Neutral Sphingomyelinase Activity Research Articles

421 - Changes in Ceramide Levels in Various Diseases Are Associated with Oxidative Stress

Ceramide is a bioactive lipid that induces cellular apoptosis, proliferation, and differentiation. Deficiency of acid sphingomyelinase, which synthesizes ceramide, has been reported to result in smaller aortic root lesions in Western diet-fed Ldl -/- mice. Thus, ceramide is predicted to play a role in atherosclerosis pathogenesis. Therefore, using animal models, we investigated whether ceramide levels were affected by oxidative stress in diseases such as atherosclerosis, diabetes, and fulminant hepatitis. The experiments were performed under acute oxidative stress caused by carbon tetrachloride (CCl 4 ) intoxication and chronic oxidative stress in diabetic rats and in aged apolipoprotein E knock out (apoE -/- ) mice. Total ceramide concentrations significantly increased in the liver, brain, kidney, and plasma of CCl 4 -intoxicated rats. This increase was primarily derived from an increase in neutral sphingomyelinase activity in the liver 2 hours after intoxication with CCl 4 and was consistent with the decrease in the GSH/GSSG ratio in the liver. In contrast, secretory sphingomyelinase was stimulated during prolonged oxidative stress associated with diabetes and atherosclerosis. After 2 weeks of streptozotocin administration, plasma secretory sphingomyelinase activity was significantly increased, resulting in elevated ceramide concentrations, whereas neutral sphingomyelinase activity was unchanged in the kidney and liver. Plasma ceramide concentrations and secretory sphingomyelinase activity also increased in aged apoE -/- mice. In conclusion, ceramide levels increased in both acute and chronic oxidative stress-related diseases; however, the enzymes responsible for these changes differed. Based on these results, we propose that antioxidants can prevent ceramide production and ameliorate the exacerbation of oxidative stress-related diseases.

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.

Role of sphingomyelinase in mitochondrial ceramide accumulation during reperfusion

Ceramide accumulation in mitochondria has been associated with reperfusion damage, but the underlying mechanisms are not clearly elucidated. In this work we investigate the role of sphingomyelinases in mitochondrial ceramide accumulation, its effect on reactive oxygen species production, as well as on mitochondrial function by using the sphingomyelinase inhibitor, tricyclodecan-9-yl-xanthogenate (D609). Correlation between neutral sphingomyelinase (nSMase) activity and changes in ceramide content were performed in whole tissue and in isolated mitochondria from reperfused hearts. Overall results demonstrated that D609 treatment attenuates cardiac dysfuncion, mitochondrial injury and oxidative stress. Ceramide was accumulated in mitochondria, but not in the microsomal fraction of the ischemic-reperfused (I/R) group. In close association, the activity of nSMase increased, whereas glutathione (GSH) levels diminished in mitochondria after reperfusion. On the other hand, reduction of ceramide levels in mitochondria from I/R+D609 hearts correlated with diminished nSMase activity, coupling of oxidative phosphorylation and with mitochondrial integrity maintenance. These results suggest that mitochondrial nSMase activity contributes to compartmentation and further accumulation of ceramide in mitochondria, deregulating their function during reperfusion.

Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase

BackgroundClostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK) cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization. MethodsWe tested the role of nSMase in the oligomerization of epsilon-toxin using specific inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining. ResultsEpsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in ACHN cells. GW4869, an inhibitor of nSMase, inhibited ceramide production induced by the toxin. GW4869 and knockdown of nSMase blocked toxin-induced cell death and oligomer formation of epsilon-toxin. Confocal microscopy images showed that the toxin induced ceramide clustering and colocalized with ceramide. ConclusionsThese results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of nSMase caused by the toxin. General significanceInhibitors of nSMase may confer protection against infection.

37 Imbalance of bioactive sphingolipids rheostat in preeclampsia

Introduction Preeclampsia (PE) affects 5–8% of all pregnancies worldwide and is a leading cause of maternal and fetal morbidity and mortality. It is manifested in 2nd trimester by new onset maternal hypertension, proteinuria, edema, and in severe cases, seizures and end-organ failure. PE is often associated with fetal complications such as intrauterine growth restriction (IUGR), stillbirth, birth hypoxia and impaired neurodevelopment. The physiopathological process of PE begins with inadequate trophoblast invasion early in pregnancy (1st trimester) and reduced uteroplacental perfusion that results in local oxidative stress and maternal systemic inflammatory response. There is growing evidence for the involvement of bioactive sphingolipids, angiogenic and pro-survival Sphingosine-1-phosphate (S1P) and pro-apoptotic Ceramide (Cer) and their synthesizing/metabolizing enzymes in trophoblast differentiation and invasion, uterine angiogenesis; all the cellular and physiological processes necessary to establish healthy placenta. We hypothesize that dysregulation of sphingolipid homeostasis (Cer-S1P rheostat) during early placentation process may have effect on downstream signaling and may contribute to the onset of the disease. Objectives Demonstrate differential expression and enzymatic activity of key sphingolipids’ enzymes (SPHK, NSMase) in in vitro and ex vivo model of placental hypoxia (JAR cells or villous explants +/− hypoxia) and in term placentae and term chorionic villous explants from PE and normotensive patients. Methods The human term placentae and chorionic villous tissue were obtained from PE and normotensive patients (Clinica Davila, Santiago, Chile). The hypoxia was induced in JAR cells (human choriocarcinoma) and villous explants by oxygen deprivation (1% or 8% O2) for 3 up to 24h. The normoxic conditions (21% O2) were used as a control. Protein and mRNA levels in human placental and chorionic tissues were determined by Western blot and real-time PCR, respectively. Neutral sphingomyelinase (NSMase) activity was measured on HPLC by using labelled exogenous substrate (6-NBD-sphingomyelin). Results Our results demonstrate that synthetic/metabolic pathways of S1P and Cer are altered in human term placentae and placental chorionic villous explants of preeclamptic patients as compared to normotensive subjects. Angiogenic Sphingosine kinase-1 (SPHK1) and Sphingosine-1-phosphate receptors (S1PR1, −3) mRNA and protein are down-regulated in PE placentae and villous explants, suggesting that they might be involved in challenged neo-angiogenesis observed in preeclampsia. Moreover, in vitro and ex vivo studies clearly show up-regulation of SPHK1 under low oxygen tension (1% O 2 ) in contrast to lack of SPHK1 stimulation under the conditions of placental physiological hypoxia (8% O 2 ). This may indicate that SPHK and its product, S1P might be an important angiogenic mediators during early placentation process, which is severely affected in PE. On the other hand, ceramide synthase 5 (CerS5) of de novo pathway is increased in PE placentae, while Neutral sphingomyelinase-2 (NSMase-2) protein expression and enzymatic activity is decreased in PE placental speicemen. These results suggest that pro-apoptotic Cer is augmented via de novo pathway in PE complicated pregnancies, while NSMase-2 derived ceramide may serve other functions. Conclusion We postulate that bioactive sphingolipids and their synthetic/metabolic pathways may contribute to pathogenesis of preeclampsia and may serve in the future as novel early biomarkers of this disease.

Biochemical and biophysical methodologies open the road for effective schistosomiasis therapy and vaccination

BackgroundSchistosomiasis caused by blood-dwelling flukes, namely Schistosoma mansoni and Schistosoma haematobium is a severe debilitating disease, widespread in sub-Saharan Africa, the Middle East, and South America. Developing and adult worms are unscathed by the surrounding immune effectors and antibodies because the parasite is protected by a double lipid bilayer armor which allows access of nutrients, while binding of specific antibodies is denied. Scope of reviewFluorescence recovery after bleaching, extraction of surface membrane cholesterol by methyl-β-cyclodextrin, inhibition and activation of sphingomyelin biosynthesis and hydrolysis, and elastic incoherent and quasi-elastic neutron scattering approaches have helped to clarify the basic mechanism of this immune evasion, and showed that sphingomyelin (SM) molecules in the worm apical lipid bilayer form with surrounding water molecules a tight hydrogen bond barrier. Viability of the parasite and permeability of the outer shield are controlled by equilibrium between SM biosynthesis and activity of a tegument-associated neutral sphingomyelinase (nSMase). Major conclusionsExcessive nSMase activation by polyunsaturated fatty acids (PUFA), such as arachidonic acid (ARA) leads to disruption of the SM molecules and associated hydrogen bond network, with subsequent access of host antibodies and immune effectors to the outer membrane and eventual parasite death. General significanceARA was predicted and shown to be a potent schistosomicide in vitro and in vivo in experimental animals and in children. Additionally, it was advocated that schistosomiasis vaccine candidates should be selected uniquely among excretory–secretory products of developing worms, as contrary to cytosolic and surface membrane antigens, they are able to activate the effector functions of the host antibodies and toxic molecules. This article is part of a Special Issue entitled “Science for Life” Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo“.

Targeting acid sphingomyelinase reduces cardiac ceramide accumulation in the post-ischemic heart

Ceramide accumulation is known to accompany acute myocardial ischemia, but its role in the pathogenesis of ischemic heart disease is unclear. In this study, we aimed to determine how ceramides accumulate in the ischemic heart and to determine if cardiac function following ischemia can be improved by reducing ceramide accumulation.To investigate the association between ceramide accumulation and heart function, we analyzed myocardial left ventricle biopsies from subjects with chronic ischemia and found that ceramide levels were higher in biopsies from subjects with reduced heart function. Ceramides are produced by either de novo synthesis or hydrolysis of sphingomyelin catalyzed by acid and/or neutral sphingomyelinase. We used cultured HL-1 cardiomyocytes to investigate these pathways and showed that acid sphingomyelinase activity rather than neutral sphingomyelinase activity or de novo sphingolipid synthesis was important for hypoxia-induced ceramide accumulation. We also used mice with a partial deficiency in acid sphingomyelinase (Smpd1+/− mice) to investigate if limiting ceramide accumulation under ischemic conditions would have a beneficial effect on heart function and survival. Although we showed that cardiac ceramide accumulation was reduced in Smpd1+/− mice 24h after an induced myocardial infarction, this reduction was not accompanied by an improvement in heart function or survival.Our findings show that accumulation of cardiac ceramides in the post-ischemic heart is mediated by acid sphingomyelinase. However, targeting ceramide accumulation in the ischemic heart may not be a beneficial treatment strategy.

Lovastatin reversed the enhanced sphingomyelin caused by 27-hydroxycholesterol in cultured vascular endothelial cells

Statins have pleiotropic properties which are involved in inhibiting the thrombogenic response. In this study, the effects of lovastatin on two phospholipids, phosphatidylcholine and sphingomyelin, were studied in cultured endothelial cells in the presence of an oxysterol, 27-hydroxycholesterol. After the cells were cultured with 50nM of lovastatin for 60h, lovastatin was found to decrease the incorporation of [3H]choline into phosphatidylcholine and sphingomyelin, inhibited CTP: phosphocholine cytidylyltransferase (CT) activity without altering the activity of sphingomyelin synthase and neutral sphingomyelinase. And lovastatin was not found to have a direct inhibitive effect on activity of CT. Exogenous mevalonic acid or cholesterol reversed the reduction of cholesterol concentration that was caused by lovastatin, but had no significant effect on the diminished [3H]sphingomyelin by lovastatin. The increase of [3H]sphingomyelin by 27-hydroxycholesterol was not detected in the presence of lovastatin. These findings suggest that (1) lovastatin can reduce sphingomyelin content by means of inhibiting phosphatidylcholine synthesis; and (2) The decrease in sphingomyelin is not related to the diminished cholesterol concentration or mevalonate-derived intermediates. This inhibitive effect of lovastatin on sphingomyelin may benefit cellular calcification caused by sphingomyelin.

TNF-R1, an immune checkpoint in melanoma?

Through binding to and activation of two receptors, TNF-R1 and TNF-R2, Tumor Necrosis Factor α (TNF) modulates various biological processes in the immune system. A growing body of evidence in the literature indicates that TNF may behave as a tumorigenic cytokine, facilitating cancer cell immune escape and tumor progression. We have recently provided evidence that host TNF signaling impairs CD8+ T cell-dependent immune response against melanoma. TNF blockade (with Etanercept) or deficiency (TNF knock out) facilitates the accumulation of CD8+ Tumor-Infiltrating Lymphocytes (TILs), thereby limiting the growth of melanoma cell lines, which express Major Histocompatibility Class 1 molecules (MHCI) at high levels. Similar findings were observed in mice lacking TNF-R1, but not TNF-R2, indicating that host TNF-R1 plays a critical role in limiting the establishment of such a CD8+ T cell-dependent immune response against melanoma under our experimental conditions [1]. We and others have documented that the factor associated with neutral sphingomyelinase activation (FAN), which is an adaptor protein of the TNF-R1 [2], enhances TNF-induced apoptosis [3], leukocyte migration [4] and antibody immune response towards a thymo-dependent antigen [5]. In addition, downregulation of FAN in B16 melanoma cells decreases TNF-induced B16 melanoma motility and invasion [6]. We recently investigated the consequences of host FAN deficiency on the tumor growth of B16 melanoma cells. In sharp contrast to what we observed in mice lacking either TNF or TNF-R1, the tumor growth of B16K1 cells was not impaired in FAN-deficient mice as compared to their wild-type counterparts (Montfort and Segui, unpublished data). The latter observation suggests that the host TNF-R1 signaling, which likely represents an immune checkpoint facilitating B16K1 melanoma growth, does not critically involve the FAN-dependent neutral sphingomyelinase activation. The role of FAN may thus be restricted to some, but not all, TNF-dependent immune responses. Accordingly, the TNF-dependent anti-infectious immune response towards Listeria monocytogenes is not compromised in mice lacking FAN [5]. One of the main mechanisms that likely accounts for the TNF-dependent immunosuppression in our experimental mouse melanoma model is the capacity of TNF to trigger cell death of activated CD8+ T cells. Whereas TNF potently induced cell death of activated CD8+ T cells as documented by others, it remained unclear which TNF receptor is required for this process. On the one hand, activated CD8+ T cells expressed TNF-R2, which mediated activation-induced cell death [7]. On the other hand, exogenous TNF elicited cell death in concanavalin-A activated CD8+ T cells in a TNF-R1-dependent manner [8]. Of note, the latter study was carried out in the presence of cycloheximide to artificially sensitize lymphocytes to TNF. The use of cycloheximide may be a source of artefact and is unlikely appropriate to evaluate the physiological role of TNF and its receptors in CD8+ T cell death. Under our experimental conditions in the absence of cycloheximide, TNF-induced cell death of activated CD8+ T cells occurred mainly through a TNF-R1-dependent process [1]. This conclusion is supported by the following findings: (i) naive CD8+ T cells, which expressed TNF-R2 but not TNF-R1, resisted TNF-induced cell death; (ii) activated CD8+ T cells, which significantly expressed both TNF-R1 and TNF-R2, were sensitive to exogenous TNF; (iii) TNF-R1-deficient CD8+ T cells were fully resistant to TNF-induced cell death; (iv) TNF-R2 deficiency minimally affected CD8+ T cell death in response to TNF. The role of TNF-R1 as an immune checkpoint in melanoma gets further credence as illustrated by our data showing that the accumulation of activated CD8+ T cells into melanoma was facilitated by TNF-R1 deficiency in an adoptive transfer experiment performed in CD8-deficient hosts [1] (Figure ​(Figure11). Figure 1 TNF triggers TNF-R1-dependent cell death of activated CD8+ T cells, limiting their tumor accumulation in melanoma Our recent findings demonstrate that TNF-R1-dependent TNF signaling impairs CD8+ TIL content and likely constitutes a potent immune checkpoint in melanoma, facilitating melanoma progression. Targeting either TNF or TNF-R1 may reduce the death of activated CD8+ TILs in patients affected with melanoma and thus may constitute an emerging immunotherapy to enhance CD8+ T cell-dependent immune response against melanoma. Clinical trials are necessary to evaluate the therapeutic value of TNF blockade strategy in melanoma patients and define the eligibility criteria such as (i) TNF expression in melanoma biopsies, (ii) pre-existing CD8+ T cell infiltration, (iii) MHCI expression by melanoma cells.

New oleyl glycoside as anti-cancer agent that targets on neutral sphingomyelinase

We designed and synthesized two anomeric oleyl glucosaminides as anti-cancer agents where the presence of a trifluoroacetyl group close to the anomeric center makes them resistant to hydrolysis by hexosaminidases. The oleyl glycosides share key structural features with synthetic and natural oleyl derivatives that have been reported to exhibit anti-cancer properties. While both glycosides showed antiproliferative activity on cancer cell lines, only the α-anomer caused endoplasmic reticulum (ER) stress and cell death on C6 glioma cells. Analysis of sphingolipids and glycosphingolipds in cells treated with the glycosides showed that the α-anomer caused a drastic accumulation of ceramide and glucosylceramide and reduction of lactosylceramide and GM3 ganglioside at concentrations above a threshold of 20μM. In order to understand how ceramide levels increase in response to α-glycoside treatment, further investigations were done using specific inhibitors of sphingolipid metabolic pathways. The pretreatment with 3-O-methylsphingomyelin (a neutral sphingomyelinase inhibitor) restored sphingomyelin levels together with the lactosylceramide and GM3 ganglioside levels and prevented the ER stress and cell death caused by the α-glycoside. The results indicated that the activation of neutral sphingomyelinase is the main cause of the alterations in sphingolipids that eventually lead to cell death. The new oleyl glycoside targets a key enzyme in sphingolipid metabolism with potential applications in cancer therapy.

The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization

Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein–protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

Ceramide Regulates MRN Complex‐ATM‐Dependent Apoptotic Pathway in Human Lymphoblastoids

Ceramide is a key lipid second messenger for apoptotic stresses. However, it remains unknown how ceramide is involved in genotoxic apoptotic pathway such as DNA double-strand breaks (DSBs). Ataxia telangiectasia mutated (ATM) plays crucial roles in cellular responses including DNA repair, cell cycle control and apoptosis following DNA DSBs. We herein demonstrated the requirement of ceramide for ATM signaling in genotoxic drug neocarzinostatin (NCS)-induced apoptotic pathway in lymphoblastoids. NCS treatment produces ceramide by synergistic effect of neutral sphingomyelinase (SMase) activation and sphingomyelin synthase (SMS) inactivation. The ceramide production activates ATM and leads to caspase-3-dependent apoptosis. Nuclear study indicates that ceramide regulates ATM activity through MRN (Mre11/ Rad50/NBS1) complex that is a primary factor to detect DSBs (figure). Thus, we demonstrate that ceramide acts as an early trigger for the activation of MRN complex-ATM pathway in nuclear upon DNA damage stress, and finally exert caspase-3-dependent apoptosis.

Does prostaglandin-E1 modulate d-galactosamine induced cell death in primary culture of human hepatocytes?

Background: Cell death pathway can occur under physiological or pathological conditions. In vitro and in vivo studies have shown that d-galactosamine (DGA) induces hepatocyte damage. Objective: The present study aims to evaluate the protective effect of prostaglandin E1 (PGE1) on DGA-induced apoptosis, necrosis and oxidative stress in primary culture of human hepatocytes. Methods: Normal human hepatocytes were obtained from the safety margin of liver specimens, removed during hepatectomy operation to liver cancer patients, and isolated using the classical collagenase perfusion method. After culture stabilization, PGE1 (1μM) was added 2h before DGA (5mM). Cultures were maintained for 24h before the parameters for apoptosis, necrosis and oxidative stress were measured. Apoptosis was studied by DNA-fragmentation, neutral (nSMase) and acid (aSMase) sphingomyelinase and caspase-3 activity. Necrosis was investigated by lactate dehydrogenase (LDH) and transaminases (ALT & AST) enzymes. The oxidative stress was assessed by malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSH), glutathione S-transferase (GST), glutathione peroxidase (GSPx), catalase (CAT), superoxide dismutase (SOD) and nitric oxide (NO). Results: The hepatotoxin DGA induced apoptosis and enhanced all parameters related to necrosis and intracellular oxidative stress. On the other hand, PGE1 reduced the measured values for the parameters indicative for the DAG induced apoptosis, necrosis and oxidative stress. In addition, PGE1 proved also to prevent GSH depletion. The obtained results provided evidences for the biochemical hepatotoxic effects of DGA (5mM) especially through the induction of apoptosis, necrosis and oxidative stress alterations in the cultured human hepatocytes. Conclusion: PGE1 could be a useful protective treatment against DGA-induced hepatocyte cell death.

Inhibitory Effects of Hybrid Liposomes on the Growth of Non-small Cell Lung Carcinoma Cells and Anti-invasive Activity by Ceramide Generation without any Drugs

Ceramide generation and invasive inhibition by hybrid liposomes (HL) composed of 90 mol% L-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene (23) dodecyl ether (C12(EO)23) were examined for human lung cancer (A549) cells in vitro. Ceramide generation for A549 cells treated with HL was observed through the activation of neutral sphingomyelinase. These was a reduction in sphingomyelin in A549 cells after treatment with HL. The anti-invasive effects of HL for A549 cells were obtained through the inhibition of MT1MMP/MMP14. Inhibitory effects of HL on the migration of A549 cells was obtained using scratch assay.

Hyperthyroidism Evokes Myocardial Ceramide Accumulation

Background: Thyroid hormones (THs) are key regulators of cardiac physiology as well as modulators of different cellular signals including the sphingomyelin/ceramide pathway. The objective of this study was to examine the effect of hyperthyroidism on the metabolism of sphingolipids in the muscle heart. Methods: Male Wistar rats were treated for 10 days with triiodothyronine (T₃) at a dose of 50µg/100g of body weight. Animals were then anaesthetized and samples of the left ventricle were excised. Results: We have demonstrated that prolonged, in vivo, T₃ treatment increased the content of sphinganine (SFA), sphingosine (SFO), ceramide (CER) and sphingomyelin (SM), but decreased the level of sphingosine-1-phosphate (S1P) in cardiac muscle. Accordingly, the changes in sphingolipids content were accompanied by a lesser activity of neutral sphingomyelinase and without significant changes in ceramidases activity. Hyperthyroidism also induced activation of AMP-activated protein kinase (AMPK) with subsequently increased expression of mitochondrial proteins: cytochrome c oxidase IV (COX IV), β-hydroxyacyl-CoA dehydrogenase (β-HAD), carnityne palmitoyltransferase I (CPT I) and nuclear peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α). Conclusions: We conclude that prolonged T₃ treatment increases sphingolipids metabolism which is reflected by higher concentration of SFA and CER in heart muscle. Furthermore, hyperthyroidism-induced increase in heart sphingomyelin (SM) concentration might be one of the mechanisms underlying maintenance of CER at relatively low level by its conversion to SM together with decreased S1P content.

Fingolimod Expands Human Umbilical Cord Blood Cells (UCB) in Vitro and Improves Engraftment Rate of Human UCB in Sublethally Irradiated NOD SCID Gamma (NSG) Mice

Background: Umbilical cord blood transplantation (UCBT) has emerged as an alternative for allogeneic hematopoietic stem cell transplantation for patients who don’t have a matched donor. However, cell dose remains an obstacle to successful UCBT and strategies aimed at improving rate and speed of engraftment are needed. Acid sphingomyelinase (ASM), a glycoprotein enzyme that converts sphingomyelin to ceramide, was recently shown to modulate inflammatory and cell stress responses. In vitro treatment with docosahexaenoic acid (DHA), an ASM inhibitor, restores colony formation ability of CD34+ cells from diabetic patients and that paralleled reduction of ASM in such cells (Tikhonenko et al. PLoS One 2013). Fingolimod (FTY720) is a novel immunosuppressant that is FDA approved for the treatment of relapsing multiple sclerosis. Fingolimod was shown to inhibit ASM activity in vitro through a mechanism that is similar to desipramine (a known inhibitor of ASM) yet at much lower micromolar concentrations (Dawson et al. Biochem Biophys Res Commun 2011).Hypothesis: Fingolimod expands UCB cells and improves engraftment of UCB cells in NSG mice.Methods: CD34+ cells, separated by magnetic-activated cell sorting (MACS) from pooled human UCB units, were cultured for 72 hours at 37 °C in RPMI-1640 + 10% FBS + 100 ng/ml each of SCF, TPO, and FL. Cells were be divided into 3 plates; the third plate had fingolimod added to culture (at 1 uM concentration) and the first 2 plates had no fingolimod added. Sphingomyelinase activity was measured using the Amplex Red Sphingomyelinase Assay Kit according to the manufacture protocol (Molecular Probes, Eugene, OR). Cells were then plated into methylcellulose for 7-10 days before colony forming units (CFU) were counted. Cells from each plate were injected into a corresponding group of sublethally irradiated (225cGy) NSG mice (n=5 per group) as follows: group 1 was injected with control cells, group 2 was injected with control cells, but mice received 5 mcg/kg/day fingolimod by intraperitoneal injection starting on the day of transplant for 6 weeks, and group 3 was injected with cells cultured with fingolimod. Engraftment of human cells was measured by flow cytometry for CD45 surface marker in peripheral blood of recipient mice at 3 weeks and 8 weeks. Engraftment rate of human UCB cells in NSG mice was estimated using a mixed statistical model.Results: Adding fingolimod to cell culture resulted in a robust expansion of UCB cells as evident by doubling in CFU (p<0.0001). Fingolimod also decreased acid, but not neutral sphingomyelinase activity by 25%. In NSG mice, the engraftment rate for control group was 0.106, which was not statistically significant from zero (p = 0.104). The slope for fingolimod treated group was 0.106, which was not significant from zero, either (p = 0.104). However, the slope for fingolimod injected group was 0.197, which was significant from zero (p = 0.0066) as shown in figure below.Conclusions: Fingolimod expands CFU activity of human UCB cells in culture. In addition, in vivo treatment of NSG mice with fingolimod accelerates engraftment of human CD34+ selected UCB cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Involvement of neutral sphingomyelinase in the angiotensin II signaling pathway.

The possibility that angiotensin II (ANG II) exerts its effects through the activation of neutral sphingomyelinase (nSMase) has not been tested in kidneys. The results of the present study provide evidence for the activity and expression of nSMase in rat kidneys. In isolated perfused rat kidney, ANG II-induced renal vasoconstriction was inhibited by GW4869, an inhibitor of nSMase. We used nSMase for investigating the signal transduction downstream of ceramide. nSMase constricted the renal vasculature. An inhibitor of ceramidase (CDase), N-oleoylethanolamine (OEA), enhanced either ANG II- or nSMase-induced renal vasoconstriction. To demonstrate the interaction between the nSMase and cytosolic phospholipase A2 (cPLA2) signal transduction pathways, we evaluated the response to nSMase in the presence and absence of inhibitors of arachidonic acid (AA) metabolism: arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2; 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of all AA pathways; indomethacin, an inhibitor of cyclooxygenase (COX); furegrelate, a thromboxane A2 (TxA2)-synthase inhibitor; and SQ29548, a TxA2-receptor antagonist. In these experiments, the nSMase-induced renal vasoconstriction decreased. ANG II or nSMase was associated with an increase in the release of thromboxane B2 (TxB2) in the renal perfusate of isolated perfused rat kidney. In addition, the coexpression of the ceramide with cPLA2, was found in the smooth muscle layer of intrarenal vessels. Our results suggest that ANG II stimulates ceramide formation via the activation of nSMase; thus ceramide may indirectly regulate vasoactive processes that modulate the activity of cPLA2 and the release of TxA2.

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6h followed by a more rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes. In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which peaked at 3h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing of SMPD3 gene encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in curcumin-treated cells, as all these events were inhibited by GW4869 or nSMase2 depletion. These data indicate that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling.

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.

Activation of neutral-sphingomyelinase, MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester-induced apoptosis in C6 glioma cells.

BackgroundCaffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.ResultsC6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.ConclusionsTaken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.