Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens

Ceramide (Cer) accumulating during the execution phase of apoptosis is generated from plasma membrane sphingomyelin (SM), which gains access to a sphingomyelinase due to phospholipid scrambling (Tepper, A. D., Ruurs, P., Wiedmer, T., Sims, P., Borst, J., and van Blitterswijk, W. J. (2000) J. Cell. Biol. 150, 155-164). To evaluate the functional significance of this Cer pool, we aimed to convert it to glucosylceramide (GlcCer), by constitutive overexpression of glucosylceramide synthase (GCS). Jurkat cells, retrovirally transduced with GCS cDNA, showed a 10-12-fold increase in GCS activity in vitro and a 7-fold elevated basal GlcCer level in vivo. However, Cer accumulating during apoptosis induced by ligation of the death receptor CD95, treatment with the anti-cancer drug etoposide, or exposure to gamma-radiation was not glycosylated by GCS. Likewise, Cer liberated at the plasma membrane by bacterial SMase was not converted by the enzyme. Thus, GCS, located at the Golgi, is topologically segregated from Cer produced in the plasma membrane. In contrast, de novo synthesized Cer as well as an exogenously supplied cell-permeable Cer analog were efficiently glycosylated, apparently due to different Cer topology and distinct physicochemical behavior of the synthetic Cer species, respectively. Exogenous cell-permeable Cer species, despite their conversion by GCS, effectively induced apoptosis. We also observed that GCS activity is down-regulated in cells undergoing apoptosis. In conclusion, GCS can convert de novo synthesized Cer but not SM-derived Cer, and, therefore, the ability of GCS overexpression to protect cells from possible detrimental effects of Cer accumulation is limited.     

Glucosylceramide Synthesized In Vitro from Endogenous Ceramide is Uncoupled from Synthesis of Lactosylceramide in Golgi Membranes from Chicken Embryo Neural Retina Cells

It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[³H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[³H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [³H]GlcCer from those enriched in [³H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.     

Plasma glucosylceramide and ceramide in type 1 Gaucher disease patients: correlations with disease severity and response to therapeutic intervention

The concentrations of plasma glucosylceramide (GlcCer) and ceramide (Cer) were determined in a cohort of type 1 Gaucher disease patients. In plasma of untreated patients, GlcCer concentrations were on average 3-fold increased (median Gaucher: 17.5 nmol/ml, range: 6.5-45.5 (n=27); median control: 5.9 nmol/ml, range 4.0-8.6 (n=15)). Although plasma Cer concentrations were not significantly different between the two groups (median Gaucher: 7.2 nmol/ml, range: 4.2-10.9 (n=27); median control: 7.8 nmol/ml, range 5.7-11.9 (n=15)) in individual patients plasma GlcCer/Cer ratio yields slightly better discrimination between Gaucher disease patients and normal individuals than the GlcCer levels. Positive correlations were detected between plasma GlcCer concentration and GlcCer/Cer ratio and severity of disease, plasma chitotriosidase and CCL18, surrogate markers of storage cells. Gaucher disease is treated by enzyme replacement and substrate reduction therapy. Both therapies were found to result in decreases in plasma GlcCer already within 6 months, without causing abnormal plasma GlcCer or Cer concentrations. The corrections in plasma GlcCer were most robust in patients with a pronounced clinical response. In conclusion, plasma GlcCer concentration and GlcCer/Cer ratio is of value to monitor Gaucher disease manifestation and response to therapeutic intervention.     

Up-regulation of glucosylceramide synthesis upon stimulation of axonal growth by basic fibroblast growth factor. Evidence for post-translational modification of glucosylceramide synthase

We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor (bFGF) and laminin to stimulate axonal growth in cultured hippocampal neurons (Boldin, S., and Futerman, A. H. (1997) J. Neurochem. 68, 882-885). We now demonstrate that stimulation of axonal growth by bFGF leads to an increase in the rate of GlcCer synthesis. Within minutes of incubation with bFGF, a significant increase in the rate of metabolism of [(14)C]hexanoyl ceramide to [(14)C]hexanoyl GlcCer is detected, but there are no changes in the rate of [(14)C]hexanoyl sphingomyelin synthesis. In vitro analysis of GlcCer synthase activity revealed an approximately 2-fold increase in the rate of [(14)C]hexanoyl GlcCer synthesis upon incubation with either bFGF or laminin; other growth factors, which did not effect the rate of axon growth, had no effect on the rate of [(14)C]hexanoyl GlcCer synthesis. The increased rate of [(14)C]hexanoyl GlcCer synthesis was not affected by preincubation with either cycloheximide or actinomycin, and no elevation of GlcCer synthase mRNA levels was detected, suggesting that GlcCer synthase is up-regulated by a post-translational mechanism. The relevance of these results for understanding the regulation of axonal growth is discussed.     

The induction of colonocyte differentiation in CaCo-2 cells by sodium butyrate causes an increase in glucosylceramide synthesis in order to avoid apoptosis based on ceramide

To examine the relationship between apoptosis accompanying differentiation and sphingolipid-metabolism, CaCo-2 cells were used as a model of human intestinal epithelial cells and the variation in cellular Cer/GlcCer-content and related enzyme activities during butyrate-induced differentiation were investigated. The simultaneous administration of PDMP as a GlcCer synthase inhibitor caused a significant increase in the amount of Cers, especially palmitoyl-Cer. Butyrate caused an increase in the amount of GlcCers, especially alpha-hydroxy fatty acid-GlcCers, and in cellular GlcCer synthase activity. Cellular Cer content related to apoptosis was mainly regulated by the GlcCer synthase-based metabolism of Cers.     

Regulation of sphingolipid and glycosphingolipid metabolism in extrahepatic tissues by endotoxin

The host response to infection and inflammation is associated with multiple alterations in lipid metabolism. We have shown that endotoxin [lipopolysaccharide (LPS)] stimulates hepatic sphingolipid synthesis and increases ceramide and glucosylceramide (GlcCer) content in circulating lipoproteins in Syrian hamsters. LPS also increases the activity and mRNA levels of serine palmitoyltransferase (SPT) and GlcCer synthase, the committed enzymes in sphingolipid and glycosphingolipid (GSL) synthesis, respectively, in the liver. To determine whether sphingolipid and GSL metabolism are regulated in other tissues during the host response to infection, we examined the effect of LPS on the regulation of SPT and GlcCer synthase in extrahepatic tissues in Syrian hamsters. LPS significantly increased SPT activity in spleen and kidney after 16 h of treatment, but had no effect on SPT activity in lung and brain, suggesting that the effect of LPS on sphingolipid metabolism is tissue specific. LPS also increased SPT mRNA levels in spleen and kidney by approximately 3-fold, suggesting that the increase in SPT activity is due to an increase in SPT mRNA expression. LPS significantly increased GlcCer synthase activity in spleen and kidney, and produced 4- and 15-fold increases in GlcCer synthase mRNA levels in spleen and kidney, respectively. LPS treatment increased GlcCer content by 1.3-fold in spleen and by 6.2-fold in kidney. LPS also increased the content of ceramide trihexoside by 1.7-fold in spleen. These results suggest that LPS regulates sphingolipid and GSL metabolism in spleen and kidney. An increase in GSL metabolites in spleen and kidney during the host response to infection and inflammation may be required for modulation of immune responses and regulation of cell growth. -- Memon, R. A., W. M. Holleran, Y. Uchida, A. H. Moser, C. Grunfeld, and K. R. Feingold. Regulation of sphingolipid and glycosphingolipid metabolism in extrahepatic tissues by endotoxin. J. Lipid Res. 2001. 42: 452--459.     

Glycosphingolipid composition of a renal cell line (MDCK) and its ouabain-resistant mutant

Glycosphingolipids were isolated from a canine kidney cell line (MDCK) and its ouabain-resistant mutant (MDCK-OR) by solvent extraction, mild alkaline methanolysis, a DEAE-Sephadex column, and preparative TLC. The glycolipids were characterized by their mobilities on TLC, an analysis of carbohydrates as trimethylsilyl methyl glycosides and acetates of partially methylated alditols, as well as by treatment with specific glycosidases. In the neutral glycolipid fraction of both cell lines, galactosylceramide (GalCer), glucosylceramide (GlcCer), lactosylceramide (LacCer), digalactosylceramide (Ga2Cer), globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), and the Forssman antigen (IV3GalNAc alpha-Gb4Cer) were identified. The contents of Ga2Cer (4.4 nmol/mg protein), Gb3Cer (0.6), Gb4Cer (2.9), and IV3GalNac alpha-Gb4Cer (19.5) in MDCK-OR were 1.4- to 2.1-fold higher than those in MDCK, while the concentrations of GlcCer (5.3) and LacCer (1.4) in MDCK-OR were about half of those in MDCK. Among acidic glycolipids of MDCK-OR, galactosyl sulfatide (GalCer-I3-sulfate) and lactosyl sulfatide (LacCer-II3-sulfate) were increased to 1.9 (2.7-fold) and 0.2 nmol/mg protein (2.0-fold), respectively, as compared to MDCK. However, N-acetylneuraminosyllactosylceramide (GM3), the predominant ganglioside in both cell lines, was decreased to about one third of the level (1.5 nmol/mg protein) in the parent MDCK (4.7 nmol/mg protein). The fatty acid of the glycolipids in both cell lines consisted mainly of saturated acids of 16, 18, 22, and 24 carbons.     

Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 μM adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 μM adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 μM adaGalCer, cell synthesis of only Gb(3) and Gb(4) was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb(2)), was generated, indicating substrate competition for Gb(3) synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb(3) synthesis was inhibited by adaGalCer. Metabolic labeling of Gb(3) in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb(2) was produced. AdaGb(2) in cells was 10-fold more effectively shed into the medium than the more polar Gb(3), providing an easily eliminated "safety valve" alternative to Gb(3) accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.     

Long-chain glucosylceramides crosstalk with LYN mediates endometrial cell migration

Endometriosis is a disease characterized by regurgitated lesions which are invasive and migratory, embedding at ectopic, extra-uterine locations. Extracellular glucosylceramides (GlcCers), bioactive sphingolipids potentiating signals for cell migration, are found in elevated levels in endometriosis; however underlying mechanisms that result in cellular migration are poorly defined. Here, we demonstrated that internalized GlcCer induced migratory activity in immortalized human endometrial stromal cells (HESCs), with highest potency observed in long-chain GlcCer. Long-chain ceramide (Cer) similarly induced cellular migration and mass spectrometry results revealed that the migratory behavior was contributed through glycosylation of ceramides. Cells treated with GlcCer synthase inhibitor, or RNAi-mediated knockdown of glucosylceramide synthase (GCS), the enzyme catalyzing GlcCer production attenuated cell motility. Mechanistic studies showed that GlcCer acts through stromal cell-derived factor-1 alpha and its receptor, CXC chemokine receptor 4 (SDF-1α-CXCR4) signaling axis and is dependent on phosphorylation of LYN kinase at Tyr396, and dephosphorylation of Tyr507. Migration was prominently attenuated in cells exposed to CXCR4 antagonist, AMD3100, yet can be rescued with diprotin A, which prevents the degradation of SDF-1α. Furthermore, blocking of LYN kinase activity in the presence of SDF-1α and GlcCer reduced HESC migration, suggesting that LYN acts downstream of GlcCer-SDF-1α-CXCR4 axis as part of its intracellular signal transduction. Our results reveal a novel role of long-chain GlcCer and the dialog between GlcCer, LYN^pTyr396 and SDF-1α-CXCR4 in inducing HESC migration. This finding may improve our understanding how endometriotic lesions invade to their ectopic sites, and the possibility of using GlcCer to modulate the SDF-1α-CXCR4-LYN^pTyr396 axis in endometriosis.     

AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides

The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer.     

Endogenous β-glucocerebrosidase activity in Abca12−/−epidermis elevates ceramide levels after topical lipid application but does not restore barrier function

ABCA12 mutations disrupt the skin barrier and cause harlequin ichthyosis. We previously showed Abca12^−/− skin has increased glucosylceramide (GlcCer) and correspondingly lower amounts of ceramide (Cer). To examine why loss of ABCA12 leads to accumulation of GlcCer, de novo sphingolipid synthesis was assayed using [¹⁴C]serine labeling in ex vivo skin cultures. A defect was found in β-glucocerebrosidase (GCase) processing of newly synthesized GlcCer species. This was not due to a decline in GCase function. Abca12^−/− epidermis had 5-fold more GCase protein (n = 4, P < 0.01), and a 5-fold increase in GCase activity (n = 3, P < 0.05). As with Abca12^+/+ epidermis, immunostaining in null skin showed a typical interstitial distribution of the GCase protein in the Abca12^−/− stratum corneum. Hence, we tested whether the block in GlcCer conversion could be circumvented by topically providing GlcCer. This approach restored up to 15% of the lost Cer products of GCase activity in the Abca12^−/− epidermis. However, this level of barrier ceramide replacement did not significantly reduce trans-epidermal water loss function. Our results indicate loss of ABCA12 function results in a failure of precursor GlcCer substrate to productively interact with an intact GCase enzyme, and they support a model of ABCA12 function that is critical for transporting GlcCer into lamellar bodies.     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

Transbilayer movement of ceramide in the plasma membrane of live cells

Ceramide (Cer) is the precursor for sphingolipids and functions as a second messenger in a variety of cellular processes including apoptosis. However, no direct target of Cer leading to apoptosis has been identified. Understanding the movement and trafficking of Cer is important for fully understanding Cer signaling. In this study, we identified, for the first time, the transbilayer movement of Cer in the plasma membrane (PM) of living cells. We developed a new method to monitor transbilayer Cer movement using ceramide kinase activity. To produce Cer on the extracellular leaflet of the PM, bacterial sphingomyelinase (SMase) was added to rat basophilic leukemia cells. Interestingly, the dramatic elevation of ceramide 1-phosphate (C1P), the product of CerK, was observed following the increase of Cer induced by SMase treatment. Since we determined that both the protein and catalytic activity of CerK exists in the intracellular compartment, the all conversion of Cer to C1P by CerK should be occur intracellularly. This result indicates the rapid transbilayer movement of Cer from the outer leaflet to the inner leaflet of the PM of living cells. Furthermore, protease digestion of membrane proteins, inhibition of ABC transporters (by glibencramide) and of cation channels (by carbonyl cyanide m-chlorophenylhydrozone), and modification of cholesterol content did not affect the transbilayer movement of Cer. Thus, this movement might occur spontaneously. Our findings indicate not only Cer movement in the PM, but also identify an intrinsic property of Cer enabling Cer signaling.     

Tumor Necrosis Factor-α-Induced Apoptosis in a Human Keratinocyte Cell Line (HaCaT) Is Counteracted by Transforming Growth Factor-α

The integrity of the human epidermis is guaranteed by a regulated balance of proliferation, differentiation, and physiologic cell death of its main cellular constituent, the epidermal keratinocyte. Physiologic cell death is known as apoptosis and has been recognized as an active regulatory mechanism, complementary to, but functionally opposite of, proliferation. The regulators of the delicate balance between cell death and proliferation are only partially understood in human keratinocytes. Transforming growth factor-α (TGF-α) has been identified as a positive regulator of proliferation and growth, while tumor necrosis factor-α (TNF-α) induces apoptosis. Both mediators are thought to influence epidermal keratinocytes under various physiological and pathophysiological conditions. In the current study we have begun to investigate potential regulatory interactions between these two mediators in the human keratinocyte cell line HaCaT. We have found that, when the HaCaT cells were sensitized by the translation inhibitor cycloheximide, TNF-α induced apoptosis, as evidenced by nuclear disintegration, DNA fragmentation (“DNA laddering”), and the appearance of soluble DNA/histone complexes. Moreover, we found that the induction of apoptosis was reduced by preincubation of the cells with TGF-α. The protective effect of TGF-α was abrogated by translation inhibition, indicating that it depended onde novoprotein synthesis. Moreover, the protective effect was not accompanied by a reduced surface expression of TNF receptor molecules. We postulate that TNF-α-induced apoptosis in HaCaT cells is counteracted by constitutively produced suppressors of apoptosis, the synthesis of which can be downregulated by inhibition of translation and upregulated by the cytokine TGF-α.     

Increased urinary excretion of glycosphingolipids in familial hypercholesterolemia

The content of glycosphingolipids (GSL) was studied in the urinary sediments (24-hr specimens) from seven normal subjects, a patient with Fabry's disease, and five homozygotes with familial hypercholesterolemia (FH). Normal urinary sediments contained very small amounts of GalCer, GlcCer, GaOse(2)Cer, LacCer, GbOse(3)Cer, and GbOse(4)Cer. In Fabry urinary sediment, the levels (nmole glucose/24 hr) of GaOse(2)Cer and of GbOse(3)Cer were 389 and 550, respectively. In urinary sediments from the FH subjects, the mean contents (nmol glucose/mg protein per 24 hr) of GlcCer, GalCer, and LacCer were 2.7, 1.9, and 15.8 times higher, respectively, than in normals. The mean contents ( micro g/mg protein per 24 hr) of total cholesterol and phospholipid in the urinary sediment of FH (1.1 and 224, respectively) and normals (0.8 and 220) were similar. The mean contents of GlcCer, GalCer, and LacCer, expressed in terms of the cholesterol content of urinary sediment (nmol glucose/ micro g cholesterol per 24 hr), were increased 3.4-, 1.6-, and 5.4-fold, respectively, in the FH homozygotes. Of the five FH homozygotes, only one, who had undergone a portacaval shunt and was also receiving lipid-lowering therapy, had a normal value of LacCer. The other four FH homozygotes had levels of LacCer that were 3- to 55-fold higher (nmol glucose/mg protein per 24 hr) and 5.5- to 7.3-fold higher (nmol glucose/ micro g cholesterol per 24 hr) than the mean of the normals. One homozygote underwent plasma exchange therapy that reduced both the baseline urinary (nmol glucose/24 hr) and plasma (nmol/100 ml) LacCer levels from 86 to 7 and from 1491 to 852, respectively. Eleven days after plasma exchange, the urinary LacCer levels approached pre-exchange levels (59 nmol glucose/24 hr). The data indicate that there is an abnormality of GSL metabolism associated with familial hyper-cholesterolemia and that the urinary excretion of GSL can be modified by plasma exchange therapy.-Chatterjee, S., C. S. Sekerke, and P. O. Kwiterovich, Jr. Increased urinary excretion of glycosphingolipids in familial hypercholesterolemia.     

Epidermal sphingomyelins are precursors for selected stratum corneum ceramides

Epidermal ceramides (Cer) comprise a heterogeneous family of seven species, including two unique omega-hydroxylated Cer, that are key components of the stratum corneum (SC) intercellular lamellar membranes responsible for the epidermal permeability barrier. Although both glucosylceramide (GlcCer) and the phospho-sphingolipid sphingomyelin (SM) are potential precursors of SC Cer, based on reported chemical structures of epidermal GlcCer and SC Cer, it is assumed that all major subfractions of SC Cer are generated from lamellar body-derived GlcCer. Yet, we and others have shown that SM-derived Cer are required for normal barrier homeostasis. Moreover, two pools of SM, one from plasma membrane, the other from lamellar body-derived contents, are potentially available for Cer production. To clarify the role of SM as a potential precursor of bulk or specific SC Cer, we compared Cer moieties in epidermal SM, Cer generated from epidermal SM by sphingomyelinase treatment, Cer within SC, and Cer that persist in Gaucher SC, where GlcCer cannot generate Cer due to an absence of beta-glucocerebrosidase. Using gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance for Cer characterization, epidermal SM comprise three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) compositions: that is, either long-chain FA (SM-1; C(22;-26)), short-chain FA (SM-2; primarily C(16)), and short-chain alpha-hydroxy FA (SM-3; C(16;-18)). In contrast, only trace quantities of omega-hydroxy FA were present. For each SM subfraction, the sphingoid base was either sphingosine or sphinganine, but phytosphingosine was not detected. Comparison of these SM with corresponding sphingomyelinase-generated epidermal Cer and SC Cer revealed that the Cer moieties of SM-1 and SM-3 are equivalent to Cer 2 (NS) and Cer 5 (AS), respectively. Moreover, both Cer 2 and Cer 5 occurred in Gaucher SC, whereas other Cer subfractions did not occur.These results indicate that two epidermal SM, that is, SM-1 and SM-3, are important precursors of two corresponding Cer in mammalian SC, that is, Cer 2 and Cer 5, but other Cer species, including the omega-hydroxy Cer species, do not derive from SM.     

Changes in the glycolipid composition and characteristic activation of GM3 synthase in the thymus of mouse after administration of dexamethasone

Glycolipids in the thymus of mice after administration of dexamethasone were compared with those in control mice. In parallel with a decrease in the tissue weight due to the disappearance of immature thymocytes in the cortex, the amounts of GlcCer, Gg₄Cer and GM1 decreased from 18 h after intraperitoneal administration of dexamethasone, but those of Gb₄Cer and Forssman glycolipid did not change, indicating the differential distribution of ganglio- and globo-series glycolipids in the thymus, GlcCer, Gg₄Cer and GM1 being on dexamethasone-sensitive cortical thymocytes, and Gb₄Cer and Forssman glycolipid on dexamethasone-resistant cells including thymic stromal cells, respectively. At the same time, a characteristic increase in GM3, whose amount per thymus and concentration per mg of thymus were increased 4-fold and 13-fold compared to those in the control mice, respectively, was observed at the onset of the decrease in tissue weight and was due to the increased activity of LacCer sialyltransferase with the enhanced expression of its gene and the concomitant decrease in cytosolic sialidase activity. One can suggest that endogenous accumulation of GM3 is involved in the dexamethasone-induced apoptosis of cortical thymocytes. On radiolabeling of the thymus with CMP-[¹⁴C]-NeuAc, the incorporation of radioactivity into GM3 was preferentially observed in the thymuses of dexamethasone-administered mice, but not in those of control mice, suggesting the possible involvement of plasma membrane-associated sialytransferase in GM3 synthesis in the thymuses of dexamethasone-administered mice. Published in 2005.     

Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects ofD-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and itsL-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found thatD-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse₃Cer and GbOse₄Cer. It also directly inhibited the activity of UDP-glucose:ceramide 1 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer 1 4 galactosyltransferase (GalT-2). In contrast,L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse₃Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse₄Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited byD-PDMP and stimulated byL-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding toD-PDMP and, in the case of GbOse₄Cer synthase, byL-PDMP as well. The stimulatory effects ofL-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Abbreviations GalT-2 UDP-galactose:GlcCer -galactosyltransferase - GbOse₃Cer Gal1 4Gal1 GlcCer - GbOse₄Cer GalNAc1 3Gal1 4Gal1 GlcCer - GlcCer glucosylceramide - GlcT-1 UDP-glucose:ceramide -glucosyltransferase - GSLs glycosphingolipids - LacCer lactosylceramide - PDMP threo-1-phenyl-2-decanolyamino-3-morpholino-1-propanol