Cell-permeable Ceramide Analog Research Articles

Clostridium perfringens α-toxin up-regulates plasma membrane CD11b expression on murine neutrophils by changing intracellular localization

Gas gangrene caused by Clostridium perfringens type A infection is a highly lethal infection of soft tissue characterized by rapid spread of tissue necrosis. This tissue destruction is related to profound attenuation of blood flow accompanied by formation of platelet-leukocyte aggregates in the blood vessels. Several studies have identified α-toxin, which has both sphingomyelinase and phospholipase C activities, as a major virulence factor in the aggregate formation via activation of the platelet gpIIbIIIa. Here, we show that α-toxin greatly and rapidly increases plasma membrane localization of CD11b, which binds to the platelet gpIIbIIIa via fibrinogen, in mouse neutrophils. Interestingly, short-term treatment of α-toxin has little effect on gene expression profiles in neutrophils, and the toxin does not change the total protein expression levels of CD11b in whole cell lysates. The following analysis demonstrated that CD11b localizes to intracellular vesicles in intact cells, but the localization changed to the cytoplasmic membrane in α-toxin-treated cells. These results suggest that CD11b is recruited to the cytoplasmic membrane by α-toxin. Previously, we reported that α-toxin promotes the formation of ceramide by its sphingomyelinase activity in mouse neutrophils. Interestingly, a synthetic cell-permeable ceramide analog, C2-ceramide, increases plasma membrane localization of CD11b, suggesting that ceramide production by α-toxin recruits CD11b to the cytoplasmic membrane to promote platelet-leukocyte aggregation. Together, our results illustrate that the increase of cell membrane CD11b expression by α-toxin might be crucial for the pathogenesis of C. perfringens to promote formation of platelet-leukocyte aggregates, leading to rapid tissue necrosis due to ischemia.

Clostridium perfringens α-toxin inhibits myogenic differentiation of C2C12 myoblasts

Clostridium perfringens type A is the causative agent of clostridial myonecrosis, and α-toxin has been reported to be responsible for the pathogenesis. Recently, it was reported that regeneration of skeletal muscle after C. perfringens-induced muscle disorders is delayed, but the detailed mechanisms have not been elucidated. Here, we tested whether α-toxin impairs the differentiation of C2C12 myoblasts, a useful cell line to study muscle growth, maturation, and regeneration in vitro. α-Toxin dose-dependently inhibited myotube formation in C2C12 cultures after induction of their differentiation by horse serum. Also, immunoblot analysis revealed that α-toxin dose-dependently decreases the expressions of two skeletal muscle differentiation markers, myogenic differentiation 1 (MyoD) and myogenin. These results demonstrate that α-toxin impairs the myogenic differentiation of C2C12 myoblasts. To reveal the mechanism behind α-toxin-mediated impairment of myogenic differentiation, we focused on ceramide production since α-toxin is known to promote the formation of ceramide by its sphingomyelinase activity. Immunofluorescent analysis revealed that ceramide production is accelerated by treatment with α-toxin. Furthermore, a synthetic cell-permeable ceramide analog, C2-ceramide, inhibited myotube formation in C2C12 cells and decreased the expressions of MyoD and myogenin, suggesting that accelerated ceramide production is involved in the α-toxin-mediated blockage of myogenic differentiation. Together, our results illustrate that the impairment of myogenic differentiation by α-toxin might be crucial for the pathogenesis of C. perfringens to delay regeneration of severely damaged skeletal muscles.

Ceramide Induces Human Hepcidin Gene Transcription through JAK/STAT3 Pathway.

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked with actinomycin D treatment. Reporter assays also confirmed the activation of 0.6 kb HAMP promoter by ceramide. HepG2 cells treated with ceramide displayed increased phosphorylation of STAT3, JNK, and NF-κB proteins. However, ceramide induced the binding of STAT3, but not NF-κB or c-Jun, to HAMP promoter, as shown by the chromatin immunoprecipitation assays. The mutation of STAT3 response element within 0.6 kb HAMP promoter region significantly inhibited the stimulatory effect of ceramide on HAMP promoter activity. Similarly, the inhibition of STAT3 with a pan-JAK kinase inhibitor and STAT3 siRNA pool also diminished the induction of both HAMP promoter activity and mRNA expression by ceramide. In conclusion, we have shown a direct role for ceramide in the activation of hepatic HAMP transcription via STAT3. Our findings suggest a crosstalk between lipid and iron metabolism in the liver, which may contribute to the pathogenesis of obesity-related fatty liver disease.

The thiol proteinase inhibitor E-64-d ameliorates amyloid-β-induced reduction of sAPPα secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells

In Alzheimer’s disease (AD), enhancing α-secretase processing of amyloid precursor protein (APP) is an important pathway to decrease neurotoxic amyloid β (Aβ) secretion. The α-secretase is reported to be regulated by protein kinase C (PKC) and various endogenous proteins or cell surface receptors. In this report, we first examined whether Aβ reduces α-secretase activity, and showed that Aβ peptide 1–40 (0.001 and 0.01μM) reduced the secretion of soluble amyloid precursor protein α (sAPPα) in carbachol-stimulated SH-SY5Y neuroblastoma cells. E-64-d (3μM), which is a potent calpain inhibitor that prevents PKC degradation, ameliorated the Aβ-induced reduction of sAPPα secretion. In addition, we observed that Aβ significantly enhanced ceramide production by activating neutral sphingomyelinase. The cell-permeable ceramide analog, C2-ceramide (1μg/mL), also reduced sAPPα secretion, and in addition, E-64-d eliminated the observed decrease of sAPPα secretion. C2-ceramide induced down-regulation of PKC-α, -β1, and -β2 isozymes in SH-SY5Y cells. These findings suggest that ceramide may play an important role in sAPPα processing by modulating PKC activity.

Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF- B signaling in human platelets

Thrombin activates platelets mainly through protease-activated receptor (PAR)1 and PAR4. However, downstream platelet signaling between PAR1 and PAR4 is not yet well understood. This study investigated the relationship between nSMase/ceramide and the NF-κB signaling pathway in PARs-mediated human platelet activation. The LC-MS/MS, aggregometry, flow cytometry, immunoprecipitation, and mesenteric microvessels of mice were used in this study. Human platelets stimulated by thrombin, 3-OMS (a neutral sphingomyelinase [nSMase] inhibitor) and Bay11-7082 (an NF-κB inhibitor) significantly inhibited platelet activation such as P-selectin expression. Thrombin also activated IκB kinase (IKK)β and IκBα phosphorylation; such phosphorylation was inhibited by 3-OMS and SB203580 (a p38 MAPK inhibitor). Moreover, 3-OMS abolished platelet aggregation, IKKβ, and p38 MAPK phosphorylation stimulated by PAR4-AP (a PAR4 agonist) but not by PAR1-AP (a PAR1 agonist). Immunoprecipitation revealed that nSMase was directly associated with PAR4 but not PAR1 in resting platelets. In human platelets, C24:0-ceramide is the predominant form of ceramides in the LC/MS-MS assay; C24:0-ceramide increases after stimulation by thrombin or PAR4-AP, but not after stimulation by PAR1-AP. We also found that C2-ceramide (a cell-permeable ceramide analog) activated p38 MAPK and IKKβ phosphorylation in platelets and markedly shortened the occlusion time of platelet plug formation in vivo. This study demonstrated that thrombin activated nSMase by binding to PAR4, but not to PAR1, to increase the C24:0-ceramide level, followed by the activation of p38 MAPK-NF-κB signaling. Our results showed a novel physiological significance of PAR4-nSMase/ceramide-p38 MAPK-NF-κB cascade in platelet activation.

P-glycoprotein antagonists confer synergistic sensitivity to short-chain ceramide in human multidrug-resistant cancer cells

P-glycoprotein (P-gp) antagonists inhibit ceramide metabolism at the juncture of glycosylation. The purpose of this study was to test whether targeting P-gp would be a viable alternative to targeting glucosylceramide synthase (GCS) for enhancing ceramide cytotoxicity. A2780 wild-type, and multidrug-resistant 2780AD and NCI/ADR-RES human ovarian cancer cell lines and the cell-permeable ceramide analog, C6-ceramide (C6-cer), were employed. Compared to P-gp-poor A2780 cells, P-gp-rich 2780AD cells converted 3.7-fold more C6-cer to nontoxic C6-glucosylceramide (C6-GC), whereas cell-free GCS activities were equal. 2780AD cells displayed resistance to C6-cer (10 μM) that was reversed by inclusion of the P-gp antagonist tamoxifen (5 μM) but not by inclusion of a GCS inhibitor. Co-administration of C6-cer and P-gp antagonists was also effective in NCI/ADR-RES cells. For example, C6-cer, VX-710 (Biricodar), and cyclosporin A (cyc A) exposure resulted in viabilities of ~ 90% of control; however, C6-cer/VX-710 and C6-cer/cyc A additions were synergistic and resulted in viabilities of 22% and 17%, respectively. Further, whereas C6-ceramide and cyc A imparted 1.5- and 0-fold increases in caspase 3/7 activity, the combination produced a 3.5-fold increase. Although the upstream elements of cell death have not been elucidated, the novel C6-ceramide/P-gp antagonist combination merits further study and assessment of clinical translational potential.

Down-regulation of expression and function of nucleoside diphosphate kinase in insulin-secreting β-cells under in vitro conditions of glucolipotoxicity

Previously, we reported a significant reduction in expression and the activity of nucleoside diphosphate kinase (NDP kinase) in islets derived from the Goto-Kakizaki rat (GK rat), an animal model for type 2 diabetes. Herein, we examined the effects of chronic exposure of insulin-secreting beta-(INS 832/13) cells to high glucose (a model for glucotoxicity), palmitate (a model for lipotoxicity), or glucose plus palmitate (a model for glucolipotoxicity) on the expression and activity of nm23-H1 (NDP kinase A) and nm23-H2 (NDP kinase B). Our findings indicate a marked reduction in the expression of both nm23-H1 and nm23-H2 and the associated NDP kinase activity under each of these conditions. A cell-permeable analog of ceramide (CER) also mimicked the effects of palmitate in significantly reducing the expression of nm23-H1 and nm23-H2 and NDP kinase activity in these cells. These findings suggest that de novo generation of intracellular CER from palmitate might represent at least one of the signaling steps involved in lipid-induced effects on NDP kinase expression and function in beta-cells. Based on these data, we conclude that glucolipotoxic conditions significantly impair expression and function of NDP kinase in pancreatic beta-cells. Potential significance of these findings, specifically at the level of abnormal G-protein activation and impaired insulin secretion under glucolipotoxic conditions is discussed.

A novel non-transcriptional pathway mediates the proconvulsive effects of interleukin-1

Interleukin-1beta (IL-1beta) is overproduced in human and rodent epileptogenic tissue and it exacerbates seizures upon brain application in rodents. Moreover, pharmacological prevention of IL-1beta endogenous synthesis, or IL-1 receptor blockade, mediates powerful anticonvulsive actions indicating a significant role of this cytokine in ictogenesis. The molecular mechanisms of the proconvulsive actions of IL-1beta are not known. We show here that EEG seizures induced by intrahippocampal injection of kainic acid in C57BL6 adult mice were increased by 2-fold on average by pre-exposure to IL-1beta and this effect was blocked by 3-O-methylsphingomyelin (3-O-MS), a selective inhibitor of the ceramide-producing enzyme sphingomyelinase. C2-ceramide, a cell permeable analog of ceramide, mimicked IL-1beta action suggesting that ceramide may be the second messenger of the proconvulsive effect of IL-1beta. The seizure exacerbating effects of either IL-1beta or C2-ceramide were dependent on activation of the Src family of tyrosine kinases since they were prevented by CGP76030, an inhibitor of this enzyme family. The proconvulsive IL-1beta effect was associated with increased Tyr(418) phosphorylation of Src-family of kinases indicative of its activation, and Tyr(1472) phosphorylation of one of its substrate, the NR2B subunit of the N-methyl-d-aspartate receptor, which were prevented by 3-O-MS and CGP76030. Finally, the proconvulsive effect of IL-1beta was blocked by ifenprodil, a selective NR2B receptor antagonist. These results indicate that the proconvulsive actions of IL-1beta depend on the activation of a sphingomyelinase- and Src-family of kinases-dependent pathway in the hippocampus which leads to the phosphorylation of the NR2B subunit, thus highlighting a novel, non-transcriptional mechanism underlying seizure exacerbation in inflammatory conditions.

Ceramide Disrupts HLA Class II‐restricted Antigen Processing and Presentation

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effect of ceramide on antigen processing and HLA class II‐mediated delivery to CD4+ T cells remains unclear. We have previously shown that intracellular ceramide levels can be regulated by ceramide anlogs that directly interfere with ceramide‐metabolizing enzymes such as acid ceramidases. In this study, we examined the actions of these cell permeable ceramide analogs on professional antigen presenting cells (APC) that constitutively express HLA class II proteins and functionally present antigens to CD4+ T cells. Our results show that the treatment of APC with ceramide analogs perturbs Ag processing and presentation in the context of HLA class II molecules. Cellular uptake of ceramide analogs into the endolysosomal compartments did not alter intracellulaer class II protein levels as confirmed by confocal microscopy and western blot analysis. Mass spectroscopic analysis showed that ceramide analogs decreased the activity of acid ceramidase elevating intracellular ceramide levels in the APC. Western blot and flow cytometric analysis confirmed that the ceramide analogs diminished acid ceramidase protein expression in APC, but not cell surface class II molecules. Data also showed that the elevation of intracellular ceramide modulates endolysosomal proteases as well as the components of the HLA class II pathway. These findings suggest that ceramide regulates multiple pathways in APC including antigen processing and presentation via HLA class II molecules.Supported by the Leukemia and Lymphoma Society (3024), American Lung Association (RG‐10435‐N), MUSC HCC Funds and NC1 PO1 CA97132

Thermoresponsive and biodegradable linear-dendritic nanoparticles for targeted and sustained release of a pro-apoptotic drug

Ceramide is a bioactive sphingolipid-derived second messenger that has been demonstrated to induce apoptosis and cell cycle arrest in various cancer cell culture systems. Although in vitro tumor cell culture models have illuminated the potential therapeutic utility of a cell-permeable analog of ceramide, C 6, in vivo delivery is impeded by the extreme hydrophobicity and physical-chemical properties of this bioactive lipid. Previously, we have demonstrated that the incorporation of C 6 into pegylated liposomal vesicles is an effective anti-cancer drug delivery strategy in vitro and in vivo. Here, we report the utilization of a novel multi-functional polymeric drug delivery system designed to therapeutically target C 6 to solid tumor tissue. This delivery system is a hydrolytically degradable and temperature-sensitive linear-dendritic nanoparticle with a lower critical solution temperature (LCST) of 30 °C. C 6 was effectively loaded into the nanoparticles, and released continuously for at least 1 month i n vitro, measured by mass spectroscopy. The preferential uptake of fluorescein isothiocyanate-labeled linear-dendritic nanoparticles into human MDA-MB-231 breast adenocarcinoma cells at temperature above the LCST (37 °C) was confirmed by confocal microscopy and quantified by flow cytometry. The accumulation of NBD-C 6 into MDA-MB-231 cells was highly enhanced by the thermoresponsive linear-dendritic nanoparticles, but not by non-thermoresponsive liposome and PEG-dendritic polymer, at temperature above the LCST (37 °C). The linear-dendritic nanoparticles alone were not toxic, but their complexes with C 6 caused significant growth inhibition and apoptosis to MDA-MB-231 cells at 37 °C. The designed thermoresponsive and biodegradable linear-dendritic nanoparticles have great potential for thermally targeted and sustained release of C 6 for the treatment of solid tumors with hyperthermia.

Ceramide and Adenosine 5′-Monophosphate-Activated Protein Kinase Are Two Novel Regulators of 11β-Hydroxysteroid Dehydrogenase Type 1 Expression and Activity in Cultured Preadipocytes

Increased activity of intracellular glucocorticoid reactivating enzyme, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in obese adipose tissue contributes to adipose dysfunction. As recent studies have highlighted a potential role of preadipocytes in adipose dysfunction, we tested the hypothesis that a variety of metabolic stress mediated by ceramide or AMP-activated protein kinase (AMPK) would regulate 11beta-HSD1 in preadipocytes. The present study is the first to show that 1) expression of 11beta-HSD1 in 3T3-L1 preadipocytes was robustly induced when cells were treated with cell-permeable ceramide analogue C(2) ceramide, bacterial sphingomyelinase, and sphingosine 1-phosphate, 2) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced activation of AMPK augmented the expression and enzyme activity of 11beta-HSD1, and 3) these results were reproduced in human preadipocytes. We demonstrate for the first time that C(2) ceramide and AICAR markedly induced the expression of CCAAT/enhancer-binding protein (C/EBP) beta and its binding to 11beta-HSD1 promoter. Transient knockdown of C/EBPbeta protein by small interfering RNA markedly attenuated the expression of 11beta-HSD1 induced by C(2) ceramide or AICAR. The present study provides novel evidence that ceramide- and AMPK-mediated signaling pathways augment the expression and activity of 11beta-HSD1 in preadipocytes by way of C/EBPbeta, thereby highlighting a novel, metabolic stress-related regulation of 11beta-HSD1 in a cell-specific manner.

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms. Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [ 14CO 2]-liberation. A dominant-negative PPARγ mutant was transfected in Caco-2 cells to suppress PPARγ-mediated functions. Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides ( p < 0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity ( p < 0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc ( p < 0.05) and ODC ( p < 0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARγ-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.

Key Role for Ceramides in Mediating Insulin Resistance in Human Muscle Cells

Elevated non-esterified fatty acids, triglyceride, diacylglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide.

Transcriptional regulation of intronic calcium-activated potassium channel SK2 promoters by nuclear factor-kappa B and glucocorticoids

Small-conductance Ca(2+)-activated K(+) channels (SK) of the SK2 subtype are widely expressed in the central nervous system where they contribute to the control of neuronal excitability. Two SK2 isoforms, SK2-S and SK2-L, the latter representing an N-terminally extended protein of SK2-S, are expressed in similar patterns in the brain. However, our understanding of mechanisms by which the expression of SK2 is regulated is limited. We identified one functional glucocorticoid response element (GRE) at position -2248 bp and two functional nuclear factor-kappB (NF-kappaB) response elements at positions -1652 and -1586 bp in the SK2-S promoter. An increase in SK2-S promoter activity was observed in PC12 cells transiently transfected with a wild-type SK2-S promoter-luciferase reporter gene construct and treated with aldosterone or dexamethasone. The mineralocorticoid receptor (MR) antagonist spironolactone or the glucocorticoid receptor (GR) antagonist mifepristone fully inhibited aldosterone or dexamethasone activation of the SK2-S promoter, respectively. SK2-S promoter activity was also induced by the cell-permeable ceramide analog, N-acetylsphingosine (C2-ceramide). Antisense oligonucleotides directed to NF-kappaB p65 or p50 suppressed SK2-S transcription induced by C2-ceramide. Deletion studies showed that only the -1586 bp NF-kappaB binding site was necessary for maximum C2-ceramide response. Finally, we showed that activation of GRs but not of MRs repressed the NF-kappaB-mediated induction of SK2-S transcription. These findings suggest a possible transcriptional cross talk between GRs and NF-kappaB in the intronic promoter regulation of SK2-S channel gene transcription.

Activation of Protein Phosphatase 2A by Palmitate Inhibits AMP-activated Protein Kinase

Elevated levels of free fatty acids contribute to cardiovascular diseases, but the mechanisms remain poorly understood. The present study was aimed to determine if free fatty acid inhibits the AMP-activated kinase (AMPK). Exposure of cultured bovine aortic endothelial cells (BAECs) to palmitate (0.4 mM) but not to palmitoleic or oleic acid (0.4 mM) for 40 h significantly reduced the Thr(172) phosphorylation of AMPK-alpha without altering its protein expression or the phosphorylation of LKB1-Ser(428), a major AMPK kinase in BAECs. Further, in LKB1-deficient cells, palmitate suppressed AMPK-Thr(172) implying that the inhibitory effects of palmitate on AMPK might be independent of LKB1. In contrast, 2-bromopalmitate, a non-metabolizable analog of palmitate, did not alter the phosphorylation of AMPK and acetyl-CoA carboxylase. Further, palmitate significantly increased the activity of protein phosphatase (PP)2A. Inhibition of PP2A with either okadaic acid, a selective PP2A inhibitor, or PP2A small interference RNA abolished palmitate-induced inhibition on AMPK-Thr(172) phosphorylation. Exposure of BAECs to C(2)-ceramide, a cell-permeable analog of ceramide, mimicked the effects of palmitate. Conversely, fumonisin B1, which selectively inhibits ceramide synthase and decreases de novo formation of ceramide, abolished the effects of palmitate on both PP2A and AMPK. Inhibition of AMPK in parallel with increased PP2A activity was founded in C57BL/6J mice fed with high fat diet (HFD) rich in palmitate but not in mice fed with HFD rich in oleate. Moreover, inhibition of PP2A with PP2A-specific siRNA but not scrambled siRNA reversed HFD-induced inhibition on the phosphorylation of AMPK-Thr(172) and endothelial nitric-oxide synthase (eNOS)-Ser(1177) in mice fed with high fat diets. Taken together, we conclude that palmitate inhibits the phosphorylation of both AMPK and endothelial nitric-oxide synthase in endothelial cells via ceramide-dependent PP2A activation.

Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent

Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 microM (53%; P < 0.05). At 40 microM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 microM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.

Involvement of Sphingolipids in Apoptin-Induced Cell Killing

The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin–ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 μM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.

IL‐1β induces a MyD88‐dependent and ceramide‐mediated activation of Src in anterior hypothalamic neurons

The proinflammatory cytokine interleukin 1beta (IL-1beta), acting at IL-1R1 receptors, affects neuronal signaling under both physiological and pathophysiological conditions. The molecular mechanism of the rapid synaptic actions of IL-1beta in neurons is not known. We show here that within minutes of IL-1beta exposure, the firing rate of anterior hypothalamic (AH) neurons in culture was inhibited. This effect was prevented by pre-exposure of the cells to the Src family inhibitor, PP2, suggesting the involvement of Src in the hyperpolarizing effects of IL-1beta. The IL-1beta stimulation of neurons induced a rapid increase in the phosphorylation of the tyrosine kinase Src and kinase suppressor of Ras (ceramide activated protein kinase (CAPK)/KSR) in neurons grown on glia from IL-1RI(-/-) mice. These effects of IL-1beta were dependent on the association of the cytosolic adaptor protein, MyD88, to the IL-1 receptor, and on the activation of the neutral sphingomyelinase, leading to production of ceramide. A cell-permeable analog of ceramide mimicked the effects of IL-1beta on the cultured AH neurons. These results suggest that ceramide may be the second messenger of the fast IL-1beta actions in AH neurons, and that this IL-1beta/ceramide pathway may underlie the fast non-transcription-dependent, electrophysiological effects of IL-1beta observed in AH neurons in vivo.

Lactoferricin-induced apoptosis in estrogen-nonresponsive MDA-MB-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces caspase-dependent apoptosis in human leukemia and carcinoma cell lines. Ceramide is a second messenger in apoptosis signaling that has been shown to increase the cytotoxicity of various anti-cancer drugs. In this study, we determined whether manipulation of intracellular ceramide levels enhanced LfcinB-induced apoptosis of estrogen-nonresponsive MDA-MB-435 breast carcinoma cells. LfcinB caused DNA fragmentation and morphological changes consistent with apoptosis in MDA-MB-435 breast cancer cell cultures, but did not affect the viability of untransformed mammary epithelial cells. MDA-MB-435 breast cancer cells also exhibited DNA fragmentation and morphological changes consistent with apoptosis following exposure to the cell-permeable ceramide analog C6. An additive increase in DNA fragmentation was observed when both LfcinB and C6 ceramide were added to MDA-MB-435 breast cancer cell cultures. A greater than additive increase in DNA fragmentation was seen when LfcinB was used in combination with tamoxifen, which prevents the metabolism of endogenous ceramide to glucosylceramide by glucosylceramide synthase, as well as blocking estrogen receptor signaling. However, a selective inhibitor of glucosylceramide synthase,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, failed to further increase DNA fragmentation by LfcinB, suggesting that tamoxifen enhanced LfcinB-induced apoptosis in breast cancer cells via a mechanism that did not involve glucosylceramide synthase inhibition. We conclude that combination therapy with LfcinB and tamoxifen warrants further investigation for possible use in the treatment of breast cancer.

C2-ceramide exhibits antiproliferative activity and potently induces apoptosis in endometrial carcinoma

The purpose of the present study was to investigate the effects of an exogenously administered cell-permeable synthetic ceramide analogue, C(2)-ceramide (N-acetyl-sphingosine) on the growth, cell cycle, and death of Ishikawa human endometrial carcinoma cells. We investigated the effects of C(2)-ceramide on Ishikawa endometrial cancer cell lines in vitro. The cells were treated with C(2)-ceramide, and its effects on cell growth, cell cycle, apoptosis, and related measurements were investigated. MTT assays showed that C(2)-ceramide, a cell-permeable analogue of ceramide, significantly induced dose- and time-dependent death in human endometrial carcinoma Ishikawa cells. Cell-cycle analysis indicated that their exposure to C(2)-ceramide decreased the proportion of cells in S phase and increased the proportion in G0/G1 and/or G2/M phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis, including cleavage of poly-ADP ribose polymerase. Furthermore, we demonstrated that the amount of phosphorylated Akt was decreased by C(2)-ceramide. These results raise the possibility that C(2)-ceramide may prove particularly effective in the treatment of endometrial cancers.