2'-deoxyuridine incorporation assay, and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, we demonstrated that S1P promoted the activation of signal transducers and activators of transcription 3 (STAT3) through sphingosine-1-phosphate receptor 2 (S1PR2), and subsequently upregulated the expression of the microRNA miR-135b, which further reduced the expression of E3 ubiquitin ligase β-transduction repeat-containing protein and led to a reduction in yes-associated protein (YAP) ubiquitinated degradation in PASMCs. YAP is the core effector of the Hippo pathway and mediates the expression of particular genes. The accumulation of YAP further increased the expression and activation of Notch3 and ultimately promoted the proliferation of PASMCs. In addition, we showed that preblocking S1PR2, prior silencing of STAT3, miR-135b, or YAP, and prior inhibition of Notch3 all attenuated S1P-induced PASMCs proliferation. Taken together, our study indicates that S1P stimulates PASMCs proliferation by activation of the S1PR2/STAT3/miR-135b/β-transduction repeat-containing protein/YAP/Notch3 pathway, and our data suggest that targeting this cascade might have potential value in ameliorating PASMCs hyperproliferation and benefit pulmonary arterial hypertension., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)