Deletion of Sphingosine Kinase 2 Attenuates Acute Kidney Injury in Mice with Hemolytic-Uremic Syndrome.

Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli. Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1-/-) or SphK2 (SphK2-/-) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2-/- mice, but exacerbated in the SphK1-/- mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1-/- and SphK2-/- in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research.

The Impact of Sphingosine Kinases on Inflammation-Induced Cytokine Release and Vascular Endothelial Barrier Integrity.

Sphingosine kinases type 1 and 2 (SphK1/2) are required for the production of the immune modulator sphingosine 1-phosphate (S1P). SphK1 deficient mice (SphK1-/-) revealed 50% reduced S1P in plasma, while SphK2-/- mice demonstrated 2-3 times increased S1P levels in plasma. Since plasma S1P is a potent inducer of vascular endothelial cell (VEC) barrier stability, we hypothesized that higher and lower levels of S1P in SphK2-/- and SphK1-/- mice, respectively, compared to wild type (wt) mice should translate into decreased and increased severity of induced systemic inflammation due to improved or damaged VEC barrier maintenance. To our surprise, both SphK1-/- and SphK2-/- mice showed improved survival rate and earlier recovery from inflammation-induced weight loss compared to wt mice. While no difference was observed in VEC barrier stability by monitoring Evans blue leakage into peripheral tissues, SphK1-/- mice demonstrated a distinct delay and SphK2-/- mice an improved resolution of early pro-inflammatory cytokine release in plasma. Ex vivo cell culture experiments demonstrated that bone marrow-derived dendritic cells (BMDC) generated from SphK1-/- and SphK2-/- mice responded with decreased interferon-γ (IFN-γ) production upon stimulation with lipopolysaccharides (LPS) compared to wt BMDC, while activation-induced cytokine expression of lymphocytes and macrophages was not majorly altered. Ex vivo stimulation of macrophages with IFN-γ resulted in increased cytokine release. These results suggest that SphK1/2 are involved in production and secretion of IFN-γ by DC. DC-derived IFN-γ subsequently stimulates the production and secretion of a large panel of inflammatory cytokines by macrophages, which belong to the main cytokine-releasing cells of the early innate immune response. Inhibitors of SphK1/2 may therefore be attractive targets to dampen the early cytokine response of macrophages as part of the innate immune response.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Aggravates Atherosclerosis and Induces Plaque Rupture in ApoE-/-Mice.

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE-/-) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE-/-/S1PR3-/- mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.

Targeting defective sphingosine kinase 1 in Niemann-Pick type C disease with an activator mitigates cholesterol accumulation.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.

Erythrocytes increase endogenous sphingosine 1-phosphate levels as an adaptive response to SARS-CoV-2 infection.

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Development and validation of a QTrap method for sensitive quantification of sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid and ligand for five G protein-coupled cell-surface receptors designated S1PR1-5. The determination of low levels of S1P remains a challenge and usually requires sophisticated analytical instrumentation and methodology. This report describes a technique using the linear ion trap mode of a basic QTrap triple-quadrupole mass spectrometer. S1P was extracted from acidified biological samples using a modified Folch extraction procedure. After the addition of C17-sphingosine as an internal standard, a step gradient LC method was used to separate the analytes on a reversed-phase C18 MultoHigh analytical column. After the internal standard C17-sphingosine was detected by multiple reaction monitoring (MRM), the detection mode was switched to enhanced product ion (EPI) mode for the detection of S1P. The mode was switched back to MRM again for the detection of other analytes. Using this QTrap method, we reached a limit of detection of 1 nM and a limit of quantification of 3 nM for S1P, which was up to 30 times more sensitive than the MRM mode with the same instrument. Intra-day precision ranged between -3.8 and 6.3%, and inter-day precision was between -13.8 and 3.3%, depending on the spiked S1P concentration.

Sphingosine-1 Phosphate Lyase Regulates Sensitivity of Pancreatic Beta-Cells to Lipotoxicity.

Elevated levels of free fatty acids (FFAs) have been related to pancreatic beta-cell failure in type 2 diabetes (T2DM), though the underlying mechanisms are not yet fully understood. FFAs have been shown to dysregulate formation of bioactive sphingolipids, such as ceramides and sphingosine-1 phosphate (S1P) in beta-cells. The aim of this study was to analyze the role of sphingosine-1 phosphate lyase (SPL), a key enzyme of the sphingolipid pathway that catalyzes an irreversible degradation of S1P, in the sensitivity of beta-cells to lipotoxicity. To validate the role of SPL in lipotoxicity, we modulated SPL expression in rat INS1E cells and in human EndoC-ßH1 beta-cells. SPL overexpression in INS1E cells (INS1E-SPL), which are characterized by a moderate basal expression level of SPL, resulted in an acceleration of palmitate-mediated cell viability loss, proliferation inhibition and induction of oxidative stress. SPL overexpression affected the mRNA expression of ER stress markers and mitochondrial chaperones. In contrast to control cells, in INS1E-SPL cells no protective effect of oleate was detected. Moreover, Plin2 expression and lipid droplet formation were strongly reduced in OA-treated INS1E-SPL cells. Silencing of SPL in human EndoC-ßH1 beta-cells, which are characterized by a significantly higher SPL expression as compared to rodent beta-cells, resulted in prevention of FFA-mediated caspase-3/7 activation. Our findings indicate that an adequate control of S1P degradation by SPL might be crucially involved in the susceptibility of pancreatic beta-cells to lipotoxicity.

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis.

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Niemann-Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1-/- mice and evaluated specific effects of treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1-/- mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1-/- mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

Regulation of ABCA1-mediated cholesterol efflux by sphingosine-1-phosphate signaling in macrophages.

Sphingolipid and cholesterol metabolism are closely associated at the structural, biochemical, and functional levels. Although HDL-associated sphingosine-1-phosphate (S1P) contributes to several HDL functions, and S1P signaling regulates glucose and lipid metabolism, no study has addressed the involvement of S1P in cholesterol efflux. Here, we show that sphingosine kinase (Sphk) activity was induced by the LXR agonist 22(R)-hydroxycholesterol and required for the stimulation of ABCA1-mediated cholesterol efflux to apolipoprotein A-I. In support, pharmacological Sphk inhibition and Sphk2 but not Sphk1 deficiency abrogated efflux. The involved mechanism included stimulation of both transcriptional and functional ABCA1 regulatory pathways and depended for the latter on the S1P receptor 3 (S1P3). Accordingly, S1P3-deficient macrophages were resistant to 22(R)-hydroxycholesterol-stimulated cholesterol efflux. The inability of excess exogenous S1P to further increase efflux was consistent with tonic S1P3 signaling by a pool of constitutively generated Sphk-derived S1P dynamically regulating cholesterol efflux. In summary, we have established S1P as a previously unrecognized intermediate in LXR-stimulated ABCA1-mediated cholesterol efflux and identified S1P/S1P3 signaling as a positive-feedback regulator of cholesterol efflux. This constitutes a novel regulatory mechanism of cholesterol efflux by sphingolipids.

Neural sphingosine 1-phosphate accumulation activates microglia and links impaired autophagy and inflammation.

Microglia mediated responses to neuronal damage in the form of neuroinflammation is a common thread propagating neuropathology. In this study, we investigated the microglial alterations occurring as a result of sphingosine 1-phosphate (S1P) accumulation in neural cells. We evidenced increased microglial activation in the brains of neural S1P-lyase (SGPL1) ablated mice (SGPL1fl/fl/Nes ) as shown by an activated and deramified morphology and increased activation markers on microglia. In addition, an increase of pro-inflammatory cytokines in sorted and primary cultured microglia generated from SGPL1 deficient mice was noticed. Further, we assessed autophagy, one of the major mechanisms in the brain that keeps inflammation in check. Indeed, microglial inflammation was accompanied by defective microglial autophagy in SGPL1 ablated mice. Rescuing autophagy by treatment with rapamycin was sufficient to decrease interleukin 6 (IL-6) but not tumor necrosis factor (TNF) secretion in cultured microglia. Rapamycin mediated decrease of IL-6 secretion suggests a particular mechanistic target of rapamycin (mTOR)-IL-6 link and appeared to be microglia specific. Using pharmacological inhibitors of the major receptors of S1P expressed in the microglia, we identified S1P receptor 2 (S1PR2) as the mediator of both impaired autophagy and proinflammatory effects. In line with these results, the addition of exogenous S1P to BV2 microglial cells showed similar effects as those observed in the genetic knock out of SGPL1 in the neural cells. In summary, we show a novel role of the S1P-S1PR2 axis in the microglia of mice with neural-targeted SGPL1 ablation and in BV2 microglial cell line exogenously treated with S1P.

Hepatocyte nuclear factor 1A deficiency causes hemolytic anemia in mice by altering erythrocyte sphingolipid homeostasis.

The hepatocyte nuclear factor (HNF) family regulates complex networks of metabolism and organ development. Human mutations in its prototypical member HNF1A cause maturity-onset diabetes of the young (MODY) type 3. In this study, we identified an important role for HNF1A in the preservation of erythrocyte membrane integrity, calcium homeostasis, and osmotic resistance through an as-yet unrecognized link of HNF1A to sphingolipid homeostasis. HNF1A-/- mice displayed microcytic hypochromic anemia with reticulocytosis that was partially compensated by avid extramedullary erythropoiesis at all erythroid stages in the spleen thereby excluding erythroid differentiation defects. Morphologically, HNF1A-/- erythrocytes resembled acanthocytes and displayed increased phosphatidylserine exposure, high intracellular calcium, and elevated osmotic fragility. Sphingolipidome analysis by mass spectrometry revealed substantial and tissue-specific sphingolipid disturbances in several tissues including erythrocytes with the accumulation of sphingosine as the most prominent common feature. All HNF1A-/- erythrocyte defects could be simulated by exposure of wild-type (WT) erythrocytes to sphingosine in vitro and attributed in part to sphingosine-induced suppression of the plasma-membrane Ca2+-ATPase activity. Bone marrow transplantation rescued the anemia phenotype in vivo, whereas incubation with HNF1A-/- plasma increased the osmotic fragility of WT erythrocytes in vitro. Our data suggest a non-cell-autonomous erythrocyte defect secondary to the sphingolipid changes caused by HNF1A deficiency. Transcriptional analysis revealed 4 important genes involved in sphingolipid metabolism to be deregulated in HNF1A deficiency: Ormdl1, sphingosine kinase-2, neutral ceramidase, and ceramide synthase-5. The considerable erythrocyte defects in murine HNF1A deficiency encourage clinical studies to explore the hematological consequences of HNF1A deficiency in human MODY3 patients.

Acid Sphingomyelinase Inhibition Stabilizes Hepatic Ceramide Content and Improves Hepatic Biotransformation Capacity in a Murine Model of Polymicrobial Sepsis.

Liver dysfunction during sepsis is an independent risk factor leading to increased mortality rates. Specifically, dysregulation of hepatic biotransformation capacity, especially of the cytochrome P450 (CYP) system, represents an important distress factor during host response. The activity of the conserved stress enzyme sphingomyelin phosphodiesterase 1 (SMPD1) has been shown to be elevated in sepsis patients, allowing for risk stratification. Therefore, the aim of the present study was to investigate whether SMPD1 activity has an impact on expression and activity of different hepatic CYP enzymes using an animal model of polymicrobial sepsis. Polymicrobial sepsis was induced in SMPD1 wild-type and heterozygous mice and hepatic ceramide content as well as CYP mRNA, protein expression and enzyme activities were assessed at two different time points, at 24 h, representing the acute phase, and at 28 days, representing the post-acute phase of host response. In the acute phase of sepsis, SMPD1+/+ mice showed an increased hepatic C16- as well as C18-ceramide content. In addition, a downregulation of CYP expression and activities was detected. In SMPD1+/- mice, however, no noticeable changes of ceramide content and CYP expression and activities during sepsis could be observed. After 28 days, CYP expression and activities were normalized again in all study groups, whereas mRNA expression remained downregulated in SMPD+/+ animals. In conclusion, partial genetic inhibition of SMPD1 stabilizes hepatic ceramide content and improves hepatic monooxygenase function in the acute phase of polymicrobial sepsis. Since we were also able to show that the functional inhibitor of SMPD1, desipramine, ameliorates downregulation of CYP mRNA expression and activities in the acute phase of sepsis in wild-type mice, SMPD1 might be an interesting pharmacological target, which should be further investigated.

Modulating sphingosine 1-phosphate signaling with DOP or FTY720 alleviates vascular and immune defects in mouse sepsis.

Sepsis is a systemic inflammatory response to pathogens and a leading cause of hospital related mortality worldwide. Sphingosine 1-phosphate (S1P) regulates multiple cellular processes potentially involved in the pathogenesis of sepsis, including antigen presentation, lymphocyte egress, and maintenance of vascular integrity. We thus explored the impact of manipulating S1P signaling in experimental polymicrobial sepsis in mice. Administration of 4-deoxypyridoxine (DOP), an inhibitor of the S1P-degrading enzyme S1P-lyase, or of the sphingosine analog FTY720 that serves as an S1P receptor agonist after phosphorylation ameliorated morbidity, improved recovery from sepsis in surviving mice, and reduced sepsis-elicited hypothermia and body weight loss. Treated mice developed lymphopenia, leading to an accumulation of lymphocytes in peripheral lymph nodes, and reduced bacterial burden in liver, but not in blood. Sepsis-induced upregulation of mRNA expression of cytokines in spleen remained unchanged, but reduction of IL-6, TNF-α, MCP-1, and IL-10 in plasma was evident. DOP and FTY720 treatment significantly reduced levels of Evans blue leakage from blood into liver and lung, decreased hematocrit values, and lowered plasma levels of VEGF-A in septic mice. Collectively, our results indicate that modulation of S1P signaling showed a protective phenotype in experimental sepsis by modulating vascular and immune functions.

Tumor specific regulatory T cells in the bone marrow of breast cancer patients selectively upregulate the emigration receptor S1P1.

Regulatory T cells (Treg) hamper anti-tumor T-cell responses resulting in reduced survival and failure of cancer immunotherapy. Among lymphoid organs, the bone marrow (BM) is a major site of Treg residence and recirculation. However, the process governing the emigration of Treg from BM into the circulation remains elusive. We here show that breast cancer patients harbour reduced Treg frequencies in the BM as compared to healthy individuals or the blood. This was particularly the case for tumor antigen-specific Treg which were quantified by MHCII tumor peptide loaded tetramers. We further demonstrate that decreased Treg distribution in the BM correlated with increased Treg redistribution to tumor tissue, suggesting that TCR triggering induces a translocation of Treg from the BM into tumor tissue. Sphingosine-1-phosphate receptor 1 (S1P1)-which is known to mediate exit of immune cells from lymphoid organs was selectively expressed by tumor antigen-specific BM Treg. S1P1 expression could be induced in Treg by BM-resident antigen-presenting cells (BMAPCs) in conjunction with TCR stimulation, but not by TCR stimulation or BMAPCs alone and triggered the migration of Treg but not conventional T cells (Tcon) to its ligand Sphingosine-1-phosphate (S1P). Interestingly, we detected marked S1P gradients between PB and BM in breast cancer patients but not in healthy individuals. Taken together, our data suggest a role for S1P1 in mediating the selective mobilization of tumor specific Treg from the BM of breast cancer patients and their translocation into tumor tissue.

Adjustment of Dysregulated Ceramide Metabolism in a Murine Model of Sepsis-Induced Cardiac Dysfunction.

Cardiac dysfunction, in particular of the left ventricle, is a common and early event in sepsis, and is strongly associated with an increase in patients' mortality. Acid sphingomyelinase (SMPD1)-the principal regulator for rapid and transient generation of the lipid mediator ceramide-is involved in both the regulation of host response in sepsis as well as in the pathogenesis of chronic heart failure. This study determined the degree and the potential role to which SMPD1 and its modulation affect sepsis-induced cardiomyopathy using both genetically deficient and pharmacologically-treated animals in a polymicrobial sepsis model. As surrogate parameters of sepsis-induced cardiomyopathy, cardiac function, markers of oxidative stress as well as troponin I levels were found to be improved in desipramine-treated animals, desipramine being an inhibitor of ceramide formation. Additionally, ceramide formation in cardiac tissue was dysregulated in SMPD1+/+ as well as SMPD1-/- animals, whereas desipramine pretreatment resulted in stable, but increased ceramide content during host response. This was a result of elevated de novo synthesis. Strikingly, desipramine treatment led to significantly improved levels of surrogate markers. Furthermore, similar results in desipramine-pretreated SMPD1-/- littermates suggest an SMPD1-independent pathway. Finally, a pattern of differentially expressed transcripts important for regulation of apoptosis as well as antioxidative and cytokine response supports the concept that desipramine modulates ceramide formation, resulting in beneficial myocardial effects. We describe a novel, protective role of desipramine during sepsis-induced cardiac dysfunction that controls ceramide content. In addition, it may be possible to modulate cardiac function during host response by pre-conditioning with the Food and Drug Administration (FDA)-approved drug desipramine.

Acid Sphingomyelinase Promotes Endothelial Stress Response in Systemic Inflammation and Sepsis.

The pathophysiology of sepsis involves activation of acid sphingomyelinase (SMPD1) with subsequent generation of the bioactive mediator ceramide. We herein evaluated the hypothesis that the enzyme exerts biological effects in endothelial stress response. Plasma-secreted sphingomyelinase activity, ceramide generation and lipid raft formation were measured in human microcirculatory endothelial cells (HMEC-1) stimulated with serum obtained from sepsis patients. Clustering of receptors relevant for signal transduction was studied by immuno staining. The role of SMPD1 for macrodomain formation was tested by pharmacological inhibition. To confirm the involvement of the stress enzyme, direct inhibitors (amino bisphosphonates) and specific downregulation of the gene was tested with respect to ADAMTS13 expression and cytotoxicity. Plasma activity and amount of SMPD1 were increased in septic patients dependent on clinical severity. Increased breakdown of sphingomyelin to ceramide in HMECs was observed following stimulation with serum from sepsis patients in vitro. Hydrolysis of sphingomyelin, clustering of receptor complexes, such as the CD95L/Fas-receptor, as well as formation of ceramide enriched macrodomains was abrogated using functional inhibitors (desipramine and NB6). Strikingly, the stimulation of HMECs with serum obtained from sepsis patients or mixture of proinflammatory cytokines resulted in cytotoxicity and ADAMTS13 downregulation which was abrogated using desipramine, amino bisphosphonates and genetic inhibitors. SMPD1 is involved in the dysregulation of ceramide metabolism in endothelial cells leading to macrodomain formation, cytotoxicity and downregulation of ADAMTS13 expression. Functional inhibitors, such as desipramine, are capable to improve endothelial stress response during sepsis and might be considered as a pharmacological treatment strategy to favor the outcome.

Deficiency of sphingosine-1-phosphate lyase impairs lysosomal metabolism of the amyloid precursor protein.

Progressive accumulation of the amyloid ß protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid ß is generated during sequential cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca(2+) from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.

Evaluating sphingosine and its analogues as potential alternatives for aggressive lymphoma treatment.

BACKGROUND: Ceramide (Cer) and sphingosine (Sph) interfere with critical cellular functions relevant for cancer progression and cell survival. While Cer has already been investigated as a potential drug target for lymphoma treatment, information about the potency of sphingosine is scarce. The aim of this study therefore was to evaluate Sph and its synthetic stereoisomer L-threo-sphingosine (Lt-Sph) as potential treatment options for aggressive lymphomas. METHODS: Diffuse large B cell lymphoma (DLBCL) cell lines were incubated with Sph and Lt-Sph and consequently analysed by flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to triple-quadrupole mass spectrometry (LC/MS/MS), electron microscopy, and Western blot. RESULTS: Sph induced cell death and blocked cell growth independently of S1P receptors in different DLBCL cell lines. Three different modes of Sph-mediated cell death were observed: Apoptosis, autophagy, and protein kinase C (PKC) inhibition. Generation of pro-apoptotic Cer accounted only for a minor portion of the apoptotic rate. CONCLUSION: Sph and its analogues could evolve as alternative treatment options for aggressive lymphomas via PKC inhibition, apoptosis, and autophagy. These physiological responses induced by different intracellular signalling cascades (phosphorylation of JNK, PARP cleavage, LC3-II accumulation) identify Sph and analogues as potent cell death inducing agents.