Neutral Ceramidase Research Articles

ITRAQ-based quantitative proteomic analysis of mycelium in different predation periods in nematode trapping fungus Duddingtonia flagrans

Duddingtonia flagrans, as a representative species of nematode-trapping fungi, has a strong environmental tolerance and nematode-trapping ability and is thought of as the most promising candidate for use as a biological agent. To clarify the proteomic differences during different predation periods and to explore the molecular background of the nematode-trapping mechanism, we carried out an iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomic analysis for Duddingtonia flagrans during three predatory stages (0 h, 12 h, and 48 h). A total of 4244 proteins were detected; 474 of these showed differential protein abundance from 0 h to 12 h, while 119 showed differential protein abundance from 12 h to 48 h. The 474 proteins from 0 h to 12 h were primarily associated with catalytic activity, molecular binding function, transport activity, biological regulation, stress response, biolocation and carbon utilization, while the 119 proteins from 12 h to 48 h were involved in substance and energy metabolism. Further bioinformatics analysis showed that these proteins could be mapped to various pathways, eight of which were significantly enriched after mapping to KEGG pathways from 0 h to 12 h, and substance and energy metabolism were enriched from 12 h to 48 h. Interestingly, many of these differentially expressed proteins were involved in the significantly enriched pathways of ubiquitin-mediated proteolysis, endoplasmic reticulum protein processing, sphingolipid metabolism, adhesion processes, MAPK signaling pathway, AMPK signaling pathway, energy and carbon metabolism, peroxidase, and oxidative phosphorylation, including acid phosphatase, neutral ceramidase, sphingomyelin phosphodiesterase, tyrosinase, and serine protease. These findings not only deepen our understanding of the proteome during different predation periods in Duddingtonia flagrans but also provide a basis for screening and identification of predation-related proteins to reveal the biological processes of the fungus and its action on nematodes, thereby helping to elucidate the nematode-trapping mechanism of D. flagrans.

Ca2+ Signaling and Barrier Function of Lung Microvascular Endothelial Cells are Modulated by Mesenchymal Stromal Cell Microparticles

IntroductionAcute respiratory distress syndrome (ARDS) is a fatal condition characterized by hyperinflammation and pulmonary microvascular leak. With no pharmacological cure, treatment remains only supportive. Mesenchymal stromal cells (MSCs) have recently generated excitement as a potential therapy, presumably functioning through paracrine mechanisms which may rely on the release of extracellular vesicles like microparticles (MPs). However, the mechanism by which MPs modulate the pathology of ARDS is poorly understood yet may pave the way to develop cell‐free cell therapeutics.ObjectiveTo identify the mechanism(s) by which MSC derived MPs enhance pulmonary microvascular barrier function.MethodsMPs were purified from conditioned medium produced by MSCs either stimulated with Ca2+ ionophore or unstimulated. Monolayers of primary human pulmonary microvascular endothelial cells (HPMECs) were injured with thrombin to induce barrier disruption, measured by transendothelial electrical resistance (TEER), mimicking lung microvascular leak as observed in ARDS. MPs were administered to treat thrombin‐injured HPMECs and changes in intracellular Ca2+ concentration ([Ca2+]i) were determined by ratiometric imaging of Fura‐2.ResultsThrombin administered to HPMECs caused an initial, brief spike in [Ca2+]i followed by a second phase characterized as a prolonged, gradual increase in [Ca2+]i. Following the thrombin‐induced [Ca2+]i spike, treatment with MPs from unstimulated MSCs eliminated the second phase and caused a sustained, decrease in [Ca2+]i below baseline. In parallel, MPs from unstimulated MSCs enhanced TEER recovery and VE‐cadherin integrity in intercellular junctions of HPMECs following thrombin‐induced permeability. Conversely, treatment with MPs from Ca2+ ionophore stimulated MSCs amplified the second phase of the [Ca2+]i response to thrombin and prevented recovery of HPMEC barrier function. HPLC‐MS revealed MPs from unstimulated MSCs had lower ceramide and higher sphingosine‐1‐phosphate (S1P) content than MPs from Ca2+ ionophore stimulated MSCs. S1P degradation by S1P lyase or blockade of the S1P receptor 1 attenuated the barrier‐protective effect of MPs from unstimulated MSCs, while ceramide degradation by neutral ceramidase improved barrier recovery following treatment with MPs from Ca2+ ionophore stimulated MSCs.ConclusionMPs from unstimulated MSCs therapeutically enhance lung capillary barrier function, presumably by attenuating endothelial [Ca2+]i responses by resetting the ceramide/S1P rheostat. These findings provide a mechanistic basis for development and optimization/enrichment of MSC derived MPs as a promising cell‐free cell therapy for ARDS.Support or Funding InformationThis work was supported by the Ontario Research Fund and Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Imbalance of Sphingolipids Synthesis/Degradation Pathway in Preeclamptic Mouse Placenta and Kidney of Intrauterine Growth Restricted Mouse Fetus

Preeclampsia (PE) is a pregnancy hypertensive disorder that results in intrauterine growth restriction (IUGR) of the offspring. Adult IUGR individuals have increased risks of developing cardiovascular and renal diseases due to poor kidney development during embryonic stage. The treatment of PE remains ineffective and the mechanism of PE‐induced poor kidney growth of IUGR fetus has not been conclusive leading to a persistent clinical challenge. Bioactive sphingolipids ceramide (Cer) and sphingosine‐1‐phosphate (S1P) function as key regulators of cellular homeostasis. The reduced levels of circulating angiogenic S1P and elevated levels of pro‐apoptotic Cer have been demonstrated in PE women. Ceramidase hydrolyzes Cer to produce sphingosine. Sphingosine can then be phosphorylated by sphingosine kinase (Sphk) 1 and 2 to form S1P. Whether sphingolipids and sphingolipids‐related protein implicate as potential key factors involved in the pathogenesis of PE and nephrogenesis in IUGR fetus is unknown. Here, we tested the hypothesis that the synthesis/degradation pathway of S1P and Cer is altered in PE placenta and kidneys of IUGR fetus. We used reduced uterine perfusion (RUP) mouse model to represent PE in this study. C57bl/6J mice underwent sham or RUP surgeries at day 13 of gestation. At day 18 of gestation, placentas from both sham (control) and RUP mice, as well as kidneys from control and IUGR pups were collected. Using commercial ELISA kits, S1P in RUP placenta was significantly lower than in sham placenta (P<0.05, n=3) despite no significant difference was found between control and IUGR pup kidneys. mRNA levels was measured using qRT‐PCR and GAPDH was used as the control housekeeping gene. Placenta results (n=5): mRNA of neutral ceramidase (Asah2) and alkaline ceramidase 2 (Acer2) but not acid ceramidase (Asah1) were three folds lower in RUP placentas suggesting the conversion of Cer to sphingosine is impaired in RUP. Lower conversion from sphingosine to S1P as the level of sphingosine kinase ‐2 (Sphk‐2) was significantly decreased in RUP placentas. Significantly lower in ceramide kinase (Cerk), and S1P phosphatase 2 (Sgpp2) mRNA levels were also observed in RUP. These data suggest that the synthesis and degradation pathways of S1P in RUP placentas are decreased which coincide with the lower angiogenic S1P. Pup kidneys results (n=7): Asah2 and Acer2 were two‐fold decreased in IUGR kidneys (P<0.05). No significant difference of Asah1, Sphk1, Sphk2, Cerk, and Sgpp2 was found between control and IUGR. However, S1P receptor 1 (S1PR1), that plays important role in nephrogenesis, was significantly lower in IUGR kidneys. We conclude that the alteration of synthesis and degradation pathways of S1P in placentas may play a role in PE pathology. Lower in Asah2, Acer2 and S1PR1 may underlie the impairment of nephrogenesis in IUGR which will impact the renal health in later of life.Support or Funding InformationDr. Intapad was supported by funding from the AHA 16SDG27770041, ASN Kidney Research Career Development grant, and Start‐up funds from Tulane University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pancreatic and mucosal enzymes in choline phospholipid digestion.

The digestion of choline phospholipids is important for choline homeostasis, lipid signaling, postprandial lipid and energy metabolism, and interaction with intestinal bacteria. The digestion is mediated by the combined action of pancreatic and mucosal enzymes. In the proximal small intestine, hydrolysis of phosphatidylcholine (PC) to 1-lyso-PC and free fatty acid (FFA) by the pancreatic phospholipase A2 IB coincides with the digestion of the dietary triacylglycerols by lipases, but part of the PC digestion is extended and must be mediated by other enzymes as the jejunoileal brush-border phospholipase B/lipase and mucosal secreted phospholipase A2 X. Absorbed 1-lyso-PC is partitioned in the mucosal cells between degradation and reacylation into chyle PC. Reutilization of choline for hepatic bile PC synthesis, and the reacylation of 1-lyso-PC into chylomicron PC by the lyso-PC-acyl-CoA-acyltransferase 3 are important features of choline recycling and postprandial lipid metabolism. The role of mucosal enzymes is emphasized by sphingomyelin (SM) being sequentially hydrolyzed by brush-border alkaline sphingomyelinase (alk-SMase) and neutral ceramidase to sphingosine and FFA, which are well absorbed. Ceramide and sphingosine-1-phosphate are generated and are both metabolic intermediates and important lipid messengers. Alk-SMase has anti-inflammatory effects that counteract gut inflammation and tumorigenesis. These may be mediated by multiple mechanisms including generation of sphingolipid metabolites and suppression of autotaxin induction and lyso-phosphatidic acid formation. Here we summarize current knowledge on the roles of pancreatic and mucosal enzymes in PC and SM digestion, and its implications in intestinal and liver diseases, bacterial choline metabolism in the gut, and cholesterol absorption.

Functions of neutral ceramidase in the Golgi apparatus

Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); thus, ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Neutral ceramidase (nCDase) is highly expressed in colorectal tissues, and a recent report showed that nCDase activity is involved in Wnt/β-catenin signaling. In addition, the inhibition of nCDase decreases the development and progression of colorectal tumor growth. Here, to determine the action of nCDase in colorectal cancer cells, we focused on the subcellular localization and metabolic functions of this enzyme in HCT116 cells. nCDase was found to be located in both the plasma membrane and in the Golgi apparatus, but it had minimal effects on basal levels of ceramide, sphingosine, or S1P. Cells overexpressing nCDase were protected from the cell death and Golgi fragmentation induced by C6-ceramide, and they showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine. Furthermore, compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial ceramidase (bCDase). The results showed that Golgi-specific bCDase also demonstrated resistance against the cell death stimulated by C6-ceramide, and it catalyzed the metabolism of ceramides and produced sphingosine in the Golgi. Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, also supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.

Neutral ceramidase: Advances in mechanisms, cell regulation, and roles in cancer.

Extensive research conducted in the last three decades has identified the roles for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) as key regulators of cellular homeostasis, growth and death. One of the major groups of enzymes in the ceramide pathway, ceramidases, converts ceramide into sphingosine and fatty acids, with sphingosine being further metabolized to S1P. Thus, these enzymes play important roles in the network controlling the functions associated with these bioactive sphingolipids. Among the family of ceramidases, neutral ceramidase (nCDase), which is named according to its optimal pH for catalytic activity, has received increased attention in the last decade. The goal of this review is to provide a brief background on bioactive sphingolipids and the ceramidases. We then describe more recent advances on nCDase, specifically the resolution of its crystal structure and understanding its roles in cell biology and physiology.

AKT as a key target for growth promoting functions of neutral ceramidase in colon cancer cells.

Despite advances in the field, colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Research into bioactive sphingolipids over the past two decades has played an important role in increasing our understanding of the pathogenesis and therapeutics of CRC. In the complex metabolic network of sphingolipids, ceramidases (CDases) have a key function. These enzymes hydrolyze ceramides into sphingosine (SPH) which in turn is phosphorylated by sphingosine kinases (SK) 1 and 2 to generate sphingosine-1 phosphate (S1P). Importantly, we have recently shown that inhibition of neutral CDase (nCDase) induces an increase of ceramide in colon cancer cells which decreases cellular growth, increases apoptosis and modulates the WNT/β-catenin pathway. We have also shown that the deletion of nCDase protected mice from the onset and progression of colorectal cancer in the AOM carcinogen model. Here we demonstrate that AKT is a key target for the growth suppressing functions of ceramide. The results show that inhibition of nCDase activates GSK3β through dephosphorylation, and thus is required for the subsequent phosphorylation and degradation of β-catenin. Our findings show that inhibition of nCDase also inhibits the basal activation status of AKT, and we further establish that a constitutively active AKT (AKT T308D, S473D; AKTDD) reverses the effect of nCDase on β-catenin degradation. Functionally, the AKTDD mutant is able to overcome the growth suppressive effects of nCDase inhibition in CRC cells. Moreover, nCDase inhibition induces a growth delay of xenograft tumors from control cells, whereas xenograft tumors from constitutively active AKT cells become resistant to nCDase inhibition. Taken together, these results provide important mechanistic insight into how nCDase regulates cell proliferation. These findings demonstrate a heretofore unappreciated, but critical, role for nCDase in enabling/maintaining basal activation of AKT and also suggest that nCDase is a suitable novel target for colon cancer therapy.

Mesenchymal Stromal Cell Microparticles Enhance Lung Endothelial Barrier Through CD44 and the S1P/ceramide Rheostat

IntroductionAcute respiratory distress syndrome (ARDS) is a fatal condition characterized by hyperinflammation and pulmonary microvascular leak. Treatment is only supportive with no pharmacological cure. Recently, mesenchymal stromal/stem cells (MSCs) have emerged as potential therapy, but greater knowledge on their mechanism(s) of action could yield development of cell‐free therapeutics.ObjectiveTo determine if MSC‐produced microparticles (MPs) ameliorate endothelial barrier disruption and identify their biomolecular contents responsible for such therapeutic effect.MethodsTo re‐capitulate lung capillary leak in ARDS, primary human pulmonary microvascular endothelial cell (HPMEC) monolayers grown on Transwell inserts were injured with thrombin to induce barrier disruption as measured by transendothelial electrical resistance (TEER). MPs were purified out of conditioned medium produced by MSCs either stimulated with Ca2+ ionophore or unstimulated. MPs were administered to treat thrombin‐injured HPMEC monolayers; surface markers were compared by flow cytometry and lipid content analyzed by HPLC‐MS.ResultsMPs from unstimulated MSCs enhanced HPMEC barrier function and TEER recovery after thrombin injury, while MPs from stimulated MSCs were injurious. Stimulated MSCs produced MPs with significantly less surface CD44. Antibody blockade of CD44 on unstimulated MPs eliminated their therapeutic effect on HPMEC monolayers. Moreover, stimulated MPs had significantly more total ceramides and less sphingosine‐1‐phosphate (S1P) than unstimulated MPs. Ceramide degradation by neutral ceramidase blunted the injurious effect of stimulated MPs. The therapeutic function of unstimulated MPs was eliminated with ceramide over‐production by acid sphingomyelinase or S1P degradation by S1P lyase.ConclusionThese results show MSC‐produced MPs were therapeutic after lung capillary endothelial barrier disruption. Beneficial effects were associated with surface CD44 and greater ratio of S1P:ceramide content. This knowledge provides a mechanistic basis for development of MSC‐based cell‐free therapeutics.Support or Funding InformationThis work was supported by Ontario Research Fund and Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gene cloning of a neutral ceramidase from the sphingolipid metabolic pathway based on transcriptome analysis of Amorphophallus muelleri.

Amorphophallus is a perennial herbaceous plant species mainly distributed in the tropics or subtropics of Asia and Africa. It has been used as a traditional medicine for a long time and now is utilized for the pharmaceutical, chemical and agriculture industries as a valued economic crop. Recently, Amorphophallus has attracted tremendous interest because of its high ceramide content. However, the breeding and genome studies are severely limited by the arduous whole genome sequencing of Amorphophallus. In this study, the transcriptome data of A. muelleri was obtained by utilizing the high-throughput Illumina sequencing platform. Based on this information, the majority of the significant genes involved in the proposed sphingolipid metabolic pathway were identified. Then, the full-length neutral ceramidase cDNA was obtained with the help of its candidate transcripts, which were acquired from the transcriptome data. Furthermore, we demonstrated that this neutral ceramidase was a real ceramidase by eukaryotic expression in the yeast double knockout mutant Δypc1 Δydc1, which lacks the ceramidases—dihydroCDase (YDC1p), phytoCDase (YPC1p). In addition, the biochemical characterization of purified A. muelleri ceramidase (AmCDase) exhibited classical Michaelis-Menten kinetics with an optimal activity ranging from pH 6.5 to 8.0. Based on our knowledge, this study is the first to report the related information of the neutral ceramidase in Amorphophallus. All datasets can provide significant information for related studies, such as gene expression, genetic improvement and application on breeding in Amorphophallus.

A neutral ceramidase, NlnCDase, is involved in the stress responses of brown planthopper, Nilaparvata lugens (St\xe5l)

Ceramidases (CDases) are vital enzymes involved in the biosynthesis of sphingolipids, which are essential components of eukaryotic membranes. The function of these enzymes in insects, however, is poorly understood. We identified a neutral ceramidase (NlnCDase) from the brown planthopper, Nilaparvata lugens, one of the most destructive hemipteran pests of rice. The C12-ceramide was the most preferred substrate for the NlnCDase enzyme. The activity of the NlnCDase enzyme was highest in the neutral-pH range (pH 6.0). It was inhibited by EGTA, Cs+ and Fe2+, while stimulated by EDTA and Ca2+. Moreover, the NlnCDase has higher transcript level and activity in adults than in eggs and nymphs, and in the reproductive organs (ovaries and spermaries) than in other tissues (i.e. heads, thorax, legs, midguts), which suggested that the NlnCDase might be elevated to mediate developmental process. In addition, transcripts and activity of the NlnCDase were up-regulated under abiotic stresses including starvation, abnormal temperature, and insecticides, and biotic stress of resistant rice varieties. Knocking down NlnCDase by RNA interference increased female survival under starvation and temperature stresses, suggesting that NlnCDase might be involved in the stress response in N. lugens.

Endogenous acid ceramidase protects epithelial cells from Porphyromonas gingivalis-induced inflammation in vitro

Ceramidases are a group of enzymes that degrade pro-inflammatory ceramide by cleaving a fatty acid to form anti-inflammatory sphingosine lipid. Thus far, acid, neutral and alkaline ceramidase isozymes have been described. However, the expression patterns of ceramidase isoforms as well as their role in periodontal disease pathogenesis remain unknown. In this study, expression patterns of ceramidase isoforms were quantified by real-time PCR and immunohistochemistry in gingival samples of patients with periodontitis and healthy subjects, as well as in EpiGingivalTM-3D culture and OBA-9 gingival epithelial cells both of which were stimulated with or without the presence of live Porphyromonas gingivalis (ATCC 33277 strain). A significantly lower level of acid ceramidase expression was detected in gingival tissues from periodontal patients compared to those from healthy subjects. In addition, acid-ceramidase expression in EpiGingival™ 3D culture and OBA-9 cells was suppressed by stimulation with P. gingivalis in vitro. No significant fluctuation was detected for neutral or alkaline ceramidases in either gingival samples or cell cultures. Next, to elucidate the role of acid ceramidase in P. gingivalis-induced inflammation in vitro, OBA-9 cells were transduced with adenoviral vector expressing the human acid ceramidase (Ad-ASAH1) gene or control adenoviral vector (Ad-control). In response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells showed significantly lower mRNA expressions of caspase-3 as well as the percentage of Annexin V-positive cells, when compared with OBA-9 cells transduced with Ad-control vector. Furthermore, in response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells produced less TNF-α, IL-6, and IL1β pro-inflammatory cytokines than observed in OBA-9 cells transduced with Ad-control vector. Collectively, our data show the novel discovery of anti-inflammatory and anti-apoptotic effects of acid ceramidase in host cells exposed to periodontal bacteria, and the attenuation of the expression of host-protective acid ceramidase in periodontal lesions.

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

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

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, Crassostrea virginica.

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 52kDa 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 88kDa 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.

Neutral ceramidase activity inhibition is involved in palmitate-induced apoptosis in INS-1 cells.

Neutral ceramidase (NCDase) is a class of ceramidases, a key enzyme in ceramide degradation. Recently, it was observed that NCDase activity was suppressed by saturated fatty acids to increase ceramide content in rat muscle. However, little is known about its changes in activity and roles in palmitate (Palm)-induced lipotoxicity in pancreatic β cells. Here, we demonstrated that Palm treatment significantly down-regulated NCDase activity, mRNA and protein levels in rat INS-1 cells. In addition, Palm caused a significant accumulation of ceramide, while SPH level remained unchanged, suggesting that inhibition of NCDase activity led to no change of SPH level after treatment with Palm for 24 h. Furthermore, NCDase overexpression significantly reduced Palm-induced apoptosis in INS-1 cells. Conversely, NCDase siRNA knockdown markedly exacerbated Palm-induced apoptosis. In conclusion, Palm treatment suppressed the activity of NCDase and down-regulated its mRNA and protein expression. Furthermore, NCDase inhibition was involved in Palm-induced apoptosis by blocking ceramide degradation in INS-1 cells.

Neutral Ceramidase Secreted Via Exosome Protects Against Palmitate-Induced Apoptosis in INS-1 Cells.

Aims: To investigate the effects of neutral ceramidase (NCDase) packaged in exosomes that are secreted from β-cells on free fatty acid (FFA)-induced β-cells apoptosis and its role in regulation of sphingolipid-mediated signaling pathway. Methods: HPLC and Western blotting were performed to determine NCDase activity and expression. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis. Electrospray ionization tandem mass spectrometry was used for ceramide (Cer), sphingosine-1-phosphate (S1P), and sphingosine (SPH) determination. Results: INS-1 cells over-expressed NCDase secreted active NCDase via exosomes. Exosomes isolated from the cultured medium of INS-1 cells that oxpressed NCDase could ameliorate palmitate-induced apoptosis. Furthermore, the results showed that exosome-derived NCDase treatment reduced intracellular Cer/S1P ratio. Conclusions: β-cell secreted active NCDase via exosome, the exosome-packaged-NCDase protected β-cells from FFA-induced apoptosis through regulating sphingolipid metabolites and it might be a potential treatment for β-cell lipotoxicity and type 2 diabetes.

Neutral ceramidase-enriched exosomes prevent palmitic acid-induced insulin resistance in H4IIEC3 hepatocytes.

Oversupply of free fatty acids such as palmitic acid (PA) from the portal vein may cause liver insulin resistance. Production of reactive oxygen species plays a pivotal role in PA‐induced insulin resistance in H4IIEC3 hepatocytes. Recently, we found that exosomes secreted from INS‐1 cells that were transfected with neutral ceramidase (NCDase) plasmids had raised NCDase activity; these NCDase‐enriched exosomes could inhibit PA‐induced INS‐1 cell apoptosis. Here, we showed that PA reduced insulin‐stimulated tyrosine phosphorylation of insulin receptor substrate 2 and decreased insulin‐stimulated uptake of the fluorescent glucose analog 2‐NBDG, confirming that insulin resistance occurred in PA‐treated H4IIEC3 cells. Moreover, NCDase‐enriched exosomes from INS‐1 cells rescued PA‐induced H4IIEC3 insulin resistance and blocked PA‐induced reactive oxygen species production in which ceramide was involved.

Ceramidases, roles in sphingolipid metabolism and in health and disease

Over the past three decades, extensive research has been able to determine the biologic functions for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) (Hannun, 1996; Hannun etal., 1986; Okazaki etal., 1989). These studies have managed to define the metabolism, regulation, and function of these bioactive sphingolipids. This emerging body of literature has also implicated bioactive sphingolipids, particularly S1P and ceramide, as key regulators of cellular homeostasis. Ceramidases have the important role of cleaving fatty acid from ceramide and producing sphingosine, thereby controlling the interconversion of these two lipids. Thus far, five human ceramidases encoded by five different genes have been identified: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). These ceramidases are classified according to their optimal pH for catalytic activity. AC, which is localized to the lysosomal compartment, has been associated with Farber's disease and is involved in the regulation of cell viability. Neutral ceramidase, which is localized to the plasma membrane and primarily expressed in the small intestine and colon, is involved in digestion, and has been implicated in colon carcinogenesis. ACER1 which can be found in the endoplasmic reticulum and is highly expressed in the skin, plays an important role in keratinocyte differentiation. ACER2, localized to the Golgi complex and highly expressed in the placenta, is involved in programed cell death in response to DNA damage. ACER3, also localized to the endoplasmic reticulum and the Golgi complex, is ubiquitously expressed, and is involved in motor coordination-associated Purkinje cell degeneration. This review seeks to consolidate the current knowledge regarding these key cellular players.

Role of neutral ceramidase in colon cancer.

Alterations in sphingolipid metabolism, especially ceramide and sphingosine 1-phosphate, have been linked to colon cancer, suggesting that enzymes of sphingolipid metabolism may emerge as novel regulators and targets in colon cancer. Neutral ceramidase (nCDase), a key enzyme in sphingolipid metabolism that hydrolyzes ceramide into sphingosine, is highly expressed in the intestine; however, its role in colon cancer has not been defined. Here we show that molecular and pharmacological inhibition of nCDase in colon cancer cells increases ceramide, and this is accompanied by decreased cell survival and increased apoptosis and autophagy, with minimal effects on noncancerous cells. Inhibition of nCDase resulted in loss of β-catenin and inhibition of ERK, components of pathways relevant for colon cancer development. Furthermore, inhibition of nCDase in a xenograft model delayed tumor growth and increased ceramide while decreasing proliferation. It is noteworthy that mice lacking nCDase treated with azoxymethane were protected from tumor formation. Taken together, these studies show that nCDase is pivotal for regulating initiation and development of colon cancer, and these data suggest that this enzyme is a suitable and novel target for colon cancer therapy.-García-Barros, M., Coant, N., Kawamori, T., Wada, M., Snider, A. J., Truman, J.-P., Wu, B. X., Furuya, H., Clarke, C. J., Bialkowska, A. B., Ghaleb, A., Yang, V. W., Obeid, L. M., Hannun, Y. A. Role of neutral ceramidase in colon cancer.

Chemokine Receptors, CXCR1 and CXCR2, Differentially Regulate Exosome Release in Hepatocytes

Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.

Abstract 2479: Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation

Abstract Background: Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in “high risk” disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear. Objectives: In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells. Methods: SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined. Results: Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. Since perturbation in complex sphingolipid levels is associated with apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Curcumin has been demonstrated to induce ROS generation. Pre-treatment with the antioxidants N-acetylcysteine or glutathione abrogated curcumin mediated apoptosis and sphingolipid generation in SMS-KNCR cells. Furthermore, curcumin mediated SMS and GCS inhibition was blocked by these antioxidants. Conclusions: ROS plays a key role in sphingolopid and curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma. Citation Format: Mehrdad Rahmaniyan, Li Li, Julio Garcia, A A. Qudeimat, Jacqueline M. Kraveka. Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2479.