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1.
3-Keto derivatives were prepared in good yield by the oxidative procedure with 2,3-dichloro-5,6-dicyanobenzoquinone from N-acetyl sphingosine, N-palmitoyl sphingosine, N-lignoceroyl sphingosine, and N-lignoceroyl psychosine. None of these 3-keto derivatives, except the one from N-acetyl sphingosine, have been previously reported. Ceramides were isolated from a calf brain and reacted with 2,3-dichloro-5, 6-dicyanobenzoquinone. Ceramides containing sphingosine (4-sphingenine) were converted to 3-keto derivative, while those containing dihydrosphingosine (sphinganine) remained intact under these conditions. The 3-keto ceramides were then separated from the ceramides containing dihydrosphingosine by preparative thin layer chromatography. Similarly cerebrosides from the same calf brain were oxidized and fractionated to 3-ketocerebrosides (from cerebrosides containing sphingosine) and unreacted cerebrosides (cerebrosides containing dihydrosphingosine). The fatty acid composition of these four sphingolipids were determined. Both the ceramides and the cerebrosides containing sphingosine had more unsaturated fatty acids than the corresponding dihydrosphingosine-containing compounds. The ratio of C16-C20 fatty acids to C22-C26 acids was higher in the ceramides containing sphingosine than in ceramides containing dihydrosphingosine, while the ratio was reversed in cerebrosides. The possible precursor-product relationship among these lipids is discussed. 相似文献
2.
Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis. 相似文献
3.
Jeffrey S. Monette Luis A. Gómez Régis F. Moreau Brett A. Bemer Tory M. Hagen 《Biochemical and biophysical research communications》2010,398(2):272-25362
Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (∼10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (∼70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other. 相似文献
4.
Sphingolipids are key lipid regulators of cell viability: ceramide is one of the key molecules in inducing programmed cell death (apoptosis), whereas other sphingolipids, such as ceramide 1-phosphate, are mitogenic. The thermotropic and structural behavior of binary systems of N-hexadecanoyl-D-erythro-ceramide (C16-ceramide) or N-hexadecanoyl-D-erythro-ceramide-1-phosphate (C16-ceramide-1-phosphate; C16-C1P) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with DSC and deuterium nuclear magnetic resonance (2H-NMR). Partial-phase diagrams (up to a mole fraction of sphingolipids X = 0.40) for both mixtures were constructed based on DSC and 2H-NMR observations. For C16-ceramide-containing bilayers DSC heating scans showed already at Xcer = 0.025 a complex structure of the main-phase transition peak suggestive of lateral-phase separation. The transition width increased significantly upon increasing Xcer, and the upper-phase boundary temperature of the mixture shifted to ∼65°C at Xcer = 0.40. The temperature range over which 2H-NMR spectra of C16-ceramide/DPPC-d62 mixtures displayed coexistence of gel and liquid crystalline domains increased from ∼10° for Xcer = 0.1 to ∼21° for Xcer = 0.4. For C16-C1P/DPPC mixtures, DSC and 2H-NMR observations indicated that two-phase coexistence was limited to significantly narrower temperature ranges for corresponding C1P concentrations. To complement these findings, C16-ceramide/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and C16-C1P/POPC mixtures were also studied by 2H-NMR and fluorescence techniques. These observations indicate that DPPC and POPC bilayers are significantly less perturbed by C16-C1P than by C16-ceramide and that C16-C1P is miscible within DPPC bilayers at least up to XC1P = 0.30. 相似文献
5.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1996,1299(2):259-266
Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C2-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O2−) production. The preincubation of PMN for 15 min with C2-ceramide increased by up to almost 3-fold the amounts of O2− generated in response to 0.1 and 1 μM fMLP. Priming was detected at C2-ceramide concentrations of 2 μM to 4 μM per million PMN. Though less potent than C2-ceramide, C6-ceramide (N-hexanoylsphingosine) could prime for O2− generated in response to 0.1 μM fMLP, with maximal effects obtained at 10–20 μM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O2− generation. As expected, TNF-α (1000 U/ml), also primed for fMLP-induced O2− production; however, the combination of TNF-α and C2-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C2-ceramide and TNF-α. C2-ceramide at 2 μM did not enhance the production of O2− induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B4 (LTB4), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C2-ceramide (2 μM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A2 or D (PLC, PLA2, PLD) were involved in the priming effect by C2-ceramide. However, C2-ceramide inhibited in a dose-related manner the production of O2− induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C2-ceramide. The priming O2− production by C2-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-α have different mechanisms than C2-ceramide. 相似文献
6.
Kai Wang Ruijuan Xu Jennifer Schrandt Prithvi Shah Yong Z. Gong Chet Preston Louis Wang Jae Kyo Yi Chih-Li Lin Wei Sun Demetri D. Spyropoulos Soyoung Rhee Mingsong Li Jie Zhou Shaoyu Ge Guofeng Zhang Ashley J. Snider Yusuf A. Hannun Lina M. Obeid Cungui Mao 《PLoS genetics》2015,11(10)
Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration. 相似文献
7.
Aiping Bai Zdzislaw M. Szulc Jacek Bielawski Nalini Mayroo Xiang Liu James Norris Yusuf A. Hannun Alicja Bielawska 《Bioorganic & medicinal chemistry》2009,17(5):1840-1848
Novel ω-N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid ceramidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and ACDase proteolytic degradation properties of LCL204 [Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.; Liu, X.; Norris, J. S.; Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015].Representative analog LCL464, (1R,2R)-2-N-(12′-N,N-dimethylaminododecanoyl amino)-1-(4″-nitrophenyl)-1,3-propandiol, inhibited ACDase activity in vitro, with a similar potency as B13 but higher than LCL204. LCL464 caused an early inhibition of this enzyme at a cellular level corresponding to decrease of sphingosine and specific increase of C14- and C16-ceramide. LCL464 did not induce lysosomal destabilization nor degradation of ACDase, showed increased cell death demonstrating inherent anticancer activity in a wide range of different cancer cell lines, and induction of apoptosis via executioner caspases activation. LCL464 represents a novel structural lead as chemotherapeutic agent acting via the inhibition of ACDase. 相似文献
8.
Zhou Y Lin XW Yang Q Zhang YR Yuan JQ Lin XD Xu R Cheng J Mao C Zhu ZR 《Biochimie》2011,93(7):1124-1131
Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C6, C12, C16, C18:1, and C24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe2+. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species. 相似文献
9.
《Prostaglandins & other lipid mediators》2010,93(1-4):62-66
There is a growing body of evidence indicating that bioactive sphingolipids play a key role in cancer development, progression and metastasis. However, sphingolipid metabolism in malignant tumors is poorly investigated. Therefore, the aim of the present study was to examine the content of selected intermediates of ceramide metabolism and the activity of key enzymes of ceramide de novo synthesis and sphingosine-1-phosphate (S1P) production in the endometrial cancer. The specimens of cancer tissue and healthy endometrium were obtained from women undergoing surgery because of the cancer (n = 23) and because of myomas (n = 18), respectively. The content of sphinganine, dihydroceramide, ceramide, sphingosine and S1P was measured using high pressure liquid chromatography. The activity of the enzymes was determined using radioactive substrates. It has been found that the content of each examined sphingolipid was markedly elevated in the cancer tissue compared with the healthy endometrium. Namely, sphinganine, sphingosine and dihydroceramide by 3–4.6-fold, ceramide and S1P by 1.9- and 1.6-fold, respectively. Interestingly, the ratio of S1P to ceramide remained stable. The activity of serine palmitoyltransferase and sphingosine kinase 1 was increased by 2.3- and 2.6-fold, respectively. We conclude that endometrial carcinoma is characterized by profound changes in sphingolipid metabolism that likely contribute to its progression and chemoresistance. 相似文献
10.
《Bioorganic & medicinal chemistry letters》2019,29(18):2681-2685
Inspired by the cytotoxicity of perphenazine toward cancer cells and its ability to activate the serine/threonine protein phosphatase 2A (PP2A), we prepared series of ether-carbon linked analogs of a constrained synthetic sphingolipid analog 3, known for its cytotoxicity, nutrient transporter down-regulation and vacuolation properties, incorporating the tricyclic neuroleptics phenoxazine and phenothiazine to represent hybrid structures with possible synergistic cytotoxic activity. While the original activity of the lead compound 3 was diminished by fusion with the phenoxazine or phenothiazine tethered moieties, the corresponding 3-pyridyltetryl ether analog 10 showed cytotoxicity and nutrient transporter down-regulation similar to the lead compound 3, although it separated these PP2A-dependent phenotypes from that of vacuolation. 相似文献
11.
Slavica Ribar Marko Mesari Mirela Bauman 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,754(2)
Endemic nephropathy (EN) is a chronic renal disease present as an endemic in Brodska Posavina, Croatia. The aim of the study was to assess the possible role of fumonisins, i.e., mycotoxins produced by Fusarium moniliforme, as causative agents for EN. Fumonisins inhibit ceramide synthase, the enzyme of de novo synthesis of sphingolipids, which leads to an increase in the sphinganine/sphingosine ratio. In the present study, a modified method has been used for the determination of the sphinganine/sphingosine ratio in human serum and urine of healthy subjects and EN patients from the endemic area. Free sphingoid bases, sphinganine and sphingosine, were obtained by base hydrolysis. Afterwards, precolumn ortho-phthaldialdehyde derivatisation, HPLC separation and quantification by fluorescence detection were performed. The results thus obtained pointed to a sphingolipid metabolism impairment, which may have been induced by fumonisins or fumonisin-like mycotoxins. As statistically significant differences were recorded in the subjects not yet affected with EN, an impairment in the metabolism of sphingolipids might be considered as an early indicator of EN. 相似文献
12.
Masashi Tsunoda Raghubir P. Sharma Ronald T. Riley 《Journal of biochemical and molecular toxicology》1998,12(5):281-289
Fumonisin B1 is a mycotoxin produced by Fusarium moniliforme, a common fungus in corn. It is known to cause a variety of diseases, including hepatic and renal degeneration in many species of laboratory and domestic animals. The known biochemical events in fumonisin B1 toxicity involve inhibition of ceramide synthase leading to disruption of sphingolipid metabolism. The effect of fumonisin B1 on ceramide and more complex sphingolipids in mice is not known. Groups of five male BALB/c mice each were injected with fumonisin B1 subcutaneously at doses of 0, 0.25, 0.75, 2.25, and 6.75 mg/kg body weight daily for 5 days. This protocol has been shown to produce a dose-dependent increase in apoptosis in liver and kidney of these animals. In the present study, liver, kidney, and brain were sampled and analyzed for free sphingoid bases and complex sphingolipids one day after the last treatment. A dose-related accumulation of free sphinganine and sphingosine was observed in liver and kidney, but not brain. The maximal increase in free sphinganine in kidney was 10-fold greater than in liver. Total phospholipids increased only in liver, whereas ceramide levels were not consistently altered in liver, kidney, or brain. In liver and kidney, fumonisin B1 treatment increased the sphinganine-containing complex sphingolipids, but no effect was observed on sphingosine-containing complex sphingolipids. No changes in complex sphingolipids were observed in brain. In liver, there was a close correlation between the extent of free sphinganine accumulation, and apoptosis and hepatopathy. This correlation was also evident in kidney but to a lessor extent. Nonetheless, the apoptosis and nephropathy occurred with little or no change in the levels of ceramide or more complex sphingolipids. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 281–289, 1998 相似文献
13.
Toshiro Sagisaka Masuo Nakano Yasuhiko Fujino 《Bioscience, biotechnology, and biochemistry》2013,77(11):1983-1987
The reactivity of sphingosine bases as substrates in the enzymatic synthesis of ceramide was in decreasing order of erythro-4-sphingenine, erythro-sphinganine, threo-4-sphingenine and thero-sphinganine in the microsomal fraction obtained from chicken liver. Unsaturated base-containing ceramide was enzymatically formed from 4-sphingenine and saturated base-containing ceramide from sphinganine. The formation of unsaturated base-containing ceramide proceeded similar to that of saturated base-containing ceramide. The data explain in part, the fact that 4-sphingenine is generally superior to sphinganine as the constituent sphingosine base in sphingolipids, the biochemical derivatives of ceramide. 相似文献
14.
Hila Zigdon Aviram Kogot-Levin Joo-Won Park Ruth Goldschmidt Samuel Kelly Alfred H. Merrill Jr. Avigdor Scherz Yael Pewzner-Jung Ann Saada Anthony H. Futerman 《The Journal of biological chemistry》2013,288(7):4947-4956
Ceramide is a key intermediate in the pathway of sphingolipid biosynthesis and is an important intracellular messenger. We recently generated a ceramide synthase 2 (CerS2) null mouse that cannot synthesize very long acyl chain (C22-C24) ceramides. This mouse displays severe and progressive hepatopathy. Significant changes were observed in the sphingolipid profile of CerS2 null mouse liver, including elevated C16-ceramide and sphinganine levels in liver and in isolated mitochondrial fractions. Because ceramide may be involved in reactive oxygen species (ROS) formation, we examined whether ROS generation was affected in CerS2 null mice. Levels of a number of anti-oxidant enzymes were elevated, as were lipid peroxidation, protein nitrosylation, and ROS. ROS were generated from mitochondria due to impaired complex IV activity. C16-ceramide, sphingosine, and sphinganine directly inhibited complex IV activity in isolated mitochondria and in mitoplasts, whereas other ceramide species, sphingomyelin, and diacylglycerol were without effect. A fluorescent analog of sphinganine accumulated in mitochondria. Heart mitochondria did not display a substantial alteration in the sphingolipid profile or in complex IV activity. We suggest that C16-ceramide and/or sphinganine induce ROS formation through the modulation of mitochondrial complex IV activity, resulting in chronic oxidative stress. These results are of relevance for understanding modulation of ROS signaling by sphingolipids. 相似文献
15.
K Wang R Xu A J Snider J Schrandt Y Li A B Bialkowska M Li J Zhou Y A Hannun L M Obeid V W Yang C Mao 《Cell death & disease》2016,7(3):e2124
Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C18:1-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C18:1-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C18:1-ceramide, but not C18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C18:1-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C18:1-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C18:1-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.Ceramides are the central lipid in the metabolic network of sphingolipids, and are generated through the de novo, catabolic and salvage pathways.1 In the de novo pathway, ceramides are synthesized through multiple steps catalyzed sequentially by serine palmitoyltransferase (SPT), keto-dihydrosphingosine reductase, (dihydro)ceramide synthases (CerSs) and dihydroceramide desaturases. In the catabolic pathways, ceramides are derived from the hydrolysis of sphingomyelins by sphingomyelinases (SMases) or the hydrolysis of glycosphingolipids. In the salvage pathway, ceramides are synthesized from sphingosine (SPH) and fatty acyl-CoA by CerSs. As CerSs (CerS1-6) have distinct specificity toward acyl-CoA chain length and degree of unsaturation, ceramides with various acyl-chains are found in mammalian cells. Upon generation, ceramides can be hydrolyzed by five ceramidases encoded by five distinct genes (ASAH1, ASAH2, ACER1, ACER2 and ACER3). These ceramidases vary in pH optimum for catalytic activity, tissue distribution, cellular localization and substrate specificity,2 allowing for regulation of specific ceramides in a cell- or tissue-specific manner.Recent studies have implicated ceramides in regulating the innate immune response. Sakata et al.3 demonstrated that lipopolysaccharides (LPS), a potent inducer of the innate immune response, increases C16-ceramide by activating acid SMase and that inhibition of SMase attenuates LPS-induced production of pro-inflammatory cytokines in THP-1 macrophages. Andreyev et al.4 found that ceramides are increased by Toll-like receptor 4 (TLR4)-specific LPS in RAW 264.7 macrophages. Schilling et al.5 revealed that LPS and palmitic acid synergistically increase C16-ceramide in primary mouse peritoneal macrophages (PMs) by activating de novo biosynthesis of ceramides and that inhibiting the C16-ceramide increase attenuates LPS-induced production of TNF-α and IL-1β in PMs. A recent study found that LPS increases ceramides in Raw 264.7 macrophages through nuclear factor kappa B (NF-κB)-dependent upregulation of SPT long chain base subunit 2 Sptlc2, a regulator of SPT.6 These results suggest that ceramides mediate the immune response in part by enhancing the production of pro-inflammatory cytokines in innate immune cells.Emerging evidence suggests that dysregulation in the innate immune response in inflammatory bowel disease (IBD) contributes to the pathogenesis of the disease.7 Consistent with the role of ceramides in potentiating the innate immune response, several studies found that ceramides may have a role in the pathogenesis of IBD. Sakata et al.3 demonstrated that blocking the generation of ceramides with the SMase inhibitor hinders mouse colitis. Fischbeck et al.8 showed that increasing ceramides in the gut by supplying mice with dietary sphingomyelins, a precursor of ceramides, aggravates mouse colitis. These results suggest that increased levels of ceramides may contribute to the pathogenesis of IBD.Although the role of ceramides and their generating enzymes in the innate immune response have been well studied, much remains unclear about the role of ceramidases involved in the catabolism of ceramides in this biological response. In this study, we investigated the role of alkaline ceramidase 3 (ACER3)/Acer3 and its substrates in immune response. We demonstrated that Acer3 is downregulated, whereas its substrate, C18:1-ceramide, is upregulated in murine immune cells and colonic epithelial cells (CECs) during the innate immune response to LPS. Using Acer3 null mice (Acer3−/−) and their wild-type (Acer3+/+) littermates, we further discovered that the inverse regulation of Acer3 and C18:1-ceramide potentiates LPS-induced production of pro-inflammatory cytokines in innate immune cells. More importantly, we found that Acer3 deficiency aggravates dextran sulfate sodium (DSS)-induced colitis and colitis-associated colorectal cancer (CAC) in a murine model. These findings indicate that Acer3/ACER3 and C18:1-ceramide are novel modulators in the innate immune response and that their dysregulation may contribute to the pathogenesis of inflammatory diseases. 相似文献
16.
Kazuyuki Kitatani Kely Sheldon Vinodh Rajagopalan Viviana Anelli Russell W. Jenkins Ying Sun Gregory A. Grabowski Lina M. Obeid Yusuf A. Hannun 《The Journal of biological chemistry》2009,284(19):12972-12978
Activation of protein kinase C (PKC) promotes the salvage pathway of
ceramide formation, and acid sphingomyelinase has been implicated, in part, in
providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007)
J. Biol. Chem. 282, 11549–11561). In the present study, we
examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes
glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated
formation of ceramide from recycled sphingosine. Glucosylceramide levels
declined after treatment of MCF-7 cells with a potent PKC activator, phorbol
12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs
significantly attenuated acid glucocerebrosidase activity and decreased
PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced
degradation of glucosylceramide and generation of sphingosine, the source for
ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased
ceramide levels. These observations indicate that GBA1 activation can generate
the source (sphingosine) for PMA-induced formation of ceramide through the
salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in
the formation of ceramide was determined. By attenuating expression of
PKCδ, cells failed to trigger PMA-induced alterations in levels of
ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is
suggested to stimulate the degradation of both sphingomyelin and
glucosylceramide leading to the salvage pathway of ceramide formation.
Collectively, GBA1 is identified as a novel source of regulated formation of
ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which
are emerging as bioactive lipid mediators thought to play crucial roles in
cellular responses (1,
2). Ceramide, a central
sphingolipid, serves as the main precursor for various sphingolipids,
including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of
formation of ceramide has been demonstrated through the action of three major
pathways: the de novo pathway
(3,
4), the sphingomyelinase
pathway (5), and the salvage
pathway
(6–8).
The latter plays an important role in constitutive sphingolipid turnover by
salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that
serve as sphingolipid backbones for ceramide and dihydroceramide as well as
all complex sphingolipids (Fig.
1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and
inhibition of PMA induction of ceramide by fumonisin B1. A, the
scheme of the sphingosine salvage pathway of ceramide formation. B,
previously published data as to effects of fumonisin B1 on ceramide mass
profiles (23) are re-plotted
as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or
without 100 μm fumonisin B1 for 2 h followed by treatment with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Results are expressed as sum of
increased mass of ceramide species. Dotted or open columns
represents C16-ceramide or sum of other ceramide species
(C14-ceramide, C18-ceramide, C18:1-ceramide,
C20-ceramide, C24-ceramide, and
C24:1-ceramide), respectively. The data represent mean ±
S.E. of three to five values.Metabolically, ceramide is also formed from degradation of
glycosphingolipids (Fig.
1A) usually in acidic compartments, the lysosomes and/or
late endosomes (9). The
stepwise hydrolysis of complex glycosphingolipids eventually results in the
formation of glucosylceramide, which in turn is converted to ceramide by the
action of acid β-glucosidase 1
(GBA1)2
(9,
10). Severe defects in GBA1
activity cause Gaucher disease, which is associated with aberrant accumulation
of the lipid substrates
(10–14).
On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also
form ceramide (15,
16). Either process results in
the generation of lysosomal ceramide that can then be deacylated by acid
ceramidase (17), releasing
sphingosine that may escape the lysosome
(18). The released sphingosine
may become a substrate for either sphingosine kinases or ceramide synthases,
forming sphingosine 1-phosphate or ceramide, respectively
(3,
19–21).In a related line of investigation, our studies
(20,
22,
23) have begun to implicate
protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage
pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol
ester (PMA) was shown to stimulate the salvage pathway resulting in increases
in ceramide. All the induced ceramide was inhibited by pretreatment with a
ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating
acute activation of the de novo pathway and establishing a role for
ceramide synthesis (20,
23). Moreover, labeling
studies also implicated the salvage pathway because PMA induced turnover of
steady state-labeled sphingolipids but did not affect de novo labeled
ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream
molecule for the activation of acid sphingomyelinase in the salvage pathway
(22). Interestingly, the
PKCδ isoform induced the phosphorylation of acid sphingomyelinase at
serine 508, leading to its activation and consequent formation of ceramide.
The activation of acid sphingomyelinase appeared to contribute to ∼50% of
the salvage pathway-induced increase in ceramide
(28) (also, see
Fig. 4C). This raised
the possibility that distinct routes of ceramide metabolism may account for
the remainder of ceramide generation. In this study, we investigated
glucocerebrosidase GBA1 as a candidate for one of the other routes accounting
for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide
after PMA treatment. A, MCF-7 cells were transfected with 5
nm siRNAs of each of four individual sequences (SCR, GBA1-a,
GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA
for 1 h. Lipids were extracted, and then the levels of the
C16-ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to nine values. B, MCF-7 cells were transfected with 5
nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
individual ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to five values. C14-Cer,
C14-ceramide; C16-Cer,
C16-ceramide; C18-Cer;
C18-ceramide; C18:1-Cer,
C18:1-ceramide; C20-Cer,
C20-ceramide; C20-Cer,
C24-ceramide; C24:1-Cer,
C24:1-ceramide. C, MCF-7 cells were transfected with 5
nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a
(GBA1) for 48 h following stimulation with (PMA) or without
(Control) 100 nm PMA for 1 h. Lipids were extracted, and
then the levels of ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Levels of C16-ceramide are
shown. The data represent mean ± S.E. of four to five values.
Significant changes from SCR-transfected cells treated with PMA are shown in
A–C (*, p < 0.02; **,
p < 0.05; ***, p < 0.01). 相似文献
17.
Hiroko Hamai Fannie Keyserman Lynne M. Quittell Tilla S. Worgall 《Journal of lipid research》2009,50(6):1101-1108
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that affect protein structure and channel function. CFTR, localized in the apical membrane within cholesterol and sphingomyelin rich regions, is an ABC transporter that functions as a chloride channel. Here, we report that expression of defective CFTR (ΔF508CFTR or decreased CFTR) in human lung epithelial cell lines increases sphingolipid synthesis and mass of sphinganine, sphingosine, four long-chain saturated ceramide species, C16 dihydroceramide, C22, C24, C26-ceramide, and sphingomyelin, and decreases mass of C18 and unsaturated C18:1 ceramide species. Decreased expression of CFTR is associated with increased expression of long-chain base subunit 1 of serine-palmitoyl CoA, the rate-limiting enzyme of de novo sphingolipid synthesis and increased sphingolipid synthesis. Overexpression of ΔF508CFTR in bronchoalveolar cells that do not express CFTR increases sphingolipid synthesis and mass, whereas overexpression of wild-type CFTR, but not of an unrelated ABC transporter, ABCA7, decreases sphingolipid synthesis and mass. The data are consistent with a model in which CFTR functions within a feedback system that affects sphingolipid synthesis and in which increased sphingolipid synthesis could reflect a physiological response to sequestration of sphingolipids or altered membrane structure. 相似文献
18.
Guiliano Gazzotti Sandro Sonnino Riccardo Ghidoni Paolo Orlando Guido Tettamanti 《Glycoconjugate journal》1984,1(2):111-121
Gangliosides GM2, GM1 and GD1b were radiolabelled at C-6 of the terminal galactose orN-acetylgalactosamine by the galactose oxidase/[3H]NaBH4 method; gangliosides GM2, GM1, Fuc-GM1 and GD1a were radiolabelled at C-3 of the long chain base by the 2,3-dichloro-5,6-dicyanobenzoquinone/[3H]NaBH4 method.By application of an original HPLC procedure, eight different molecular species were prepared from each labelled ganglioside. Each of these species was characterized by the presence of one of the following long chain bases:erythro C18 sphingosine,threo C18 sphingosine,erythro C18 sphinganine,threo C18 sphinganine,erythro C20 sphingosine,threo C20 sphingosine,erythro C20 sphinganine andthreo C20 sphinganine.From GD1b only the species containing theerythro forms of long chain bases were obtained.The individual molecular species were more than 99% homogeneous and had a radiopurity better than 99%. The molecular species of the same ganglioside, radiolabelled at C-3 of the long chain base, had identical specific radioactivity, namely 1.17, 1.25, 0.85 and 1.28 Ci/mmol for GM2, GM1, Fuc-GM1 and GD1a respectively. The molecular species of the same ganglioside, radiolabelled at C-6 of terminal galactose orN-acetylgalactosamine, had similar specific radioactivity, namely 1.34–1.40, 1.44–1.51, 1.37–1.44 Ci/mmol for GM2, GM1 and GD1b respectively. 相似文献
19.
The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2–3.3. This enzyme prefers to hydrolyze the acidic GSLs, II3SO3LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4–4.5. Since oyster ceramidase greatly prefers ceramides with C6 to C8 fatty acids, C6-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C16 and C18 fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source. 相似文献
20.
Regulation of Insulin-Stimulated Glucose Transporter GLUT4 Translocation and Akt Kinase Activity by Ceramide 总被引:8,自引:2,他引:6
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Scott A. Summers Luis A. Garza Honglin Zhou Morris J. Birnbaum 《Molecular and cellular biology》1998,18(9):5457-5464
The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C2-ceramide inhibited insulin-stimulated glucose transport by ~50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C2-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C2-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C2-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide’s inhibition of Akt. These studies demonstrate ceramide’s capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis. 相似文献