Ceramide generated by acidic sphingomyelinase contributes to tumor necrosis factor-alpha-mediated apoptosis in human colon HT-29 cells through glycosphingolipids formation. Possible role of ganglioside GD3

In the present study we assessed the contribution of acidic sphingomyelinase (ASMase), a ceramide generating enzyme, in tumor necrosis factor (TNF)-mediated apoptosis in human colon HT-29 cells. TNF induced apoptosis in HT-29 cells in a time- and dose-dependent fashion. Downregulation of the active endogenous ASMase form prevented TNF-stimulated ASMase activity and apoptosis. Furthermore, inhibition of glucosylceramide synthase, which blunted TNF-stimulated GD3 levels, abolished TNF-mediated cell death. Immunocytochemical staining revealed the co-localization of GD3 with mitochondria induced by TNF. The knockdown of targeted GD3 synthase by antisense expression vector protected HT-29 cells against TNF-induced cell death. Thus, ASMase plays a key role in TNF-induced cell death in human colon epithelial cells possibly through GD3 generation.     

GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion.

Lipid and glycolipid diffusible mediators are involved in the intracellular progression and amplification of apoptotic signals. GD3 ganglioside is rapidly synthesized from accumulated ceramide after the clustering of death-inducing receptors and triggers apoptosis. Here we show that GD3 induces dissipation of DeltaPsim and swelling of isolated mitochondria, which results in the mitochondrial release of cytochrome c, apoptosis inducing factor, and caspase 9. Soluble factors released from GD3-treated mitochondria are sufficient to trigger DNA fragmentation in isolated nuclei. All these effects can be blocked by cyclosporin A, suggesting that GD3 is acting at the level of the permeability transition pore complex. We found that endogenous GD3 accumulates within mitochondria of cells undergoing apoptosis after ceramide exposure. Accordingly, suppression of GD3 synthase (ST8) expression in intact cells substantially prevents ceramide-induced DeltaPsim dissipation, indicating that endogenously synthesized GD3 induces mitochondrial changes in vivo. Finally, enforced expression of bcl-2 significantly prevents GD3-induced mitochondrial changes, caspase 9 activation, and apoptosis. These results show that mitochondria are a key destination for apoptogenic GD3 ganglioside along the lipid pathway to programmed cell death and indicate that relevant GD3 targets are under bcl-2 control.     

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.     

Apoptosis of human breast carcinoma cells in the presence of disialosyl gangliosides: II. Treatment of SKBR3 cells with GD3 and GD1b gangliosides

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le(x) biosyntheses [Basu, S (1991) Glycobiology, 1, 469-475; and ibid, 427-435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of (14)C-L-Serine. At lower concentrations (0-20 microM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both (14)C-sphingolipid and (14)C-ceramide was higher. However, at higher concentrations (20-100 microM), wherein apoptosis occurred in high frequency, the (14)C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death.     

High levels of exogenous C2-ceramide promote morphological and biochemical evidences of necrotic features in thyroid follicular cells

CD95 and ceramide are known to be involved in the apoptotic mechanism. The triggering of CD95 induces a cascade of metabolic events that progressively and dramatically modifies the cell shape by intense membrane blebbing, leading to apoptotic bodies production. Although the CD95 pathway has been abundantly described in normal thyrocytes, the effects of cell permeable synthetic ceramide at morphological and biochemical levels are not fully known. In the present study, we show that thyroid follicular cells (TFC) exposed to 20 microM of C(2)-ceramide for 4 h are characterized by morphological features of necrosis, such as electron-lucent cytoplasm, mitochondrial swelling, and loss of plasma membrane integrity without drastic morphological changes in the nuclei. By contrast, TFC treated with 2 microM of C(2)-ceramide for 4 h are able to accumulate GD3, activate caspases cascade, and induce apoptosis. Furthermore, we provide evidence that 20 microM of C(2)-ceramide determine the destruction of mitochondria and are not able to induce PARP cleavage and internucleosomal DNA fragmentation, suggesting that the apoptotic program is not activated during the death process and nuclear DNA is randomly cleaved as the consequence of cellular degeneration. Pretreatment with 30 microM of zVAD-fmk rescued TFC from 2 microM of C(2)-ceramide-induced apoptosis, whereas, 20 microM of C(2)-ceramide exposure induced necrotic features. Deltapsi(m) was obviously altered in cells treated with 20 microM of C(2)-ceramide for 4 h (75% +/- 3.5%) compared with the low percentage (12.5% +/- 0.4%) of cells with altered Deltapsi(m) exposed to 2 microM of C(2)-ceramide. Whereas, only 20% +/- 1.1% of cells treated with anti-CD95 for 1 h showed altered Deltapsi(m). Additionally, Bax and Bak, two pro-apoptotic members, seem to be not oligomerized in the mitochondrial membrane following ceramide exposure. These results imply that high levels of exogenous ceramide contribute to the necrotic process in TFC, and may provide key molecular basis to the understanding of thyroid signaling pathways that might promote the apoptotic mechanism in thyroid tumoral cells.     

GD3 Prevalence in Adult Rat Retina Correlates with the Maintenance of a High GD3-/GM2-Synthase Activity Ratio Throughout Development

Unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosyl ceramide series but also maintain a prevalence of GD3, a ganglioside of the lactosylceramide series characteristic of proliferative neural cells, when they are fully differentiated. We show here that GD3 is prevalent at all developmental periods of the rat retina from birth [50% of total gangliosidic N-acetylneuraminic acid (NeuNAc)] to adult (30% of total gangliosidic NeuNAc). GD3-synthase specific activity increased about 1.5-fold from birth to day 7 and essentially plateaued thereafter. The GD3-/GM2-synthase specific activity ratio was compared in rat and chicken retina at early and late developmental stages. In chicken retina the ratio was about 0.7 at early (when GD3 is prevalent) and decreased to 0.07 at late (when GD1a is prevalent) developmental stages. In rat retina the ratio was about 13 and 6 at, respectively, early and late developmental stages. These findings suggest that the prevalence of GD3 and of other "b" pathway gangliosides in adult rat retina neurons could be due in part to the maintenance of a high GD3-/GM2-synthase activity ratio throughout development of the tissue.     

Tissue-specific ceramide response in the chronically hypoxic rat model mimicking cyanotic heart disease

BACKGROUND AND OBJECTIVE: Acute hypoxia is associated with apoptosis and increase in ceramide levels in various organs. To assess the effect of chronic hypoxia on ceramide accumulation in the lungs and kidneys, we utilized an animal model mimicking cyanotic heart disease. METHODS: Rats were placed in a hypoxic environment at birth and oxygen levels were maintained at 10% in an air-tight Plexiglas chamber. Controls remained in room air. Animals were sacrificed and the lung and kidneys were harvested and weighed at 1 and 4 weeks, respectively. Ceramide levels were measured using a modified diacylglycerol kinase assay. RESULTS: Significant polycythemia developed in the hypoxic rats at 1 and 4 weeks. Indexed lung and kidney masses were significantly increased in the hypoxic animals as compared to controls at 1 and 4 weeks, respectively. The ceramide levels in the hypoxic lungs and kidneys were not significantly different from control groups at 1 and 4 weeks. [Ceramide/phosphate ratio in the kidneys was 1.28 +/- 0.17 (C) versus 1.18 +/- 0.12 (H) at 1 week; P = 0.39, and 1.46 +/- 0.08 (C) versus 1.33 +/- 0.15 (H) at 4 weeks (P = 0.44)] and [ceramide/phosphate ratio (pmol/nmol) in the lungs was 2.29 +/- 0.14 (C) versus 1.98 +/- 0.12 (H) at 1 week (P = 0.17), and 2.42 +/- 0.16 (C) versus 2.30 +/- 0.05 (H) at 4 weeks, P = 0.34]. CONCLUSION: The response of lungs and kidneys to chronic hypoxia includes increase in indexed mass and lack of ceramide accumulation. This is similar to the response previously reported in the chronically hypoxic brain and heart. Thus, various organs appear to have similar ceramide response pattern to chronic hypoxia.     

A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease

Acid ceramidase (aCDase) is one of several enzymes responsible for ceramide degradation within mammalian cells. As such, aCDase regulates the intracellular levels of the bioactive lipid ceramide. An inherited deficiency of aCDase activity results in Farber disease (FD), also called lipogranulomatosis, which is characterized by ceramide accumulation in the tissues of patients. Diagnosis of FD is confirmed by demonstration of a deficient aCDase activity and the subsequent storage of ceramide. Existing methods include extremely complex assays, many of them using radiolabeled compounds. Therefore, the aCDase assay and the in vitro enzymatic diagnosis of FD are still performed in only a very limited number of specialized laboratories. Here, the new fluorogenic substrate Rbm14-12 was synthesized and characterized as a new tool to determine aCDase activity. The resulting optimized assay was performed in 96-well plates, and different fibroblast and lymphoid cell lines derived from FD patients and controls were tested to measure aCDase activity. As a result, the activity in cells of FD patients was found to be very low or even null. This new fluorogenic method offers a very easy and rapid way for specific and accurate determination of aCDase activity and, consequently, for diagnosis of FD.     

Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts

To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)-/- knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/-) did have a 36% reduced content of qualitatively normal gangliosides (7.0+/-0.8 nmol LBSA/mg cell protein; control: 11+/-1.6 nmol). However, two unexpected findings characterized the homozygous (GM3-/-) MEF. Despite complete knockout of GM3S, (i) GM3-/- MEF retained substantial ganglioside content (21% of normal or 2.3+/-1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc-GM1b, and GD1alpha, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.     

Expression and distribution of S-100 protein, CD83 and apoptosis-related proteins (Fas, FasL and Bcl-2) in thyroid tissues of autoimmune thyroid diseases

Previous studies have shown that dendritic cells (DCs) and apoptosis-related proteins play a critical role in the pathogenesis of autoimmune thyroid diseases (ATD).This study was designed to investigate the expression and distribution of S-100 protein, CD83 and apoptosis-related proteins (Fas, FasL and Bcl-2) in the thyroid tissues of ATD and their role in ATD pathogenesis as determined by immunochemical staing techniques and other methods. Pathological tissues of 30 patients with Hashimoto's thyroiditis (HT), 30 patients with Graves' disease (GD) and 30 cases of thyroid follicular adenoma (TFA, as control) were used for this study. A higher expression of S-100 in HT (4.2+/-3.1%) and GD (3.9+/-2.8%) vs TFA (0.95+/-0.64%) (p<0.001). was observed as well as a higher expression of CD83 in HT (22.58+/-13.96% and GD (29.92+/-14.43%) vs TFA (5.19+/-8.08%) (p<0.001). HT thyrocytes adjacent to thyroid infiltrating lymphocytes (TILs) showed greater increases in the levels of Fas and FasL than did the GD thyrocytes while HT TILs exhibited lower expression of Fas and FasL than did the GD TILs. GD thyrocytes expressed increased levels of the antiapoptotic protein Bcl-2 as compared to the low levels detected in HT thyrocytes. An opposite pattern was observed in the TILs in GD (low expression of Bcl-2) and HT (high expression of Bcl-2). The findings suggest that the high expression of DC markers is related to the pathogenesis of HT and GD. Up-regulation of both the number and matured functions of DCs may lead to the presentation of more antigens to lymphocytes which are related to the development of autoimmune thyroid diseases. The regulation of Fas/FasL/Bcl-2 in GD favors apoptosis of infiltrating lymphocytes and thyrocyte survival. The regulation of Fas/FasL/Bcl-2 in HT may promote thyrocyte apoptosis leading to hypothyroidism.     

Aberrant expression of the insulin-like growth factor-1 receptor by T cells from patients with Graves' disease may carry functional consequences for disease pathogenesis

Graves' disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like growth factor receptor-1 (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD. IGF-1R on fibroblasts, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-16 and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 +/- 4% (mean +/- SE; n = 33) in patients with GD compared with 15 +/- 3% (n = 21; p < 10(-8)) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and IGF-1R+CD4+CD3+ cells as those found in the peripheral circulation. GD-derived peripheral T cells express durable, constitutive IGF-1R expression in culture and receptor levels are further up-regulated following CD3 complex activation. IGF-1 enhanced GD-derived T cell incorporation of BrdU (p < 0.02) and inhibited Fas-mediated apoptosis (p < 0.02). These findings suggest a potential role for IGF-1R displayed by lymphocytes in supporting the expansion of memory T cells in GD.     

JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction leading to cell death, yet remarkably little is known about the mechanisms underlying mitochondrial injury in cerebral ischemia/reperfusion (IR). Ceramide, a naturally occurring membrane sphingolipid, functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. In this study, cerebral IR-induced ceramide elevation resulted from ceramide biosynthesis rather than from hydrolysis of sphingomyelin. Investigation of intracellular sites of ceramide accumulation revealed the elevation of ceramide in mitochondria because of activation of mitochondrial ceramide synthase via post-translational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. The effect of ceramide on mitochondria was somewhat specific; dihydroceramide, a structure closely related to ceramide, did not inflict damage. Stimulation of ceramide biosynthesis seems to be under control of JNK3 signaling: IR-induced ceramide generation and respiratory chain damage was abolished in mitochondria of JNK3-deficient mice, which exhibited reduced infarct volume after IR. These studies suggest that the hallmark of mitochondrial injury in cerebral IR, respiratory chain dysfunction, is caused by the accumulation of ceramide via stimulation of ceramide synthase activity in mitochondria, and that JNK3 has a pivotal role in regulation of ceramide biosynthesis in cerebral IR.     

Critical modifier role of membrane-cystic fibrosis transmembrane conductance regulator-dependent ceramide signaling in lung injury and emphysema

Ceramide accumulation mediates the pathogenesis of chronic obstructive lung diseases. Although an association between lack of cystic fibrosis transmembrane conductance regulator (CFTR) and ceramide accumulation has been described, it is unclear how membrane-CFTR may modulate ceramide signaling in lung injury and emphysema. Cftr(+/+) and Cftr(-/-) mice and cells were used to evaluate the CFTR-dependent ceramide signaling in lung injury. Lung tissue from control and chronic obstructive pulmonary disease patients was used to verify the role of CFTR-dependent ceramide signaling in pathogenesis of chronic emphysema. Our data reveal that CFTR expression inversely correlates with severity of emphysema and ceramide accumulation in chronic obstructive pulmonary disease subjects compared with control subjects. We found that chemical inhibition of de novo ceramide synthesis controls Pseudomonas aeruginosa-LPS-induced lung injury in Cftr(+/+) mice, whereas its efficacy was significantly lower in Cftr(-/-) mice, indicating that membrane-CFTR is required for controlling lipid-raft ceramide levels. Inhibition of membrane-ceramide release showed enhanced protective effect in controlling P. aeruginosa-LPS-induced lung injury in Cftr(-/-) mice compared with that in Cftr(+/+) mice, confirming our observation that CFTR regulates lipid-raft ceramide levels and signaling. Our results indicate that inhibition of de novo ceramide synthesis may be effective in disease states with low CFTR expression like emphysema and chronic lung injury but not in complete absence of lipid-raft CFTR as in ΔF508-cystic fibrosis. In contrast, inhibiting membrane-ceramide release has the potential of a more effective drug candidate for ΔF508-cystic fibrosis but may not be effectual in treating lung injury and emphysema. Our data demonstrate the critical role of membrane-localized CFTR in regulating ceramide accumulation and inflammatory signaling in lung injury and emphysema.     

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.     

Induction of Ganglioside Biosynthesis and Neurite Outgrowth of Primary Cultured Neurons by l-threo-1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol

Abstract: We reported previously that stereoisomers of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the d - threo and l - threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with l - or d - threo -PDMP. These isomers exhibited opposite effects on neurite outgrowth: d -PDMP was inhibitory at concentrations ranging from 5 to 20 µ M , whereas l -PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µ M . Rat neocortical explants were doubly labeled with [¹⁴C]serine and [³H]galactose at 15 µ M l - or d -PDMP. l -PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas d -PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of l -PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N -acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of l -PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides.     

An early increase in the disialoganglioside GD3 contributes to the development of neuronal apoptosis in culture

We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.     

Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells

Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as H(2)O(2), C(6)-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitor dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-d-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by C(6)-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2.     

Poster Session 3

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of β-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of apoptosis that was induced with the novel ceramide analog (NCA) N-oleoyl serinol (S18). This was most likely due to an 80% reduced expression of prostate apoptotis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 MAPK-kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone, which only weakly stained for complex gangliosides. Complex gangliosides may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression. NCAs may be useful to analyse the molecular mechanisms that underlie or counteract ceramide-induced apoptosis during neuronal development.
Acknowledgements:   Supported by grants MH61934-04 (to E.B.) and NS11853 (to R.K.Y.).     

Differential effects of saturated and unsaturated fatty acid diets on cardiomyocyte apoptosis, adipose distribution, and serum leptin

Fatty acids are the primary fuel for the heart and are ligands for peroxisome proliferator-activated receptors (PPARs), which regulate the expression of genes encoding proteins involved in fatty acid metabolism. Saturated fatty acids, particularly palmitate, can be converted to the proapoptotic lipid intermediate ceramide. This study assessed cardiac function, expression of PPAR-regulated genes, and cardiomyocyte apoptosis in rats after 8 wk on either a low-fat diet [normal chow control (NC); 10% fat calories] or high-fat diets composed mainly of either saturated (Sat) or unsaturated fatty acids (Unsat) (60% fat calories) (n = 10/group). The Sat group had lower plasma insulin and leptin concentrations compared with the NC or Unsat groups. Cardiac function and mass and body mass were not different. Cardiac triglyceride content was increased in the Sat and Unsat groups compared with NC (P < 0.05); however, ceramide content was higher in the Sat group compared with the Unsat group (2.9 +/- 0.2 vs. 1.4 +/- 0.2 nmol/g; P < 0.05), whereas the NC group was intermediate (2.3 +/- 0.3 nmol/g). The number of apoptotic myocytes, assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining, was higher in the Sat group compared with the Unsat group (0.28 +/- 0.05 vs. 0.17 +/- 0.04 apoptotic cells/1,000 nuclei; P < 0.04) and was positively correlated to ceramide content (P < 0.02). Both high-fat diets increased the myocardial mRNA expression of the PPAR-regulated genes encoding uncoupling protein-3 and pyruvate dehydrogenase kinase-4, but only the Sat diet upregulated medium-chain acyl-CoA dehydrogenase. In conclusion, dietary fatty acid composition affects cardiac ceramide accumulation, cardiomyocyte apoptosis, and expression of PPAR-regulated genes independent of cardiac mass or function.     

Ceramide increases steroid hormone production in MA-10 Leydig cells.

Ceramide is known to have major roles in the control of cell proliferation, differentiation, and apoptosis. Recent studies also have shown that ceramide affects steroid production by JEG-3 choriocarcinoma cells, acutely dispersed rat Leydig cells, and ovarian granulosa cells, but the mechanism by which this occurs is unknown. Because ceramide induces apoptosis in many different cell types, we hypothesized that ceramide might affect steroidogenesis and/or induce apoptosis in MA-10 murine Leydig cells. To test this, MA-10 cells were incubated with either the water soluble C2-ceramide, (N-acetyl-sphingosine, 0.01-10 cm); bacterial sphingomyelinase (1-100 mU/ml); or C2-dihydroceramide (N-acetyl-sphinganine, 0.1-10 microM). The data show that N-acetyl-sphingosine significantly increased basal (0.87 +/- 0.2 vs. 0.42 +/- 0.09 ng/mg cell protein, P < 0.01) and human chorionic gonadotropin (hCG) stimulated progesterone (P) synthesis (204 +/- 12 vs. 120 +/- 5 ng/mg cell protein, P < 0.001); as did sphingomyelinase (basal P = 0.83 +/- 0.1 ng/mg cell protein, P < 0.01; hCG stimulated P = 173 +/- 7 ng/mg cell protein, P < 0.001). C2-dihydroceramide also increased basal P synthesis but was less effective than ceramide on a molar basis. Neither sphingomyelinase (100 mU/ml) nor ceramide (10 microM) had any effect on cAMP production or human chorionic gonadotropin binding; and neither induced any signs of apoptosis (FragEL DNA fragmentation assay and electron microscopy). Cells incubated with anti-Fas (300 ng/ml) demonstrated DNA fragmentation, nuclear condensation, and frequent apoptotic bodies, but had no change in P synthesis. These data show that ceramide significantly increases MA-10 Leydig cell P synthesis but does not induce apoptosis. The mechanism by which ceramide increases steroid hormone synthesis remains unknown but does not appear to be linked to the induction of apoptosis in MA-10 cells.