The regulation of p53, p38 MAPK, JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells.

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells.

Control of CD1d-restricted antigen presentation and inflammation by sphingomyelin.

Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann-Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.

Analysis of 1-Deoxysphingoid Bases and Their N-Acyl Metabolites and Exploration of Their Occurrence in Some Food Materials.

Most common sphingolipids are comprised of "typical" sphingoid bases (sphinganine, sphingosine, and structurally related compounds) and are produced via the condensation of l-serine with a fatty acyl-CoA by serine palmitoyltransferase. Some organisms, including mammals, also produce "atypical" sphingoid bases that lack a 1-hydroxyl group as a result of the utilization of l-alanine or glycine instead of l-serine, resulting in the formation of 1-deoxy- or 1-desoxymethylsphingoid bases, respectively. Elevated production of "atypical" sphingolipids has been associated with human disease, but 1-deoxysphingoid bases have also been found to have potential as anticancer compounds, hence, the importance of knowing more about the occurrence of these compounds in food. Most of the "typical" and "atypical" sphingoid bases are found as the N-acyl metabolites (e.g., ceramides and 1-deoxyceramides) in mammals, but this has not been uniformly assessed in previous studies nor determined in consumed food. Therefore, we developed a method for the quantitative analysis of "typical" and "atypical" sphingoid bases and their N-acyl derivatives by reverse-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. On the basis of these analyses, there was considerable variability in the amounts and molecular subspecies of atypical sphingoid bases and their N-acyl metabolites found in different edible sources. These findings demonstrate that a broader assessment of the types of sphingolipids in foods is needed because some diets might contain sufficient amounts of atypical as well as typical sphingolipids that could have beneficial or possibly deleterious effects on human health.

Native and Polyubiquitinated Forms of Dihydroceramide Desaturase Are Differentially Linked to Human Embryonic Kidney Cell Survival.

There is controversy concerning the role of dihydroceramide desaturase (Degs1) in regulating cell survival, with studies showing that it can both promote and protect against apoptosis. We have therefore investigated the molecular basis for these opposing roles of Degs1. Treatment of HEK293T cells with the sphingosine kinase inhibitor SKi [2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole] or fenretinide, but not the Degs1 inhibitor GT11 {N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octan-amide}, induced the polyubiquitination of Degs1 (Mr = 40 to 140 kDa) via a mechanism involving oxidative stress, p38 mitogen-activated protein kinase (MAPK), and Mdm2 (E3 ligase). The polyubiquitinated forms of Degs1 exhibit "gain of function" and activate prosurvival pathways, p38 MAPK, c-Jun N-terminal kinase (JNK), and X-box protein 1s (XBP-1s). In contrast, another sphingosine kinase inhibitor, ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], at concentrations of 25 to 50 µM failed to induce formation of the polyubiquitinated forms of Degs1. In contrast to SKi, ABC294640 (25 µM) promotes apoptosis of HEK293T cells via a Degs1-dependent mechanism that is associated with increased de novo synthesis of ceramide. These findings are the first to demonstrate that the polyubiquitination of Degs1 appears to change its function from proapoptotic to prosurvival. Thus, polyubiquitination of Degs1 might provide an explanation for the reported opposing functions of this enzyme in cell survival/apoptosis.

Critical Role for Very-Long Chain Sphingolipids in Invariant Natural Killer T Cell Development and Homeostasis.

The role of sphingolipids (SLs) in the immune system has come under increasing scrutiny recently due to the emerging contributions that these important membrane components play in regulating a variety of immunological processes. The acyl chain length of SLs appears particularly critical in determining SL function. Here, we show a role for very-long acyl chain SLs (VLC-SLs) in invariant natural killer T (iNKT) cell maturation in the thymus and homeostasis in the liver. Ceramide synthase 2-null mice, which lack VLC-SLs, were susceptible to a hepatotropic strain of lymphocytic choriomeningitis virus, which is due to a reduction in the number of iNKT cells. Bone marrow chimera experiments indicated that hematopoietic-derived VLC-SLs are essential for maturation of iNKT cells in the thymus, whereas parenchymal-derived VLC-SLs are crucial for iNKT cell survival and maintenance in the liver. Our findings suggest a critical role for VLC-SL in iNKT cell physiology.

Regulation of very-long acyl chain ceramide synthesis by acyl-CoA-binding protein.

Ceramide and more complex sphingolipids constitute a diverse group of lipids that serve important roles as structural entities of biological membranes and as regulators of cellular growth, differentiation, and development. Thus, ceramides are vital players in numerous diseases including metabolic and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very-long-chain acyl-CoA esters, which is required for its ability to stimulate CerS activity. We also show that high-speed liver cytosol from wild-type mice activates CerS3 activity, whereas cytosol from ACBP knock-out mice does not. Consistently, CerS2 and CerS3 activities are significantly reduced in the testes of ACBP-/- mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial importance in understanding the regulation of ceramide metabolism in pathogenesis.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain.

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.

Neuronal accumulation of glucosylceramide in a mouse model of neuronopathic Gaucher disease leads to neurodegeneration.

Gaucher disease has recently received wide attention due to the unexpected discovery that it is a genetic risk factor for Parkinson's disease. Gaucher disease is caused by the defective activity of the lysosomal enzyme, glucocerebrosidase (GCase; GBA1), resulting in intracellular accumulation of the glycosphingolipids, glucosylceramide and psychosine. The rare neuronopathic forms of GD (nGD) are characterized by profound neurological impairment and neuronal cell death. We have previously described the progression of neuropathological changes in a mouse model of nGD. We now examine the relationship between glycosphingolipid accumulation and initiation of pathology at two pre-symptomatic stages of the disease in four different brain areas which display differential degrees of susceptibility to GCase deficiency. Liquid chromatography electrospray ionization tandem mass spectrometry demonstrated glucosylceramide and psychosine accumulation in nGD brains prior to the appearance of neuroinflammation, although only glucosylceramide accumulation correlated with neuroinflammation and neuron loss. Levels of other sphingolipids, including the pro-apoptotic lipid, ceramide, were mostly unaltered. Transmission electron microscopy revealed that glucosylceramide accumulation occurs in neurons, mostly in the form of membrane-delimited pseudo-tubules located near the nucleus. Highly disrupted glucosylceramide-storing cells, which are likely degenerating neurons containing massive inclusions, numerous autophagosomes and unique ultrastructural features, were also observed. Together, our results indicate that a certain level of neuronal glucosylceramide storage is required to trigger neuropathological changes in affected brain areas, while other brain areas containing similar glucosylceramide levels are unaltered, presumably because of intrinsic differences in neuronal properties, or in the neuronal environment, between various brain regions.

Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses.

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

Age-dependent changes in the sphingolipid composition of mouse CD4+ T cell membranes and immune synapses implicate glucosylceramides in age-related T cell dysfunction.

To determine whether changes in sphingolipid composition are associated with age-related immune dysfunction, we analyzed the core sphingolipidome (i.e., all of the metabolites through the first headgroup additions) of young and aged CD4(+) T cells. Since sphingolipids influence the biophysical properties of membranes, we evaluated the compositions of immune synapse (IS) and non-IS fractions prepared by magnetic immuno-isolation. Broadly, increased amounts of sphingomyelins, dihydrosphingomyelins and ceramides were found in aged CD4(+) T cells. After normalizing for total sphingolipid content, a statistically significant decrease in the molar fraction of glucosylceramides was evident in both the non-IS and IS fractions of aged T cells. This change was balanced by less dramatic increases in the molar fractions of sphingomyelins and dihydrosphingomyelins in aged CD4(+) T cells. In vitro, the direct or enzymatic enhancement of ceramide levels decreased CD4(+) T cell proliferation without regard for the age of the responding T cells. In contrast, the in vitro inhibition of glucosylceramidase preferentially increased the proliferation of aged CD4(+) T cells. These results suggest that reductions in glucosylceramide abundance contribute to age-related impairments in CD4(+) T cell function.

Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry.

This study describes the use of a stable-isotope labeled precursor ([U-¹³C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (¹³C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-¹³C]palmitic acid, ∼60% of the total palmitoyl-CoA was ¹³C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ∼1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.

A method for visualization of "omic" datasets for sphingolipid metabolism to predict potentially interesting differences.

Sphingolipids are structurally diverse and their metabolic pathways highly complex, which makes it difficult to follow all of the subspecies in a biological system, even using "lipidomic" approaches. This report describes a method to use transcriptomic data to visualize and predict potential differences in sphingolipid composition, and it illustrates its use with published data for cancer cell lines and tumors. In addition, several novel sphingolipids that were predicted to differ between MDA-MB-231 and MCF7 cells based on published microarray data for these breast cancer cell lines were confirmed by mass spectrometry. For the data that we were able to find for these comparisons, there was a significant match between the gene expression data and sphingolipid composition (P < 0.001 by Fisher's exact test). Upon considering the large number of gene expression datasets produced in recent years, this simple integration of two types of "omic" technologies ("transcriptomics" to direct "sphingolipidomics") might facilitate the discovery of useful relationships between sphingolipid metabolism and disease, such as the identification of new biomarkers.

Characterization of mutant serine palmitoyltransferase 1 in LY-B cells.

CHO-LY-B cells have been useful in studies of sphingolipid metabolism and function because they lack serine palmitoyltransferase (SPT) activity. Cloning and sequencing of the SPT1 transcript of LY-B cells identified the mutation as a guanine to adenine change at nucleotide 738, causing a G246R transformation. Western blots revealed low expression of the mutant SPT1 peptide, but activity was not detectable by mass spectrometric analysis of [(13)C]-palmitate incorporation into sphinganine, sphingosine, 1-deoxysphinganine, or 1-desoxymethylsphinganine. Treatment of LY-B cells with chemical chaperones (DMSO or glycerol) increased the amounts of mutant SPT1 as well as SPT2, but SPT activity was not restored. This study has established that G246R mutation in hamster SPT1 results in the loss of SPT activity.

Human cytomegalovirus regulates bioactive sphingolipids.

Sphingolipids are present in membranes of all eukaryotic cells. Bioactive sphingolipids also function as signaling molecules that regulate cellular processes such as proliferation, migration, and apoptosis. Human cytomegalovirus (HCMV) exploits a variety of cellular signaling pathways to promote its own replication. However, whether HCMV modulates lipid signaling pathways is an essentially unexplored area of research in virus-host cell interactions. In this study, we examined the accumulation of the bioactive sphingolipids and the enzymes responsible for the biosynthesis and degradation of these lipids. HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK), the enzyme that generates sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). We also utilized a mass spectrometry approach to generate a sphingolipidomic profile of HCMV-infected cells. We show that HCMV infection results in increased levels of dhS1P and ceramide at 24 h, suggesting an enhancement of de novo sphingolipid synthesis. Subsequently dihydrosphingosine and dhS1P decrease at 48 h consistent with attenuation of de novo sphingolipid synthesis. Finally, we present evidence that de novo sphingolipid synthesis and sphingosine kinase activity directly impact virus gene expression and virus growth. Together, these findings demonstrate that host cell sphingolipids are dynamically regulated upon infection with a herpes virus in a manner that impacts virus replication.